foods and diets in disease

Foods and Diets in Health

Part 1: Basic food science

Part 2: Diet and disease

Part 3: Foods, diets and disease

A compilation of lecture series delivered for

Food Science at College of Human Health, FSU, Tallahassee

Innovations And Solutions Inc.

3945 West Pensacola Street, Tallahassee, Florida 32304

Web-based Selected Lecture Series

Foods and Diets in Health Part 3: Foods, diets and disease

________________________________________________

Course Number: Human Health HU 4657 and BCE 4003c and 4004c Florida State University and Florida A&M University Elective Course at: Maharana Pratap A&T University, Udaipur, Rajasthan, and Uttar Pradesh Technical University, Lucknow, Uttar Pradesh, India Part 1: Basic food science Part 2: Diet and disease Part 3: Foods, diets and disease Editors: Rakesh Sharma,Ph.D, M.Sc-Ph.D(IIT-D) Bharati D Shrinivas, Ph.D(Nagpur)

Sponsoring Institution: Innovations and Solutions Inc. USA(R) Address: Center of Nanoscience and Biotechnology, Innovations and Solutions Inc. 901 West Jefferson Street, Tallahassee A26, Florida 32304 Copyright© 2009. The material here is a compilation from web-based information and research articles solely for sharing and educational purposes. Prohibited the material to use for any profit making business without prior permission from sole editors or original authors by email. Cited text and materials is from web sources: Sribd.com, Novapublishers.com, nsti.org. Cited material sources: NSTI 2007,2008 and 2009 Conference CDs Free distributed material from scribd.com Contact Email: [email protected] [email protected]

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Table of contents

Part 1: Basic food science

Lecture 1: Food science Lecture 2: Principles of food and health Lecture 3: Food components and diets Lecture 4: Classification of foods Lecture 5: Chemistry of foods I Lecture 6: Chemistry of foods II Lecture 9: Metabolism of carbohydrates in food Lecture 10: Metabolism of lipids in food Lecture 11: Metabolism of proteins in food Lecture 12: Metabolism of vitamins and minerals in food Lecture 13: Metabolism of nucleic acids in food Lecture 14: Physiological relationship of food metabolism in the body Lecture 15: Applied techniques: Analysis of foods Lecture 16: Applied techniques: Evaluation of foods and nutrition Lecture 17: Applied techniques: Nutrition assessment and dietetics

Part 2: Diet and disease

Lecture 1: Principles of diet in health and disease Lecture 2: Dietary groups Lecture 3: Dietary surveys in community Lecture 4: Nutritional diseases and disorders Lecture 5: Therapeutic diets and nutraceuticals Lecture 6: Analytical methods of nutritional analysis and diet preparation Lecture 7: Clinical dietetics in hospital practice Lecture 9: Public health nutrition Lecture 10: Ditetics as profession

Part 3: Foods, diets and disease

Lecture 1: A survey of foods and diets in disease Lecture 2: Dietary fibers and gut motility Lecture 3: Prification, structure, health benefits: Feruloyl Arabinoxylans in fibers Lecture 4: Rhinacanthus nasutus: antimutagen properties Lecture 5: Dietary fibers in cultured cells Lecture 6: Bamboo shoots as dietary fibers Lecture 7: Tropical and temperate fruits Lecture 8: Relationship of physical activity and dietary fiber consumption Lecture 9: Cardiovascular prevention by soluble fiber Lecture 10: Processing techniques and their effect on fruit phytochemicals Lecture 11: Grafting of biofibers

Lecture 12: Bioactive foods and nutraceutical supplementation criteria in cardiovascular protection

Lecture 13: Comparison of cholesterol lowering diets: Apple, Casein

Appendix Paper 1: Cholesterol 7a-Hydroxylase Activity Is Increased by Dietary Modification with Psyllium Hydrocolloid, Pectin, Cholesterol and Cholestyramine in Rats. Few words on 3D imaging Paper 2: Cholesterol 7a-Hydroxylase Activities From Human and Rat Liver Are Modulated In Vitro Posttranslationally by Phosphorylation/Dephosphorylation

Foods, Diets and Disease Editors: Rakesh Sharma, Bharati D Shrinivas © 2009 Innovations And Solutions, Inc. ___________________________________________________________________________

Lecture 2

DIETARY FIBERS AND GUT MOTILITY

Rakesh Sharma, Bharati D Shrinivas

ABSTRACT The relationship between dietary fibers and motility of the digestive tract is essential for the accomplishment of their functions, but is somewhat complex, as there may be reciprocal influences. In fact, it is well known that fibers can modify gastrointestinal motility, but it is not as much known that gastrointestinal motility may modify the functions of fibers and reverse their beneficial effects. The effects of motility on fiber functions obviously is not irrelevant with respect to the fiber characteristics (e.g. viscosity, water holding capacity, fermentability etc.) and vice-versa. Consequently the kind of fibers and the kind of gut motility should be taken into account when considering their functional interactions. Fibers may influence both the motor activity of the stomach, by delaying in most cases gastric emptying, and that of the small intestine, where the most frequent fiber effect is an acceleration of transit, whereas in the colon the transit may be variously modified. However, when the motor activity is impaired, the presence of fibers in the gut lumen may became deleterious. In fact, if there is an inefficient gastric motility with gastric stasis, the accumulation of fibers in the gastric cavity induces a further worsening of gastric motility and in some cases may lead to an abnormal fermentation of fibers and formation of bezoars. A delayed intestinal transit may favour the small intestinal bacterial overgrowth, which is responsible of an out of place fiber fermentation in the small intestine with many patho-physiological problems, and, in addition, if there is a chronic pseudo-obstruction, may provoke an episode of functional obstruction. The altered transit in the colon due to constipation or diarrhoea may produce differences in fiber fermentation, that in its turn may modify the colonic transit with an abnormal production of gases. In addition, when there is a condition of severely altered colonic transit the addition of a large quantity of fibers to diet may further worsen the motor activity leading to an impaction. For these reasons the addition of fibers to a diet, that usually is beneficial for the gut transit, should be done with caution in patients with the above mentioned alterations of gut motility.

Rakesh Sharma, Bharti D Shrinivas

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INTRODUCTION Dietary fibers carry out many physiological functions in the gastrointestinal tract aimed to health preservation through their fermentation by colonic bacteria and the maintenance of a normal escretory function. The addition of dietary fibers to a diet is found to increase the frequency of bowel movements and this fact suggests that fibers influence gut motor activity and transit. However the effect of fibers on gastrointestinal motility depends both on the segment of the gut considered and on the kind of fibers. In fact the various segments of the gastrointestinal canal have different patterns of motor activity and each kind of fiber has a different behaviour in the intestinal lumen that depends on its intrinsic characteristics. To investigate the relationships between dietary fibers and gut motility it is necessary first of all to define the characteristics of the gastroduodenal, small intestinal and colonic motor activities and afterwards those of dietary fibers.

CHARACTERISTICS OF GUT MOTILITY DURING FUNCTIONAL PERIODS Gastrointestinal motility carries out various functions during both the interdigestive and digestive periods through the performance of different motor patterns that will be briefly summarized hereafter before discussing the characteristics and the effects of fibers. Interdigestive Period The interdigestive period is characterized by a peculiar motor pattern called “interdigestive migrating

motor complex” (IMMC) [1.2] characterized by a phase of peristaltic hyperactivity (Figure 1) called phase III or activity front, lasting a few minutes and propagating from the stomach through the duodenum, jejunum and ileum down to the ileo-cecal valve every about 90 minutes. The phase III is preceded by a period of irregular motor activity, called phase II, with pressure waves that progressively increase in frequency and amplitude and is followed more or less abruptly by a phase of motor silence, called phase I, that takes up about one third or one quarter of the entire cycle. The principal function (Table 1) of the IMMC is clearing the gastrointestinal lumen from the not absorbed leftovers of the meal, bacteria and foreign stuffs with the aid of the cyclic output of gastric, duodenal, pancreatic, biliary and IgA secretions (Figure 2), that takes place just before the phase III of peristaltic activity. In this manner it performs a “washing” and a “disinfection” of the gastrointestinal tract from the

stomach down to the ileo-caecal valve, so avoiding the gastric stasis of indigestible fibers and preventing the bacterial overgrowth in the stomach and intestine that may give rise to abnormal fermentations. For this reason it is also called the “intestinal housekeeper” [3].

Dietary Fibers and Gut Motility

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Table 1. Functions of the Interdigestive Migrating Motor Complex (Immc) and Secretory Cyclic

Activities of the Gastrointestinal Tract

1. to eliminate from the gastrointestinal lumen the not digested and not absorbed substances that remain after the completion of digestive and absorptive activity. 2. to perform a cyclic washing, cleaning and “disinfection” of the

gastrointestinal lumen with the gastric, duodenal, biliary pancreatic and immuno-globulinic A secretions that increase just before the appearance of the activity front of IMMC, that sweeps them along the gastrointestinal canal. 3. to avoid the stasis and reflux of secretions from the duodenum to the stomach and from the cecum to the ileum.

Figure 1. Prolonged gastrointestinal manometric recording during fasting. Trace 1 is from the gastric corpus, traces 2 and 3 are from the antrum and traces 4-6 from the duodenum.

Note in the top tracing two periods of gastroduodenal intense motor activity at about 90 min interval starting in the gastric corpus and propagating through the antrum in the duodenum. In the bottom panel one can see that this motor activity is represented by strong peristaltic contractions that reach the maximal frequency of 3/min in the stomach and 12/min in the duodenum. This cyclic motor activity is called phase III or activity front of the interdigestive migrating motor complex (IMMC), is preceded by a period of increasing motor activity (phase II) and is followed by a period of motor silence (phase I). (Modified from Bortolotti M. La dispepsia. Piccin Nuova Libraria, Padova, 1996)


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Digestive Period When a meal is ingested, the IMMC cycling is immediately interrupted al all levels of the gastrointestinal tract (Figure 3) and is replaced by a motor activity similar to that of phase II with phasic waves of variable amplitude. The duration of interruption is proportional to the caloric content of the meal, being about 4-5 hours for a meal of 500 Kcal, and is longer for lipids with respect to proteins and carbohydrates. Indigestible fibers do not stop the IMMC cycling [4,5]. The reappearance of IMMC takes place first in the small intestine and afterwards in the stomach. The ingested meal is received and processed by the stomach until it is ready for emptying in the intestine. Gastric emptying is controlled by various gastric and intestinal neuro-hormonal mechanisms that are activated by the physico-chemical characteristics of the meal.

Figure 2. Secretory cyclic activities that takes place in the upper gut in correspondence with the phase III of IMMC.

Note: The gastric, duodenal, bilio-pancreatic and IgA secretions that are pushed forward by the peristaltic contractions of the IMMC activity front and carry out a “washing” and “disinfection” of the gastrointestinal lumen during the interdigestive period.

Liquids are emptied more rapidly than solids, while meals containing proteins, lipids and carbohydrates are emptied more slowly than inert ingesta and the emptying rate depends on the caloric density, being slower as the latter is higher [6]. Solid meals requires a mechanical treatment by the gastric motor activity aimed to reduce the volume of the meal particles to a diameter <1-2 mm.


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Figure 3.

Note: The intake of a meal stops the occurrence of IMMC phases at all levels of the entire gastrointestinal tract with appearance of an irregular motor activity that continues until the processes of digestion and absorption are carried out. At this point the cycling activity reappears, before in the intestine and after in the stomach, to clean up the not digested and not absorbed leftovers of the meal.

The pylorus works as a filter blocking the particles with a diameter >1-2 mm, so that the antral motor activity may grind and triturate them to a suitable diameter [7]. However, if the viscosity of the liquid phase is high, the pylorus lazes the capacity of discriminating the particles with higher diameter that, consequently are abnormally emptied [8,9] and may induce maldigestion. The solid particles, which have a specific weight inferior or superior to that of water, are emptied with more difficulty, as well as the hard particles with respect to those which are compressible. The emptying of non digestible solids is very important, because they include dietary fibers. The latter ones, that are not grinded and reduced to a diameter <1-2 mm, remain in the stomach until the other foods adequately triturated are emptied and the digestive and absorptive processes are accomplished. At this point the activity front of the IMMC reappears and cleans up the gastric lumen from the residual solid stuff by means of peristaltic contractions with a great propulsive capacity that propels the particles of the gastric content into the intestine even when their diameter is >2 mm. Hence the gastric emptying


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of fibers requires this kind of motor activity and for this reason is emptied with a more or less substantial delay with respect to the other more digestible components of a meal.

Table 2. Functions of the Digestive Motor Activity of the Gastrointestinal Tract

1. to receive and store the meal in the gastric fundus with receptive relaxation and reservoir function (reception and storage of the meal), 2. to deliver progressively the meal with gastric secretions from the fundus to the antrum with adaptive contractions (intragastric distribution of

the meal),

3. to triturate solids in particles of 1-2 mm with strong antral contractions against the closed pylorus (mixing and grinding of the meal).

4. to selectively and progressively deliver the properly treated gastric chyme into the duodenum (emptying of the meal).

5. to mix the nutrients with the digestive secretions and to distribute the chyme on the maximal possible mucosal surface of the intestine for a rapid and complete absorption (mixing and absorption).

The digestive motor activity of the small intestine is represented by segmenting waves the majority of which (80%) are not propulsive. In fact they have the task of mixing the nutrients with the digestive secretions and distributing the chyme on the maximal possible mucosal surface of the intestine for a rapid and complete absorption. Once the digestion and absorption of the meal is accomplished, about 5 hours after a normal meal, the not absorbed residues, including dietary fibers, are swept away together bacteria, insoluble material, etc by the peristaltic activity of the IMMC phase III. The motor activity of the ileo-colonic junction has the function of regulating the passage of intestinal contents into the colon [10], preventing at the same time the reflux of colonic content back into the ileum, to avoid the small intestinal bacterial overgrowth (SIBO). The terminal ileum shows a reflex clearing activity in connection with some products of bacterial fermentation refluxed from the caecum, as, for example, the short chain fatty acids. The ileocolonic transit is rapid post prandially and slow and erratic during fasting [11]. The ileo-caecal sphincter does not discriminate between solids and liquids [12], while distal small bowel selectively retains dietary fibers [13]. In this manner the ileo-caecal junction has the task of functionally separating the small intestinal environment from that of the colon. If this functional separation is altered, the colonic bacteria growth more or less proximally in the intestinal lumen, fermenting the not absorbed nutrients, as well as dietary fibers, and cause the small intestinal bacterial overgrowth.


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The motor activity of the colon gives rise to a colonic transit far more slow than that of the other portions of gut, representing about 90% of the total transit time. The characteristic of the colonic motor activity can be easily observed with the technique of prolonged manometric monitoring in physiologic and pathophysiologic conditions [14,15].

During the interdigestive period it does not show a motor activity similar to that of the

gastrointestinal migrating motor complex, even if it presents periods of inactivity especially during the nocturnal rest [16] alternated with periods of activity [17]. The latter ones are characterized by stationary and propagated contractions, including “mass movements” that usually precede defecation.

After meal ingestion a “gastrocolic reflex” [18] takes place with segmenting and migrating

contractions that give rise to propulsion as well as retropulsion of the colonic content and may end in a mass movement. The activation of this reflex is initially due to a stimulation of mechanical receptors of the gastric wall, while the subsequent phase depends essentially on lipidic content of the meal, that acts through neuro-hormonal mechanisms [19].

The functions of the colonic motility are aimed (Table 3) to delay the transit of the chyme arriving from the ileum, to allow water, salts and short chain fatty acids absorption with segmental non propagated contractions, to store the feces allowing fermentation and to propel the colonic content into the rectum with peristaltic contractions (mass movements) for defecation.

Table 3. Functions of Colonic Motility

1. to delay the transit of the chyme arriving from the ileum, to allow water, salts and short chain fatty acids absorption with segmental non propagated contractions (mixing and absorption), 2. to store the feces allowing the fermentation by colonic bacteria (storage), 3. to propel the colonic content into the rectum with peristaltic contractions (mass movements) for defecation (propulsion and defecation).

The colonic transit conditions both water absorption and bacterial fermentation. So one of the consequences of slow transit is a greater production of gas, that, if there is a presence of only saccharolytic bacteria, may be hydrogen, or, if there is the presence of methanogenic bacteria, also methane, as happens in the 30-50% of the European population [20]. The time interval between the arrival of the intestinal content in the cecum and the expulsion of feces from the rectum is that allowed for water absorption and bacterial fermentation of the not absorbed nutrients and dietary fibers. More the transit is rapid, less time is allowed for water absorption and bacterial fermentation, while slower the transit is, more time is available for water absorption and bacterial fermentation. The not fermented dietary fibers are propelled along the colon and represent the bulk of feces together with the bacterial mass and other not digested residues.


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CHARACTERISTICS OF FIBERS THAT INFLUENCE GUT MOTILITY

Dietary fibers may modify gut motor activity and transit in different manners that depend on the tract of the gut considered and on the intrinsic characteristics of the fibers, such as viscosity, solubility, water holding capacity, fecal bulking activity and fermentability (Table 4).

Table 4. Characteristics of fibers that may Influence Gut Motility

1. Viscosity: capacity of a fiber dispersed or dissolved in water to gelatinize and form a dense and viscid solution. 2. Solubility. capacity of dissolve in water without leaving particles in suspension. 3. Water holding capacity (WHC): capacity of swelling and retain water in its matrix 4. Fecal bulking index (FBI): index that measures the ability of swelling and retain water. 5. Fermentability: capacity of being destroyed by intestinal bacteria forming other chemical compounds.

Viscosity and Solubility Viscosity is the capacity of a fiber dispersed or dissolved in water to gelatinize and form a dense viscid solution, whereas solubility is the capacity of a fiber of dissolving in water, without leaving particles in suspension. The fibers that give viscous solutions are in general soluble (glucomannan, guar gum, psyllium, etc.) (Table 5), while insoluble fibers give dispersions in water that are less viscous. The viscosity of a fiber solution depends on various factors including pH. In fact fibers as pectin and alginate tend to gelatinize, when exposed to a pH less than 3, consequently, when the gastric pH is higher than 3, a condition that occurs during the intake of a meal or during a treatment with antisecretory drugs, the gelification of some fiber may be more difficult. In addition the viscosity may change in a non linear fashion, when the “shear rate” of the fluid varies, as

happen in the gastrointestinal canal, in consequence of the diameter of the lumen and the motility patterns [21]. An interesting experiment was conducted by Dikeman et al [22] to determine the viscosities of both soluble and insoluble dietary fibers during gastric and small intestinal digestive simulation and multiple shear rates, obtaining different values of viscosity for the same fiber depending on the gastrointestinal tract and digestive period.

Table 5. Solubility and Viscosity of the Most Common Dietary Fibers

Fiber Solubility Viscosity

Pectin yes yes Psyllium yes yes


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Glucomannan yes yes Guar gum yes yes Linseed yes yes Guar gum partially hydrolized yes no Maltodestrin resistant partially hydrolized yes no Inuline yes no Cellulose no no Bran no no

Water Holding Capacity and Fecal Bulking Index Water holding capacity (WHC) of a fiber is the capacity of swelling and retain water in its matrix. This contributes to increase the volume of intestinal content. With regard to the colonic content the point of reference is the fecal bulking index (FBI), that measures the real effect of a fiber on fecal swelling and differs strongly among the various fibers (Table 6). WHC influences remarkably not only the fecal mass, but also the fermentability of the fiber and the absorption of nutrients in the intestine Fermentability Fermentability is the capacity of being destroyed by intestinal bacteria with formation of other chemical compounds. Some of these products, such as short chain fatty acids and gases may influence gut motility. There are significant differences in fermentability among the various fibers (Table 7 ), that depend not only on the chemical composition, but also on the water holding capacity that favors the penetration of bacteria inside the matrix of the fiber.

Table 6. Fecal Bulking Index (FBI) of Some Fibers

Fiber FBI Pectin 3.6 Oat bran 13.1 Guar gum 16.1 Linseed grounded 33.1 Ispaghula (90% fiber) 441

Table 7. Degree of Fermentability of Some Fibers

Low or partial High

Cellulose Pectins Hemicellulose Gums Lignin Inulin Resistant stark Oligosaccharides


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EFFECT OF FIBERS ON GUT MOTILITY The influence of fibers on motility may be reciprocal. In fact, while on one hand, it is well accepted that the fibers may modify gut motility, on the other hand, is not as much known that the gut motility may interfere with the beneficial effects of fibers. There are motility alterations that may involve either tract of the alimentary canal and may be influenced by the characteristics of the fibers. Consequently, when the dietary fibers are administered to a subject or a patient, the kind of fiber and the kind of gut motility should be taken into account, keeping care to their functional interactions. Stomach The characteristic of the digestive motility of the stomach, that does not empty the particles of the meal with a diameter >1-2 mm, produces a retention in the gastric lumen of the dietary fibers that cannot be reduced to a diameter <1-2 mm by the antral motor activity during gastric digestion. For this reason this kind of fibers affects gastric motility, delaying gastric emptying. Different preparations of the same fiber may influence gastric motility in different manners. In fact coarse bran delays gastric emptying, while fine bran does not [23]. This fact probably may account for different results obtained in various studies with the same kind of fibers. The fibers that form viscous solutions delay gastric emptying, while the fibers that are insoluble have less effect on gastric emptying, provided that are reduced to a diameter <1-2 mm, otherwise they are emptied at the end of the digestive period. As the viscosity of a solution of fibers depends on pH, when the pH of the gastric juice is less than 3, they tend to gelatinize, as happens with pectin and alginate, but when the gastric pH is higher than 3, as during the intake of a meal or during a treatment with antisecretory drugs, the gelification of some fibers may occur with more difficulty. In addition the viscosity may change in a non linear fashion when the “shear rate” of the fluid varies, as

happen in the gastrointestinal canal, in consequence of the diameter of the lumen and the motility patterns during interdigestive and digestive periods [21). Clinical Corollary: Stomach

The restraining effect the fibers on gastric emptying may be beneficial, as, when taken in correspondence of a meal, they induces early satiety [24,25] and, consequently, may help to lose weight. Another beneficial effect of fibers that delay gastric emptying may be the prevention of dumping

syndrome in patients who underwent gastric resection [26] and the improvement of the glucose

tolerance in non insulin-dependent diabetes, because they slow down the glucose absorption [27,28]. However the fiber effect that delays gastric emptying may become dangerous in particular conditions connected with gastric dysmotility. In fact, in patients with delayed gastric emptying due to impairment of neuromuscular functions, especially if this impairment is so severe as to induce gastroparesis (Figure


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4), the absence of the interdigestive cyclic motor activity that cleans the gastric lumen at the end of the digestive period, may cause the retention of dietary fibers in the gastric lumen. This fact worsen

dyspeptic symptoms and, in particular conditions of anacidity, favors an abnormal fermentation with gas production, that determines gastric distension and bloating. Another consequence of the retention of fibers is the formation of bezoars and, in particular, of pharmacobezoars comprised of medications in a background of pathologic gastric motility or excessive use of anticholinergics [29].

Figure 4. A) Radiogram performed about 6 hours after ingestion of barium in a patient with both gastroparesis and intestinal pseudo-obstruction. Note that the barium is still present in the stomach and small intestine.


12 B) Gastrointestinal manometric recording in a patient with both gastroparesis and intestinal pseudo-obstruction. The recording sites (1-6) are the same of Figure1, whereas the trace n. 7 is from the jejunum. Note that during the entire recording time lasting about 120 min there is a complete absence of motor activity in the stomach (traces 1-3) and small intestine (traces 4-7), with only sporadic and low amplitude contractions in the intestine. Intestine The fibers that produce viscous solution, such as guar gum, delay intestinal transit [30], whereas other fibers, such as bran, does not modify or even hasten it [31]. We performed a study on this aspect [32], testing the effect on gastrointestinal transit of a balanced mixture of dietary fibers (Fitomagra Plus, ABOCA), expressly chosen to hasten small intestinal transit and delay gastric emptying. In a series of 10 patients with a slight overweight the effects of dietary fibers mixture on gastric emptying and intestinal transit of a meal were investigated with a scintigraphic method. The values the half gastric emptying time (T1/2) and of the intestinal transit time of a semiliquid and bromatologically equilibrated meal of 300 kcal, measured without the previous intake of the preparation were compared with those obtained after the fiber mixture intake in a different day and the results were statistically compared by using the ANOVA, Wilcoxon and Student t test for paired data. The intestinal transit was significantly (p<0.05) accelerated by the fiber intake, while gastric emptying was delayed, but not significantly (Figure 5 ). Clinical Corollary : Intestine

The effect of the acceleration of intestinal transit may decrease the intestinal absorption of nutrients [33,34]. This effect is added to the increase in satiety due to decrease in gastric emptying and contributes with other factors to lose body weight of patients overweight, as it has been demonstrated after a month intake of the fiber mixture above described [32].


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Figure 5. Values (mean + SD) in minutes (min) of gastric emptying (T 1/2) and intestinal transit of a meal with a scintigraphic technique in basal conditions (black bars-A) and after a dose of dietary fiber mixture (white bars-B).

Note that the intestinal transit was significantly accelerated, whereas the gastric emptying was delayed, although not significantly. * = statistically significant [p<0.05] in comparison with the basal period with ANOVA test " = statistically significant [p<0.05] in comparison with the basal period with Wilcoxon test ^ = statistically significant [p<0.05] in comparison with the basal period with Student t test However, in patients with a delayed intestinal transit, that in some cases may reach the severity of a chronic pseudo-obstruction (Figure 4), the continuous intake of a large quantity of dietary fibers, especially those that delay intestinal transit, may create a problem. In fact, if the peristaltic activity front (phase III) of the IMMC, that sweeps through the intestine the not digested fibers, is absent or impaired, an accumulation of fibers may take place in the intestinal lumen, favoring in some cases the functional block of the transit. In addition, in patients with small intestinal bacterial overgrowth due to intestinal stasis, the abundant intake of dietary fibers may be responsible of an excessive out of place small intestinal fermentation with many patho-physiological problems. These range from fat and vitamins malabsorption [35] with consequent malnutrition, osteoporosis and anemia [36], to a secondary alteration of intestinal motility [37] and visceral hypersensitivity, through the production of a liposaccharide and short chain fatty acids [38, 39]. Colon Dietary fibers affect colonic transit both by increasing fecal mass and through specific effects of the substances produced during bacterial fermentation. The increase of the endoluminal volume of intestinal


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content influences the intestinal peristaltic motor activity and hence the transit especially of the colon. Many factors contribute to determine the volume of the fecal mass. The amount of fibers added to the diet is of great importance in increasing the volume of the feces and hence the velocity of the transit. In fact the addition of 20 g. of psyllium, guar gum or ispaghula accelerates the total or colonic transit in a patient with constipation, while dosages lower than 5 g. are ineffective on transit velocity, but nevertheless increase the quantity of feces and the number of defecations, that become easier through a decrease in fecal consistency [31,40-44]. The increase in fecal mass is not only due to the quantity of a non fermented fiber that remains in the feces, but also to the “ fecal bulking index” of a fiber [45), that is related to its water holding capacity, the capacity of retain water into its matrix. The fecal bulking index may differ remarkably among fibers [Table 5], being, for example, 446 for ispaghula and 3.6 for pectin [45]. This property of some fibers remarkably influences the fecal mass, and also facilitates their fermentability, as bacteria may penetrate more easily into its matrix. The fiber fermentability may contribute, although not strikingly, to increase the fecal mass, by favoring the bacterial growing [46]. In fact bacteria represent a quote of the volume of the feces that may exceed the 60% [47-49]. More a fiber is fermentable, larger is the fecal mass produced and the level of fermentability shows marked differences among fibers (Table 6). The cabbage fiber, for example, the 92% of which is fermentable, increases the fecal mass more than the wheat fiber, that has only the 36% fermentable [50], whereas the resistant starch which is easily fermented reaches the colon in a considerable quantity that ranges between 8 and 40 g a day [51] and increases the fecal mass of 2-3 g for each gram of it [52].. Finally, the water retained in the lumen by the products of fermentation (i.e. short chain fatty acids), that exert an osmotic effect, may contribute to increase the volume of feces. The amount of fecal mass is strictly correlated with intestinal transit. A variation in fecal mass may influence transit and viceversa [52]. A slow transit may cause an increase in water absorption, hence a decrease in fecal mass, while an accelerated transit increases the amount of feces. Some products of fiber fermentation by the colonic flora are able to influence the motor activity of the colon. In fact, the presence of methane may slow the colonic transit [53], as it stimulates non propulsive contractions, especially in irritable bowel syndrome (IBS) patients [54], worsening the constipation. Short chain fatty acids, instead, stimulate propulsive colonic motor activity [55,56] through a release of 5HT and Ach [57]. If the production of short chain fatty acids takes place in the proximal colon, the effect on colonic transit is more evident. The relationship between short chain fatty acids and motility is bidirectional: in fact, on one hand, they stimulate transit and, on the other hand, the acceleration of transit increases the concentration of them in stools [58]. Moreover the fibers may indirectly influence motility through a stimulation of mucin and gas production.

Mucin is essential for the propulsion of feces and is produced by a diet rich in fibers [59]: The production of mucin in the small intestine and colon is correlated with the fiber “bulking property”,

while in the caecum it depends on fiber fermentability [60]. The gas produced in consequence of


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bacterial fermentation and trapped in the fiber matrix, increases the volume of the intestinal content leading to a stimulation of motility and transit. The gas represents itself a stimulus for propulsive motility, when is not absorbed by the mucosa or is consumed by other bacteria through methanogenesis [61] or acetogenesis [62]. The gut motor activity that determines the transit of gas is somewhat different from that responsible of the transit of liquids and solids. The gas propulsion takes place more easily in the erect rather than supine position [63], is independent from the motor activity that propels solid and liquid contents, as is not carried out by propagated phasic waves, but by tonic contractions [64]. Particularly effective are in the colon the giant propulsive contractions with consequent gas evacuation [65], probably activated by a reflex response to the distension of the colonic lumen induced by gas [66]. However the motor activity, on one hand, is stimulated by gas, but, on the other hand, influences the gas absorption by the mucosa, the gas production through fermentation and the gas consumption by bacteria. The consequences of an increase and decrease of colonic propulsive motility on the colonic gas

absorption are opposite, under the same conditions of gas production and consumption. In fact, if there is a high propulsive motor activity, the time allowed for gas absorption is less, whereas a slow propulsion favors a higher gas absorption. In the absence of methanogenic bacteria, an increase of propulsive motility shortens the time allowed for fiber fermentation and, consequently, the gas production is lower, whereas, if there is a low propulsive motility, the fiber fermentation and gas production is increased. In the case of consistent presence of methanogenic bacteria that take place in 30-50% of the population [67] is necessary to make other considerations. If there is an increase of propulsive motor activity, the time available for gas consumption by the bacteria is reduced and more gas is available in the colon for excretion. On the other hand a decrease of propulsive motility, with consequent stasis of gas in the lumen, favors a higher gas consumption by methanogenic bacteria. In conclusion, the amount of gas present in the colon is the result not only of the production, but also of the interrelations among motor activity, absorption and consumption. Clinical Corollary : Colon

The presence of fibers in the gut lumen is most important to maintain a normal colonic motility. In fact an experiment with a diet devoid of fibers for 27 days impairs the myogenic and neurogenic intestinal contractions through a decrease of enterochromaffin cells, that are important for the transduction of mechanical and chemical stimuli in the colonic lumen necessary for the motor activity [68].


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Figure 6. Manometric recording of the rectosigmoid tract in a normal subject (top tracing) and in two patients with constipation (middle and bottom tracings).

In the first patient (1) the stimulation with bisacodyl induces high pressure contractions, while in the other (2) the drug is completely ineffective (colonic inertia). (From: Preston JE and Lennard-Jones JE. Dig Dis Sci 1985;30:289). On the other hand, if the colonic motility is severely impaired for neuromuscular alterations (Figure 6) with consequent delay in content transit, the addition of a high amount of fibers to the diet further impairs colonic motility and worsens the symptoms [69]. In fact in 80% of patients with constipation due to slow colonic transit and in 60% of those with obstructed defecation, the effect of fibers is scarce or absent [70] with worsening of symptoms [71]. In addition the consumption of a high fiber diet delays intestinal gas transit, promoting gas retention and the appearance of symptoms due to abdominal distension in some individuals [72]. In particular the insoluble fibers, as corn and wheat bran, may


17

worsen the clinical outcome in some cases of IBS [69]. On the other hand a soluble fiber supplementation, while does not affect the oro-cecal transit, significantly prolongs colonic transit [73].To date there are few studies about the relationship between viscosity and laxative effect [21], but a study with soluble fibers (psyllium, ispaghula, etc) demonstrated an improvement of symptoms of irritable bowel syndrome [69].

EFFECT OF SINGLE FIBERS ON GUT MOTILITY The effects of some most used dietary fibers on gut motility is summarized in Table 8 and is described below with more details. Gums form viscous solutions that delay gastric emptying, intestinal transit and nutrient absorption from the small bowel [4,11,74] at a dose ≥ 9 g [75], whereas with lower doses other investigators found that guar gum does not have any influence on gastric emptying and small intestinal transit time with scintigraphy and breath test [44,76] . In addition, guar gum at a dose of 30 gr accelerates the frequency of antral contractions and the postprandial pattern of motility in duodenum and jejunum, increases the jejunal transit time [77] and markedly prolongs the duration of postprandial motor activity in the human small bowel [30]. Partially hydrolized guar gum decreases colonic transit time [78] . Glucomannan (Amorphophallus konjac), which is highly viscous, delays gastric emptying and decreases nutrient absorption [79]. It also increases stool frequency and restores to normal a delayed oro-cecal transit in patients with constipation in studies with radio-opaque markers and H2 breath test [80,81]. Psyllium (Plantago psyllium) increases stool frequency, delays gastric emptying and decreases total intestinal transit time [40,82-84], but does not modify colonic and rectal motor activity [85]. Ispaghula (Plantago ovata) seeds do not influence gastric emptying, small intestine transit and colonic transit [86], ispaghula husks do not alter total intestinal transit time in IBS patients [87] and oro-cecal transit time with breath test [31,44], but decreases the colonic transit time with radiopaque markers [42]. Pectin for some investigators delays gastric emptying [11,88-90], whereas for others does not significantly influence gastric emptying [82,91-93], but decreases total transit time [94].

Table 8. Effect of Some Fibers on Gut Motility

Gastric emptying

Intestinal transit

Oro-caecal transit

Colonic transit

Oro-anal transit

Guar gum delays (dose>5 g)

delays (dose>5 g)

delays (dose>5 g) / /

no effect no effect no effect / /


18

(dose<5 g) (dose<5 g) (dose<5 g)

Bran

delays (coarse)

hastens (coarse) hastens hastens hastens

no effect (fine)

no effect (fine) / / /

Lignin / no effect no effect hastens no effect Cellulose little effect delays hastens / Hemicellulose / / delays / Ispaghula no effect no effect / hastens / Glucomannan delays / hastens / / Inulin / hastens / / / Pectin delays * / / / hastens Psyllium delays / / / hastens Linseed / hastens / / / Calcium polycarbophil

no effect^ or hastens§ / / hastens*

or delays° /

Resistant starch hastens / / no effect or

shortens /

^ = in man ; § = in dog; * = in IBS constipation; ° = in IBS diarrhea Linseed (Linum usitatissimum) accelerates intestinal transit [95,96]. Bran has an effect on motility that depends on the dimension of its particles. In fact coarse bran, but not fine bran, delays gastric emptying [23,31, 93] and accelerates small bowel transit [ 23,31,97], as well as whole gut transit [98,99]. Wheat bran increased the duration of postprandial pattern of duodenal motility [77] and decreases the mean retention time in the small intestine and colon [100]. Rice bran and barley bran flour accelerate total gut transit [101-103]. Lignin has a little effect on gastric emptying and small intestinal transit, but accelerates colonic transit [11,104]. Cellulose has little influence on gastric emptying and small bowel transit, but accelerates colonic transit [11,104], increases the duration of postprandial pattern in duodenum and jejunum and markedly increases jejunal transit time [77]. Hemicellulose does not modify the gastric emptying of lipids [105], but delays gastrointestinal transit time [106], while purified hemicellulose (xilan) decreases transit time [94]. Inulin, which is soluble but not viscous, increases the frequency and velocity of the activity front of the interdigestive migrating motor complex [107], likely because it produces short chain fatty acids with fermentation. Other fructo-oligosaccharides accelerate gastric emptying [108].


19

Calcium polycarbophil does not delays the gastric emptying of radiolabeled pellets in man [109], but stimulates a fed-like gastric motility and accelerates the gastric emptying of radiolabeled pellets in dog [110]. It accelerates colonic transit in constipation of IBS patients [111] and patients with spinal cord disorders [109,112], while in patients with IBS and diarrhea it prolongs mean colonic transit time measured with radiopaque markers [111]. Resistant starch hastens gastric emptying [113], does not influence whole gut transit time in man [101] or slightly shortens transit time in rats [114],while in association with wheat bran further shortens transit time in humans [115]. The differences observed in the above listed effects on gut motility of some fibers may be due to differences in methods used for measuring the gut transit, to different fiber dosages and preparations, as well as to different subjects used for experiments (animals, healthy subjects or patients).

CONCLUSION The gastrointestinal and colonic motility play a pivotal role in the accomplishment of the physiological functions of dietary fibers. The fibers influence and in some cases determine the kind of motor activity of the stomach, small intestine and colon, through their characteristics, such as viscosity, water holding capacity, fermentability and production with their fermentation of substances acting on gut motility. It is necessary to know the effect of the fibers in each gut segment, because some fibers go fast through some bowel segments and slow through others. Consequently the total transit time is not a good predictor of the rate of transit through particular gut segments. Besides other beneficial effects, fibers are necessary for a correct gut motility performance, but in some conditions may be dangerous especially if there is an impairment of gut motor activity. In this case the beneficial fiber functions are replaced by noxious effects that lead to pathological conditions. This may happens, for example, in patients with markedly delayed gastric emptying and intestinal transit, as in the case of gastroparesis and pseudo-obstruction, respectively, as well as in patients with small intestine bacterial overgrowth and colonic inertia. In these conditions fibers may worsen motor activity and symptoms, leading to potentially dangerous effects. For these reasons it is important to keep in mind the reciprocal influences that take place between fibers and gut motility, when a diet with a high amount of fibers is prescribed to a patient with the above mentioned motility problems.

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Foods, Diets and Disease Editor: Rakesh Sharma, Bharati D Shrinivas ©2009 Innovations And Solutions, Inc. ___________________________________________________________________________

Lecture 3

PURIFICATION, STRUCTURE AND HEALTH BENEFITS: FERULOYL ARABINOXYLANS IN FIBERS

Rakesh Sharma, Bharti D Shrinivas The nature of carbohydrates present in the food is growing field of interest

within the food industry due to the potential of some of them to help prevent diseases of lifestyle. Non-glycemic carbohydrates, i.e., those carbohydrates (or their components) that are not absorbed in the small intestine and, therefore, transit down to become fermented in the colon, have drawn lot of attention. In fact, food carbohydrates can be broadly classified on the basis of their in vivo digestibility into digestible and non-digestible carbohydrates (Table1) (Asp, 1996; Englyst et al., 1992). Non-digestible carbohydrates have been collectively referred to as „dietary fibre‟ (Hipsley, 1953). Some of these

carbohydrates are of particular interest to the food industry for the purpose of developing „functional foods‟, i.e., foods that are able to exert positive health

effects. Non-digestible oligo/polysaccharides are considered as prebiotics, which stimulate the growth of bifidobacteria in the colon.

A distinction was established between insoluble DF and soluble DF. The

effects of insoluble DF are of limited interest because of their low functionality and fermentability (Hsu and Penner, 1989). By contrast, soluble DF in general has a wide functionality due to its ability to interact with water,


2

and is almost fully fermented by the large intestine micro flora, bringing about much desired physiological/metabolic effects (Lopez et al., 1999). Cereals, the staple food for millions of people across the world, are the chief source of both soluble and insoluble DF (Plaami, 1997). Arabinoxylans, along with some amount of -D-glucans, are the major components of soluble DF (Rao and Muralikrishna, 2004).

Table 1. Classification of Carbohydrates Based on Their in Vivo

Digestibility

Subgroup Components

Digestible

Monosaccharides Glucose, galactose, mannose, fructose (ketose), Arabinose, xylose Sorbitol, mannitol

Disaccharides Sucrose, maltose, lactose, Oligosaccharides malto-oligosaccharides Polysaccharides Starch – amylose and amylopectin

Non-digestible

Disaccharides Trehalose

Oligosaccharides Raffinose, stachyose, verbascose, fructo- and xylo-oligosacchairdes

Polysaccharides Starch - modified and resistant, Non-starch – cellulose, ligno-cellulose, arabino-xylans, mixed glucans, mannans, pectins

DISEASES OF LIFESTYLE/CIVILIZATION – ROLE OF

DIETARY FIBRE Interest in carbohydrates/polysaccharides is increasing due to the recent

worldwide concern about the continuously increasing rates of many common diseases, known as diseases of lifestyle/civilization. Some of these common diseases in western countries are linked to the deficiency of complex carbohydrates/dietary fibre in food. The list includes obesity, diabetes, atherosclerosis and chronic heart problems, increased cholesterol in the body, hypertension, constipation and diverticulosis, colorectal cancer and many

Purification, Structure and Benefits…

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more. Obesity in particular, is raising in adults and now in children as well (FAO/WHO, 1998). Although obesity as a significant phenomenon has usually been associated with developed countries, it is now also on the rise in the developing countries.

Cereals form the quantitatively most important source of DF. Consuming cereals and cereal based products are known to have beneficial roles in human nutrition and health and have been linked to their phytochemical profiles (Adom and Liu, 2002; Adom et al., 2003; Charalampopoulos et al., 2002; Mori et al., 1999). After more than 30 years of research into many and varied claims for its benefits, it is now clear that fibre has uniquely significant physical/physiological effects. Accumulating evidence favors the view that increased intake of DF can have positive health effects against chronic diseases, such as cardiovascular diseases, diverticulosis, diabetes and colon cancer. Prevention of constipation and regulation of transit time are mainly caused by the bulking effect of DF. It is also partly fermented in large intestine by a mixed flora of anaerobic bacteria and most of the physiological effects of DF are thought to be based on this property (Scheeman, 1998).

A daily intake of approximately 30 g of dietary fibre is encouraged to promote health benefits associated with fibre. Because of the increased nutritional awareness, the food industry is facing the challenge of developing new food products with special health enhancing characteristics (Charalampopoulos et al., 2002). To meet this challenge, it must identify new sources of neutraceuticals and other natural and nutritional materials with the desirable functional characteristics (Izydorczyk et al., 2001). In view of the therapeutic potential of DF, more fibre incorporated food products are being developed all over the world. However, consumer acceptability of these functional foods depends not only on the nutritional property, but also on the functional and sensory quality. These factors are considered while developing functional foods.

CHARACTERIZATION OF POLYSACCHARIDES Simple monosaccharides can be built into giant molecules called complex

polysaccharides that rival DNA and proteins in size and complexity. It‟s a

testament to the importance of sugars that scientists have granted them an „ome‟ of their own. Just as the „genome‟ and „proteome‟, the „glycome‟ of an

organism or cell encompasses all the sugars it makes. Still in its infancy,


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glycomics is slowly revealing its huge cast of sugar-related characters – structure and their myriad roles. The glycome – study of carbohydrates, of a single cell type or creature is probably many thousands of times more complex than the genome (Schmidt, 2002). It is the polysaccharides – their structure or bonding that makes the characterization very difficult. Even chemically synthesizing oligosaccharides by capping sugar molecules with „protecting‟

groups at all but one branch point leaves compounds with a mixture of bonds formed in different orientations, requiring extensive purification procedures after each new sugar building block is added.

A polysaccharide may contain between ten and a million sugar residues. Polysaccharides are rarely homogeneous and usually have a very wide molecular weight distribution; often they are regarded as group of very closely related molecular species varying in both molecular architecture and size („polydisperse‟). Structural characterization of polysaccharides usually requires extensive purification procedures.

ISOLATION, FRACTIONATION AND PURIFICATION Characterization of polysaccharides first requires them to be isolated from

biological samples. They may be extracted with various extractants such as water (for water soluble arabinoxylans and mixed glucans), polar non-aqueous solvents (for starch and glycogen) (Leach and Schoch, 1962), chelating agents (for pectins) (Selvendran, 1985), N-methyl morpholine-N-oxide (MMNO, for cellulose) (Chanzy et al., 1979) and alkali (for hemicellulose A and B) (Wilkie, 1979). Water extraction at different temperatures can be carried out to obtain gums and mucilages.

Polysaccharides thus isolated from the biological samples are rarely homogeneous and require extensive fractionation and purification steps before proceeding further with structural characterization. Polysaccharides differ in their molecular size, shape and charge, and can be fractionated using various methods such as fractional precipitation with solvents (ethanol, acetone), salts (ammonium sulphate) or methods such as ion exchange/affinity/gel permeation chromatographies.

HOMOGENEITY CRITERIA


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Polysaccharides are highly complex and diverse, and unlike proteins, they are heterogeneous in their chemical characteristics like molecular weight and composition which in turn affects their physical properties. The heterogeneity arises because their biosynthesis, which is controlled indirectly by glycosyltransferase genes, unlike direct templates in case of DNA and proteins. Glycosyltransferases, the enzymes with individual specificities, are responsible for the transfer of sugar residues from particular glycosyl donor to the growing polysaccharide chain. Variations in polysaccharide structures may result from (a) departure from absolute specificity of the transferases, (b) incomplete formation of segments/side chains and (c) post polymerization changes. If these variations are continuous with respect to parameters such as molecular size, proportions of sugar constituents and linkage type, separation into discrete molecular species will be impossible and the polysaccharide sample would be called „polydisperse‟. If the heterogeneity lies in their

molecular size, but not in their chemical composition, they are called „polymolecular‟ (Aspinall, 1980).It is difficult to establish the purity of any

polysaccharide sample unambiguously. Showing the absence of heterogeneity by as many independent criteria as possible is considered to be sufficient to go ahead with the structural characterization.

There are number of methods to show the absence of overall heterogeneity of a polysaccharide sample. Some of them are (a) consistency in chemical composition and physical properties such as optical rotation and viscosity, (b) chromatographic methods such as ion exchange, affinity and gel filtration, (c) ultra centrifugation pattern, (d) electrophoretic methods such as cellulose acetate and capillary electrophoresis and (e) spectroscopic methods such as IR and NMR. Purified sample is then subjected to the structural characterization (Aspinall, 1982).

Structural Characterization Sugar residues forming the polysaccharide chain may either be in linear or

branched arrangements. They may all be of the same type (homoglycan) or of different types (heteroglycan). The length of a polymer chain, called degree of polymerization, is specified by the number of structural units it contains. The structural units may either in pyranose or in furanose ring form.

The structure of a polysaccharide can be organized into four different levels similar to that of proteins; they are (a) primary, (b) secondary, (c) tertiary and (d) quaternary.


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The covalent sequence of monomeric units along with the respective glycosidic linkages is known as „primary structure‟. Depending on the primary

structures, polysaccharide chains may also adopt characteristic shapes such as ribbons, extended helices and hollow helices, which are known as „secondary

structures‟. Energetically favored interactions between chains of well defined

secondary structures result in ordered organizations, which are known as „tertiary structures‟. Further associations between well-defined entities result in higher levels of organizations, known as „quaternary structures‟ (Perez and

Kouwijzer, 1999). The major problems in the determination of the molecular structure of

complex carbohydrates/polysaccharides are to establish (a) the molecular weight and nature of constituent sugar residues including their ring size, (b) the position and anomeric configurations of the inter-glycosidic linkages, (c) the sequence of residues/linkages and (d) overall arrangement of polymeric chains.

Structural elucidation of plant polysaccharides is a very tough task due to their non-periodic repeating units unlike microbial polysaccharides. However, several methods are available for the determination of the polysaccharide structure and are broadly categorized into three main classes: (a) chemical, (b) enzymatic and (c) spectroscopic methods (Aspinall, 1982).

Chemical Methods

Molecular Size

As the polysaccharides are „polydisperse‟ in nature, their molecular size is

represented as average of either weight (Mw) or number (Mn). The determination of both weight and number average gives an indication of the molecular size distribution, greater the difference between Mw and Mn, greater the polydispersity of the sample. Number average can be obtained by membrane osmometry (Mn > 20 kDa) and vapor pressure osmometry (van Dam and Prins, 1965) and weight average can be obtained by light scattering (Manley, 1963). Similarly, weight average can also be obtained by ultracentrifugation (sedimentation equilibrium or approach to equilibrium). These methods, however, are based mainly on the theoretically calculated average values.

Gel filtration chromatography, on the other hand, is a simple and widely used method to obtain the average molecular weight of the polysaccharide


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sample. The column needs to be pre-calibrated with known molecular weight markers to determine the molecular weight of the unknown samples.

Sugar Composition

Determination of the sugar composition of the polysaccharides involves the identification and quantification of sugar constituents. De-polymerization of the polysaccharide is a prerequisite, for which various methods have been developed and complete acid hydrolysis is the most common and widely used one. Aldose containing polysaccharides can be completely hydrolyzed with minimum loss of constituent sugars either by 0.5 or 1.0 molar sulfuric acid at 100C for about 6 h (Selvendran et al., 1979) or by 1.0 molar trifluoro acetic acid at 120C for 1 h (Albersheim et al., 1967). However, ketose containing sugars are very unstable under these conditions and thus mild acid hydrolysis either by 0.1 molar oxalic acid at 70C for 1 h (Aspinall et al., 1953) or by 0.05 molar sulfuric acid at 80C for 1 h (Codington et al., 1976) is followed for their de-polymerization. Incomplete hydrolysis takes place when a polysaccharide contains either amino sugars or uronic acid residues. Amino sugar containing polysaccharides require stronger acid and they can be completely hydrolyzed by 4 molar HCl at 100C for about 6 h (Spiro, 1972). Uronic acid containing polysaccharides undergo decomposition (liberate carbon dioxide) upon acid hydrolysis by 12% HCl (Whyte and Englar, 1974). This can be circumvented by reducing the carboxyl group with water soluble carbodiimide/sodium borohydride mixture followed by acid hydrolysis. Determining the difference in the sugar composition before and after carboxyl reduction gives the amount of uronyl residues (Lindberg et al., 1972).

Monosaccharides released upon acid hydrolysis can easily be identified and quantified either by HPLC of GLC method. HPLC method is non-destructive, requires no derivatization and sample can be recovered after the analysis. Separation of individual sugar residues is based either on cation/anion exchange (water) or on partition (acetonitrile: water) chromatography. Detection of sugars is done using refractive index (RI) detector (McGinnis and Fang, 1980). However, low sensitivity of RI detector requires large amount of sample (in micrograms) for analysis.

GLC is a destructive method and requires sample derivatization. However, it is much more sensitive than HPLC and requires very less amount of sample (in nanograms) for analysis. Trimethylsilyl (TMS) ethers, trifluoroacetyl (TFA) esters and alditol acetates are the most commonly prepared derivatives. Constituent monosaccharides are reduced with sodium boro-hydride or


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deuteride to obtain acyclic form and then acetylated using either acetic anhydride and pyridine (1:1) (Sawardekar et al., 1965) or acetylated aldonitrilation (hydroxylamine/pyridine and acetic anhydride, PAAN derivatives) (Dmitriev et al., 1971). Unlike TMS derivatization, acetylation eliminates the formation of multiple derivatives when different rings are formed (pyranose or furanose) or different anomeric forms are generated from reducing sugars or from equilibrium mixture of methyl glycosides. The acyclic derivatives can be identified by their retention time and if necessary by mass.

The enantiomeric forms of the constituent sugars can not be distinguised by the above mentioned methods. Although majority of the sugars are in D form, sugars such as rhamnose (in pectins) and arabinose (in arabinoxylans) are in L-form. These enantiomeric forms can be distinguished by converting into equilibrium mixtures of glycosides of chiral alcohols (+/- 2-butanol or +/- 2-octanol) followed by GC analysis using capillary columns (Leontein et al., 1978).

Linkage Analysis

Methylation (conversion of all the free hydroxyl groups into methoxyl groups) is the most versatile and widely used technique for the determination of linkages in polysaccharides. It gives information on linkage positions, ring size (pyranose or furanose), non-reducing end groups, and kind and extent of branching (Hirst and Percival, 1965). The method involves complete etherification of free/un-substituted hydroxyl groups; i.e., those not involved in ring formation, inter sugar glycosidic linkages, or carrying substitutions stable at conditions used for methylation of the polysaccharides and for subsequent hydrolysis of the methylated derivatives (Lindberg, 1972).

In particular, Hakomori methylation is the one which is most reliable and extensively used method for the complete methylation of the polysaccharides (Hakomori, 1964). In this method, polysaccharide is dispersed in dimethyl sulfoxide (DMSO), treated with sodium methyl sulfinyl methanide (sodium dimsyl) and then reacted with methyl iodide. Hakomori‟s method is very

effective compared to other methods wherein etherification may not be achieved in a single step or complete alkoxide formation may not take place. Haworth (dimethyl sulfate as alkylating agent and aqueous 30% sodium hydroxide as base), Purdie (methyl iodide as both solvent and alkylating agent and silver oxide as base) and Kuhn (N,N-dimethyl formamide/DMSO as dipolar aprotic solvent, methyl iodide/dimethyl sulfate as alkylating agent and silver/barium oxide as base) methylation are some other methods, which have restricted use and can be employed when the sample is partially methylated.


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The completeness of methylation can be ascertained either by methoxyl group determination or by the absence of O-H stretching vibrations in the IR spectrum.

The characterization of per-methylated polysaccharides requires identification and quantification of all the sugar derivatives formed upon de-polymerization and is performed by GLC-MS (Dutton, 1973). Per-methylated polysaccharides are hydrolyzed and reduced to form acyclic derivatives. Since per-methylated polysaccharides are less soluble in aqueous solvents, initial partial hydrolysis is done with organic solvents such as formic acid and then complete hydrolysis with dilute aqueous acids is performed. Acyclic derivatives obtained after reduction are acetylated to obtain alditol acetates, which is the most widely used derivatization method for the characterization of per-methylated sugars. The mass spectra of per-methylated alditol acetates are generally simple to interpret, with fragmentation patterns characteristic of constituent sugars and their substitution pattern. However, methylation analysis does not give information on stereo-chemical nature (/) of the constituent sugars (Lonngren and Svensson, 1974).

Primary fragment ions from per-methylated alditol acetates arise by -cleavage with preferred formation of (a) ions with lower molecular weight, (b) ions from cleavage between two methoxyl bearing carbon atoms, (c) ions from cleavage of a methoxyl bearing and an acetoxyl bearing carbon atom with marked preference for the methoxyl bearing species to carry the positive charge and (d) ions from very low abundance of cleavage between two acetoxyl bearing carbon atoms. Primary fragment ions undergo a series of subsequent elimination reactions to give secondary fragment ions which include losses by (a) β-elimination of acetic acid (m/e 60) or methanol (m/e 32), (b) -elimination of acetic acid but not methanol and (c) via cyclic transition states of formaldehyde, methoxy-methyl acetate or acetoxy-methyl acetate (Jansson et al., 1976).

Oxidation Characterization of the products obtained from oxidative cleavage of

polysaccharides can give details about the mode of linkage, substitution pattern and configuration of sugar residues/linkages (/β). Two important methods of

oxidative cleavage are chromium trioxide and periodate oxidations.


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Cro3 Oxidation

The configuration of glycosidic linkages in polysaccharides can be determined by chromium trioxide oxidation, which is shown to preferentially oxidize -linked polysaccharides over -linked ones. The difference is attributed to the easy formation of a keto-ester by cleavage at the bridge oxygen of β-anomeric compounds (Lindberg et al., 1975).

Periodate Oxidation

It is the widely used method for the determination of linkages in polysaccharides. Glycol cleavage via oxidation by sodium metaperiodate gives formic acid (usually from triol cleavage in pyranose) or formaldehyde (from exocyclic diol, CHOH-CH2OH groups) and the oxidant is reduced to iodate. The liberated products can be estimated by various methods such as titrimetry and spectrophotometry for the oxidant reduced, acid-base titration for the formic acid liberated or colorimetry for the formaldehyde formed (Hay et al., 1965).

Smith Degradation The aldehydes liberated upon periodate oxidation, and the sugar residues in

the polysaccharide, which are resistance to oxidation are reduced with sodium borohydride and hydrolyzed to obtain monosaccharides along with residual stubs of oxidized units; either glycerol (from pentitol) or erythritol and threitol (from 1→4 and 1→6 linked hexitols, respectively) (Goldstein et al., 1965). Smith degradation products are identified and quantified by GLC-MS.

Oligosaccharide Analysis Polysaccharides can be partially fragmented/de-polymerized and the

analysis of the oligosaccharides thus obtained can give information regarding the distribution of side chains (random/uniform or non-random) in turn providing complete structural information of the parent polysaccharides. Oligosaccharides can be obtained by several chemical methods such as partial acid hydrolysis, acetolysis, trifluoroacetolysis, mercaptolysis and methanolysis. They can be fractionated/purified and characterized by following similar methods employed for polysaccharides. In particular, MALDI-TOF-MS and FAB-MS are useful for oligosaccharide


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characterization, providing molecular mass and sequence of the constituent sugar residues, respectively (York et al., 1990).

Enzymatic Method Oligosaccharides can be obtained by fragmentation/de-polymerization of

polysaccharides with the use of specific polysaccharide degrading enzymes. Enzymes cleave the polysaccharides with the varying degree of polymerization. By characterizing the oligosaccharides released and also the leftover polysaccharides, one can get information regarding the structure of parent polysaccharides. Based on the mode of action, polysaccharide degrading enzymes – glycosidases are classified into two groups: exo- and endo-glycosidases. Exo-glycosidases act on polysaccharides and release mono/disaccharide units from the non-reducing terminal, whereas endo-glycosidases cleave the polysaccharides randomly (at the un-branched regions of both main and side chains), resulting in the release of oligosaccharides with varying degree of polymerization. Enzymatic method of obtaining oligosaccharides has several advantages over chemical method, viz. (a) their specificity, both to linkage type and substitution pattern, (b) lack of by-products, (c) high reaction rates and (d) control over the reaction. Various cell wall polysaccharides such as arabinoxylans (Hoffmann et al., 1992; Subba Rao and Muralikrishna, 2004) and xyloglucans (Lerouxel et al., 2002) have been characterized by analyzing the oligosaccharides obtained on enzymatic method.

Spectroscopic Methods Spectroscopic methods are much easier to perform compared to chemical

and enzymatic methods of oligo/polysaccharide analysis and they complement the data obtained from other two methods. Some of the important spectroscopic methods are: infra red (IR), mass spectrometry (MS), optical rotatory dispersion (ORD), circular dichroism (CD) and X-ray diffraction and nuclear magnetic resonance(NMR).

NMR Spectrometry

NMR spectrometry is the rapid and non-destructive method to study the structure of polysaccharides, requiring no modification or degradation of the


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sample. 13C and 1H NMR together can give the details on molecular complexity and fine structure of the polysaccharides. 13C NMR can give details about the composition, linkage and conformation of polysaccharides (Jennings and Smith, 1978) and can also ascertain the purity of the polysaccharide sample. However, it can not differentiate the enantiomeric configuration of sugars. Various plant polysaccharides like arabinoxylans (Hoffmann et al., 1991; Izydorczyk and Biliaderis, 1993; Subba Rao and Muralikrishna, 2004), mixed glucans (Uzochukwu et al., 2002) and pectins (Ryden et al., 1989) have been characterized using 13C NMR spectroscopy( ShyamaPrasad Rao & Muralikrishna 2007).

IR Spectroscopy

Infrared waves are absorbed by the vibrating chemical bonds in the polysaccharides giving characteristic IR spectra (vibrational) in the frequency range of 4000 to 400 cm-1. IR spectroscopy can be used for the detection of functional groups, configuration of sugar residues and to know the substitution pattern. It is used to characterize arabinoxylans and their oligosaccharides (Kacurakova et al., 1998).

Mass Spectrometry

Mass spectrometry is the indispensable technique in the characterization of oligo/polysaccharides. In the conventional mass spectrometry, polysaccharide sample can not be analyzed directly and hence it is separated into small molecules/constituent sugar residues and derivatized with acetylation/alkylation in order to make them volatile. Mass spectrometry is based on the principle that ions of different mass: charge ratio (m/e) are separated due to their differential diffraction in the combined electric and magnetic fields. Chemical ionization and electron ionization are the two important methods by which ionization can be achieved. In chemical ionization, molecular ions remain intact and spectra obtained are simple to interpret. On the other hand, electron ionization may result in complicated spectra because ions entering the analyzer may get fragmented by the high energy transferred from the bombarding electrons.

The advent of recent mass spectrometric techniques such as matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and fast atom bombardment-mass spectrometry (FAB-MS) have revolutionized the oligo/polysaccharide analysis. These techniques do not need laborious sample derivatization steps, but provide valuable information on the molecular mass and sequence of constituent residues. Many plant


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oligosaccharides including arabinoxylans have been characterized using these techniques (Jacobs et al., 2003; Lerouxel et al., 2002; Subba Rao and Muralikrishna, 2004).

Cereal Polysaccharides

The major constituent (60 – 80%) of cereals is starch, a storage

polysaccharide, which is made up of two constituents: a linear 1→4 linked

amylose and branched 1→4 linked amylopectin. Apart from starch, cereals

also contain other polysaccharides known as non-starch polysaccharides, which include cellulose, hemicelluloses, arabinoxylans, 1-3/1-4 -D-glucans, glucomannans, pectins and arabinogalactans (Fincher, & Stone, 1986; Izydorczyk, & Biliaderis, 1995). These non-starch polysaccharides mainly occur in the cell walls, where they play both structural and growth-regulating role and are divided into two types: „fibrillar‟ and „matrix‟ polysaccharides. Cellulose, a β 1→4 linked polymer of glucose forms the micro-fibrils in the cell wall. All other non-starch polysaccharides belong to the „matrix‟

polysaccharide group, are very heterogeneous in structure. They form complexes with each other and with other cell-wall components such as cellulose, proteins (extensins, rich in hydroxy proline residues), lignin (polymer of cinnamyl alcohol) and other phenolic constituents.

Together, alkali extractable matrix polysaccharides have been termed „hemicelluloses‟ as they were considered to be chemically and structurally related to cellulose.

The rigidity and strength of the cell wall is related to the integrity of cellulose/hemicellulose network. During cell growth, however, wall expansion has been found to be dependent on the enzymatic modification of the hemicellulosic component (Pauly et al., 2001).

Arabinoxylans/Feruloyl Arabinoxylans In 1927, non-starchy, gummy polysaccharides were isolated from bread

wheat flours and shown to consist predominantly of pentoses, arabinose and xylose (Freeman and Gortner, 1932; Hoffman and Gortner, 1927). Similar polysaccharides were also found in durum wheat, rye and barley, and were initially referred to as pentosans and later as arabinoxylans. Pentosans, in general represent a heterogeneous group of polysaccharides which, in addition


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to pentose sugars, may also contain hexoses, hexuronic acids and some proteins, and hence current nomenclature is more structure descriptive, identifying several polymeric components such as arabinoxylans or arabinogalactan peptides, depending on the molecular constitution of the polysaccharides.

Arabinoxylans have been identified in a variety of tissues of the main cereals of commerce: wheat, rye, barley, oat, rice and sorghum (Fincher and Stone, 1986) as well as in some other plants: rye grass (Hartley and Jones, 1976), pangola grass (Ford, 1989) and bamboo shoots (Ishii, 1991). Although arabinoxylans are minor components (but some times up to 10% as in barley grain) of entire cereal grains, they play important structural and functional role in plant cell.

Arabinoxylan consists of a linear backbone of β-(1→4)-D-xylopyranosyl residues, partly substituted with single -L-arabinofuranosyl residues at O-2, and O-3, or at both O-2 and O-3 positions of the xylose residues (McNeil et al., 1975; Vietor et al., 1994). The presence of arabinosyl substituents and their distribution over the xylan backbone affect such arabinoxylan properties as solubility and interaction with other polymeric cell wall components (Andrewartha et al., 1979; McNeil et al., 1975) as well as restrict the enzymic degradation by endoxylanase (Vietor et al., 1994). Some arabinose residues are covalently linked through ester linkages to ferulic acid (Smith and Hartley, 1983). General structure of feruloyl arabinoxylan is depicted in the Figure 1.

In type II walls, which are present in grasses, arabinoxylans are the major non-cellulosic polysaccharides in the primary walls. The arabinoxylans in type II walls have abundant arabinosyl side chains. They are also substituted with galactose and high amounts of glucuronic acid. In general, arabinoxylans were divided into water extractable and water un-extractable, based on the extractability, and this difference largely arises from their degree and pattern of substitution, feruloylation and non-covalent interactions with other wall components.

Ferulic acid, a hydroxycinnamic acid is the major bound phenolic acid in cereals arabinoxylans, and is synthesized by phenylpropanoid pathway. It is concentrated mainly in the aleurone layer (~ 75%) of the grain and comprises about 0.5% in wheat and 0.14% in barley grains.

Ferulic acid is a strong antioxidant and known to protect cells from UV radiation. Ferulic acid groups present in the arabinoxylans, on oxidative coupling yield diferulates/dimers which link adjacent polymers together, tightening the structure of the cell wall and thus restricting cell expansion.


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Ferulic acid protects the polysaccharides against enzymic hydrolysis. It also protects the plants from microbial/pathogen invasion.

Figure 1. General structure of feruloyl arabinoxylan.

Biosynthesis of Arabinoxylan

The cell wall component-polysaccharide biosynthesis takes place in different sub-cellular compartments. Cellulose and callose are made at the plasma membrane, whereas pectin and hemicelluloses (arabinoxylans) are believed to be synthesized in the Golgi apparatus (Carpita and Gibeaut, 1993).

Xylans are common polysaccharides in plant cell walls, particularly in secondary cell walls where they are deposited as the major non-cellulosic polysaccharides. The xylans in type II walls, which are present in grasses and some related plants, have abundant -L-arabinofuranosyl side chains attached through (1→3) and (1→2) linkages apart from a small amount of glucuronosyl

and other side chains (Aspinall, 1980; McNeil et al., 1984). This type of xylan, a heteropolysaccharide is known as arabinoxylan.

Heteropolysaccharide biosynthesis can be divided into four steps: chain or backbone initiation, elongation, side chain addition, and termination and extracellular deposition (Iiyama et al., 1993; Waldron and Brett, 1985). Our understanding of these different steps in biosynthesis is still very incomplete. The main enzymes responsible for heteropolysaccharide biosynthesis are glycosyltransferases, but only very few genes for these have been identified, and the enzymes responsible for synthesizing the backbone of xylans are only partially characterized (Porchia et al., 2002). The backbone-synthesizing enzymes may belong to the cellulose synthase-like proteins, but this assumption may be false as it is now known that callose synthase does not resemble cellulose synthase (Hong et al., 2001).

The biosynthesis of (1→4) linked β-xylosyl backbones in xylans is catalyzed by β-1,4-xylosyltransferase. This enzyme has been investigated in many plants, including wheat seedlings wherein the activity was characterized


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from microsomal membranes (Porchia and Scheller, 2000). An UDP-D-glucuronate decarboxylase (E.C. 4.1.1.35) was shown to catalyze the synthesis of UDP-D-xylose from UDP-D-glucuronate in an essentially irreversible reaction that is believed to commit glycosyl residues to heteroxylan synthesis (Zhang et al., 2005).

The addition of side chains to xylans has been less investigated and little is known about the way in which the different glycosyltransferases interact to form the complete polysaccharide. A study of glucuronosyltransferase has shown an interaction with xylosyltransferase (Baydoun et al., 1989). Although arabinose is a common monosaccharide in plant polysaccharides and glycoproteins, there are few reports on the arabinosyltransferases involved in polysaccharide synthesis. Recently, Porchia et al. (2002) reported the presence of arabinoxylan arabinosyltransferase (AX-AraT) in microsomal and Golgi membranes isolated from wheat seedlings and showed that AX-AraT is dependent on the synthesis of unsubstituted xylan acting as acceptor. They have also demonstrated the formation of a single arabinosylated protein and its possible role in arabinoxylan biosynthesis.

Biosynthesis of Ferulic Acid

Being a secondary metabolite, biosynthesis of ferulic acid is fairly well understood. Ferulic acid, a hydroxy-cinnamic acid derivative, is synthesized in plants via shikimate/phenylpropanoid pathway from phenylalanine or L-tyrosine. Shikimate/arogenate pathway leads, through phenylalanine, to the majority of plant phenolics, the phenylpropane (C6-C3) derivatives (phenylpropanoids). p-Coumaric acid is formed as an intermediate in the ferulic acid biosynthesis.

Feruloylation of Arabinoxylans

One of the characteristic features of arabinoxylans is their high content of bound ferulic acid (and small amount of p-coumaric acid), chiefly ester linked to -L-arabinofuranose usually at O-5 position. The feruloylation and p-coumaroylation occur on highly specific hydroxyl groups of polysaccharides. However, there is no complete agreement on to the site of feruloylation of wall polysaccharides or the nature of the feruloyl donor. Fry and Miller (1989) administered (3H) arabinose into spinach cultured cells and traced its incorporation into arabinose units of the major wall polysaccharides. The authors showed that arabinosylation and feruloylation occurred co-synthetically and intracellularly. Similarly, Obel et al. (2003) showed the


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intracellular feruloylation of arabinoxylans in wheat suspension-cultured cells. On the other hand, Yamamoto et al. (1989) suggested that feruloylation site is located within the matrix of barley coleoptile cell walls.

Meyer et al. (1991) showed that feruloyl-CoA is a donor for feruloylation. A microsomal preparation from suspension cultured parsely (Petroselinum

crispum) cells was able to transfer ferulic acid from feruloyl-CoA to uncharacterized endogenous wall polysaccharides. An alternative feruloyl donor may be the glycosidic ester of ferulic acid (1-O-feruloyl--D-glucose). Mock and Strack (1993) demonstrated that 1-O-sinapoyl--D-glucose is formed by UDP-glucose: hydroxycinnamate D-glucosyltransferase (E.C. 2.4.1.120).

OXIDATIVE GELATION IN VIVO Feruloyl arabinoxylans are known to undergo oxidative phenolic coupling

(dimerization) (Figure 2) reactions in walls; the coupling reactions themselves in vivo would be remarkably specific. To permit a coupling reaction, feruloyl groups on the same or different polysaccharide chains must be juxtaposed. Matrix polysaccharides could be imagined in gelatinous form and they would have enough mobility to place feruloyl residues in close proximity. But at present there is no definite proof for this theory. Peroxidases are candidates for the catalysis of the dehydrogenative dimerization of feruloyl residues in the cell wall. The peroxidases not only generate free radical intermediates of ester-linked feruloyl residues, but may also generate the hydrogen peroxide needed to achieve this from various hydrogen donors. Several mechanisms have been proposed for hydrogen donor generation. Ogawa et al. (1996) showed that one of the physiological functions of the cytosolic CuZn-superoxide dismutase is supplying hydrogen peroxide for lignification.

Obel et al. (2003) have observed the intracellular formation of ferulic acid dimer, which is limited to 8,5‟-diferlulic acid, while other dimers appeared to be formed extracellularly in wheat suspension-cultured cells. Similarly, Fry et al. (2000) reported the intraprotoplasmic and wall-localized formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures. It is argued that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively and have control on cell expansion.


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Figure 2. Covalent diferulate cross-link between arabinoxylan molecules.

FUNCTIONS OF ARABINOXYLANS AND FERULOYL ARABINOXYLANS IN VIVO

Feruloyl arabinoxylans (feraxans) are the major polysaccharides in the type

II walls, which are present in grasses. With the very complex and diverse structure, arabinoxylans may have roles in the cross-linking of cellulose microfibrils and may thereby regulate cell development, expansion and strengthen the wall by mechanical resistance (Carpita, 1996). These polysaccharides, by means of oxidative coupling, also become polymerized into the lignin macromolecules. Such polymerizations decrease wall extensibility and may ultimately be involved in the control of cell growth. They also limit biodegradation/digestibility of polysaccharides, thus forming an effective barrier


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against microbial invasion. Feruloyl oligosaccharides are known as signal molecules between plants and microorganisms (Darvill et al., 1992).

Figure 3. Feraxan – feraxanases system. Each arrow represents a different enzyme: xylanase (1), xylo-pyranosidase (2), arabino-furanosidase (3), galacto/gluco-pyranosidases (4), glucuronidase (5), feruloyl esterase (6), p-coumaroyl esterase (7) and O-acetyl esterase (8).

DEGRADATION OF ARABINOXYLANS AND FERULOYL ARABINOXYLANS IN VIVO

Feruloyl arabinoxylans (feraxans) (Nishitani and Nevins, 1989) are highly

complex and diverse in structure and therefore require an array of hydrolytic enzymes for their degradation (Figure 3). Collectively these enzymes are referred to as feraxanases (Nishitani and Nevins, 1989). Xylanase and feruloyl esterase are perhaps the key enzymes involved in the biodegradation of feraxans and they need to act synergistically. Xylanase would break the long-chain xylans into feruloyl-arabino-xylo-oligosaccharides, which in turn would be easily accessed by feruloyl esterase for the de-esterification of ferulic acid. On the other hand ferulic acid esterase may act upon feraxans to cleave the feruloyl moieties, thus facilitating their degradation by xylanase. Arabinofuranosidase, xylopyranosidase, glucuronidase, galactosidase and acetyl esterase are some of the other enzymes in the feraxanase group which are required for the complete biodegradation of feraxans.

Feraxan biodegradation is supposed to be a constant/continuous process in the cellular maintenance. However, during seed germination/malting, their biodegradation is hastened in the endosperm and aleurone cell wall by the induced feraxanases/xylanolytic enzymes. There are some reports on the in

vivo biodegradation of feraxans during malting/germination of cereals such as


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wheat, barley, rye and ragi (Autio et al., 2001; Obel et al., 2002; Rao and Muralikrishna, 2004; Subba Rao and Muralikrishna, 2004).

Figure 4. One of the (partial) biodegradation pathways for ferulic acid leading to vanillin via β-oxidation.( Source:- Gasson etal, 1998))

The ferulic acid degradation is not well understood, however, it may take place by chain shortening via β-oxidation process (Figure 4) (Gasson et al., 1998) directly analogous to the well known β-oxidation pathway of fatty acids. Vanillin, a highly valued flavor compound, is the main degradation product of ferulic acid.

Fine Structure of Arabinoxylans Although arabinoxylans have been of interest to cereal chemists and

technologists for many years, structural studies initiated in 1951 by Perlin were taken up only in the 1990s when a number of workers focused on the detailed structural characteristics of these polysaccharides. General structure of arabinoxylans is now well known. However, these polymers are highly heterogeneous in chemical structure and molecular weight. They vary not only


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from source to source, but also in different parts and fractionation and purification methods employed. This prompts arabinoxylans to be studied from different cereal sources both from structural and functional viewpoint.

In general, arabinoxylans from various cereals and/or other plants share the same basic chemical structure. However, they differ in the manner of substitution of the xylan backbone. The main differences are found in the ratio of arabinose to xylose, in the relative proportions and sequence of the various linkages between these two sugars, and in the presence of other substituents.

The ratio of Ara/Xyl in arabinoxylans from wheat endosperm may vary from 0.50 to 0.71 (Rattan et al., 1994) but it is usually lower than that found in bran (Shiiba et al., 1993) (figures 5A and 5B). Similarly rye endosperm arabinoxylans are less substituted (0.48 – 0.55) (Bengtsson and Aman, 1990) than their bran counterparts (0.78) (Ebringerova et al., 1990). In general rice (Shibuya and Iwasaki, 1985) and sorghum (Vietor et al., 1994) seem to consist of more highly branched xylan backbones than those from wheat, rye and barley (figures 5E and 5F), and they may contain galactose and glucuronic acid substituents, in addition to the pentose sugars.

Figure 5. Structural models for cereal arabinoxylans. Less branched endosperm/ insoluble (A) and more branched bran/soluble (B) arabinoxylans. Highly branched


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(region A) (C) and less branched (region B) (D) arabinoxylans. Less branched wheat (E) and more branched rice (F) arabinoxylans.

With a relatively low degree of branching, arabinoxylans from wheat, rye and barley contain a rather high amount of un-substituted Xylp residues and a relatively low amount of mono-substituted Xylp residues, compared to the more highly branched arabinoxylans from rice and sorghum. The proportion of doubly substituted residues seems not to be related to the arabinose to xylose ratio and varies substantially among various arabinoxylans; highest amount has been reported for wheat bran arabinoxylans. The presence of O-2 mono-substituted xylose residues has been verified in all cereal arabinoxylans except those of rye endosperm. This type of xylose substitution appears to be a structural feature characteristic especially of barley arabinoxylans; a close to one ratio of O-3 to O-2 mono-substituted Xylp residues suggest almost equal distribution of both linkages in the polysaccharide (Vietor et al., 1992).

Cereal arabinoxylans exhibit a high degree of endogenous micro-heterogeneity. It is, therefore, not possible to assign a single structure to arabinoxylans. In order to get better insight into the structural characteristics of individual homogeneous arabinoxylans, several investigators extensively fractionated arabinoxylans using ethanol or ammonium sulphate graded precipitation techniques (Gruppen et al., 1992a; Gruppen et al, 1992b; Izydorczyk and Biliaderis, 1992; Vietor et al., 1992 SubbaRao& Muralikrishna 2004). Increased concentration of ethanol/ammonium sulphate resulted in arabinoxylan fractions in continuously increasing Ara/Xyl ratios. The higher degree of branching was also accomplished by variations in the relative proportions of un-, mono- and di-substituted Xylp residues. Highly substituted arabinoxylan fractions contained less un-substitued Xylp residues.

The distribution of arabinosyl substituents along the xylan backbone is probably of greater importance than the degree of substitution itself, since it affects the conformation (Andrewartha et al., 1979) and the capacity of arabinoxylans to interact with each other and/or with other polysaccharides. According to the early work by Perlin and co-workers (Ewald and Perlin, 1959; Goldschmid and Perlin, 1963), wheat endosperm arabinoxylans consist branched regions where O-3 or O-2,3 substituted xylose residues are separated by single un-substituted xylose residues. At lengths of approximately 20 – 25 xylose units, relatively smooth domains of at least two to five (and possibly more) un-substituted xylosyl residues may be present.

Based on ammonium sulphate fractionation and oligosaccharide analysis upon xylanase hydrolysis, wheat (endosperm) water-soluble arabinoxylans are


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reported to have three structural domains. Region I is highly substituted (more of O-2,3), and periodate oxidation/Smith degradation studies demonstrated that substituted xylose residues are present either isolated, in pairs or even as three contiguous residues, which may in large be limited by steric hindrance. Region II is similarly substitution, but contains more of O-3 xylose residues. Region III, which separates highly substituted domains, contains sequence of 2 – 6 or more un-substituted xylose residues. Different fractions differ in the proportion/ratio of these regions.

Wheat alkali-extractable arabinoxylans differ in their fine structure from water-soluble arabinoxylans and presumed to have two regions (A and B) (Figure 5C and 5D). The highly branched region A composed mostly of repeating tetrameric units of un- and di-substituted xylose residues. This region also contains some O-2 substituted xylose residues. The less dense region B, which alternates with region A, includes at least seven contiguous un-substituted xylose residues.

The structure of arabinoxylans from barley endosperm (Vietor et al, 1992) was shown to be more regular than that from wheat. The major region, mono- (enriched with O-2) and di-substituted xylose residues are separated by un-branched xylose residue, and the clusters are separated by regular un-branched region of at least four xylose units.

Rye arabinoxylans have a different structure; the major polymer structure (arabinoxylan I) has xylose chain substituted exclusively at O-3, and minor polymer (arabinoxylan II) contains di-substituted O-2,3 xylose residues.

Rice and sorghum arabinoxylans are highly substituted and overall they resemble branched regions of other cereal arabinoxylans. Structural elucidation purified arabinoxylans isolated from finger millet and its malt by methylation, GLC–MS, periodate oxidation, Smith degradation, NMR, IR, optical rotation, and oligosaccharide analysis indicated that the backbone was a 1,4-β-d-xylan, with the majority of the residues substituted at C-3. The major oligosaccharide generated by endo xylanase treatment was homogeneous with a molecular weight of 1865 Da corresponding to 14 pentose residues as determined by MALDI-TOF-MS and gel filtration on Biogel P-2. The structural analysis of this oligosaccharide showed that it contained 8 xylose and 6 arabinose residues, substituted at C-3 (monosubstituted) and at both C-2 and C-3 (disubstituted SubbaRao & Muralikrishna 2004).

Water-soluble feruloyl arabinoxylans (feraxans), isolated from native and malted (96 h) rice (Oryza sativa) and ragi (Eleusine coracana) grains, were fractionated on DEAE-cellulose, followed by purification on Sephacryl S-300 and the homogeneity was ascertained by high performance size exclusion


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chromatography, cellulose acetate and capillary electrophoresis. Structural characterization of the purified polysaccharides by methylation, followed by GLC–MS, and also by 1H NMR and 13C NMR spectroscopy, indicated very high branching and presence of high amounts of O-2 substituted xylans.

The amount of O-2, 3 disubstituted xylopyranosyl residues and the arabinose:xylose ratio was higher in malt feraxans. All feraxan samples consumed almost equal amounts of Periodate (4.02–4.30 μmol/mg).

High mountf xylose (40%), as identified by Smith degradation, further substantiated the high branching of feraxans. A model is presented depicting the structure of water-soluble feraxans from rice and ragi and their changes upon malting.( Shyama Prasad Rao& Muralikrishna 2007)

PHYSICOCHEMICAL/FUNCTIONAL ROLES OF ARABINOXYLANS IN RELATION TO FOOD AND NUTRITION

In the past few decades, arabinoxylans have stimulated research interest

since they have been proven to have significant influence on the water balance (Jelaca and Hlynka, 1971) and rheological properties of dough (Meuser and Suckow, 1986; Michniewicz et al., 1991), retrogradation of starch (Biliaderis and Izydorczyk, 1992; Gudmundsson et al., 1991) and bread quality (Delcour et al., 1991; McCleary, 1986,SubbaRao etal 2004,Shyma prasad Rao etal 2007).

The chemical nature, including the subtle difference in the structure of the polysaccharides, is important in knowing their exact functional roles. Further, the multitude of free hydroxyl groups occurring in any polysaccharide allow for an infinite amount of hydrogen bonding (intra and inter-bonding), which again influence the physical behavior of the polysaccharides. The distribution of arabinosyl substituents along the xylan backbone is known to affect the conformation of arabinoxylans (Andrewartha et al., 1979) and the intermolecular associations, which in turn have a direct bearing on certain physical and functional properties of these macromolecules.

Cereal arabinoxylans widely vary in their molecular weight and different methods of determination of molecular weight may give different values for the same arabinoxylan population (Fincher and Stone, 1986). Very high molecular weight of up to 5,000,000 has been reported for barley endosperm arabinoxylans (MacGregor and Fincher, 1993). The conformation of arabinoxylans, which can be determined by X-ray diffraction analysis, is


25

dependent on substitution patterns. Arabinoxylans are shown to have a 3-fold, left handed helix and in the solid state they appear as an extended, twisted ribbon when xylan backbone is un-substituted (Fincher and Stone, 1986). This conformation is relatively flexible, supported by one H-bond between adjacent xylose residues and forms aggregates into insoluble complexes, stabilized by intermolecular H-bonding. Presence of arabinosyl substitution stiffens the molecule by maintaining the xylan backbone more extended and thus prevents its aggregation. Flexibility of xylan backbone is limited by the steric hindrance/interaction of arabinose side groups (Yui et al., 1995).

As a result of their rather stiff conformation, arabinoxylans exhibit very high viscosity in aqueous solutions, compared to the intrinsic viscosity of other polysaccharides such as dextran and gum arabica (Fincher ad Stone, 1986). In general, increased arabinose substitution was associated with increased asymmetry of arabinoxylan molecules and thus with higher hydrodynamic volume/viscosity. However, other factors such as xylan chain length, presence of ferulic acid and specific arrangement of arabinose residues along the xylan backbone influence this property.

In the presence of free radical-generating agents (e.g. hydrogen peroxide/peroxidase, ammonium persulphate, ferric chloride, linoleic acid/lipoxygenase), arabinoxylans are capable of forming three-dimensional networks (gels or viscous solutions). This unique property, now known as „oxidative gelation‟ of water extracts of wheat flour was first described by Durham (1925). A number of factors such as molecular weight and substitution of the arabinoxylans influence the gelling property. However, presence of ferulic acid is prerequisite for the gelling ability of the polysaccharide and numerous hypotheses concerning the mechanism of this reaction have been developed. Detection of diferulic acid in oxidized arabinoxylan systems indicates that cross linking occurs through the coupling of two adjacent ferulic acid residues (Geissmann and Neukom, 1973).

Arabinoxylans are known to influence the quality of bakery products due to their physicochemical properties like viscosity and water holding capacity (Izydorczyk and Biliaderis, 1995). They absorb high amounts of water (6 – 8 times their weight) and when added to wheat flour, they compete with other constituents of dough for water. Studies showed significant increase in the farinograph water absorption, dough development time and loaf volume when arabinoxylans are added to the bread dough (Biliaderis et al., 1995; Vanhamel et al., 1993). However, at very high concentrations, due to the increase in viscosity, arabinoxylan addition adversely affected the bread quality (Biliaderis et al., 1995). Arabinoxylans are shown to protect protein foams


26

against thermal disruption and retain gas in the dough (Hoseney, 1984). Viscosity of arabinoxylans adds to the strength and elasticity of gluten-starch films surrounding the gas bubbles and slows down the rate of CO2 diffusion from dough during baking, affecting firmness and homogeneity of crumb texture. The presence of minor groups such as feruloyl and acetyl groups in arabinoxylans do have an affect on their functional properties such as viscosity,foam stabilization and gelling as revealed by a recent study(Madhavilatha& Muralikrishna 2009)

Arabinoxylans are now considered to be prebiotics and used especially as arabinoxylo-oligosaccharides in functional foods for actively managing the colonic micro-flora with the aim of improving host health. A prebiotic is defined as „a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health‟ (Gibson and

Roberfroid, 1995 Chithra& Muralikrishna 2009)). They are also emerging as natural antioxidants, immuno-modulators and

components of edible films (ShyamaPrasadRao& Muralikrishna 2006.).

Uses of Ferulic Acid There have been studies showing potential health benefits of ferulic acid,

such as anti-carcinogenic and anti-inflammatory properties. Ferulic acid is a strong UV absorber and constitutes the active ingredient in many skin lotions and sunscreens. It is also part of the gel matrix of wound healing, in a chemical form similar to the diferulated cross-links between arabinoxylan polymers in the cell walls. In the food industry, it is extracted from agro-industrial waste and bio-converted using fungi to vanillin, a much valued flavor compound. Its ability to inhibit peroxidation of fatty acids finds as natural food preservative and antioxidant.

Future Perspectives

Dietary fibre is a combination of several polysaccharides varying in their (a) Composition (b) molecularweight (c) charge(uronicacid) (d) acetyl/feruloyl groups (e)associated proteins and lipids ( varying with respect to covalent, hydrogen,hydrophobic and vanderwaal inter actions). Eventhough many studies have been carried out with respect to their purification,structural characterization and their combined effect on alleviate disease symptoms pertaining to diabetus,atherosclerosis and colon cancer, however the same is not true with respect to individual purified polysaccharides .Various cereal


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brans and pulse husks which are rich in dietary fibre components are the ideal sources to do in depth work and also to address the various molecular mechanisms by which the dietary fibre components alleviate disease symptoms of various diseases .

Dietary fibres and their resultant degradation products such as oligosacchrides can modulate the beneficial bacterial populations present in the colon to exert health benefits to the host and they are also responsible to decrease the growth of harmful bacteria such as Clostridium Perfringens. Overall the dietary fibre research did not see the end of the tunnel wherein one can conclusively say that the destination is reached and there is no more path to traverse. Future research holds lot of promise and many unanswered and intriguing questions will be addressed with respect to the role of dietary fibre components and oligosaccharides with and without ferulic acid in health and disease.

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Lecture 4

RHINACANTHUS NASUTUS: ANTIMUTAGEN PROPERTIES


ABSTRACT

Rhinacanthus nasutus (Hattkaku-Reishi-Soh, Thong-Pun-Chang) are widely cultivated in tropical and subtropical regions of South Asia just like Japanese tea trees and have been used in treatments and preventions of diverse diseases as a folklore medicines. The phytochemicals of Rhinacanthus nasutus have been noticed for their healthy effects. Recently, their phytochemicals have been isolated from various parts such as leaves, stems, roots and total plants and also their effects Rhinacanthus

nasutus found by researchers. The purpose of this review is to represent their components and their actions known by now.

Keywords: Rhinacanthus nasutus (Hattkaku-Reishi-Soh, Thong-Pun-Chang), benzoquinone, naphthoquinones, anthraquinones, anti-mutagenicity

ABBREVIATIONS USED lawsone (1) lawson methyl ether (2) potassium lawson methyl ether (2’)

Rakesh Sharma, Bharati D Shrinivas 2

clotrimazole (3) chlorhexidine (4) 4-acetonyl-3,5-dimethoxy-p-quinol (5) β-amyrin (6) glutinol (7) lupeol (8) stigmasterol (9) sitosterol (β-sitosterol, 10) 2-methoxy-4-propionyl-phenol (11) umbelliferone (12) 2-methylanthraquinone (13) 2,6-dimethoxybenzoquinone (14) 3,4-Dihydro-3,3-dimethyl-2H-naphtho[2,3-b]pyran-5,10-dione（15） rhinacanthin-A (16) rhinacanthin-B (17) psychorubrin (3-hydroxy-1H-3,4-dihydronaphtho[2,3-c]pyran-5,10-dione, 18) rhinacanthin C (19) rhinacanthin D (20) rhinacanthin-O (21) rhinacanthin-P (22) rhinacanthin-G (23) rhinacanthin-H (24) rhinacanthin-I (25) rhinacanthin-J (26) rhinacanthin-K (27) rhinacanthin-L (28) rhinacanthin-M (29) rhinacanthin-N (30) rhinacanthin Q (31) wogonin (32) adriamycin (33) oroxylin (34) (+)-praeruptorin (35) allantoin (36) aflatoxin B1 (37) furylfuramid (38) ganciclovir (39) amantadine (40) rhinacanthin-E (41) rhinacanthin-F (42) quercetin (43)

Rhinacanthus Nasutus 3

1. INTRODUCTION Rhinacanthus nasutus (L.) Kurz of Hattkaku-Reishi-Soh (Japanese name),

Thong-Pun-Chang (China name) and Thong Phan Chang (Thai name) belongs to Acanthaceae, and is native in tropical and subtropical regions in South-East Asia such as Taiwan, Southern China, Thailand and India. Rhinacanthus

nasutus (L.) Kurz or Justicia nasutus L. is mainly a shrub around 1-2 m just like Japanese tea trees (Photo 1).

Photograph 1. Whole plant of Rhinacanthus nasutus.


In these regions, Rhinacanthus nasutus (L.) Kurz has been widely used in their treatment of hepatitis, diabetes, hypertension, herpes virus infections, cancer, skin diseases and others since old times [Gotoh et al., 2004; Sendl et

al., 1996; Wu et al., 1998a]. Acanthus mollis L.. of the same family could be found in the wide tropical

regions from Assam or Punjab of India to Sri Lanka (Ceylon) or Singapore. Surprisingly, Acanthus mollis L.. could be found as an ornamental plant even in outer cultivations without green house in Tokyo of temperate zone such Japan (Photo 2).

Photograph 2. Whole plant of Acanthus mollis L.. belonging to Acanthaceae. Photographed by Noboru Motohashi, 5/20/2009 Wed. at Itabashi Botanical Gardens, Itabashi-ku, Tokyo.


Generally, their diverse phytochemicals of functional components could be widely found in kingdom. Among these phytochemicals, their oxidative phytochemicals such as carotenoids, flavonoids and anthocyanins could especially be found highly abundant in human daily fruits and vegetables [Motohashi, 2008a; Motohashi, 2009b].

Interestingly, Rhinacanthus nasutus (L.) Kurz contains more highly abundant their quinoid structure molecules such as benzoquinones, naphthoquinones and anthraquinones when compared to other edible plants such as fruits and vegetables. Then, the diverse phytochemicals of Rhinacanthus nasutus (L.) Kurz has been isolated by many researchers with their great interests. Therefore, the purpose of this review is to reveal their diverse phytochemicals containing the phytoquinones and healthy effects on Rhinacanthus nasutus (L.) Kurz.

2. PHYTOCHEMICALS AND HEALTH EFFECTS OF RHINACANTHUS NASUTUS

Rhinacanthus nasutus has been used as the folkrore medicine in Taiwan

and often administrated in their remedies of mainly hepatitis, diabetes, hypertension and skin disease.

It has been known that the components of Rhinacanthus nasutus are mainly generally flavonoids，naphthoquinones, and anthraquinones [Tian-shung et al., 1995a].

1. Phytochemicals in Leaves of Rhinacanthus Nasutus

(1) Naphthoquinones

In 2000, group of Pharmaceutical Sciences, Prince of Songkla University in Thailand isolated lawson methyl ether (2) of a derivative of lawsone (1) from leaves of Rhinacanthus nasutus. In Thailand, Rhinacanthus nasutus has been used a mouthrinse.

Lawson methyl ether (2) had their antifungal activity with a minimum inhibitory concentration (MIC) of 512 μg/mL, and also had their antibacterial activity for Staphylicoccus aureus.

Lawson methyl ether (2) had the very low acute toxicity with 50% lethal dose (LD50) 70.7 mg/kg by intraperitoneal (ip) administration in mice. It appeared that an oral administration of 0.5% lawson methyl ether (2) in


sodium carboxymethyl cellulose (CMC) oral base might be stable under a heating-cooling cycle test.

In antifungal activity against Candida albicans in in vitro test, lawson methyl ether (2) represented the similar activity when compared to 1.0% clotrimazole (3) cream [Panichayupakaranant et al., 2000a](Figure 1).

In 2000, the same research group of Pharmaceutical Sciences, Prince of Songkla University in Thailand studied their chemical stability and skin irritation to skins on lawson methyl ether (2) in sodium carboxymethyl cellulose (SCMC) oral base. A mouthrinse lawson methyl ether (2) in SCMC represented the low acute toxicity with 50% lethal dose (LD50) 70.7 mg/kg by intraperitoneal (ip) administration in mice.

A mouthrinse of 0.5% lawson methyl ether (2) in sodium carboxymethyl cellulose (CMC) oral base was stable under a heating-cooling cycle test.

An oral administration of lawson methyl ether (2) in sodium carboxymethyl cellulose (CMC) oral base did not cause any skin irritation under both their primary skin irritation test (patch test) and cumulative skin irritation test.

The solution of potassium lawson methyl ether (2’) produced the erythema with some papulosquamous in cumulative skin irritation test [Blignaut et al., 2006; Panichayupakaranant et al., 2002b](Figure 1).

In 2006, a dentistry group of Prince of Songkla University in Thailand studied the effect of a mouthrinse potassium lawson methyl ether (2’) against Candida albicans isolated from 51 human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) subjects. Their antifugal activity of 0.5% potassium lawson methyl ether (2’) was compared to the activity of 0.12% and 0.2% chlorhexidine (4), which an antibacterial, effective against a wide variety of Gram-negative and Gram-positive organisms (Figure 2).

Figure 1. Lawson methyl ether (2) of napthoquinoes in the leaves of Rhinacanthus

nasutus (L.) Kurz] and lawsone (1) of its related napthoquinones, and a mouthrinse clotrimazole (3)


Figure 2. Lawson methyl ether (2) of napthoquinoes in the leaves of Rhinacanthus

nasutus (L.) Kurz] and lawsone (1) of its related napthoquinones, and a mouthwash clorhexidine (4)

Then, The positive culture of Candida albicans was observed in 13 out of

51 plates (25.4%) on 0.12% chlorhexidine (4) mouthwash, in 5 out of 51 plates (9.8%) on 0.2% chlorhexidine (4) mouthwash and in 4 out of 51 plates (7.8%) on 0.5% potassium lawson methyl ether (2’).

The mean number of Candida albicans colonies for three mouthwashes of 0.12% chlorhexidine (4), 0.2% chlorhexidine (4) and 0.5% potassium lawson methyl ether (2’) were 3.08 (range 0-40), 0.35 (range 0-13), and 0.84 (range 0-24), respectively. Their antifungal activity was statistically found to be significant different between 0.5% potassium lawson methyl ether (2’) and 0.12% chlorhexidine (4), and between the 0.12% chlorhexidine (4) and 0.2% chlorhexidine (4). However, there is no difference between antifungal activity of 0.5% potassium lawson methyl ether (2’) and 0.2% chlorhexidine (4) [Blignaut et al., 2006; Motohashi, 2009c; Prasirst et al., 2006].


2. Phytochemicals in Leaves and Stems of Rhinacanthus Nasutus

(1) Quinols

In 1995, a group of National Cheng Kung University, Taipei of Taiwan, isolated a novel 4-acetonyl-3,5-dimethoxy-p-quinol (5) from leaves and stems of Rhinacanthus nasutus [Tian-shung et al., 1995a](Figure 3).

(2) Triterpenoids

In 1995, a group of National Cheng Kung University, Taipei of Taiwan, isolated three known triterpenoide ofβ-amyrin (6), glutinol (7) and lupeol (8) from leaves and stems of Rhinacanthus nasutus [Choudhary et al., 2005; Tian-shung et al., 1995a](Figure 4).

Figure 3. A novel 4-acetonyl-3,5-dimethoxy-p-quinol (5) in the leaves and stems of Rhinacanthus nasutus (L.) Kurz]

Figure 4. Three triterpenods of β –amyrin (6), glutinol (7) and lupeol (8) in the leaves and stems of Rhinacanthus nasutus (L.) Kurz]


(3) Steroids

In 1995, a group of National Cheng Kung University, Taipei of Taiwan, isolated two known steroids of stigmasterol (9), sitosterol (β- sitosterol, 10) and their two derivatives from leaves and stems of Rhinacanthus nasutus

[Tian-shung et al., 1995a](Figure 5).

(4) Benzenoids

In 1995, a group of National Cheng Kung University, Taipei of Taiwan, isolated 2-methoxy-4-propionyl-phenol (11) of benzenoids from leaves and stems of Rhinacanthus nasutus [Tian-shung et al., 1995a](Figure 6).

Figure 5. Two steroids of stigmasterol (9) and sitosterol (β-sitosterol, 10) in the leaves and stems of Rhinacanthus nasutus (L.) Kurz]

Figure 6. A benzonoid of 2-methoxy-4-propionyl-phenol (11) in the leaves and stems of Rhinacanthus nasutus (L.) Kurz


(5) Coumarins

In 1995, a group of National Cheng Kung University, Taipei of Taiwan, isolated umbelliferone (12) of coumarins from leaves and stems of Rhinacanthus nasutus [Tian-shung et al., 1995a](Figure 7).

(6) Anthraquinones

In 1995, a group of National Cheng Kung University, Taipei of Taiwan, isolated 2-methylanthraquinone (13) of anthraquinones from leaves and stems of Rhinacanthus nasutus [Tian-shung et al., 1995a](Figure 8).

(7) Benzoquinones

In 1995, a group of National Cheng Kung University, Taipei of Taiwan, isolated 2,6-dimethoxybenzoquinone (14)of benzoquinones from leaves and stems of Rhinacanthus nasutus [Tian-shung et al., 1995a](Figure 9).

]

Figure 7. Umbelliferone (12) of coumarins in the leaves and stems of Rhinacanthus

nasutus (L.) Kurz

Figure 8. An anthraquinoe of 2-methylanthraquinone (13) in the leaves and stems of Rhinacanthus nasutus (L.) Kurz


Figure 9. A benzoquinoe of 2,6-dimethoxybenzoquinone (14) in the leaves and stems of Rhinacanthus nasutus (L.) Kurz

(8)Glycosides

In 1995, a group of National Cheng Kung University in Taipei of Taiwan isolated four glycosides from leaves and stems of Rhinacanthus nasutus [Tian-shung et al., 1995a].

Sitosterol-β-D-glucopyranoside Stigmasterol-β-D-glucopyranoside 3,4-Dimethoxyphenol-β-D-glucopyranoside 3,4,5-Trimethoxyphenol-β-D-glucopyranoside

(9) Carbohydrates

In 1995, a group of National Cheng Kung University in Taipei of Taiwan isolated methyl-α-D-galactopyranoside from leaves and stems of Rhinacanthus

nasutus [Tian-shung et al., 1995a].

(10) Chlorophyll

In 1995, a group of National Cheng Kung University, Taipei of Taiwan, isolated methyl phenophorbide-a of chlorophyll from leaves and stems of Rhinacanthus nasutus [Tian-shung et al., 1995a].

(11) Naphthopyrans

In 1993, a collaboration group with Kasetsart University, Bangkok of Thailand, isolated 3,4-dihydro-3,3-dimethyl-2H-naphtho[2,3-b]pyran-5,10-dione（15）of naphthopyrans from leaves and stems of Rhinacanthus nasutus (Figure 10).


Figure 10. 3,4-Dihydro-3, 3-dimenthyl-2 H-naptho[2, 3-b] pyran-5,10-dione (15) of napthopyrans in the stems and leaves of Rhinacanthus nasutus (L.) Kurz

The antifungal activity on 3,4-dihydro-3,3-dimethyl-2H-naphtho [2,3-b]

pyran-5,10-dione（15）was determined by observation of their inhibition effect for spore germination of Pyricularia oryzae. The 50% effective dose (ED50) value of 3,4-dihydro-3,3-dimethyl-2H-naphtho[2,3-b]pyran-5,10-dione（15） was 0.4 ppm. The inhibitory value of 3,4-dihydro-3,3-dimethyl-2H-naphtho[2,3-b]pyran-5,10-dione（15）for rice blast diseases was 82.3% at 100 ppm. It is expected that 3,4-dihydro-3,3-dimethyl-2H-naphtho[2,3-b]pyran-5,10-dione（15）will be antifungal agents for human, together with the regions of agrichemicals [Kodama et al., 1993].

(12) Antitumor activity on extracts of leaves and stems of Rhinacanthus

nasutus

In 2004, a collaboration group of medicine of Chiang Mai University of Thailand and Unversity of Leeds of UK extracted their leaves and stems of Rhinacanthus nasutus. Generally, it is known that Nitric oxide (NO) produced from the activated macrophages play a role in both inflammatory processes and anti-inflammatory processes. Then, it is studied that whether the extracts from Rhinacanthus nasutus might modulate the production of an oxygen radical NO and tumor necrosis factor- (TNF- ) using J774.2 mouse macrophages. Also, it is examined that whether the extracts from Rhinacanthus nasutus could represent their expression of inducible nitric oxide synthase (iNOS) which were not controlled by calcium and their expression of tumor necrosis factor- (TNF- ) genes.


When the ethanol extracts from Rhinacanthus nasutus were used in combination with a macrophage activator lipopolysaccharide (LPS) which enhance their enzyme activity, their productions of the oxygen radical NO was significantly increased. The secretion of tumor necrosis factor- (TNF- ) was parallel with their productions of the oxygen radical NO. The increases of secretion of tumor necrosis factor- (TNF- ) were also associated with their elevation of tumor necrosis factor- messenger RNA (TNF-α mRNA). These

results mean that their extracts from Rhinacanthus nasutus could either increase or decrease their productions of the oxygen radical NO by the increase of macrophages. It suggests that these effects might be mainly mediated through the effect by the expression of tumor necrosis factor- (TNF- ) [Punturee et al., 2004a].

3. Phytochemicals in Roots of Rhinacanthus Nasutus In 2006, a group of Tropical Medicine, Mahidol University, Bangkok in

Thailand, tried methanol extracts from roots of Rhinacanthus nasutus and examined their antivirus effects on two formulations of tablet with the extract at 5% and 10% concentration. Their mosquito larvicidal activity of root extracts from Rhinacanthus nasutus against Ades aegypti which mediate the yellow fever virus with 5-10% lethal percentage and the chikungunya virus with severe arthralgia, or against Culex quinquefasciatus (Anopheles spp.) of mosquito which mediate the yellow fiber virus, dengue virus, chikungunya virus was studied.

First, their 50% lethal concentration (LC 50) values, mosquito larvicidal activity against Ades aegypti by two Rhinacanthus nasutus root methanol extracts at 5% and 10% concentration in water, were 13.6 mg/L and 14.2 mg/L, respectively, and these are not significant activity.

While their 50% lethal concentration (LC 50) values, mosquito larvicidal activity against Culex quinquefasciatus by two Rhinacanthus nasutus root methanol extracts at 5% and 10% concentration in water, were 18.7 mg/L and 17.3 mg/L, respectively, and similarly in the Ades aegypti, these also are not significant activity. There is no any observations on their larval mortality in the two control groups such as lactone solution and dechlorinated water.

Second, their toxicity of two Rhinacanthus nasutus root methanol extracts at 5% and 10% concentration was tested by Poecilia reticulata of female and male guppies belong to Poeciliidae (Family), Poecilia (Genus).


Then, their 50% lethal concentration (LC50) values after 48 against female guppy by two Rhinacanthus nasutus root methanol extracts at 5% and 10% concentration in water, were 105.2 mg/L and 110.8 mg/L, respectively.

Following, their 50% lethal concentration (LC50) values after 48 against male guppy by two Rhinacanthus nasutus root methanol extracts at 5% and 10% concentration in water, were 99.1 mg/L and 103.4 mg/L, respectively.

Interestingly, these tests, the female and male guppies were almost sensitive to the same dose against the Rhinacanthus nasutus root methanol extracts. There is no any observations on their larval mortality in the two control groups such as lactone solution and dechlorinated water.

In these two acute-toxiciy bioassays, 50% lethal concentration (LC50) values after 48 hours exposures against guppies by the Rhinacanthus nasutus root methanol extracts at 5% and 10% concentration in water were 5-fold to 10-fold higher than the LC50 of Rhinacanthus nasutus root methanol extracts against mosquito larvae. Therefore, it suggested that Rhinacanthus nasutus root methanol extracts might be safe for both human and fishes, and moreover, these extracts might be effective for prevention against mosquitoes which mediate the mosquito-related diseases such as yellow fiber [Rongsriyam et al., 2006].

(1) Naphthoquinones

In 1988, a group of Applied Chemistry, Province College of Arts and Sciences, Taichung Hsien of Taiwan, isolated two novel naphthoquinones of rhinacanthin-A (16) and rhinacanthin-B (17) from the roots of Rhinacanthus

nasutus [Tian-Shung et al., 1988b](Figure 11). In 1972, a group of NIH in USA represented that rhinacanthin-B (17) had

significantly 50% effective dose (ED50=3.0μg/mL against KB cells in tissue culture assay. However, rhinacanthin-A (16) exhibited no cytotoxicity for KB cells [Geran et al., 1972].

The significant difference of their cytotoxicity between rhinacanthin-A (16) and rhinacanthin-B (17) also could be supported rhinacanthin-B (17) has a lipophilic site in the molecule. Therefore, the lipophilic site of rhinacanthin-B (17) might play an important role to contribution of the lipophilicity to their enhanced cytotoxicity. Similarly, this could be also observed in the case of a cytotoxic psychorubrin (3-hydroxy-1H-3,4-dihydronaphtho[2,3-c]pyran-5,10-dione, 18) of naphthoquinones from Psychotria rubra [Hayashi et al., 1987](Figure 11).


Figure 11. Two novel napthoqionones of rhinacanthin-A (16) and rhinacanthin-B (17) from the roots of Rhinacanthus nasutus and psychorubrin (3-hydroxy-1H-3,4-dihydronaptho[2, 3-c]pyran-5,10-dione, 18)


Figure 12. Rhinacanthin-A(16), rhinacanthin-B (17), rhinacanthin-C (19) and rhinacanthin-D (20) of napthoquinones in the roots of Rhinacanthus nasutus (L.) Kurz


Figure 12. (Continued 1) Rhinacanthin-O (21), rhinacanthin-P (22) of two dimethyldihydropyrano-1,4-naphthoquinones and rhinacanthin-G (23), rhinacanthin-H (24), rhinacanthin-I (25), rhinacanthin-J (26), rhinacanthin-K (27), rhinacanthin-L (28), rhinacanthin-M (29), rhinacanthin-N (30) of eight 2-hydroxy-1,4-naphthoquinones in the roots of Rhinacanthus nasutus (L.) Kurz)


Figure 13. Psychorubrin (3-hydroxy-1 H-3,4-dihydronaptho [2,3-c] pyran-5, 10-dione, 18) of napthoquinones

In 1998, a group of National Cheng Kung University, Tainan in Taiwan,

isolated four known rhinacanthins such as rhinacanthin-A (16)], rhinacanthin-B (17), rhinacanthin-C (19) and rhinacanthin-D (20), two novel dimethyldihydropyrano-1,4-naphthoquinones such as rhinacanthin-O (21) and rhinacanthin-P (22), and eight novel 2-hydroxy-1,4-naphthoquinones such as rhinacanthin-G (23), rhinacanthin-H (24), rhinacanthin-I (25), rhinacanthin-J (26), rhinacanthin-K (27), rhinacanthin-L (28) and rhinacanthin-M (29), rhinacanthin-N (30) [Tian-Shung et al., 1988b; Wu et al., 1998a](Figure 12)(Figure 12(continued 1).

Interestingly, in 1987, a group of School of Pharmacy, University of North Carolina, Chapel Hill, confirmed that psychorubrin（18）of a new naphthoquinone from the alcoholic extract of Psychotria rubra, represented the significant cytotoxicity for KB cells (ED50=3.0 g/mL). However, when a hydrophilic hydroxy group was present in such compounds, their reduced in

vitro activity was significantly observed [Hayashi et al., 1987]. In 1998, a group of Chemistry, National Cheng Kung University, Tainan

in Taiwan, isolated a novel rhinacanthin-Q (31) of novel naphthoquinones and ten known rhinacanthins such as rhinacanthin-A (16)，rhinacanthin-B (17)，rhinacanthin-C (19), rhinacanthin-D (20)，rhinacanthin-G (23), rhinacanthin-H (24), rhinacanthin-I (25)，rhinacanthin-K (27)，rhinacanthin-M (29) and rhinacanthin-N (30) [Wu et al., 1998b](Figure 14).


Figure 14. New rhinacanthin-Q (31) and 10 unknown napthoquinonesin the roots of Rhinacanthus nasutus (L.) Kurz]

In 1998, the same group of Chemistry, National Cheng Kung University,

Tainan in Taiwan, studied their cytotoxic evaluation against five carcinogenic cell strains such as KB cell of human oral epidermoid carcinoma, P388 murine leukemia cells, A549 cells of carcinomic human alveolar basal epithelial cells, HT-29 human intestinal cancer cells and human promyelocytic leukemia cells (HL-60) on these eleven naphthoquinones (16, 17, 19, 20, 23, 24, 25, 27, 29, 30, 31). Among these five carcinogenic cell strains used in this evaluation, all


1,4 naphthoquinones (16, 17, 19, 20, 23, 24, 25, 27, 29, 30, 31) showed their significant cytotoxicity against four carcinogenic cell strains of P388 murine leukemia cells, A549 cells of carcinomic human alveolar basal epithelial cells, HT-29 human intestinal cancer cells and human promyelocytic leukemia cells (HL-60), excepting only KB cell of human oral epidermoid carcinoma.

Generally, it is known that when agglutinins such as collagen, and thrombin were added to normal platelet, the direct agglutination is happened and a platelet aggregation is observed with their decrease of turbidity.

It is also know that arachidonic acid of straight chain unsaturated fatty acids bond with peroxisome proliferator-activated receptor (PPAR) in a nuclear receptor of platelet and activate, and finally inhibit their cell differentiation. Similarly, it is thought that arachidonic acid also might form their direct agglutination of platelet.

From these facts, their antiplatelet aggregation activity was examined on nine rhinacanthins of 1,4-naphthoquinones such as rhinacanthin-A (16), rhinacanthin-B (17), rhinacanthin-C (19), rhinacanthin-G (23), rhinacanthin-H (24), rhinacanthin-I (25), rhinacanthin-K (27), rhinacanthin-M (29), rhinacanthin-Q (31) at each 100μg/mL concentration. These nine rhinacanthins

used in this study showed antiplatelet aggregation activity in wide ranges from rhinacanthin-B (17, 7% inhibition) to rhinacanthin-A (16, 100% inhibition)，rhinacanthin-C (19, 100 inhibition) and rhinacanthin-M (29, 100% inhibition) against arachidonic acid (100 μM)-induced rabbit platelet aggregation.

Similarly, their antiplatelet aggregation activity by rhinacanthins against collagen (100 μM)-induced rabbit platelet aggregation were in wide ranges from rhinacanthin-M (29, 5% inhibition) to rhinacanthin-A (16, 100% inhibition)，rhinacanthin-B (17, 100% inhibition) and rhinacanthin-M (29, 100% inhibition).

Surprisingly, rhinacanthin-B (17, 100% inhibition) only showed antiplatelet-activating factor (anti-PAF) at 2 ng/mL concentration. Then, it is thought that rhinacanthis such as rhinacanthin-B (17) might decompose platelet-activating-factor (PAF) acetylhydrolase, which exists in plasma and which happens allergy diseases such as allergic airway disease and anaphylaxis, or inflammation [Wu et al., 1998b].

In 2004, a group of Kesetsart University, Bangkok in Thailand, examined their antitumor activity against three cancer cell lines of KB human epiderimoid carcinoma, HeLa human cervical carcinoma and HepG2 human hepatocellular carcinoma and antivirus activity against Vero cell line with their higher sensitivity as many human viruses among the African green monkey


kidney cells by three rhinacanthis such as rhinacanthin-M (29), rhinacanthin-N (30)] and rhinacanthin-Q (31), using adriamycin (33) as control (Figure 15).

Figure 15. The cytotoxicity of rhinacanthin-M (29), rhinacanthin-N (30), rhinacanthin-Q (31) in the roots of Rhinacanthin nasutus (L.) Kurz, and adriamycin (33) of a control in biossay.


First, among three rhinacanthins (29, 30, 31), their 50% median inhibition concentration (IC50) against KB human epiderimoid carcinoma was rhinacanthin-M (29) (IC50=1.53 μM), rhinacanthin-N (30) (IC50<0.22 μM),

rhinacanthin-Q (31) (IC50=0.35 μM) and adriamycin (33)(IC50=0.033 μM),

respectively. Second, among three rhinacanthins (29, 30, 31), their 50% median

inhibition concentration (IC50) against HeLa human cervical carcinoma was rhinacanthin-M (29) (IC50=3.02 μM), rhinacanthin-N (30) (IC50<0.30 μM),

rhinacanthin-Q (31) (IC50=1.09 μM) and adriamycin (33) (IC50=0.33 μM),

respectively. Third, among three rhinacanthins (29, 30, 31), their 50% median

inhibition concentration (IC50) against HepG2 human hepatocellular carcinoma was rhinacanthin-M (29) (IC50=4.85 μM), rhinacanthin-N (30) (IC50=0.38 μM), rhinacanthin-Q (31) (IC50=0.97 μM) and adriamycin (33) (IC50=0.40 μM), respectively.

Finally, among three rhinacanthins (29, 30, 31), their 50% median inhibition concentration (IC50) against Vero cell line was rhinacanthin-M (29) (IC50=48.64 μM), rhinacanthin-N (30) (IC50=12.65 μM), rhinacanthin-Q (31) (IC50=32.70 μM) and adriamycin (33) (IC50=23.94 μM), respectively.

From these four cytotoxicity examinations, two rhinacanthins such as rhinacanthin-N (30) (IC50=12.65 μM) and rhinacanthin-Q (31) (IC50=32.70 μM) could be expected as higher antitumor and antivirus agents by further

modifications of their rhinacanthins. Additionally, the group synthesized their derivatives of three

rhinacanthins（29, 30, 31）and examined the similar biological tests [Kongkathip et al., 2004].

In 2006, a collaboration group of National Cancer Institute, Bangkok in Thailand, examined their induction of apoptosis against HeLaS3 human cervical carcinoma cells by TUNEL method on three rhinacanthins such as rhinacanthin-C (19), rhinacanthin-N (30) and rhinacanthin-Q (31) from the roots of Rhinacanthus nasutus (L.) Kurz.

First, their increase of the carcinoma cells was dose-dependently inhibited when these three rhinacanthins of rhinacanthin-C (19), rhinacanthin-N (30) and rhinacanthin-Q (31) are added to HeLaS3 human cervical carcinoma cells. At the same time, their productions of caspase-3 in HeLaS3 human cervical carcinoma cells also increased dose-dependently. Among three rhinacanthins, rhinacanthin-N (30) showed significantly antitumor activity

Second, the breakage of DNA fragmentation of HeLaS3 cells was observed as an apparent morphological change by agarose gel electrophoresis.


From these facts, their inhibitions of the increase against HeLaS3 human cervical carcinoma cells by three rhinacanthins of rhinacanthin-C (19), rhinacanthin-N (30) and rhinacanthin-Q (31) were caused by apoptosis. It seems that activation of caspase-3 might activation of caspase apperas to be directly responsible for many of molecular and structural changes in apoptosis. Then, it is thought that these rhinacanthins might induce the apoptosis against HeLaS3 human cervical carcinoma cells by mediating caspase-3. Therefore, it is expected that these rhinacanthins will be one of novel anticancer drugs in the near future [Siripong et al., 2006a](Figure 16).

Figure 16. Rhinacanthin-C (19), rhinacanthin-N (30) and rhinacanthin-Q (31) in the roots of Rhinacanthin nasutus (L.) Kurz]


Further, in 2006, a collaboration group of National Cancer Institute, Bangkok in Thailand, examined their anticancer effect by liposome injection of three rhinacanthins of rhinacanthin-C (19), rhinacanthin-N (30) and rhinacanthin-Q (31) from the roots of Rhinacanthus nasutus (L.) Kurz. In 2006, the same research group have already confirmed the induction of apoptosis against HeLaS3 human cervical carcinoma cells by three same rhinacanthis [Siripong et al., 2006a]．However, these antitumor avtivity of three rhinancanthins is limited in only water medium. Therefore, their liposomalization enabled the injection of three rhinacanthins and the rhinacanthins could be expected to transfer to lipoproteins in the bloodstream.

First, the 50% lethal dose（LC50）against HeLaS3 human cervical carcinoma cells) by their liposomal formulation of three rhinacanthins of rhinacanthin-C (19), rhinacanthin-N (30) and rhinacanthin-Q (31) was 32 μM,

17 μM and 70 μM, after 24 hour injection, 19 μM, 17 μM and 52 μM after 48

hour injection, and 2.7 μM, 2.0 μM and 5.0 μM after 72 hour injection, respectively.

Second, three rhinacanthins of rhinacanthin-C (19), rhinacanthin-N (30) and rhinacanthin-Q (31) were intraperitoneally (i.p.) administrated the Meth-A murine sarcoma ascites cells to male BALB/c mice. After 8-10 days, their increased ascites cells were taken. These ascites cells were implanted subcutaneously (s.c) to five week-old male BALB/c mice. At the same time, 5.0 mg/kg/day of three rhinacanthins of rhinacanthin-C (19), rhinacanthin-N (30) and rhinacanthin-Q (31) were intraperitoneally (i.p.) administrated to five week-old male BALB/c mice fro 10 days. Among three rhinacanthins, rhinacanthin N (30) inhibited significantly the growth of the solid tumor.

From these facts, it suggests that rhinacanthin N (30) could inhibit apparently the growth of tumors in in vitro examination. Therefore, the liposome with the injectable formation of higher hydrophobic drugs might be expected their higher effective anticancer drugs, just like a sample as the liposome of hydrophobic rhinacanthin N (30)[Siripong et al., 2006b] (Figure 16).

(2) Triterpenoids

In 1988, a collaboration group of Applied Chemistry of Province College of Arts and Sciences, Taichung Hsien in Taiwan, isolated lupeol (8) of triterpenoids from the roots of Rhinacanthus nasutus (L.) Kurz [Tian-Shung et

al., 1988b](Figure 17).


In 1998, the group of National Cheng Kung University, Tainan in Taiwan, also isolated lupeol (8) from the roots of Rhinacanthus nasutus (L.) Kurz [Wu et al., 1998a](Figure 17).

(3) Steroids

In 1988, a group of Applied Chemistry, Province College of Arts and Sciences, Taichung in Taiwan, isolated two known steroids such as stigmasterol (9) and sitosterol (β- sitosterol (10)), and also the glucosides of two their steroid from the roots of Rhinacanthus nasutus (L.) Kurz [Tian-Shung et al., 1988b](Figure 18).

Figure 17. Lupeol (4) of triterpenoids in the roots of Rhinacanthus nasutus (L.) Kurz

Figure 18. Two steroids of stigmasterol (9) and sitosterol (β -sitosterol, 10) in the roots of Rhinacanthus nasutus (L.) Kurz


(4) Flavonoids

In 1998, a group of National Cheng Kung University, Tainan in Taiwan, isolated two known antioxidative flavonoids such as wogonin (32) and oroxylin (34) from the roots of Rhinacanthus nasutus (L.) Kurz [Shih et al., 2009; Wu et al., 1998a](Figure 19).

In 1998, the same group of Chemistry, National Cheng Kung University, Tainan in Taiwan, studied their cytotoxic evaluation against five carcinogenic cell strains such as KB cell of human oral epidermoid carcinoma, P388 murine leukemia cells, A549 cells of carcinomic human alveolar basal epithelial cells, HT-29 human intestinal cancer cells and human promyelocytic leukemia cells (HL-60) on an oxidative flavonoid wogonin (32).

Among these five carcinogenic cell strains used in this evaluation, wogonin (32) showed their significant cytotoxicity against four carcinogenic cell strains of P388 murine leukemia cells, A549 cells of carcinomic human alveolar basal epithelial cells, HT-29 human intestinal cancer cells and human promyelocytic leukemia cells (HL-60), excepting only KB cell of human oral epidermoid carcinoma.

Generally, it is known that when agglutinins such as collagen, and thrombin were added to normal platelet, the direct agglutination is happened and a platelet aggregation is observed with their decrease of turbidity.

It is also know that arachidonic acid of straight chain unsaturated fatty acids bond with peroxisome proliferator-activated receptor (PPAR) in a nuclear receptor of platelet and activate, and finally inhibit their cell differentiation. Similarly, it is thought that arachidonic acid also might form their direct agglutination of platelet.

From these facts, their antiplatelet aggregation activity was examined on wogonin (32) at 100μg/mL concentration. Wogonin (32) used in this study showed antiplatelet aggregation activity (100% inhibition) against arachidonic acid (100 μM)-induced rabbit platelet aggregation.

Similarly, their antiplatelet aggregation activity by Wogonin (32) against collagen (100 μM)-induced rabbit platelet aggregation showed the 73% inhibition [Wu et al., 1998b].

(5) Coumarins

In 1998, a group of Chemistry of National Cheng Kung University isolated a (+)-praeruptorin (35) of coumarins from the roots of Rhinacanthus

nasutus [Wu et al., 1998a](Figure 20).


Figure 19. Wogonin (32) and oroxylin (34) of two flavonoids in the roots of Rhinacanthus nasutus (L.) Kurz

Figure 20. (+)-praeruptorin (35) of coumarins from the root of Rhinacanthjus nasutus

(6) Allantoin

In 1998, a group of National Cheng Kung University, Tainan in Taiwan, isolated allantoin (36) of the diureide of glyoxylic acid as a metabolite of purines from the roots of Rhinacanthus nasutus (Figure 21).


Figure 21. Allantoin (36) of purine metabolite in the roots of Rhinacanthus nasutus (L.) Kurz

Widely, allantoin (36) could find in many plants and also in allantoic fluid and fetal urine. Interestingly, allantoin (36) could be found as a urinary excretion product of purine metabolism in most mammals, however, allantoin (36) could not find in man or the higher apes. Allantoin (36) also could be chemically synthetized by the oxidation of uric acid. Allantoin (36) had once used to encourage the epithelial formation in mounds and ulcers, and also in osteomyelitis [Wu et al., 1998a].

(7) Antimutagenicity on extracts of the roots of Rhinacanthus nasutus

In 1990, a group of National Cancer Institute, Bangkok in Thailand, examined their mutagenicity (carcinogenicity) by Ames test on the extracts from the roots of Rhinacanthus nasutus (Honma et al., 1999).

First, the mutagenic potential on the extracts from the roots of Rhinacanthus nasutus was tested. For the test of whether the extracts from the roots of Rhinacanthus have their mutagenicity or have not, their mutagenicity test was examined against two Salmonella typhimurium tester strains such as TA98 and A100 together with their PCB-induced S-9 mix, which the mutagenic polychlorinated biphenyls (PCBs) were added in a single lot of post-mitochondrial supernatant fractions of rat liver homogenates (S-9 mix) of a rat liver homogenate. By the test, the extracts from the roots of Rhinacanthus

nasutus showed no mutagenicity against two Salmonella typhimurium of TA98 and A100.

Second, the antimutagenicity of the extracts from the roots of Rhinacanthus nasutus was examined by Ames test.

Generally, it is known that the domestic fowl and other animals fed with their infected peanut meal to aflatoxin B1 (AFB1, 37) might die by the cause of afflatoxicosis, hepatocellular carcinoma, or cholangiocarcinoma, and has


also been implicated as a cause of human hepatic carrcinoma. Furylfuramid (AF-2, 38) was also known to be their higher positive reverse mutation. Therefore, two standard mutagens such as aflatoxin B1 (AFB1, 37) and furylfuramid (AF-2, 38) were used on the antimutagenicity by the extracts from the roots of Rhinacanthus nasutus (Figure 22). First, their mutagenicity against Salmonella typhimurium tester strarin TA100 was examined by adding two hexane and chloroform extracts from the roots of Rhinacanthus nasutus to AFB1 (37) S-9 mix, or alone S-9 mix. Then, both of two hexane and chloroform extracts from the roots of Rhinacanthus nasutus showed significantly their antimutagenicity of Salmonella typhimurium strarin TA100 by aflatoxin B1 (AFB1, 37) of an indirect mutagen. However, methanol extract from the roots of Rhinacanthus nasutus was not antimutagenic on Salmonella typhimurium TA100 by aflatoxin B1 (AFB1, 37). Second, three methanol, hexane and chloroform extracts from the roots of Rhinacanthus

nasutus were examined on their antimutagenicity of Salmonella typhimurium TA100 in S-9 mix with furylfuramid (AF-2, 38) and alone S-9 mix. Then, all three methanol, hexane and chloroform extracts from the roots of Rhinacanthus nasutus were not antimutagenic on Salmonella typhimurium TA100 against furylfuramid (AF-2, 38) of a direct mutagen. Additionally, the methanol extract from the roots of Rhinacanthus nasutus was no antimutagenic on Salmonella typhimurium TA100 against both two aflatoxin B1 (AFB1, 37) and furylfuramid (AF-2, 38) [Rojanapo et al., 1990].

Third, their extracts from the roots of Rhinacanthus nasutus were examined on the enzyme inhibition of rat liver aniline hydroxylase. Aniline hydroxylase is one of oxygenases, which a drug-metabolizing, cytochrome P-450 enzyme which catalyzes the hydroxylation of aniline to hydroxyaniline in the presence of reduced flavoprotein and molecular oxygen. For example, aryl 4-hydroxylase catalizes aniline to aminophenol.

Figure 22. Two mutagens of aflatoxin B1 (AFB1, 37) and furylfuramid (AF-2, 38)


The enzyme inhibitory effects by their extract from the roots of Rhinacanthus nasutus were measured by using the PCB-induced S-9 fractions as the source of aniline hydroxylase. Among three extracts of methanol, hexane and chloroform extracts, the chloroform extract showed the highest inhibition effect against aniline hydroxylase, followed by hexane extract. However, the methanol extract was no inhibitory against aniline hydroxylase.

It suggests that from these three facts of Ames test and their enzyme inhibitory effects of rat liver aniline hydroxylase, their qualitative and quantative inhibition of mutagenicity by their extract from the roots of Rhinacanthus nasutus might be greatly corresponding to their enzyme inhibition of the rat liver aniline hydroxylase [Rojanapo et al., 1990].

4. Phytochemicals in Leaves and Roots of Rhinacanthus Nasutus

(1) Naphthoquinones

In 2004, a collaboration group of Kobe University isolated rhinacanthin-C (19) of naphthoquinones from the leaves and roots of Rhinacanthus nasutus [Gotoh et al., 2004; Sendl et al., 1996](Figure 23).

When rhinacanthin-C (19) containing in ethanol extract (500 mg/kg/day, pa administration) from the roots of Rhinacanthus nasutus and water extract (500 mg/kg/day, pa injection) from the leaves of Rhinacanthus nasutus and mitomycin C (MMC) (1 mg/kg/day, ip administration) have been administrated to sarcoma 180 mice for 14 days, their inhibition ratio % (ir %) were significantly 52.5, 44.2 and 52.7%, respectively [Gotoh et al., 2004].

Figure 23. Rhinacanthin-C (19) of napthoquinones in the leaves and roots of Rhinacanthus nasutus (L.) Kurz


5. Phytochemicals of Whole Plant of Rhinacanthus Nasutus In 2005, a group of Medicine of Chiang Mai University, Chiang Mai in

Thailand, examined the influence of cell-mediated and humoral immune response by extracts of whole plants of Rhinacanthus nasutus.

First, the water and ethanol extracts of whole plants of Rhinacanthus

nasutus influenced significantly on the lymphocytes of human peripheral blood mononuclear cells (PBMCs) which are widely distributed in bloods and enhanced their activity of the NK cells. Moreover, the extracts of whole plants of Rhinacanthus nasutus enhanced their increases and productions of their interleukin 2 (IL-2) which induced their production of interferon- (IF- ) having the antitumor effects, and also enhanced their increases and productions of their tumor necrosis factor-α (TNF-α) which cause their

necrosis against solid tumor cells. Second, the water and ethanol extracts of whole plants of Rhinacanthus

nasutus enhanced only the secondary antibody response against BALB/c mice are useful for research into both cancer and immunology (100 mg/kg body weight) [Punturee et al., 2005b].

(1) Naphthoquinones

In 1996, a group of Shaman Pharmaceuticals, California in USA, isolated two novel naphthoquinones such as rhinacanthin-C (19) and rhinacanthin-D (20) from whole plant of Rhinacanthus nasutus [Sendl et al., 1996](Figure 24).

Two rhinacanthin-C (19) and rhinacanthin-D (20) showed high antivirus activity. For example, their median inhibition concentration (IC50) of rhinacanthin-C (19), rhinacanthin-D (20) and a derivative of acyclovir, ganciclovir (39) with in vitro activity against human herpesviruses and used for the treatment of cytomegalovirus infections were 0.56μg/mL, 0.75μg/mL

and 1000μg/mL, respectively and showed their significant antivirus activity

against human CMN virus (hCMV)(Figure 25). Similarly, their median inhibition concentration (IC50) of rhinacanthin-C

(19), rhinacanthin-D (20) and amantadine (40) (Figure 25) of an antiviral compounds used in the prophylaxis and management of type A influenza also were 0.2μg/mL，0.78μg/mL and >56μg/mL, respectively. Their antivirus effects against type A influenza by rhinacanthin-C (19) and rhinacanthin-D (20) were higher than that of amantadine (40) [Sendl et al., 1996].


Figure 24. Rhinacanthin-C (19) and rhinacanthin-D (20) of two novel napthoquinones in the whole plants of Rhinacanthus nasutus (L.) Kurz]

Figure 25. Ganciclovir (39) of anti-human herpes virus agent and amantadine (40) of anti-influenza type A


Figure 26. Rhinacanthin-E (41) and rhinacanthin-F (42) of two novel lignans in the whole plant of Rhinacanthus nasutus (L.) Kurz, and amantadine (40) of antiviral (Influenza A) agent

(2) Anti-influenza virus

In 1997, group of Sharnan Pharmaceuticals of South San Francisco in California USA, isolated two novel rhinacanthins (lignans) such as rhinacanthin-E (41) and rhinacanthin-F (42) from whole plant of Rhinacanthus

nasutus and examined their antivirus activity of rhinacanthin-E (41) and rhinacanthin-F (42) by anti-Flu-A cytopathic effect assay (CPE) in in vitro assay.

Their antiviral activity (μg/mL) against Influenza virus type A by

rhinacanthin-E (41) was 7.4μg/mL at 50% effective concentration (EC50), 102μg/mL at 50% inhibitory concentration (IC50)and 15 at selective index (IC50/EC50, SI), respectively.

Similarly, Their antiviral activity (μg/mL) against Influenza virus type A by rhinacanthin-E (41) was 3.1μg/mL at 50% effective concentration (EC50), 21μg/mL at 50% inhibitory concentration (IC50)and 6.8 at selective index


(IC50/EC50, SI), respectively. Then, the two rhinacanthins (41, 42) showed significantly their antiviral activity.

Their antiviral activity (μg/mL) against Influenza virus type A by a control

amantadine (40) which used in the prevention and treatment of type A influenza was 0.054μg/mL at 50% effective concentration (EC50), 56μg/mL at 50% inhibitory concentration (IC50)and 1000 at selective index (IC50/EC50, SI), respectively [Kernan et al., 1997](Figure 26).

6. Phytochemicals from Flowers of Rhinacanthus Nasutus

(1) Flavonols

In 1981, a group of Chemistry Department of University of Madras, Tiruchirapalli in India, isolated a yellow pigment quercetin (43) of oxidative flavonols from fresh flowers of Rhinacanthus nasutus [Subramanian et al., 1981] (Figure 27).

Figure 27. Quercetin (43) of flavonols in the flowers of Rhincanthus nasutus (L.) Kurz


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Honma, M; Hayashi, M; Shimada, H; Tanaka, N; Wakuri, S; Awogi, Tv Yamamoto, KI; Kodani, N; Nishi, Y; Nakadate, M; Sofuni, T; Evaluation of the mouse lymphoma tk assay (microwell method) as an alternative to the in vitro chromosomal aberration test. Mutagenesis 14, 5-22, 1999.

Kernan, MR; Sendl, A; Chen, JL; Jolad, SD; Blanc, P; Murphy, JT; Stoddart, CA; Nanakorn, W; Balick, MJ; Rozhon, EJ. Two new lignans with activity against influenza virus from the medicinal plant Rhinacanthus

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Isolation and identification of an antifungal naphthopyran derivative from Rhinacanthus nasutus. J Nat Prod 56, 292-294, 1993.

Kongkathip, N; Luangkamin, S; Kongkathip, B; Sangma, C; Grigg, R; Kongsaeree, P; Prabpai, S; Pradidphol, N; Piyaviriyagul, S; Siripong, P. Synthesis of novel rhinacanthins and related anticancer naphthoquinone esters. J Med Chem 47, 4427-4438, 2004.

Motohashi N (ed.). Bioactive Heterocycles VI. Flavonoids and Anthocyanins

in Plants, and Latest Bioactive Heterocycles I. 2008a, Springer, Heidelberg, Germany.


Motohashi, N (ed.). Bioactive Heterocycles VII. Flavonoids and Anthocyanins

in Plants, and Latest Bioactive Heterocycles II. 2009b, Springer, Heidelberg, Germany.

Motohashi, Noboru. Kampoh Kenkyu (Chinese Medicine), (8), in press, (in Japanese), 2009c.

Panichayupakaranant, P; Reanmongkol, W. Evaluation of chemical stability and skin irritation of Lawsone methyl ether in oral base. Pharm Biol 40, 429-432, 2002b.

Panichayupakaranant, P; Yuenyongsawad, S; Reanmongkol, W. Lawsone methyl ether in oral base and its chemical stability. Songklanakarin J Sci

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Panichayupakaranant, P; Teanpaisan, R; Nittayananta, W. Antifungal activity of potassium lawsone methyl ether mouthwash in comparison with chlorhexidine mouthwash on oral Candida isolated from HIV/AIDS subjects (abstract). Adv Dent Res 19 170, 2006.

Punturee, K; Wild, CP; Kasinrerk, W; Vinitketkumnuen, U. Immunomodulatory activities of Centella asiatica and Rhinacanthus

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nitric oxide and tumor necrosis factor-alpha in J774.2 mouse macrophages. J Ethnopharmacol 95, 183-189, 2004a.

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Rongsriyam, Y; Trongtokit, Y; Komalamisra, N; Sinchaipanich, N; Apiwathnasorn, C; Mitrejet, A. Formulation of tablets from the crude extract of Rhinacanthus nasutus (Thai local plant) against Aedes aegypti and Culex quinquefasciatus larvae: a preliminary study. Southeast Asian J

Trop Med Public Health 7, 265-271, 2006. Sendl, A; Chen, JL; Jolad, SD; Stoddart, C; Rozhon, E; Kernan, M; Nanakorn,

W; Balick, M. Two new naphthoquinones with antiviral activity from Rhinacanthus nasutus. J Nat Prod 59, 808-811, 1996.

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Siripong, P; Yahuafai, J; Shimizu, K; Ichikawa, K; Yonezawa, S; Asai, T; Kanokmedakul, K; Ruchirawat, S; Oku, N. Induction of apoptosis in tumor cells by three naphthoquinone esters isolated from Thai medicinal plant: Rhinacanthus nasutus KURZ. Biol Pharm Bull 29, 2070-2076, 2006a.


Siripong, P; Yahuafai, J; Shimizu, K; Ichikawa, K; Yonezawa, S; Asai, T; Kanokmedakul, K; Ruchirawat, S; Oku, N. Antitumor activity of liposomal naphthoquinone esters isolated from Thai medicinal plant: Rhinacanthus nasutus Kurz. Biol Pharm Bull 29, 2279-2283, 2006b.

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Tian-shung, Wu; Chien-Chin, Yang; Pei-Lin, Wu; Ling-Kang, Liu. A quinol and steroids from the leaves and stems of Rhinacanthus nasutus, Phytochemistry 40, 1247-1249, 1995a.

Tian-Shung, Wu; Hsien-Ju, Tien; Mou-Yung, Yeh; Kuo-Hsiung, Lee. Isolation and cytotoxicity of Rhinacanthin-A and -B, two naphthoquinones, from Rhinacanthus nasutus. Phytochemistry 27, 3787-3788, 1988b.

Wu, TS; Hsu, HC; Wu, PL; Leu, YL; Chan, YY; Chern, CY; Yeh, MY; Tien, HJ. Naphthoquinone esters from the root of Rhinacanthus nasutus. Chem

Pharm Bull 46, 413-418, 1998a. Wu, TS; Hsu, HC; Wu, PL; Teng, CM; Wu, YC. Rhinacanthin-Q, a

naphthoquinone from Rhinacanthus nasutus and its biological activity. Phytochemistry 49, 2001-2003, 1998b.


Lecture 5

DIETARY FIBERS IN CULTURED CELLS EXPERIMENTAL IN VITRO DIGESTION AND FERMENTATION MODELS OF FIBER

HEALTH EFFECTS


ABSTRACT

To study health effects of different foodstuffs and nutrients, e.g. dietary fibres, on the large intestine, it is a prerequisite to obtain samples that resemble contents of the gut after digestion of the respective foodstuffs. Since the content of the human large bowel is inaccessible for routine investigations, several different in vitro systems have been established to simulate digestion and bacterial fermentation in the human gastrointestinal tract. The simplest form of these in vitro models is the batch style fermentation using defined populations of bacteria or faecal material to simulate digestive processes in the large intestine. More sophisticated models involve multistage systems simulating the whole gastrointestinal tract, including mouth, stomach as well as small and large intestine. In vitro fermentation models have been extensively used to analyse the metabolites generated by digestion and fermentation of different dietary fibres and other foodstuffs and how these metabolites influence the gut microbiota. Additionally these in vitro fermentation systems have been also used to produce samples resembling gut contents and to analyse possible health effects of these samples in in vitro cell culture studies. Since dietary fibres reach the colon undigested, simple


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batch fermentation models of the large intestine are sufficient to study possible health effects of dietary fibres on cultured cell lines. Dietary fibres are recognised for their potential to prevent cancer, e.g. colorectal cancers. Therefore a lot of research has been done to analyse effects of fermentation supernatants from dietary fibres on colon cancer prevention using different colon cell models. Chemical analyses of fermentation supernatants obtained by in vitro fermentation of dietary fibres, provided information on the respective concentrations of different components, e.g. short chain fatty acids (SCFA). Synthetic mixtures of the different components were then used to investigate which components are especially active. Dietary fibres are typically ingredients of foodstuffs that have numerous other ingredients. To analyse possible synergistic effects of dietary fibres and these ingredients, more complex in vitro systems, taking into account earlier stages of digestion, e.g. mouth, stomach or small intestine, have to be used. These multi-stage fermentation systems have therefore been modified to be used in cell culture studies. Such models will improve studies analysing the effects of foodstuffs on prevention of colon cancer and gut health in general. The following chapter summarises recent progressions on the use of in vitro models to study health effects of dietary fibres and other nutrients using in vitro colon cell models.

INTRODUCTION The gastrointestinal tract (GIT) is one of the major organ systems of

multicellular animals. It is important for food uptake, digestion and extraction of energy and nutrients as well as excretion of the remaining waste. In humans the GIT can be roughly divided into the upper and lower gastrointestinal tract. Mouth, pharynx, oesophagus and stomach make up the upper GIT, whereas the lower GIT is comprised of small intestine, large intestine and anus. The small intestine can be structurally divided into duodenum, jejunum and ileum. In the small intestine the majority of digestion takes place and nutrients reach the blood stream after absorption by the epithelium. Caecum, colon and rectum form the large intestine. The colon consists of the ascending, transverse, descending, and sigmoid part. It is important for storage and disposal of waste as well as maintenance of water balance. Furthermore the colon hosts the majority of the gut flora. The most important function of the gut flora is the fermentation of dietary fibre, resulting in short chain fatty acids (SCFA), mainly acetate, propionate and butyrate and gases (e.g. CO2, CH4, H2). Additionally colonic microorganisms also play a part in vitamin synthesis

Digestion and Fermentation Models…

3

and in absorption of calcium, magnesium and iron. Diverse functions are attributed to SCFA and have been reviewed in great detail before (Topping and Clifton, 2001). To name a few, SCFA can stimulate growth of beneficial bacteria, reduce the growth of pathogenic bacteria and constitute the major energy source for small intestine and colon. In general, increased levels of SCFA are associated with improved gut health (Wong et al., 2006). Major health issues concerning the gastrointestinal tract are obstipation, inflammatory bowel disease and colorectal cancer. All of these diseases can be influenced by modulation of the fermentation profile, which is affected by the composition of the colonic microflora (Rastall, 2004; Guarner and Malagelada, 2003). In this regard the use of probiotics as functional foods has gained major interest by nutritionists and clinicians (Saxelin et al., 2005). Probiotic bacteria are resistant towards the acidic environment of the stomach and are therefore able to colonize the colon after oral ingestion. After colonisation they confer a health benefit for their host. In addition to their role in stimulation of the immune system (Delcenserie et al., 2008), which has implications for gut health in general, a possible preventive effect of probiotics against colorectal cancer is discussed (de LeBlanc et al., 2007). The growth of probiotic bacteria can be selectively stimulated by prebiotics. Prebiotics are polysaccharides which meet certain criteria, namely (1) resistance to gastric acidity, to hydrolysis by mammalian enzymes, and to gastrointestinal absorption; (2) fermentation by intestinal microflora; and (3) selective stimulation of the growth and/or activity of those intestinal bacteria that contribute to health and well-being (Gibson et al., 2004). Due to these properties prebiotics can generally improve gut health and they are specifically important for the prevention of colorectal cancer (Lim et al., 2005). One of the major mechanisms of colon cancer prevention by prebiotics seems to be the increase in SCFA, especially butyrate, and this may be the basis of preventive effects of dietary fibre which has been suggested by some, but not all epidemiologic studies (Bingham et al., 2003). On the molecular level, selective inhibition of proliferation, induction of differentiation, and apoptosis in colon cancer cells may account for the chemopreventive potential of SCFA (Kles and Chang, 2006). Colorectal cancer is a global health issue, because it is one of the most frequent cancers worldwide. The formation of colorectal cancer is promoted by frequent exposition to carcinogens which are ingested with the food and therefore contained in faeces. These carcinogens can reach the faeces directly through the GIT or indirectly after elimination via bile acids. Detoxification of carcinogens, bulking of stool, reducing faecal transit time, reducing the exposition of the colonic epithelium to carcinogens, and


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reducing growth/inducing death of initiated cells are thus the most important mechanisms of colorectal cancer prevention by nutrition, especially dietary fibres (Surh, 2003). Next to nutritional factors, physical activity is the most important factor in reducing risk for colorectal cancer, as has been recently highlighted by an expert report of the American Institute for Cancer Research and the World Cancer Research Fund (WCRF/AICR, 2007).

HOW TO STUDY EFFECTS ON GUT HEALTH? Given the significance of dietary fibres and other nutritional and

environmental factors on gut health, it is important to find suitable approaches to analyse those effects under physiological conditions. In this regard human studies analysing the effects of dietary fibres on colonic health can be considered the most relevant approach. However, access to human materials like gut contents and tissues is usually restricted for practical and ethical reasons. Hence, varying methodologies have been used to analyse the digestion and fermentation of dietary fibres and possible consequences. Macfarlane and colleagues (Macfarlane et al., 1992) analysed products from bacterial fermentation in the colon in sudden death victims within 3 hours of death. A more applicable approach is the determination of fermentation products in faecal samples, but this approach is also limited, because the majority of SCFA is absorbed in the intestine. Studies using ileostomy patients are therefore a valuable alternative to study intestinal absorption or excretion of nutrients (Knudsen and Hessov, 1995). Because of the restrictions in human studies, a lot of research on the digestion of dietary fibres has been done in animal studies (Bach Knudsen and Hansen, 1991; Elsden et al., 1946). On the one hand animal studies offer the possibility of controlled diets, direct accessibility to gut contents and tissues as well as direct administration of drugs or toxins. On the other hand rodents, which are used in many of the studies, are coprophages displaying a different physiology of digestion than humans, whereas pigs, which have also been used in a number of animal studies, display a structurally different digestive tract with a different distribution of gut bacteria. As a result of these methodical limitations diverse models simulating the digestion of the human GIT in vitro have been developed within the last two decades. These models are powerful tools to study processes of digestion and fermentation as well as effects on colonic bacterial profiles. Their main advantage is that they allow the addition and


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removal of samples at practically any step of human digestion as well as modulation of specific factors, like pH, faecal transit time, or addition of selected strains of bacteria to study the effects of these factors on digestion and fermentation. The different in vitro digestion and fermentation models will be introduced in the following paragraphs, focussing on the basic principles and applications.

OVERVIEW OF IN VITRO FERMENTATION SYSTEMS TO STUDY GUT FUNCTION

In vitro systems simulating the human digestive tract range from simple

batch fermentations to elaborate computer-controlled multi-stage continuous systems. Based on the question being addressed, the whole GIT, from mouth to colon, or selected parts of the GIT can be simulated. Barry et al. (1995), Lebet et al.( 1998a) and Olano-Martin et al. (2000) developed simple batch approaches simulating the colon to characterize prebiotic effects of different polysaccharides. All three models use faecal slurries from healthy human donors as bacterial source and incubate them with different carbohydrates under anaerobic conditions. The models differed in the media, duration of incubations or amount of substrate used. Determined endpoints to analyse the fermentation profile typically include quantification of modified SCFA concentrations, consumption of carbohydrates, gas production, pH values, bile acids or monitoring populations of predominant gut bacterial groups. To validate physiological relevance of the methods a comparison of the results to in vivo data (animal or human if available) is indicated. Thus, Barry et al. (1995) demonstrated that SCFA concentrations obtained from their batch system were in close correspondence to the data obtained from in vivo studies using rats. Instead of using complex bacterial mixtures (i.e. faecal slurries), it is also possible to use specific bacterial strains.

In general, static batch systems are inexpensive and allow a large number

of substrates to be tested. Therefore these methods represent a simple way to analyse digestion of single carbohydrates. However, they have some drawbacks, when digestion of more complex food matrices shall be investigated. For example, Lebet et al. (1998b) showed that high concentrations of starch mask the fermentation patterns of fibre fractions. They therefore expanded the fermentation system and included an in vitro


6

digestion procedure before the in vitro fermentation. The in vitro digestion included incubations with -amylase, porcine pepsin and porcine pancreatin as well as a dialysis step to remove small molecular metabolites. Hence, this in

vitro digestion simulates processes occurring in the early GIT, namely mouth, stomach and small intestine. By this modification of the model they were able to remove the majority of starch and obtained unmasked results for different fibre fractions. For the analysis of complex food matrices pre-digestion steps are therefore indicated. Another drawback of the colon models introduced above is the fact that they neglect regional differences within the colon, are static and pH values are uncontrolled. In consequence long-term fermentations with these models are not possible, because physiological conditions are not maintained possibly resulting in unphysiological bacterial profiles. To study long-term effects on bacterial profiles, multi-stage continuous models are thus required. A number of such models have been developed including models of the stomach and small intestine (Minekus et al., 1995a), the colon (Gibson et

al., 1988; Macfarlane et al., 1989; Minekus et al., 1999) as well as small intestine and colon (Molly et al., 1993). A recent review gives an excellent overview of the different models (Macfarlane and Macfarlane, 2007).

The Simulated Human Intestinal Microbial Ecosystem (SHIME) model

established in 1993 by Molly et al. (1993) is a complex 5-step multi-chamber system that is increasingly used to simulate human GIT. It consists of 5 vessels corresponding to different parts of the GIT, namely duodenum/jejunum, ileum, caecum/ascending colon, transverse colon, and descending colon. The vessels are connected via pumps to transport contents between the vessels. In addition, a pH electrode and pH control unit is attached to each vessel and ports for output of medium and sampling of headspace and liquid phase gas are part of each vessel. This setup enables simulation of varying conditions in the small intestine and colon, i.e. continuous movement of contents, different and controlled pH conditions, different retention times, different media and different sample volumes. Additionally liquid and gaseous samples can be taken from each vessel at any time to analyze metabolites and bacterial profiles. After inoculation with faecal suspensions bacteria were grown for six weeks, allowing long-term studies on bacterial profiles. The determined bacterial profiles were comparable to in vivo data. After selection of an appropriate culture medium the concentrations of SCFA after digestion of a human western diet suspension (15% protein, 20% fat, 45% carbohydrate) corresponded fairly well to in vivo data. Since absorption of metabolites is not simulated by the SHIME model, analyses of the digestion process are not


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possible and the system is limited to study microbial interactions. However, since different substances can be added to the system (e.g. bile acids, drugs, probiotics), it is a powerful tool to study influence of those substances on human gut microflora. The model was thus used to address various interesting questions. For example, the prebiotic potential of chicory inulin was confirmed using this system, as digestion of inulin resulted in a significant increase in bifidobacteria (Van de Wiele et al., 2004). The addition of Lactobacillus

acidophilus 74-2, which is used in probiotic products, and fructooligosaccharide to the second vessel (duodenum/jejunum) of the SHIME increased the amounts of bifidobacteria and SCFA (especially butyrate and propionate) in the colon (vessel 4-6) demonstrating probiotic activity of this strain (Gmeiner et al., 2000). Furthermore analysis of soygerm powder increased bacterial fermentation resulting in substantial metabolism of free isoflavones and suggesting for the first time a possible beneficial effect of soygerm powder on gut health.

Very interesting dynamic continuous multi-stage models were developed

by scientist at the TNO Nutrition and Food Research Institute in Zeist, Netherlands. These models are computer-controlled and thus probably the most sophisticated so far. Two different models were established namely TIM-1 (TNO intestinal model 1) (Minekus et al., 1995b) and TIM-2 (Minekus et al., 1999), corresponding to stomach/small intestine and colon, respectively. The technology of the two systems is very complex and a detailed description is beyond the scope of this article. However, flexible walls of the reaction container to which a pressure can be applied enabling the simulation of peristaltic motions are one of the special features. Additionally, both models contain hollow-fiber membranes to remove small molecules and simulate absorption by the intestinal epithelium. A unique feature of the TIM models is a connected computer with specifically developed software which is based on parameters from in vivo data. This computer controls quantity and duration of the meals, pH values, secretion rates, water absorption, and transit time. The physiological relevance was analyzed in comparison to available in vivo data and it was demonstrated that the models can accurately reproduce physiological parameters like meal size and duration, peristaltic motions, pH, gastric and intestinal secretions, gastrointestinal transit and absorption of digested products and water. Therefore, the TIM models are potent tools to investigate digestion of nutrients and even complete meals, the fate of ingested medicines, survival of ingested microorganisms and effects on the colonic microflora in vitro under nearly physiological conditions. Physiological


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processes that cannot be simulated are active transport and feedback mechanisms of the intestinal epithelium. In addition the instruments are highly complex and thus comparably expensive. TIM models were for example used to analyze metabolism of the glucosinolate sinigrin (Krul et al., 2002), effects of bile on survival of lactic acid bacteria (Marteau et al., 1997) or the availability of heterocyclic aromatic amines in the GIT.

Since all of the different models simulating digestion of the human GIT

have advantages and disadvantages, the selection of a suitable model decisively depends on the question being addressed (and of course on the availability of the respective method). In some instances it can be useful to study effects using more than one model. Olano-Martin et al. used a batch culture fermentation and a three-stage continuous culture system of the colon (simulating the proximal, transverse and distal parts of the colon) to analyze in depth the fermentability of dextran, oligodextran and maltodextrin (Olano-Martin et al., 2000). They were able to demonstrate the prebiotic properties of the analyzed carbohydrates. A direct comparison of the TIM-1 model with a three-step batch culture simulating digestion in mouth, stomach and small intestine revealed different digestibility of resistant starch (RS) (Fassler et al., 2006b). Depending on the type of RS fermented, the models were either comparable or gave different results, but the authors were not able to conclude which model reflected the in vivo situation more closely. However, results from both models were in general analogous to data obtained from ileostomy patients. In another study by the same authors (Fassler et al., 2006a) fermentability of different resistant starch preparations in the TIM-2 model and a static batch fermentation were compared and a substantial differences in the concentrations of SCFA and a higher reproducibility of results obtained with the batch culture was found. The authors concluded that “the dynamic

model simulates the behavior of the human colon in vivo more precisely whereas the batch model is better for screening of compounds”. On the other hand, they also highlighted that direct comparison of the two methods is difficult, because they differ in a variety of parameters.

In summary, different models are available to simulate digestion and

fermentation by the human GIT and the colonic microflora. All methods have advantages and disadvantages, but all are useful tools to investigate processes in the GIT. The selection of a suitable model strongly depends on the aim of the study.


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EFFECTS OF FERMENTATION SAMPLES ON CULTURED COLON CELLS

As explained above, in vitro simulation of digestion as well as

fermentation is important to analyse digestibility of foods and nutrients, determination of metabolites or influences of varying factors or substances on colonic microflora. Therefore these methods are important to study effects on gut health in general. Nonetheless, to determine in detail how the intestinal epithelium is influenced by the different metabolites or differences in fermentation profiles, it is necessary to study the effects of fermentation samples in cell culture experiments using colon cells. This approach is relatively new and the number of publications is sparse. However the studies that are available have yielded highly interesting results and will be introduced in the following section.

In general incubation of cultured colon cells with complex fermentation samples were used to analyse uptake of nutrients, effects on membrane integrity, antioxidative effects, cytotoxic effects or effects on gene expression and signal transduction. Many studies have analysed potential of different foods to prevent the formation of colorectal cancer, i.e. the chemopreventive potential (Beyer-Sehlmeyer et al., 2003). Since many endpoints relevant to colorectal cancer, e.g. antioxidative capacity are also relevant for other diseases, statements about gut health in general can be often derived from such studies. Other studies dealt with the uptake of nutrients. Salovaara et al. (2003) treated colon adenocarcinoma cells (Caco-2) with TIM-1 samples obtained from digestion of vegetables with white or whole meal bread. They analysed effects of different transit times on iron metabolism and demonstrated that iron absorption from white bread samples increased with prolonged transit times. Many studies investigated the effects of prebiotics and prebiotics on cultured cells. Commane et al. used different prebiotic carbohydrates and incubated them with different probiotic bacterial strains in a batch-style in vitro fermentation (Commane et al., 2005). Fermentation samples increased the strength of tight junctions as measured by trans-epithelial electrical resistance measurements with Caco-2 cells. Since disruption of tight junctions is involved in promotion of colorectal cancer, this finding suggests protective effects. Furthermore some of the samples were able to protect against the negative effects of deoxycholic acid, a potentially carcinogenic secondary bile acid, upon tight junction integrity. Batch-fermented samples of RS also increased membrane integrity of Caco-2 cells (Fassler et al., 2007).


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Additionally the same samples displayed anti-genotoxic activity against H2O2. Wheat-bran derived arabinoxylans fermented with faecal suspensions in vitro protected HT29 colon cancer cells against DNA damage induced by 4-hydroxynonenal (HNE) and enhanced the activity of Glutathione-S-Transferases (GSTs) which are important for detoxification of carcinogens (Glei et al., 2006). However, a control (fermentation without arabinoxylans) was comparably effective. In another study HT29 and Caco-2 cells were incubated with fermentation samples obtained by fermentation of Synergy1 (inulin enriched with oligofructoses, Beneo-Orafti) using a three-stage fermentation model of the colon. The samples modulated different tumour markers, i.e. cell survival, differentiation, tumour progression and invasive growth. The sample from the gut model vessel representing the distal colon, was most effective for all parameters, probably on account of higher butyrate concentrations (Klinder et al., 2004).

These are some examples for studies analysing the effects of complex

fermentation samples on colon cancer cells. It is also possible to investigate which specific compounds of the complex samples are responsible for certain effects, by comparing effects of the complex samples with single compounds or mixtures of several compounds in corresponding concentrations. This was done in a study using fermentation samples of various dietary fibre sources and samples of butyrate alone and SCFA (acetate, propionate and butyrate) in the same concentrations found in the fermentation samples. All of the employed samples had significant inhibitory effects on the growth of HT29 cells. Inhibition of cell growth was strongest in complex fermentation samples and butyrate alone was the least effective (Beyer-Sehlmeyer et al., 2003). This result suggests additive or synergistic effects of the complex samples and SCFA mixtures, respectively. GST activity was only induced by butyrate and chemoresistance towards HNE was caused by selected SCFA mixtures, but not all corresponding fermentation samples. All of the studies mentioned before used colon cancer cells, but to study effects on the healthy epithelium, primary cells isolated from healthy colon tissues need to be used. While primary colon cells are difficult to cultivate and typically die after short times, a method has been recently described incubating whole epithelial tissue stripes from clinical colon samples which were viable in vitro for more than 12 hours (Sauer et al., 2007). These tissue stripes were incubated with Synergy1 fermented by gut bacteria. The samples were not cytotoxic for primary cells, but increased metabolic activity, pointing to trophic effects. Effects on gene expression


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were analysed using a cDNA macroarray and real-time qPCR. Expression of GSTM2 and GSTM5 was increased, but the activity of total GSTs was not modulated. The induction of GST gene expression by the fermentation samples may protect the cells from carcinogenic compounds (Sauer et al., 2007).

Whereas most of the studies used dietary fibres/prebiotics as substrate

for the fermentation reaction, it is also possible to analyse the effects of other nutritional matrices. For example, fermentation samples from apple juice extracts (AEs) decreased the levels of reactive oxygen species in Caco-2 cells. However the unfermented AEs were more effective, indicating that active compounds may have been degraded by the fermentation procedure (Bellion et al., 2008). In another study polyphenolic apple extracts (PAE) were fermented in vitro with human faecal flora, resulting in increased levels of SCFA and degradation of polyphenols. The apple extracts reduced cell growth of HT29 cells and the colon adenoma cell line LT97, pointing at chemoprotective properties of apple extracts (Veeriah et al., 2007). The two latter studies used static batch fermentation models of the colon to digest apples. This is a simple approach to obtain first hints on effects of fermented apple extracts. However, since many of the apple ingredients would be degraded in vivo and not reach the colon, a more sophisticated simulation of digestion, including simulation of mouth, stomach and small intestine would be more appropriate for such substrates. A problem in this regard seems to be the presence of cytotoxic compounds in samples from simulation of small intestine. Both samples obtained by digestion of RS with TIM-1 (Fassler et al., 2007) and samples from the SHIME model (De Boever et al., 2000) supplemented with oxgall, a dry mixture of the salts of the gall of the ox, displayed cytotoxic effects on Caco-2 or Hela cells, respectively. In the latter study the effects were caused by high concentrations of oxgall. Our group has recently adapted an in vitro simulation of the GIT (including mouth, stomach, small intestine and colon) which can be used in cell culture experiments (unpublished). The method to simulate digestion of mouth, stomach and small intestine, includes a dialysis step to simulate absorption of small molecules, and is based on the model established by Aura et al. (1999). Samples using the original protocol were cytotoxic on different cultured colon cell lines. This cytotoxic effect was avoided by reducing the bile acid concentrations. The modified protocol is currently successfully used to study effects of different nutrients and foodstuffs on chemoprevention and general markers of gut health in colon cells.


12

CONCLUSION The human GIT is of major significance for uptake and digestion of

nutrients, energy supply and excretion. It is continuously exposed to xenobiotics and endogenous metabolites which can cause colorectal cancer or other diseases of the colon like inflammatory bowel disease. A key influence comes from the colonic microbiota. The composition and metabolism of those bacteria can modulate the fermentation profile and thus improve gut health. Because of its important role in the physiology of humans, it is of high interest to find ways to investigate the processes occurring in the intestine. Since tissues and gut contents from humans are not accessible and animal studies are not always representative, because of a different physiology, in vitro simulations of the human GIT are powerful tools to analyse digestion and the colonic microflora. A number of different models is available, each one with advantages and disadvantages. Simple batch techniques are inexpensive and allow testing of a large number of samples. However, especially for the digestion of complex food matrices and to analyse long-term effects on gut bacteria, pH controlled multi-stage models are of advantage. They better reflect the physiology of the GIT, because the pH value, transit time and addition of different media or bacteria can be controlled, absorption is simulated and they take regional differences of the GIT into account. Nonetheless all models are helpful to gain information of digestive processes, but the appropriate method has to be selected and the selection depends on the aim of the study. To analyse the effects of the complex fermentation samples on the intestinal epithelium different cell culture models can be used. Cell culture studies have been used to demonstrate anti-carcinogenic and anti-oxidative properties of different foodstuffs and nutrients e.g. dietary fibres. This approach is therefore very useful to study how complex fermentation samples interact with the cells of the epithelium and modulate molecular processes in the epithelial cells resulting in protection against colorectal cancer and improvement of colonic health. A further progression of the methods involved will certainly increase our understanding of the physiology and pathology of the GIT.


13

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Glei, M., Hofmann, T., Kuster, K., Hollmann, J., Lindhauer, M. G. & Pool-Zobel, B. L. (2006). Both wheat (Triticum aestivum) bran arabinoxylans and gut flora-mediated fermentation products protect human colon cells from genotoxic activities of 4-hydroxynonenal and hydrogen peroxide. J. Agric. Food Chem. 54, 2088-2095.

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Macfarlane, G. T., Cummings, J. H., Macfarlane, S. & Gibson, G. R. (1989). Influence of Retention Time on Degradation of Pancreatic-Enzymes by Human Colonic Bacteria Grown in A 3-Stage Continuous Culture System. Journal of Applied Bacteriology 67, 521-527.

Macfarlane, G. T., Gibson, G. R. & Cummings, J. H. (1992). Comparison of Fermentation Reactions in Different Regions of the Human Colon. Journal of Applied Bacteriology 72, 57-64.

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Molly, K., Woestyne, M. V. & Verstraete, W. (1993). Development of A 5-Step Multichamber Reactor As A Simulation of the Human Intestinal Microbial Ecosystem. Applied Microbiology and Biotechnology 39, 254-258.

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Gastroenterol. 40, 235-243.


Lecture 6

BAMBOO SHOOTS AS DIETARY FIBRES


ABSTRACT

Bamboo is a grass which belongs to the same family as our staple cereal crops and has multifarious uses in industry besides being used by rural people for food, housing and other domestic purposes. A little known fact is the edible characteristics of its juvenile shoots in the form of fresh, fermented and canned form. Use of juvenile, fresh and soft bamboo shoots as vegetable, and in the form of fermented food produced by traditional and industrial methods is well known, more particularly in bamboo growing Asian countries. Commercially, bamboo shoots are available in canned form, though fresh shoot is far superior in taste and texture. Bamboo shoots are considered as a health food as they are endowed with health enhancing properties being rich in nutrient components mainly proteins, carbohydrates, minerals, fibre, amino acids and vitamins, are low in fat and sugar and have no cholesterols. The shoots contain phytosterols and a high amount of fibre due to which they are labeled as nutraceuticals or natural medicines. Dietary fibre contents are cellulose, hemicelluloses and lignin. There are two broad types of dietary fibre described according to its solubility: insoluble and soluble. Nutrient detergent fibre (NDF) determines the indigestible component of the plant material. It consists of hemicellulose, cellulose and lignin. Acid detergent fibre (ADF) primarily represents cellulose and lignin. ADF is often used to calculate digestibility, while NDF is often used to predict intake potential. The major function of dietary fibre is to reduce the time


2

of release of ingested food in colon. The roughage is aids in the digestive process and elimination of waste It also functions for holding water and acts like sponge in alimentary canal. The content of dietary fibre and its components in bamboo shoots increases with age, but as the tissue grows old, the shoots become inedible. With fermentation and canning also, their content increases in the shoots. Whereas, the fermented shoots have a lesser amount of ADF, the canned as well as the fresh shoots have nearly equal amount of ADF. Lignin content in both the fresh and canned shoots was less than the fermented shoots. The canned shoots have a comparatively higher content of hemicelluloses than the fermented as well as the fresh shoots. The fermented shoots have higher amounts of cellulose than the fresh shoots while canned shoots have lower amount of cellulose than both the fresh and fermented shoot. The dietary fibre content in the fresh juvenile shoots ranges between 2.255-4.490 g/100g fresh weight and reaches upto 13.840/100gm fresh weight in 10 days old emerged shoots of some bamboo species. The recommended level of fibre for adults is 25-30 g a day, in combination with at least 2 litres of fluid to ensure thorough digestion. Foods and food products that contain 6g fibre per 100g or 100 ml are labeled as a „high fibre‟ food. Thus,

bamboo shoots can be considered as „fibre rich‟ foods and can meet the daily requirement of fibre in the diet. German and US companies Qualicel and Vitacel market fibre additives in white powder form with at least 95% fibre and bamboos being the fastest growing plants, can provide raw material for production of such fibre additives.

INTRODUCTION Bamboos are plants of global interest because of their distinctive life

form, ecological importance and the wide range of uses and values they have for humans [Bystriakova et al., 2004]. At least one third of the human race uses bamboo in one way or another. They are gaining increased attention as an alternative crop with multiple uses and benefits providing human beings with various living resources. They are intermingled with the tradition and culture of rural and tribal populations from times immemorial due to which they have been variously called as „The Cradle to Coffin Plant‟, „The Poor

man‟s Timber‟, „Friend of the People‟, „Green Gasoline‟, „The Plant with

Thousand Faces‟ and „ The Green Gold‟. This green gold is sufficiently cheap and plentiful to meet the vast needs of human populace from the “child‟s cradle to the dead man‟s bier”. As a renewable natural resource, it

plays a major role in the livelihood of rural people and is an integral part of our cultural, social and economic conditions [Tewari, 1988; Madhab, 2003].

Bamboo Shoots

3

Because of its multifarious utility, both in the traditional way for the rural people as well as in modern society, bamboo is becoming a very important plant worldwide. There are more than 1500 different documented traditional uses of bamboo [INBAR, 1997; Shrestha, 1999]. Bamboos provide food, shelter, medicine, raw materials for construction, wood substitute and paper and pulp industry. They are also used for making furniture, handicrafts, containers, tool handles, poles, musical instruments, bows and arrows, boats, rafts, fishing poles etc. In addition, because of their characteristic growth habits, bamboos have enormous potential for alleviating many environmental problems like soil erosion control, water conservation, land rehabilitation and carbon sequestration [Benzhi et al., 2005]. However, a lesser known use of bamboos is the edible value of its shoots which are excellent vegetables with tender sprouts and have a good taste. Bamboo shoots are highly popular in Asian cuisine, particularly in countries like China, Japan, Korea, Taiwan, Thailand and Philippines. Juvenile bamboo shoots, in addition to being delicious, are rich in nutrient components, mainly proteins, carbohydrates, minerals and fibre, are low in fat and sugar and have no cholesterols. Moreover, they are rich in vitamins and amino acids. Young edible shoots are harvested during the monsoons when the shoots just emerge from the ground (Figure 1a,b), and the typical “shooting season” of a species rarely exceeds two months. For consumption, hard sheaths are removed and thin slices are prepared (Figure 1. c-e). Fresh shoots have a crisp, crunchy and sweet flavour imparting a unique taste and are mostly used in soup, salads, pickles, spring rolls or other stewed and dried dishes. These are also used as an extender because it takes on the flavour of the ingredients in which it is cooked. The most common preparation involves boiling the shoots in stocks, soups or salted water for use in assorted dishes. The shoots are not only used as vegetables, but are also processed and preserved in many forms such as dried, fermented, salinised, pickled, water soaked and canned shoots. Bamboo shoots are gastronomic treats whether used fresh or in fermented or roasted form. Being endowed with many health enhancing properties, bamboo shoots are projected as a new health food.


4

Figure 1. a. Plant morphology; b. emerging juvenile shoot; c. shoots with sheaths and with sheaths removed; d. juvenile shoots cut into half; e. sliced shoots.

NUTRITIONAL VALUE OF BAMBOO SHOOTS The criterion for healthy nutrition, in so far as food intake is concerned,

constantly evolves with an understanding of food-health relationship.

Bamboo Shoots

5

Recently, the research and exploration of bamboo as a food commodity has witnessed tremendous activity. Due to this, major advances have been made in fresh shoot production and processing and analysis of nutrient component of edible shoots. This is because bamboo shoots are endowed with health enhancing properties being rich in nutrients with a high content of protein, amino acids, carbohydrate, minerals and several vitamins. They also contain phytosterols and high amount of fibre that are labeled as “nutraceuticals” or

“natural medicines”. Phytosterols have cholesterol lowering activity. Bamboos grow well without the application of fertilizers or chemicals and are hence are more likely to be free from pollution. Moreover, the shoots are well protected from pollutants by several layers of sheaths. Bamboo shoots are a good source of edible fibre (6-8%) which helps in lowering cholesterol level in the blood. Fat content is extremely low in bamboo shoots (2.46%) which are, therefore, very good for weight conscious people. The high cellulosic content of bamboo shoots stimulates appetite. Being crisp, crunchy, tender with a sweet flavour, the shoots have a unique and delicious taste that functions as an appetizer.

Bamboo Shoots as a Source of Dietary Fibre Bamboo shoots are a rich source of dietary fibre. Dietary fibre is an

important part of a health promoting diet and consists of the remnants of edible plant cells, polysaccharides, lignin and associated substances resistant to digestion by the alimentary enzymes of humans (DeVries et al. l999). It identifies a macroconstituent of foods that includes cellulose, hemicellulose,,lignin, gums, modified celluloses, mucilages, oligosaccharides, and pectins and associated minor substances, such as waxes, cutin, and suberin. They pass through the stomach and small intestine undigested and reach the large intestine virtually unchanged (Lee and Prosky, 1995; Trowell and Burkitt, 1986). Most other nutrients are digested and are being used in other parts of the body by this stage. During its passage through the large intestine some components of dietary fibre are broken down to varying degrees and absorbed by the body; the remaining components are excreted in the faeces. The analysis of dietary fibre in food is very complex and only a limited number of foods have been examined in detail. Dietary fibre from different foods, and even different samples of the same food, contain varying quantities of the components that collectively make up dietary fibre. Each of these components has different biological properties and it is frequently not clear which of these is most beneficial. Dietary fiber has been one of the most


6

enduring dietary interests worldwide and has been a topic of considerable volume of medical research and is recommended as part of the treatment and prevention of a number of diseases. The major function of dietary fibre is to reduce the time of release of ingested food in colon. It is known to be associated with reduced incidence of coronary heart diseases. It also functions for holding water and acts like sponge in alimentary canal. So the food with high dietary fibre is recommended for diet and is good for health. This roughage is essential for the mechanism of digestion and elimination of waste [Gopalan et al., 1971]. In hypercholesterolemic man, a diet rich in dietary fibre can significantly lower plasma total cholesterol and low density lipoproteins cholesterol and thus potentially lower the risk of coronary heart diseases [Whyte et al., 1992; Gold et al, 1999]. There is increasing epidemiological evidence that population groups which consume reasonable amounts of dietary fiber (20 – 35g / day) have lower risk of a number of chronic diet-related diseases such as diverticular disease, coronary heart disease, obesity, type 2 diabetes mellitus, gall stone, colonic carcinoma, hyperlipidaemia, constipation, haemorrhoids and irritable bowel syndrome [Cummings et al, 1997]. Due to these qualities, bamboo shoots have been projected as a health food.

The fibre content in bamboo shoots can be classified accordingly as Nutrient detergent fibre (NDF) which determines the indigestible component of the plant material consisting of hemicellulose, cellulose and lignin [Van Soest, 1978] and Acid detergent fibre (ADF), primarily representing cellulose and lignin. ADF is often used to calculate digestibility, while NDF is often used to predict intake potential. Sixteen species of bamboos viz. Bambusa

bambos (Linn.) Voss, B. kingiana Gamble, B. nutans Wall ex. Munro, B.

polymorpha Munro, B. tulda Roxb., B vulgaris var. vulgaris Schrad., Dendrocalamus asper (Schultes f.) Becker ex Heyne, D. brandisii (Munro) Kurz., D. giganteus Munro, D. hamiltonii Nees & Arn. ex Munro, D.

membranaceus Munro, D. strictus (Roxb.) Nees, Gigantochloa albociliata

(Munro) Kurz, G. rostrata Wong, Thyrsostachys oliveri Gamble and T.

siamensis Gamble were selected and analyzed for the dietary fibre content of their fresh edible juvenile shoots. The juvenile shoots of all the species were collected at the stage when their tips were just emerging above the ground. The fermented and canned shoots of Dendrocalamus giganteus available in the local market were also analyzed for the same component as in the case of fresh juvenile shoots. The results of dietary fibre content and its components of the bamboo species is presented below.

Bamboo Shoots

7

Table 1. Dietary Fiber (NDF) and Its Components in the Freshly Emerged

Juvenile Shoots of Various Species (G/100g Fresh Weight).

Sl

No. Names

of species NDF ADF Lignin Hemi-cellulose Cellulose

1. Bambusa

bambos

3.535 ±0.015

2.810 ±0.010

1.460 ±0.030

0.725 ±0.005

1.350 ±0.001

2. B. kingiana 4.490

±0.060 3.190

±0.043 2.010

±0.020 1.300

±0.017 1.180

±0.023

3. B. nutans

2.275 ±0.005

1.240 ±0.050

0.430 ±0.010

1.034 ±0.995

0.710 ±0.040

4. B.

polymorpha

3.815 ±0.055

2.290 ±0.012

1.300 ±0.009

1.515 ±0.043

0.990 ±0.003

5. B. tulda

3.970 ±0.020

3.360 ±0.031

2.300 ±0.010

0.609 ±0.989

1.060 ±0.021

6. B. vulgaris

var. vulgaris 4.240

±0.010 3.280

±0.022 2.400

±0.011 0.959

±0.988 0.780

±0.011

7. Dendrocalam

us asper

3.540 ±0.065

3.000 ±0.014

1.260 ±0.010

0.475 ±0.054

1.740 ±0.004

8. D. brandisii 4.027

±0.087 3.060

±0.061 2.010

±0.013 0.967

±0.026 1.050

±0.048

9. D. giganteus 2.645

±0.025 2.150

±0.009 0.560

±0.010 0.495

±0.016 1.589

±0.999

10. D. hamiltonii

3.900 ±0.030

3.230 ±0.026

2.170 ±0.017

0.670 ±0.004

1.060 ±0.009

11. D.

membranaceu

s

2.905 ±0.055

1.400 ±0.011

0.870 ±0.024

1.425 ±0.044

0.609 ±0.992

12. D. strictus

2.255

±0.005 1.380

±0.020 0.640

±0.028 0.844

±0.985 0.939

±0.992

13. Gigantochloa

albociliata

4.150 ±0.106

3.750 ±0.054

2.700 ±0.050

0.400 ±0.052

1.050 ±0.004

14. G. rostrata 4.200

±0.090 3.320

±0.023 1.820

±0.021 0.930

±0.067 1.500

±0.002

15. Thyrsostachys

oliveri

3.910 ±0.023

2.840 ±0.040

1.280 ±0.010

1.069 ±0.983

1.560 ±0.030

16. T. siamensis 3.715

±0.054 2.150

±0.014 1.200

±0.009 1.565

±0.040 1.250

±0.005 The variation in the content of cellulose in D. giganteus and T. oliveri shoots was

insignificant (Table 1).


8

Figure 2. Comparison of NDF content (g/100g fresh weight) in the freshly emerged juvenile shoots of various species.

Figure 3. Comparison of ADF content (g/100g fresh weight) in the freshly emerged juvenile shoots of various species.

Dietary Fibre (NDF)

The NDF content in juvenile shoots ranged between 2.255 - 4.490 g/100 g fresh weight (Table 1). The highest fibre content was found in B. kingiana

Bamboo Shoots

9

(4.490 g/100g fresh weight) shoots while the lowest was found in those of D.

strictus (2.255 g/100 g fresh weight) (Table 1 and Figure 2). The shoots of B.

vulgaris var. vulgaris, D. brandisii, G. albociliata and G. rostrata had also high amount of dietary fibre (Table 1 and Figure 2).

Acid Detergent Fibre (ADF)

The content of ADF ranged from 1.240-3.750 g/100 g fresh weight (Table 1) where the highest value was recorded in G. albociliata (3.750 g/100 g fresh weight) and the lowest in B. nutans (1.240 g/100 g fresh weight) (Table 1 and Figure 3). The shoots of B. tulda, B. vulgaris var. vulgaris, D. hamiltonii and G.

rostrata have nearly equal amount of ADF% (Table 1). Likewise, the variation in the shoots of B. bambos and T. oliveri was insignificant (Figure 3).

Lignin

The content of lignin in the shoots ranged from 0.430-2.700 g/100 g fresh weight (Table 1). G. albociliata has the highest amount of lignin content (2.700 g/100 g fresh weight) and that of B. nutans has the lowest (0.430 g/100 g fresh weight) (Table 1 and Figure 4). The shoots of D. giganteus, D.

membranaceus and D. strictus have low content of lignin (< 1 g/100 g fresh weight) (Table 1). The variation in lignin content in B. polymorpha, D. asper and T siamensis was insignificant (Table 1 and Figure 4). Lignin content in B.

kingiana and D. brandisii was found equal, each having 2.010 g/100 g fresh weight (Table 1 and Figure 4).


10

Figure 4. Comparison of lignin content (g/100g fresh weight) in the freshly emerged

juvenile shoots of various species.

Figure 5. Comparison of hemicellulose content (g/100g fresh weight) in the freshly emerged juvenile shoots of various species.

Hemicellulose

Hemicellulose content in the shoots varied from 0.400-1.565 g/100 g fresh weight (Table 1). The highest hemicellulose content was present in T.

siamensis (1.565 g/100 g fresh weight) and the lowest in the shoots of G.

albociliata (0.400 g/100 g fresh weight) (Table 1 and Figure 5). B. vulgaris

var. vulgaris and D. brandisii had almost equal content (Table 1).

Cellulose

The shoots have cellulose content varying from 0.609-1.740 g/100 g fresh weight (Table 1) with the maximum cellulose content in D. asper (1.740 g/100 g fresh weight) and the minimum in D. membranaceus (0.609 g/100 g fresh weight) (Table 1 and Figure 6). B. nutans, B. polymorpha, B. vulgaris var. vulgaris and D. strictus have low cellulose contents with their values being lesser than 1 g/100 g fresh weight of the shoots (Table 6). The shoots of B.

Bamboo Shoots

11

tulda and D. hamiltonii have identical amounts of cellulose (Table 1 and Figure 6).

Table 2. Dietary Fiber (NDF) and Its Components in the Freshly Emerged Juvenile (Jv) and 10 Days Old Emerged

(Old) Shoots of the Five Species (G/100g Fresh Weight)

Sl No. Names of species

NDF ADF Lignin Hemicellulose Cellulose jv old jv old jv old jv old jv old

1. Bambusa

bambos

3.535 ±0.015

9.640 ±0.105

2.810 ±0.010

7.560 ±0.092

1.460 ±0.030

4.802 ±0.041

0.725 ±0.005

2.080 ±0.013

1.350 ±0.001

2.758 ±0.051

2. B. tulda 3.970

±0.020 10.690 ±0.102

3.360 ±0.031

8.206 ±0.089

2.300 ±0.010

5.542 ±0.056

0.609 ±0.989

2.484 ±0.013

1.060 ±0.021

2.754 ±0.033

3. D. aspe 3.540

±0.065 10.850 ±0.023

3.000 ±0.014

8.270 ±0.010

1.260 ±0.010

5.680 ±0.008

0.475 ±0.054

2.580 ±0.013

1.740 ±0.004

2.690 ±0.002

4. D. giganteus 2.645

±0.025 13.840 ±0.041

2.150 ±0.009

9.520 ±0.021

0.560 ±0.010

6.320 ±0.014

0.495 ±0.016

4.320 ±0.020

1.589 ±0.999

3.200 ±0.007

5. D .hamiltonii 3.900

±0.030 8.200

±0.020 3.230

±0.026 6.105

±0.012 2.170

±0.017 3.890

±0.009 0.670

±0.004 2.095

±0.008 1.060

±0.009 2.095

±0.003 ± indicates standard deviation


14

Figure 6. Comparison of cellulose content (g/100g fresh weight) in the freshly emerged juvenile shoots of various species.

Table 3. Dietary Fibre (NDF) and Its Components in the Freshly Emerged

Juvenile, 10 Days Old Emerged, Fermented and Canned (Non-Salted)

Shoots of D. Giganteus

Nature of shoots NDF ADF Lignin Hemi-cellulose Cellulose

Freshly emerged juvenile shoots

2.645 ±0.025

2.150 ±0.009

0.560 ±0.010

0.495 ±0.016

1.589 ±0.999

10 days old emerged shoots

13.840 ±0.041

9.520 ±0.021

6.320 ±0.014

4.320 ±0.020

3.200 ±0.007

Fermented shoots 4.180 ±0.104

3.280 ±0.076

1.398 ±0.042

0.900 ±0.028

1.882 ±0.034

Canned shoots (non-salted)

3.040 ±0.108

2.020 ±0.095

0.780 ±0.038

1.020 ±0.013

1.240 ±0.057

The bamboo shoots vary in their dietary fibre quantity even within same

species with the variation in age of the shoots and the way in which the shoots were preserved (Table 2 and 3). The increase in dietary fibre content and its components in the shoots increases with age [Nirmala et al., 2007]. With

Bamboo Shoots

15

fermentation and canning also, their content increases in the shoots. Whereas, the fermented shoots have a lesser amount of ADF, the canned as well as the fresh shoots have nearly equal amount of ADF. Lignin content in both the fresh and canned shoots was less than the fermented shoots. The canned shoots have a comparatively higher content of hemicelluloses than the fermented as well as the fresh shoots. The fermented shoots have higher amounts of cellulose than the fresh shoots while canned shoots have lower amount of cellulose than both the fresh and fermented shoot.

Among some selected species of bamboo shoots, the dietary fibre content in the fresh juvenile shoots range between 2.255-4.490 g/100g fresh weight with the highest fibre content in Bambusa kingiana (4.490 g/100 g fresh weight) and the lowest in D. strictus (2.255 g/100 g fresh weight). The fibre content in the 10 days old emerged shoots increased more than 3 times to those present in their respective freshly emerged juvenile shoots (Table 2). There was significant increase of fibre components (ADF, lignin, hemicellulose and cellulose) in the old emerged shoots of all the five species. High content of upto 40% cellulose stimulates appetite. Being nutritious, crisp, crunchy and with a sweet flavor, the shoots have a unique and delicious taste that functions as an appetizer. Foods and food products that contain 6g fibre per 100g or 100 ml may be labeled as a „high fibre‟ food and the recommended level of fibre

for adults is 25-30 g a day, in combination with at least 2 litres of fluid to ensure thorough digestion. Thus, bamboo shoots can be considered as „fibre

rich‟ foods and can meet the daily requirement of fibre in the diet.

CONCLUSION Fibre has been associated with a number of health benefits such as a faster

“transit time” i.e. the time it takes for the body's waste to be moved out of the

body, reduced exposure of the body to carcinogens or cancer causing components in food and fluid, bowel protection, more increased fermentation by the microflora or “bugs” in the bowel and increased amounts of a

compound called butyrate, the preferred energy source for cells called colonocyctes. It decreases the fat products and promotes the peristalsis of intestine, which has the effect to prevent colon cancer [Cummings et al, 1997]. Bamboo shoots, due to their high nutrient content, are finding a new place in the spectrum of plants, fibre and foods used to enhance the quality of life. Tremendous progress has been made in fresh shoot production, processing and

Rakesh Sharma, Bharati D Srinivas

16

utilization of edible bamboos, exploitation of bamboo beverage, extraction and utilization of effective components from bamboo hydrolysate. Nearly 90 bamboo shoot based industries exist in Zhejiang province of China, and there are 25 canning industries in Prachinburi province of Thailand [Thammincha, 1988]. Canned bamboo shoots occupy an important place in Thailand‟s global

trade providing an export earning of about US$ 30 million annually. In contrast to being their routine component of everyday meals of Asian people, in the western countries bamboo shoots are available only in canned form, imported from other countries. This pattern is however, now gradually changing and in Europe, shoots are available, though on a small scale from groves planted near Bordeaux in France, Carasco in Italy and New South Wales in Australia. But, since the local produce is not sufficient to meet the market demand, bamboo shoots in Western markets and Australia are usually imported in processed form. Bamboo shoots with its 6-8% fibre content can be considered as “fibre rich” food and can meet the daily requirement of fibre in

the diet. German and US companies Qualicel and Vitacel market fibre additives in white powder form with at least 95% fibre and bamboos being the fastest growing plants, can provide raw material for production of such fibre additives. Hence, efforts need to be made to popularize the consumption of bamboo shoots from the health perspective.

REFERENCES

Benzhi, Z., Maoyi, F., Xiaosheng, Y. & Zhengcai, L. (2005). Ecological function of bamboo forest: Research and Application. Journal of Forestry

Research, 16(2), 143-147. Bystriakova, N., Kapos, V. & Lysenko, I. (2004). Bamboo Bioiversity- Africa,

Madagascar and the Americas. UNEP-WCMC/INBAR, A Bandson production, UK.

Cummings, J. H., Roberfroid, M. B. & Anderson, H. (1997). A new look at dietary carbohydrates: Chemistry, Physiology and Health. European

Journal of Clinical Nutrition, 51, 417-423. DeVries, J. W., Prosky, L., Li, B. & Cho, S. (1999). A Historical Perspective

on Defining Dietary Fiber. Cereal Foods World, 44(5),367-369. Gold, S. P., Man, I. L. Kader, A. A. & Keintz, C. (1999). Influence of

production, handling and storage on phyto nutrient content of foods. Nutrition Reviews, 57 (9), S46 – S52.

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17

Gopalan, C., Ramasastri, B. V. & Balasubramanian, S. C. (1971). Nutritive value of Indian foods. ICMR Publ., Hyderabad, India.

INBAR. (1997). The plant with thousand faces. 5, 13. Lee, S. & Prosky, L. (1995). International survey on dietary fibre definition,

analysis and reference materials. Journal of Association of Official

Analytical Chemists International. 78, 22- 36. Madhab, J. (2003). The Green Gold: Under Exploited Wealth of the North-

East India. Dialogue, 5 (2), 45-52. Nirmala, C., David, E. & Sharma, M. L. (2007). Changes in nutritive

components during ageing of emerging juvenile bamboo shoots. International Journal of Food Sciences and Nutrition, 58(8), 612-618.

Shrestha, K. (1999). Distribution and status of bamboos in Nepal. In: Bamboo-

Conservation, Diversity, Ecogeography, Resource, Utilization and

Technology (eds. Rao, A.N. and Rao, V.R.). Proceedings of a training course cum workshop, 10-17 May, 1998, Kunming and Xishuangbanna, Yunnan, China and IPGRI-APO, Serdang, Malaysia.

Tewari, D. N. (1988). Bamboo as poverty allevator. Indian Forester, 114 (10), 610-612.

Thammincha, S. (1988). Some aspects of bamboo production and marketing. In Bamboo: Current Research (eds. Rao, I.V.R., Gnanaharan, R. and Sastry, C.B.), Proc. International Bamboo Workshop, Cochin, 14-18, November, 1988, KFRI and IDRI, Canada, 1988, 320-326.

Trowell, H. & Burkitt, D. (1986). Physiological role of dietary fibre: a ten year review. Journal of Dentistry for Children. 53, 444-447.

Van Soest, P. J. (1978). Component analysis of fibre in foods: Summary and recommendations. American Journal of Clinical Nutrition, 31 (10), 75.

Whyte, D. L., McArthur, R., Topping, D. & Nestel P. (1992). Oat bran lowers plasma cholesterol levels in mildly hypercholesterolemic men. Journal of

the American Dietetic Association, 92 (4), 446-449.


Lecture 7

TROPICAL AND TEMPERATE FRUITS

Rakesh Sharma, Bharati D Shrinivas Epidemiological studies provide evidence that plant-based foods play a

crucial role in the prevention of chronic diseases. The association between dietary vegetable intake and chronic diseases is mainly attributed, along with the dietary fiber constituent, to a wide range of plant secondary compounds called phytochemicals. Fiber-rich foods are very good sources of these phytochemicals, which include polyphenolics, carotenoids, plant sterols and lignans. These so-called co-passengers, or co-travelers, of dietary fiber may contribute to the nutritional benefits of fiber-rich food and are an essential part of the healthful dietary fiber complex. Fruits are rich sources of various vitamins, minerals and dietary fiber required by the human body for optimal health. In addition, fruits are diverse in antioxidant composition and antioxidant activity. The objective of this chapter is to highlight the potential of tropical and temperate fruits as sources of dietary fiber with associated antioxidant compounds.

INTRODUCTION Epidemiological studies evidence that plant-based foods play a crucial role

in the prevention of chronic diseases. Specifically, consumption of fruit and vegetables has been strongly associated with a decreased risk of disease by


2

modulating DNA damage, lipoprotein and protein oxidation, platelet aggregation, leukocyte adhesion and vascular function 1-3. The association between dietary vegetable intake and chronic diseases is mainly attributed, along with the dietary fiber constituent, to a wide range of plant secondary compounds called phytochemicals 4-7. Fiber-rich foods are very good sources of these phytochemicals which include polyphenolics, carotenoids, plant sterols and lignans. These so-called co-passengers, or co-travelers, of dietary fiber contribute to the nutritional benefits of fiber-rich food and are an essential part of the healthful dietary fiber complex 8.

Fruits are rich sources of various vitamins, minerals and dietary fiber required by the human body for optimal health. In addition, fruits are diverse in antioxidant composition and antioxidant activity 9. Either fruits from the temperate zone, usually characterized by a large edible portion, or fruits from tropical and subtropical regions, with relatively high ratios of non-edible portion such as peels, seeds and stones, are rich in these antioxidant bioactive compounds 10.

Reactive oxygen species (ROS) are generated in humans under physiological conditions as a result of normal intracellular metabolism in mitochondria and peroxisomes, as well as from a variety of cytosolic enzyme systems. Oxidative stress arises from an imbalance between the production of ROS and the natural antioxidant defense mechanisms of the body in favor of the former 11-13. Increasing evidence suggests that inflammatory and oxidative stress play a pivotal role in the development of certain degenerative diseases associated with aging 14,15. Thus, the ability of exogenous antioxidants from dietary intake to scavenge ROS in the human body—and thereby to decrease the amount of free radical damage to biological molecules like lipids, proteins and DNA—may be one of the protective mechanisms of foods of plant origin.

TROPICAL AND TEMPERATE FRUITS AS A SOURCE OF

DIETARY FIBER AND BIOACTIVE COMPOUNDS Some recent studies have pointed out that fiber derived from fruits may

possess associated bioactive compounds, and thus may be partially responsible for the health benefits of high fruit intake 8. The selection of suitable sources

Tropical and Temperate Fruits

3

to provide new dietary fiber products with high antioxidant capacity derived from natural associated compounds could be an appropriate tool with which to achieve a better antioxidant status along with the recommended higher dietary fiber intake 16. The objective of this chapter is to highlight the potential of selected fruits as sources of dietary fiber with associated antioxidant compounds.

Apple (Malus domestica Borkh., Rosaceae) Apples have been characterized as a good dietary source of polyphenols,

along with dietary fiber. Polyphenol compounds are predominantly localized in the peel. The major compounds isolated and identified included catechin, hydroxycinammates, phloretin glycosides, quercetin glycosides and pro-cyanidins 17,18. The relationship of hydroxycinnamic acids in the peel and the peeled fruit of Golden delicious varieties have been studied in some detail 19. A significantly higher content in ferulic acid (16.76%), -coumaric acid (29.50%) and caffeic acid (23.27%) in the peel than in peeled apples was reported. Also, total polyphenols were significantly higher in peels (1.11 0.12 g/100 g) than in peeled apples (0.69 0.07 g/100 g) (19). These authors measured the content of total dietary fiber (AOAC method) in fractions (both peeled and peels) of fresh apple fruits. Results showed significantly lower dietary fiber (both soluble an insoluble) in peeled apples than in the peels. In a second stage, Gorinstein and co-workers 20 evaluated the influence of peel and peeled apple fruit on plasma antioxidant capacity on rats. A significant increase in the plasma antioxidant activity in the group fed with whole apple in comparison to the control group was found. In this research, when antioxidant status was evaluated in plasma, an increase in the TRAP value and a decrease in the biomarker of lipid oxidation, malondialdehyde—measured spectropho-tometrically as thiobarbituric reactive substances—was found. It is worth to mention that these authors stated that the correlation between dietary fiber and its antioxidant activity by TRAP assay was very poor (R2 = 0.3267). Interestingly, the effect of baking on dietary fiber, phenolics and total antioxidant capacity was investigated using a model system of muffins incorporated with dried apple skin powder (ASP) as a value-added food ingredient. As a result, the total dietary fibre content, total phenolic content and total antioxidant capacity of muffins were positively correlated to the amount of ASP incorporated into muffins 21.


4

Citrus Fruits Citrus fruits have a high content of phenolics, dietary fiber, ascorbic acid,

and trace elements. In order to define the antioxidant potential of certain citrus fruits and to propose the most preferable one for dietary prevention of cardiovascular and other diseases, a systematically comparison of peel and pulp of grapefruit (Citrus paradise, Macfad), oranges (Citrus sinensis, L.) and lemons (Citrus limonis, Osbeck ) was done 22. The range of the total dietary fiber content was 2.43–2.54 and 1.29–1.34 g/100 g fresh citric fruits for peels and peeled fruits, respectively. No significantly difference was found among the citrus fruits tested. The contents of total soluble, and insoluble dietary fiber in peels were significantly higher than in peeled fruits. The peeled lemons, oranges and grapefruits contained 164, 154 and 135 mg/100g, and their peels 190, 179 and 155 mg/100g of total polyphenols, respectively. The content of total polyphenols in peeled lemons and their peels was significantly higher than in peeled oranges and grapefruits and their peels, respectively.

Grape (Vitis sp., Vitaceae) Apart from oranges, grapes are the world´s largest fruit crop. About 80%

of the total crop is used in wine making and pomace (grape skins and seeds) represents approximately 20% of the weight of grapes processed 23.

Dietary fiber is a major fraction in many by-products from the agricultural industry 23. Grape pomace is a good source of dietary fiber and polyphenolic associated compounds 24. The phenolic composition of grape pomace varies considerably, depending on grape, variety and technology of wine making (specifically, duration of pomace content, cell disruption, during crushing, and pressing) 25. The antioxidant capacity of grape pomace 25,26 has led to the development of a new concept of antioxidant dietary fiber. Grape pomace is shown as a product that combines in a single material the physiological effects of both dietary fiber and antioxidants 27. This natural product should have a content of dietary fiber of up to 50% (dry matter basis), and one gram exerts the same radical scavenging activity as 50 mg of DL--tocopherol (DPPH assay). More recently, the dietary fibre content and antioxidant activity of two by-products of the vinification process—pomace and stem—of Manto Negro


5

red grape was measured. Both by-products presented high contents of total dietary fibre, comprising three-fourths of the total dry matter; remarkably, the stem by-product showed large amounts of extractable polyphenols (11.6%). The free radical-scavenging capacity of the former by-products was determined using the DPPH method, resulting in EC50 values of 0.46 g dm/g DPPH (stem) and 1.41 g dm/g DPPH (pomace) 28.

Pear (Pyrus communis L., Rosaceae) To underline the importance of using whole fruit, Gorinstein and co-

workers 26 assessed the bioactivity of pears using in vitro and in vivo

models. In line with the findings of other authors 29,30, the content of dietary fiber, phenolic acids, flavonoids and total polyphenol were significantly higher in peels than in pulps. The assays used to evaluate the potential antioxidant activity—inhibition of linoleic acid oxidation measured by the bleaching of -carotene as indicator, and scavenging of nitric oxide and DPPH- radicals—by pear fractions exerted similar trends. An inhibition percentage of 65.3%, 66.0% and 53.6% was found in the case of peels, respectively; whereas 24.5%, 20.7% and 9.9% was found in the case of pulps, respectively. That is, the antioxidant potential was significantly higher in peels than in pear pulp. In both fractions, a correlation was found between the antioxidant potentials and the total polyphenol content. On the other hand, dietary fiber content was higher in pear peel than in pulp. In contrast, a poor correlation was observed between the nitric oxide values and the content of total dietary fiber (R2 = 0.2937). In a model rat, diets supplemented with pear peel exerted a significantly higher positive influence on plasma antioxidant capacity (TRAP and malondialdehyde values) of rats than diets with fruit pulp.

Peach (Prunus persica Batsch., Rosaceae) Goristein’s group 20,21 studied the dietary fiber content and related

polyphenols in the pulp and peel fractions of peaches (Catherina variety), and compared it with their radical scavenging measured by the TRAP assay. The content of soluble and insoluble dietary fiber in pulp was lower than in peel in


6

fresh peaches. In both fractions, insoluble was higher than soluble fraction. In general, hydroxicinammic acids showed higher significant content in peel from peach than in peeled peaches, i.e., 34.96% for -coumaric acid and 25.06% for caffeic acid, whereas no difference between both fractions was observed in the case of ferulic acid. TRAP value was higher in peel than in peeled fraction.

Guava (Psidium guava L., Myrtaceae) The results of recent research 15 indicated that pulp and peel fractions of

guava showed high content of dietary fiber (48.6–49.4%) and total polyphenols (2.6–7.8%). The antioxidant activity of enriched polyphenol extracts from these fractions was studied, measuring scavenging of DPPH radical and protection towards Cu-induced oxidation of in vitro human low-density lipoprotein. Also, reducing ability was tested using ferric reducing antioxidant power assay (FRAP). All fractions tested showed a remarkable antioxidant capacity, and its activity was correlated with the corresponding total phenolic content. Peel fractions, with higher content of polyphenol than pulp, showed a better antioxidant capacity in the three models. Guava fruit was proposed as a useful ingredient rich in dietary fiber with associated antioxidant compounds. A high correlation among the assays tested and the amount of total polyphenols was found. It is worth mentioning that because guava is one of the best sources of vitamin C among fruits, it is likely that vitamin C contributed to a relatively high extent to the antioxidant activity measured in the hydrosoluble extracts in the three models tested.

Mango (Mangifera indica L., Anacardiaceae) Mango is one of the most important tropical fruits. This fruit has gained

increasing relevance in the European market 24. The peel proportions in mango fruit range from 20% to 30% of the whole fruit. Recently, the need has been stressed to develop technological processes for the preparation of high dietary fiber powders from orange or mango fruits. Specifically, the washing and drying processes are the two steps that could influence to a greater extent the losses of the associated bioactive compounds 31. Larrauri and co-workers 29 evaluated the antioxidant activity of powdered dietary fiber


7

samples obtained from mango peels. This product was obtained from fresh mango peels by successively wet-milling, washing with water and a drying process. In addition, soluble sugars were removed to increase soluble dietary fiber content and total polyphenols. As a result, an insoluble and soluble dietary fiber content of 43.4% and 28.1%, respectively, and a content of polyphenols of 7.0%—dry matter basis—were given. At a concentration of 0.05%, the inhibition of temperature-induced oxidation of linoleic of mango peel dietary fiber was 0.75 times as effective as 2-terc-butyl-4-hydroxianysol and, 3.4 times higher than that of DL--tocopherol.

Another approach deals with the incorporation of mango peel powder in soft dough biscuits to improve dietary fiber content and antioxidant properties as nutraceutical properties of the biscuits. The results indicated that wheat flour incorporated with mango peel powder (10%) yielded dietary fiber-enriched biscuits with improved antioxidant properties 32.

Pineapple (Ananas comosus L. Merr., Bromeliaceae) A dietary fiber powder obtained from pineapple fruit shell has been

characterized as having a high total dietary fiber content (70.6%) 33. The insoluble fraction amounted to 99% of the total dietary fiber. At a concentration of 0.5 of powdered sample/100 mL in the final mixture, pineapple fibers showed higher antioxidant activity (86.7%) than orange peel fiber (34.6%) in the inhibition of linoleic acid oxidation. Myricetin is the major polyphenol identified (59% of total polyphenols) in pineapple fiber, which could be responsible for the antioxidant activity. To get a balanced fiber composition, pineapple fiber could be mixed with another dietary fiber product with a high soluble dietary fiber fraction.

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[5] Liu, RH. (2003). Health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. Am J Clin

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GR; Kall, M; Breinholt, V; Castenmiller, JJM; Stagsted, J; Jakobsen, J; Skibsted, L; Rasmussen, SE; Loft, S & Sandstrom, B. (2004). The 6-a-day study: Effects of fruit and vegetables on markers of oxidative stress and antioxidative defense in healthy nonsmokers. Am J Clin Nut, 79, 1060-1072.

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[11] Witztum, JL & Berliner, JA. (1998). Oxidized phospholipids and isoprostanes in atherosclerosis. Curr Opin Lipidology, 9, 441-448.

[12] Jiménez-Escrig, A. (2003). Oxygen and Oxidation I: Reactive oxygen species and oxidative damage to biological substrates. Nutrición Clínica

y Dietética Hospitalaria, 23, 171-180. [13] Jiménez-Escrig, A. (2003). Oxygen and Oxidation II: Antioxidant

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Hospitalaria, 23, 181-191. [14] Bruunsgaard, H; Poulsen, HE; Pedersen, BK; Nyyssönen, K; Kaikkonen,


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J & Salonen JT. (2003). Long-term combined supplementations with -tocopherol and vitamin C have no detectable anti-inflammatory effects in healthy men. J Nutr, 133, 1170-1173.

[15] Jiménez-Escrig, A; Rincón, M; Pulido, R & Saura-Calixto, F. (2001). Guava fruit (Psidium guajava L.) as a new source of antioxidant dietary fiber. J Agric Food Chem, 49, 5489-5493.

[16] Lotito, SB; Frei, B. Relevance of apple polyphenols as antioxidants in human plasma: Contrasting in vitro and in vivo effects. Free Radic Biol Med, 2004 36, 201-211.

[17] Lu, Y & Foo, LY. (2000). Antioxidant and radical scavenging activities of polyphenols from apple pomace. Food Chem, 68, 81-85.

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Foods, Diets and Disease Editor: Rakesh Sharma, Bharati D shrinivas ©2009 Innovations And Solutions, Inc. ___________________________________________________________________________

Lecture 8

RELATIONSHIP OF PHYSICAL ACTIVITY AND DIETARY FIBER CONSUMPTION


ABSTRACT

Background

Increasing fruit and vegetable intake in the general population is one of the major concerns and aims of health promotion programs around the world. The teenage years are an important transition period from childhood to adulthood, when patterns of behavior and lifestyle choices are developing that will affect their current and future health. The heavy consumption of fast food and lack of adequate fruit and vegetable intake among adolescents in most developed countries are of increasing concern. Objective

The study aims to examine the fruit and vegetable consumption, physical activity levels and body mass index among teenagers. Methodology

A total of 203 adolescents (115 males and 88 females, mean age 13.5 years) from a secondary school participated in the study, in which their body mass index, dietary habits and physical exercise pattern were recorded. Results

Participants’ intake of fruits and vegetables was inadequate, with half consuming ≤ 1 serving of fruit per day and the great majority (90%)

consuming < 3 servings of vegetables per day. The reported reasons for the low consumption of fruits and vegetables included dislike of these foods, (47%), lack of availability of fruits and vegetables at home (25%), and habitually dining out (28%). Physical activity levels were far from


optimal, with almost 50% of the participants not performing any form of exercise during the previous seven days, and only 28% having done some form of exercise during the week prior. Only 22% reported doing moderate amounts of exercise. Conclusion In light of the inadequate consumption of fruits and vegetables, low physical activity level, and high prevalence of overweight and obesity found in this study, educational initiatives are urgently needed to encourage teenagers in Hong Kong to adopt a healthier diet and more active lifestyle.

INTRODUCTION Diet is one of the most potent tools for the prevention of certain types of

cancer (US Department of Health and Human Services, 2000; Willet, 2000), preventing or reversing non-communicable diseases such as diabetes, and improving cancer survival (Physicians Committee for Responsible Medicine, 2008). It is noted that lifelong dietary preferences are formed based on the eating patterns that are established in youth.

However, the dietary pattern of teenagers is found to be inadequate to meet the dietary recommendations for health. Youngsters in the United States and in Hong Kong tend to eat snack food products that are high in sugar or fat but low in disease-preventing ingredients such as fruits and vegetables (Krebs-Smith et al., 1996; Peterson and Sigman-Grant, 1997; Tse & Benzie, 2006).

The food preferences of the younger population are far from optimal, with consumption of desserts and snacks (such as cakes and ice-cream) four times or more per day, and fried food also being eaten four times or more per day (Lee & Tsang, 2004).

It is estimated that 19% of gastrointestinal cancers, 31% of ischemic heart disease and 11% of stroke worldwide is related to inadequate intake of fruits and vegetables (World Health Organization, 2003). In this regard, it is worrying that no US state reportedly achieves the national targets for fruit and vegetable consumption as recommended by WHO (World Health Organization, 2003; Center for Disease Control, 2008a). Likewise, even though the traditional Chinese diet is regarded as a healthy one, people in Hong Kong have been found to consume inadequate amounts of fruits and vegetables (Lam, Chan and Ho, 2002).

Fruit and Vegetable Consumption and Physical Activity 3

Sedentary lifestyles have also become a concern, with more than 50% of adults in the US not doing enough physical activity and 24% of them inactive for most of the day. The population in Hong Kong is also found to be physically inactive. It is reported that adults spend an average of 2.4 hours per day watching television, while teenagers spend 3 hours per day watching television (Lam, Chan and Ho, 2002; The Boys’ and Girls Club Association of Hong Kong, 2003).

Laziness and lack of interest in exercise are the main causes for physical inactivity (Center for Health Protection, 2005). It is worrying to find that teenagers in Hong Kong are physically inactive and exercise less than their counterparts in other developed countries (Hui et al., 2001). Secondary school children were found to spend too much time on computers, homework and other relevant activities, with the amount of time spent on sports and exercise low (Lam, Chan and Ho, 2002).

The teenage years are an important transition period from childhood to adulthood, when patterns of behavior and lifestyle choices are developing that will affect their current and future health (Centers for Disease Control and Prevention, 2008a). The objective of the present study was to examine health status, dietary habits and physical activity among teenagers in Hong Kong. The findings will give more insight for planning and implementing nutrition and lifestyle education programs to modulate dietary and behavioral changes for health promotion and lowering the risk of age-related disease.

MATERIALS AND METHODS

Research Design An experimental pre-post study design was selected, at the school level.

After gaining ethical approval from the Ethics Committee of the University, teenagers in a secondary school in Hong Kong were approached and invited to join our study.

Sample Description The secondary school was located in Shatin, a residential district in Hong

Kong in which 76% of the population is aged below 65. Shatin is an urban


district with public and private housing estates. The majority of the residents were middle class with a satisfactory socio-economic status.

Procedure The researcher and research assistants visited the school and performed a

health assessment for the participants, including body weight, height, body fat content, and body mass index (BMI), and subjects completed a questionnaire regarding their dietary habits and physical exercise pattern. The questionnaire was developed by the research team. To address the content validity, five experts, including two dietitians, two physiotherapists and one occupational therapist, reviewed the questionnaire; the content validity index was 0.804. The test-retest reliability was also established (r = 0.861) by repeat testing among 10 secondary two students over two weeks.

RESULTS

Characteristics of the Participants The study population included a total of 203 teens in year 8 of school

education. There were 115 males and 88 females (male: 56.7%; female: 43.3%), and the mean age was 13.5 years.

Body Mass Index Table 1 shows the Body Mass Index (BMI), calculated using the Asian

standard (Choo, 2002). It was found that 17% of the participants were overweight or obese, while body fat content was found to be slightly high to high in 26% of the participants. No significant difference was found between boys and girls in their BMI, yet more boys than girls were found to have slightly high or high body fat content, and this result was statistically significant (p<0.05). Please see that table 1.


Table 1. Body Mass Index and Fat Composition of the Participants

(n=203)

Overall % % by Gender p valuea

Boys n=115

Girls n=88

BMI Underweight 12.3 10.4 14.8 0.27 Normal 70.9 73.0 68.2 Overweight 8.4 6.1 11.4 Obese 8.4 10.5 5.6 Fat composition Low 15.3 1.7 33.0 0.00* Normal 58.6 65.2 50.0 Slightly high 15.8 20.9 9.1 High 10.3 12.2 8.0

a Chi-square test was used to compare boys’ and girls’ data. A p value of < 0.05 was considered statistically significant.

Dietary Patterns

Intake of fruits and vegetables was inadequate among the participants. As

shown in Table 2, around half of the participants (49.8%) consumed ≤ 1

serving of fruit per day and most of them (90.1%) consumed < 3 servings of vegetables per day. The reported reasons for the inadequate consumption of fruits and vegetables included dislike of eating fruits and vegetables (46.7%), lack of availability of fruits and vegetables at home (25%), and habitually dining out (28.3%).

In terms of intake of fried food and desserts, the majority of the participants

ate less than one serving per day. The overall intake for less healthy food (fish balls, candies/chocolates, French fries, and ice-cream) as snacks was higher than that of healthy food choices (low fat biscuits, bread, fruits).


Table 2. Dietary Habits Among Participants, Overall and by Gender

(n=203)

Overall (%) % by Gender p valuea Male n=115 Female n=88

Fruit consumption per day None 2.0 1.9 2.1 0.87 ≦ 1 serving 47.8 49.6 45.3 2-3 servings 45.2 44.6 45.8 ＞ 3 servings 4.4 2.3 2.1

Vegetable consumption per day None 0.4 0.0 1.1 0.71 ＜ 3 servings 89.7 89.6 89.8 3-5 servings 7.9 7.8 8.0 ＞ 5 servings 2.0 2.6 1.1

Fried food consumption per day < Once 49.5 48.2 51.2 0.66 Once 34.0 34.2 33.7 Twice 14.0 14.0 14.0

Dessert consumption per day < Once 33 37.7 29.5 0.62 Once 37.4 38.3 36.4 Twice 24.6 22.6 27.3 > Three times 4.9 3.5 6.8


Table 3. Snack Preferences among Participants (n=203)

Overall (%) % by Gender p valuea

Male n=115

Female n=88

Preferred Snacks a. Healthy food

Biscuits 6.8 6.3 6.2 0.68 Sponge cakes/bread 7.1 7.4 8.1 Fruit 12.8 12.4 13.2

b. Less healthy food Fish balls 16.8 16.4 16.7 0.77


Candies/chocolates 16 17.4 17.3 Fries/potato chips 23.7 27.1 18.3 Ice-cream 16.8 18.0 16.2

Snacks Expected to be Sold in Tuck Shop a. Healthy food

Sushi 32.2 34.8 32.2 0.46 Wheat bread 5.1 4.1 5.3 Fruit 12.1 13.2 10.1

b. Less healthy food Cup noodles 7.3 8.3 7.6 0.55 Fish balls 13.7 15.2 13.1 Fried chicken wings/legs 12.7 13.4 12.9 Ice-cream 16.9 15.0 14.8

a Chi-square test was used to compare boys’ and girls’ data. A p value of < 0.05 was considered statistically significant. No significant difference was found between boys and girls in their fruit

and vegetable consumption, intake of fried food and desserts, preference for snacks and expectations regarding items sold in the school tuck shop (p<0.05).

(tables 2 & 3 near here)

Physical Activity Table 4 shows the total hours reportedly spent performing exercise and

sitting (being sedentary) in the previous seven days. It was found that about half of the participants had not performed exercise of any form in the previous seven days, and 40% of them seldom or rarely walked up the stairs. No significant difference was found between boys and girls in the frequency of doing exercise, indicating that all participants had been quite physically inactive over the previous seven days (p>0.05). Also, the average sitting (sedentary) hours per day in the previous seven days was around 9 hours, although boys were found to be less sedentary than girls (p<0.05).

(table 4 & 5 near here)


Table 4. Physical Activity Habits Among Participants (n=203)

Overall (%) % by Gender p valuea Male n=115 Female n= 88

% Reporting Doing Exercise During Previous Seven Days None 48.8 47.2 47.6 0.50 ＜ 3.5 23.6 23.7 29.9 ≧ 3.5 27.6 29.1 22.5

Daily Physical Activity Reported a. Walking up stairs instead of taking the elevator

Always 12.8 16.5 14.8 0.00* Sometimes 42.5 38.3 47.7 Seldom 26.6 28.7 23.9 Rarely 15.2 16.5 13.8

b. Doing housework Always 12.8 7.8 19.3 0.00* Sometimes 50.2 46.6 55.7 Seldom 28.1 32.2 22.7 Rarely 8.9 13.9 1.3

c. Declining transportation or increasing walking time to destination Always 12.8 11.3 14.8 0.00* Sometimes 30.0 22.1 26.1 Seldom 32.0 27.8 37.5 Rarely 25.1 27.8 21.6


Table 5. Participants’ Daily Average Hours of Sitting During the Previous

Seven Days (n=203)

Hours Per Day (Mean ± SD) Sitting Duration Per Day in Previous Seven Days p valuea

Total 8.95 ± 4.77 0.000* Male (n=115) 7.40 ±3.93 Female (n=88) 10.98 ± 5.03

a Paired t test was used to compare boys’ and girls’ data. A p value of < 0.05 was considered statistically significant.


DISCUSSION

The present study showed inadequate intake of fruits and vegetables, as

well as less healthy snack choices, among the majority of participants. Also, the prevalence of overweight and obesity was quite high (17.5%) and around one quarter had high body fat content. It was also worrying to find that physical activity levels were far from optimal, with almost half the participants not performing any form of exercise and less than one third having done some form of exercise during the previous week. These findings are consistent with previously reported dietary habits, prevalence of obesity and physical activity levels among teenagers in Hong Kong and worldwide (World Health Organization, 2000; Department of Health of Hong Kong, 2002; Center for Disease Control and Prevention, 2008b; Centers for Disease Control and Prevention, 2008c).

The finding of inadequate consumption of fruits and vegetables indicates the need for nutrition education. People are born with great sensitivity to bitter tastes and so children tend to avoid foods with bitter flavors. Examples of such foods include cabbage, broccoli, spinach, Brussels sprouts, and grapefruit juice. Nevertheless, these foods, as well as many other fruits and vegetables, contain phytochemicals that may reduce the risk of cancer and other diseases (DeBruyne, Pinna & Whitney, 2008). The information regarding fruits and vegetables and their role in cancer prevention needs to get across to teenagers and their parents in order to encourage them to make healthier choices of food and consume more fruits and vegetables.

Adult behavior patterns and lifestyle choices are developed in the teenage years, and will subsequently affect their current and future health (Centers for Disease Control and Prevention, 2008a). It is therefore important to disseminate information regarding optimum dietary habits and physical activity among teenagers. In the Ottawa Charter (1986), health promotion is defined as the process of enabling people to increase control over and to improve their health (Bhuyan, 2004). This means that health should be seen in the context of people’s lives, and as a resource for life rather than an ‘ideal’

state free of disease. To enable us to increase control and to improve our health, strategies including building public policy, creating supportive environments, strengthening community action are needed, as well as developing personal skills via health education programs and activities (Bhuyan, 2004). Nutrition behavior is a complex process. Thus, a holistic


health education and health promotion approach would appear to be essential in successfully implementing nutrition education programs.

It is important to create a supportive environment in the school grounds by making healthy foods easily available, having strategically placed information regarding nutritional quality and food labels, providing incentives to promote dietary behavior change, and providing easy access to sound nutrition advice via professional websites. Teenagers’ independent purchases of snack foods

are very much influenced by environmental factors such as availability, advertising and marketing strategies (Hill et al., 1998). In this regard, school tuck shops should provide more healthy food, such as fruits, and reduce their stock of unhealthy food, including salt- and fat-laden food such as deep-fried chicken wings, chicken legs and potato chips.

CONCLUSION Most of the healthy teenagers who participated in the present study had a

normal BMI, but a high tendency to being overweight and obese was seen in the group. Also, their largely inactive, sedentary lifestyle implies the need for improved health behavior. It is important to provide nutrition education in the school environment. It would be beneficial to integrate structured and culturally suitable dietary and lifestyle programs into the secondary school curriculum. Likewise, an environment more supportive of health-promoting lifestyles among students should be provided by making available the resources and opportunities for increasing physical activity, enforcing healthy dietary guidelines in the school cafeteria, and providing proper weight management measures in the school grounds.

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Choo, V. (2002). WHO reassesses appropriate body-mass index for Asian populations. The Lancet, 20, 360(9328) 235.

DeBruyne, L. K., Pinna, K. & Whitney, E. (2008). Nutrition and diet therapy. principles and practice. Australia: Thomson.

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School, 1997-2002. Hong Kong: Department of Health. Hill, L., Casswell, S., Maskill, C., Jones, S. & Wyllie, A. (1998). Fruit and

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Foods, Diets and Disease Ed:Rakesh Sharma, Bharati D Shrinivas ©2009 Innovations And Solutions, Inc. ___________________________________________________________________________

Lecture 9

CARDIOVASCULAR PREVENTION BY SOLUBLE FIBER (PLANTAGO OVATA HUSK):

PLASMA TRIGLYCERIDES AND APOLIPOPROTEIN B TO APOLIPOPROTEIN A-I RATIO IN MEN


ABSTRACT

Background: New dietary strategies to reduce cardiovascular disease (CVD) risk include, as part of secondary prevention, the addition of fiber to the diet. Objective: To study the effect of treatment with soluble-fiber derived from Plantago ovata (Po) husk on lipids, in 28 men with CVD and plasma low density lipoprotein (LDL)-cholesterol concentration ≤3.35 mmol/L (≤130 mg/dL). A low-saturated-fat diet supplemented with 10.5 g/d Po husk was consumed for 8 weeks, under controlled conditions. Results: Following Po husk treatment, plasma apolipoprotein (apo) A-I increased 4.3% (P<0.01 and plasma triglycerides decreased 6.7% (P<0.02), the apo B 100/ apo A-I ratio decreased 4.7% (P<0.02) from (mean ±SD) 0.77±0.10 to 0.73±0.19. Conclusion: In secondary prevention of CVD, Po husk treatment induces a reduction in the apoB/apoA-I ratio, a measure that is identified as a strong new risk factor for CVD and which is a target for lipid-lowering therapy; the lower the apoB/apoA-I ratio, the lower is the risk. No effect on plasma LDL cholesterol was observed. Possible Po husk modes-of-action will be proposed.

INTRODUCTION Dietary fiber intake can reduce the risk of cardiovascular disease (CVD)(1) by acting, in part, on plasma lipid concentrations. Several studies have shown that soluble fibers are more effective in lowering blood cholesterol than are insoluble fibers(2).


Current advice on lowering cholesterol concentrations and reducing CVD risk by therapeutic lifestyle changes include increasing the amount of dietary fiber consumption, specifically of soluble (viscous) fiber, to 10–25 g/d(3-6). Thus, an increased soluble fiber intake, within a therapeutic lifestyle, takes on an essential modality in the clinical management of CVD risk reduction(3-6). Differences in genetic background are known to affect lipid metabolism and the response to diet(7,8). Identifying common variations in genes involved in the intestinal absorption of lipids and, hence, in the dietary response to soluble fiber is an attractive goal. We investigated Plantago ovata (Po) husk, ((Madaus, S.A., Barcelona, Spain), a source of natural, concentrated, soluble fiber obtained from the outer membranous green envelope of the Po seed. Po husk and Po seeds, the latter often used as a source of insoluble fiber for experimental control purposes in trials, are well-tolerated, safe, and effective bulk laxatives(9). The purpose of our study was to assess, in a crossover design protocol, the effects of 10.5 g/d soluble Po husk fiber compared to 10.5 g/d of an equivalent insoluble fiber added as therapeutic measures to a diet low in saturated fat and cholesterol. The target variables measured were plasma lipids, lipoproteins and apolipoproteins in patients with established coronary heart disease but with plasma low density lipoprotein (LDL)-cholesterol ≤3.35

mmol/L (130 mg/dL)(9). The potential interactions with genes involved in the response to dietary fiber therapy were explored, as well. The insoluble fiber used as control additive was hemicellulose and lignin Klason obtained from Po seeds (Madaus S.A., Barcelona, Spain). The two products are obtained from the same Po plant; Po husk consisting only of the epidermis and collapsed adjacent layers removed from the dried ripe Po seeds(9). In a controlled, single-blind, crossover study consisting of a 4 week dietary adaptation period, 28 patients were randomly assigned to two different fiber-supplement periods of 8 weeks each. We incorporated a washout period of 8 weeks between the 1st and 2nd periods of the study in order to test possible interactions between treatment and sequence order (carry-over effect)(9). In our study in men with CVD following a low saturated-fat low-cholesterol diet, the incorporation of Po husk significantly reduced plasma triglyceride concentrations by 6.7%, apo B:apo A-I ratio by 4.7% and increased the apo A-I concentration by 4.3%(9). The plasma triglyceride reduction observed with the Po husk treatment was approximately half that of the reduction (between 10% and 16%) observed with statin therapies (the most widely used LDL-cholesterol-lowering drugs) and was similar to that of ezetimibe (7% triglyceride reduction). With this modest hypotriglyceridemic effect, Po husk can be considered as an adjuvant treatment in patients with moderate hypertriglyceridemia(10). The Apo B/apo A-I ratio has been shown to be the best marker of atherogenic and anti-atherogenic particles in plasma. The lower the apoB/apoA-I ratio means the lower CVD risk. The INTERHEART study showed this ratio to be a marker of risk of myocardial infarction, irrespective of the geographic regions of the populations studied(11). Recently, the apo B/apo

Beneficial Effects of Soluble Fiber 3

A-I ratio was linked to the risk of fatal stroke(12) in a similar manner to that of myocardial infarction, and other ischemic events(12). Po husk significantly increased high density lipoprotein (HDL)-cholesterol concentrations by 6.7% relative to the insoluble fiber (9). This finding is of considerable clinical relevance because studies on the secondary prevention of CVD have shown that the recommended LDL-cholesterol lowering diets (low in saturated fat and cholesterol) have, as well, the detrimental effect of decreasing HDL-cholesterol concentrations. In our study, Po husk did not produce the LDL-cholesterol-lowering effect observed with other soluble fibers. This could have been due to several factors. For example, our patients consumed a more palatable and more tolerable low dose (10.5 g/d) than did the subjects in another study in which the dose of the same Po husk preparation was higher (14 g/d)(13). Reports indicate that the initial concentration of cholesterol is predictive of the subsequent reduction in cholesterol concentrations induced by some soluble fibers(14). In our study, the low-moderate basal LDL-cholesterol concentration of the patients (mean: 3.04 mmol/L; maximum: 3.36 mmol/L) was highly predictive of the failure of dietary Po husk supplementation to lower LDL cholesterol concentrations. Of note is the finding that both fibers (soluble and insoluble) significantly reduced the patients’

waist circumference and waist-to-hip ratio. This aspect has not been previously reported. Hence, supplementing a low-fat diet with fiber may have an additional benefit on CVD risk factors by reducing abdominal fat without appearing to have a significant effect on the BMI(9). A limitation of the present study is, probably, the small sample size of our study population. Also, an influence of the genes studied such as fatty acid binding protein (FABP-2) as well as apo A-IV, and apo E genes and their variants on the response to the dietary fiber intervention cannot be ruled out(9). However, following the Po husk treatment, the carriers of the Thr54 allele of FABP-2 had significantly higher HDL cholesterol (8%) than did those who had the insoluble fiber added to the diet. Our results indicate that soluble Po husk treatment induced a more favorable effect on the lipoprotein profile (i.e. reduction in CVD risk factors) than did a comparable insoluble fiber. The lowering of plasma triglycerides by Po husk has been observed in obese Zucker rats(15). The variables such as higher systolic blood pressure, plasma concentrations of triglycerides, total cholesterol, FFA, glucose, insulin, and TNF-α, and the hypoadinectinemia that occurred

in obese Zucker rats consuming the control diet for 25 weeks, were significantly improved in those fed the 3.5% Po husk fiber-supplemented diet. These data indicate that treatment of a Po husk-supplemented diet prevents endothelial dysfunction, hypertension, and obesity development, while ameliorating dyslipidemia and abnormal plasma concentrations of adiponectin and TNF- α in obese Zucker rats(15). The mode of action of soluble fiber remains unclear. Diet and its nutrients can modify the expression of genes involved in CVD(7,8). Therefore, an assessment of how dietary components such as fiber can affect the expression of genes and proteins involved in CVD is a relevant clinical issue and would elucidate biological action mechanism of the nutrient. The effect of soluble fiber on lipid metabolism in intestinal


cells has been poorly studied(16). Short chain fatty acids (SCFAs) produced by anaerobic bacterial intestinal fermentation of soluble fiber are proposed as regulating lipid metabolism in the intestine. However, the exact mechanism of action of SCFAs in the intestine remains unknown. The presence of SCFAs in the colon as well as the small intestine has been well documented(17-21). Intestinal anaerobic bacterial fermentation of fiber produces mainly acetate (Ac), propionate (Pr) and butyrate (Bu) in a quasi-constant proportion, and with a molar relation of 60:25:15(21). Following the consumption of a high fiber diet, 90–95% of the SCFAs produced are absorbed by the human colon(22).

To improve the knowledge of health-benefit mechanisms of SCFAs, we studied the effects of SCFAs on gene expression in a human enterocyte cell line (Caco-2/TC-7)(16). The transcriptional profiles of Caco-2/TC-7 cells following SCFA exposure in vitro were evaluated with a whole genome array (AB Human Genome Survey Array). The SCFAs used were acetate (Ac), propionate (Pr), and butyrate (Bu) at different concentrations ranging from 2 to 20 mM. SCFA concentrations have been described as ranging from 1 to 13 mM from the jejunum to the ileum(17) while other reports indicate a luminal range of Bu between 10 and 20 mM in the intestinal lumen(18). Total RNA was then isolated for microarray and quantitative real-time PCR analysis. Our results showed that treatment of human enterocytes with Pr and Bu has an impact on a wide variety of genes. These genes were classified according to the PANTHER classification system, and the results showed that 7 and 9 biological processes were significantly affected by Pr and Bu, respectively. Further, the results from the classification of genes differentially expressed by Pr and Bu showed that 4 and 11 metabolic pathways, respectively, were significantly altered; including the intestinal cholesterol biosynthesis pathway. The differential array expression analysis showed that 9 genes of this cholesterol biosynthesis pathway were down-regulated (Figure 1). These results were validated by real-time PCR. Mevalonate formation from acetyl CoA is the first fundamental step of cholesterol biosynthesis. At this step, Pr and Bu decreased the expression of 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) synthetase 2 by 2.9-fold and 4.3-fold, respectively, compared to controls. In addition, both fatty acids decreased by 1.5-fold the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR), the key enzyme in the cholesterol synthesis process. In the second fundamental step of this pathway, which is the transformation of mevalonate into squalene, Bu decreased the expression of isopentenyl diphosphate isomerase (IDI1) by 3.6-fold, the expression of dimethylallyl/geranyl trans-transferase (FDPS) by 3.2-fold, and the expression of the farnesyl-diphosphatase farnesyltransferase (FDFT1) by 2.3-fold. In the last fundamental step in the process, which is the transformation of squalene into cholesterol, Pr and Bu decreased the expression of lanosterol 5-desaturase (SC5DL) by 2.4-fold and 2.5-fold, respectively, and the expression of methylsterol mono-oxygenase (SC4MOL) by 7.8-fold and 9.8-fold, respectively. Moreover, Pr decreased the expression of the peroximal trans-2-enoyl-CoA reductase (PECR) by 2.2-fold.


Figure 1. Cholesterol biosynthesis pathway. Downregulated genes are represented in grey boxes.


We selected FDPS, SC4MOL, and HMGCoAR for validation by Real-Time PCR. In addition, we also selected SQLE because it is a novel target in the design of new hypocholesterolemic drugs. The effects of Pr and Bu on the expression of FDPS, SC4MOL, HMGCoAR, and SQLE genes were evaluated by real-time PCR. We showed that treatment of TC-7 enterocytes with Pr and Bu decreased, with variable intensities, the mRNA levels of all the genes tested. Pr and Bu had a dose-dependent effect on FDPS mRNA levels. Further, the inhibitory effects of Pr and Bu on the expression of HMGCoAR were 16% and 33% respectively. For SC4MOL and SQLE, the inhibitory effect of Pr and Bu showed variable intensity. Our results are in agreement with a previous study in which SCFAs suppress cholesterol synthesis in the intestine(23). However, from our results we are unable to conclude a definitive mechanism for the cholesterol-reducing effect of soluble fiber. Our in vitro study enabled us to identify a wide variety of biological processes and metabolic pathways affected by the SCFAs tested. Importantly, our results show that the overall effect of Pr and Bu is to down-regulate the expression of 9 key genes involved in intestinal cholesterol biosynthesis and, hence, to inhibit this pathway. Bu appears to be the SCFA with the most important impact on enterocyte gene expression. Of interest in future studies would be gene expression after treatment with SCFAs obtained from the in vitro fermentation of soluble fiber, in an intestinal fermentation model system. In

vitro fermentation is a non-invasive and time-efficient method used to assess fiber fermentability in vivo. These studies need to be conducted with triacylglycerol and apo A-I because both are, at least in part, produced in intestinal cells and are modified by Po treatment. Increasing evidence indicates that genetic factors influence the relationship between dietary components and disease risk, and that various protective and risk factors affect the incidence of disease(7,8). It is now possible to visualize differences between the genetic profiles of individuals at the molecular level and to begin to understand how they relate to differences between individuals’

responses to physiological factors at the level of the whole organism. Also, new techniques can be applied to gain an understanding of the way differences between other aspects of an individual’s molecular biology affect the response to physiological factors.

These techniques are known collectively as “omics”. The study of the total genetic makeup

(the genome) known as genomics, and the analysis of the total metabolic profile (the metabolome) defined as metabolomics, can provide ways to understand the influence of dietary components on disease. Also, transcriptomics and proteomics are the terms that describe the study of the total gene expression and the total protein complement of an organism, respectively (7,8). Our transcriptomic approach with respect to intestinal cells suggests that a relationship could exist between fiber, plasma lipids, and atherosclerosis. However, the mechanisms involved remain unknown.


Dietary fiber treatment may reduce the risk of CVD, but the processes involved would need to be elucidated by “omics” in order to clarify the new mechanisms of action of fiber. In the future, the situation will arise in which it would be possible for individuals to make truly informed choices regarding which foods provide the best opportunities for health, well-being and reduced risk of disease. Finally, it is important to focus on the overall quality of the diet, which includes fiber and other individual foods or nutrients, such that there is a balance in energy intake and expenditure, while enhancing regular physical activity to maintain health.

REFERENCES [1] Pereira, MA; O’Reilly, E; Augusstsson, K; et al. (2004). Dietary fiber and risk of coronary heart disease. A pooled analysis of cohort studies. Arch Intern Med., 164:370-6. [2] Petchetti, L; Frishman, WH; Petrillo, R & Raju, K. (2007). Nutriceuticals in cardiovascular disease: psyllium. Cardiol Rev, 15;116-22. [3] Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. (2001). Executive summary of the third report of the National Cholesterol Education Program (NCEP). JAMA, 285:2486-97. [4] Grundy, SM; Cleeman, JI; Bairey Merz, N; et al. (2004). Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III Guidelines. J

Am Coll Cardiol, 44:720-32. [5] Lichtenstein, AH; Appel, LJ; Brands, M; et al. (2006). Diet and lifestyle recommendations revision 2006: a scientific statement from the American Heart Association Nutrition Committee. Circulation, 114:82-96. [6] Graham, I; Atar, D; Borch-Johnsen, K; et al. (2007). European guidelines on cardiovascular disease prevention in clinical practice: executive summary. Eur Heart J,

28:2375-414. [7] Subbiah MT. (2008). Understanding the nutrigenomic definitions and concepts at the food-genome junction. OMICS, 12:229-35. [8] Ordovas, JM & Tai, ES. (2008). Why study gene–environment interactions? Curr Opin

Lipidol, 19:158-67. [9] Solà, R; Godàs, G; Ribalta, J; et al. (2007). Effects of soluble fiber (Plantago ovata husk) on plasma lipids, lipoproteins, and apolipoproteins in men with ischemic heart disease. Am J

Clin Nutr, 85:1157-63. [10] Brunzell JD. (2007). Hypertriglyceridemia. N Engl J Med, 357:1009-17. [11] Yusuf, S; Hawken, S; Ounpuu, S; et al. (2004). Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet, 364:937-52.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Subbiah%20MT%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus


[12] Walldius, G; Aastveit, AH & Jungner, I. (2006). Stroke mortality and the apoB/apoA-I ratio: results of the AMORIS prospective study. J Intern Med, 259:259-66. [13] Sierra, M; García, JJ; Fernandez, N; et al. (2002). Farmafibra Group. Therapeutic effects of psyllium in type 2 diabetic patients. Eur J Clin Nutr, 56:830-42. [14] Brown, L; Rosner, B; Willett, WW & Sacks, FM. (1999). Cholesterol-lowering effects of dietary fiber: a meta-analysis. Am J Clin Nutr, 69:30-42. [15] Galisteo, M; Sánchez, M; Vera, R; et al. (2005). A diet supplemented with husks of Plantago ovata reduces the development of endothelial dysfunction, hypertension, and obesity by affecting adiponectin and TNF-alpha in obese Zucker rats. J Nutr, 135:2399-404. [16] Alvaro, A: Solà, R; Rosales, R; et al. (2008). Gene expression analysis of a human enterocyte cell line reveals down regulation of cholesterol biosynthesis in response to short-chain fatty acids. IUBMB Life, 60:757-64. [17] Cummings, JH; Pomare, EW; Branch, WJ; et al. (1987). Short chain fatty acids in human large intestine, portal, hepatic and venous blood. Gut, 28:1221-7. [18] Mortensen, PB & Clausen, MR. (1996). Short-chain fatty acids in the human colon: relation to gastrointestinal health and disease. Scand J Gastroenterol, 216 (suppl.):132-48. [19] Roy, CC; Kien, CL; Bouthillier, L; et al. (2006). Short-chainfatty acids: ready for prime time? Nutr Clin Pract, 21:351-66. [20] Wong, JM; de Souza, R; Kendall, CW; et al. (2006). Colonic health: fermentation and short chain fatty acids. J Clin Gastroenterol, 40:235-43. [21] Garcia Peris, P; Breton Lesmes, I; de la Cuerda Compes, C; Camblor Alvarez, M & Colonic metabolism of fiber. (2002). Nutr Hosp, 17(Suppl 2):11-6. [22] Schmitt, MG Jr; Soergel, KH; Wood, CM; et al. (1977). Absorption of short-chain fatty acids from the human ileum. Am J Dig Dis, 22:340-4. [23] Hara, H; Haga, S; Aoyama, Y; et al. (1999). Short-chain fatty acids suppress cholesterol synthesis in rat liver and intestine. J Nutr, 129: 942-8.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Galisteo%20M%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

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Foods, Diets and Disease Rakesh Sharma, Bharati D Shrinivas © 2009 Innovations And Solutions, Inc. ___________________________________________________________________________

Chapter 10

PROCESSING TECHNIQUES AND THEIR EFFECT ON FRUIT PHYTOCHEMICALS


1. ABSTRACT

Fruits are an excellent source of essential vitamins, minerals, and dietary fiber in the human diet. They are also a rich source of secondary metabolites that proving to play an important role in protection against numerous chronic diseases. These substances are almost ubiquitous in plant-derived foods and inherently have more subtle effects than nutrients. Carotenoids and flavonoids, the mostly spread secondary metabolites in fruits, have received much attention over the past decade due to their putative health-protective effects. A significant portion of the fruits consumed are processed and many of the processed products are stored in a variety of packaging materials for extended periods of time prior to consumption. Bioactive compounds that are naturally present in fruits may undergo transformations during food processing that neither decrease their nutritional value nor bioactive value but may increase it by favoring their absorption and metabolism in the human body. Thus, in this chapter there is a significant need to understand how the different processing methods used in the food industry may modify their contents, structure, and biological activity in humans.

Key words: Fruits; Phytochemicals; Food processing


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2. INTODUCTION Broadly speaking, nutrients are classified into two groups, namely

macronutrients (also called energy producing nutrients or energy-yielding nutrients) and micronutrients (which are characterized by their essentiality for human health and the low quantities in which they need to be ingested). Energy-producing nutrients include carbohydrates, fats, and proteins. Micronutrients often refer to vitamins and minerals. Phytochemicals, also called bioactive compounds, are substances present in foods in low levels that may have a role in health maintenance in humans. Fruits have proved to be essential for a balanced diet. This is believed to be mainly due to their content of vitamins, fibers, and phytochemicals, the latter being responsible in part for the antioxidant properties of fruits and foods of fruit origin. Manufacturing processes are changing the nutritional properties of some foods. For instance, partial hydrogenation of vegetable oil results in the formation of trans-fatty acids, and heat treatment of protein solutions in an alkali environment results in the formation of lysinoalanine. Both of these have been shown to have detrimental health effects. On the other hand, some nutrients and bioactive compounds that are naturally present in fruits may undergo transformations during food processing that neither decrease their nutritional value nor bioactive value but may increase it by favoring their absorption and metabolism in the human body. Thus, in this chapter there is a significant need to understand how the different processing methods used in the food industry may modify their contents, structure, and biological activity in humans.

3. IMPORTANCE OF FOOD COMPOSITION DATABASES FOR DIETARY RECOMMENDATION

Information on the retention of phytochemicals in processed foods is

much needed to update food composition tables, commonly used for epidemiological and nutritional studies. In addition to compositional data on proximate (water, protein, total lipid, total carbohydrate, and ash), minerals, vitamins, lipid components, and amino acids databases have been developed for dietary phytochemicals namely carotenoids and flavonoids (flavonols, flavones, flavanones, flavan-3-ols and anthocyanidins), including a separate

Processing Techniques

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database for proanthocyanidins. Documentation of the effects of processing on bioactive phytochemicals would enable better interpretation of data with respect to dietary habits and human health, which is a critical step for formulating dietary recommendations. A greater understanding of such issues would also be of great interest to food processors who wish to retain or possibly boost levels of health-promoting compounds during manufacturing, as well as substantiate health claims for phytochemicals present in their products. Information on nutrient content of processed fruit is also of great interest to consumers. Consumers are becoming more aware of health benefits of fruits and are interested in knowing how fresh and processed products differ in nutritional quality. Unfortunately, a common perception among consumers is that processed fruits are inferior in nutritional quality compared to fresh products. Although fruit nutritional quality can be compromised during processing, there are instances where chemical reactions, interactions between components, and release of bound components during processing can lead to an increase in nutritional quality. Clearly, more information is needed on this topic in order to better inform consumers about dietary choices. The goal of this chapter is to provide a comprehensive review of the effects of thermal and non-thermal processing techniques, freezing, packaging, and storage on flavonoids, ascorbic acid and carotenoids, the major phytochemicals found in fruits. The potential effects of processing unit operations on phytochemicals will also be discussed and potential strategies to prevent losses identified (Tomas Barberan and Gil, 2008).

4. EFFECTS OF COMPOUND SOLUBILITY, CELLULAR AND STRUCTURAL LOCALIZATION ON PHYTOCHEMICAL

LOSSES Phytochemical structure, which influences solubility, and cellular

localization of phytochemicals are two critical factors influencing their retention during processing (Table 1). The relatively polar phytochemicals including various classes of phenolics (hydroxybenzoic and hydroxycinnamic acids) and flavonoids (primarily anthocyanins, flavonols, flavones, procyanidins) are readily leached into water during blanching and syrups/ brines during canning. In contrast carotenoids are well retained during blanching and canning due to their non-polar nature and resistance to leaching, although losses can occur due to thermal degradation and oxidation.


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Localization of phytochemicals within the plant cell can also influence losses during processing. Phenolics compartmentalized in the vacuole and apoplast are readily lost as a result of membrane disruption following thermal treatments, while cell wall bound phenolics resist leaching and may be more easily extracted or bioavailable after processing due to tissue softening (Dewanto et al., 2002a). The carotenoids are contained within chloroplasts or chromoplasts in membrane-protein complexes. After thermal processing, carotenoids are more easily extracted from plant tissues due to tissue softening and/or destruction of the membrane-protein complex. This phenomenon is commonly cited as one of the reasons why many processed fruits have higher levels of carotenoids than their fresh counterparts (Shi and Maguer, 2000; Dewanto et al., 2002b), although other factors such as leaching of soluble solids into the liquid canning medium and inactivation of carotene oxidizing enzymes may also explain the apparent increase (Sa and Rodriguez-Amaya, 2004).

Table 1. Properties of Phytochemicals in Plant Tissues

(Adapted From Kalt, 2005) Feature Carotenoids Phenolics

Subgroups

Numerous, for example, lutein, lycopene, α- carotene, β-carotene, zeaxanthin; β-cryptoxanthin

Numerous, for example, phe-nolic acids, hydrozycinna-mates, flavonoids inc. flavor-nols, catechins, anthocyanins

Solubility Lipid Water

Cellular Localization

Associated with membrane protein, complexes in chloroplast or chromoplast

Dissolved in vacuole and apoplast

Structural Localizati

Some types (for example, tomato lycopene) preferen-tially in surface tissues like peel and outer pericarp

Anthocyanins preferentially in peel; proanthocyanidins in peel and seed; hydroxycinnamates in flesh; ellagitannins in seeds


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5. EFFECTS OF PROCESSING UNIT OPERATIONS ON PHYTOCHEMICAL LOSSES

There are many potential sites where phytochemical losses can occur

during canning operations (Table 2). Raw materials must be washed thoroughly to remove dirt and debris prior to blanching and minor losses of water soluble phytochemicals may occur if epidermal tissues have been damaged. In fruit canning where blanching is typically not employed due to the delicate nature of fruit tissue, the heat-labile enzyme polyphenol oxidase can cause considerable losses of chlorogenic acid and flavonoids prior to its inactivation through pasteurization. Physical removal of tissues such as skins and seeds during processing can have a substantial effect on phytochemical retention. In many fruits, carotenoids and flavonoids located predominantly in epidermal tissues are removed by peeling operations (manual or lye assisted) prior to processing, which can greatly reduce concentrations of the compounds in processed products. In a study on peaches, manual and lye-assisted peeling methods resulted in 1.5 and two-fold reductions in total phenolics, respectively (Asami et al., 2003b). Removal of seeds prior to processing can also result in losses of phenolics residing in seeds such as procyanidins and ellagitannins. Significant quantities of lycopene and flavonols in tomatoes can be lost during peeling since the skin contains about three-fold higher levels of lycopene than the whole fruit and 98% of the flavonols are located in the skin (Stewart et al., 2000).

The byproducts of fruit processing represent promising sources of compounds with bioactive properties that could be exploited in the development of novel food ingredients and dietary supplements (Schieber et al., 2001). The size and type of fruit product (whole, halved, quartered, diced, sliced, julienne, puree) may influence phytochemical retention due to variation in the surface area of abraded surfaces. Although a study has not been conducted comparing phytochemical retention in different piece sizes of fruits, it is likely that the higher number of disrupted cells and larger surface areas of small pieces would promote greater leaching of water-soluble phytochemicals. Fruit purees are distinctly different from other canned products in that only a small amount of water is added to control product consistency. Greater retention of water-soluble phytochemicals in purees in comparison to products canned in water or syrup would be expected since in purees the compounds are trapped within the product matrix, whereas extensive leaching can occur in products canned in aqueous media. The composition of brine is another


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important consideration, since specific phenolic compounds can precipitate when exposed to acidic conditions. The type of retort method used for sterilization can also impact the phytochemical content of canned fruits.

In a static system cans or jars remain stationary throughout the sterilization process resulting in slow heat penetration throughout the can and lengthy process times. In more elaborate rotary processing systems, the cans or jars may be rotated end-over-end or allowed to rotate on their axes during the sterilization process. The agitation incurred via rotary processing increases heat penetration throughout the can and greatly reduces process time, enabling greater color and texture retention (Abbatemarco and Ramaswamy, 1994). In addition to the retort method, the sterilization time and temperature can influence the retention of phytochemicals. The degradation of anthocyanins (Ahmed et al., 2004; Kirca et al., 2006) and carotenoids (Shi et al., 2003) follow first-order reaction kinetics, hence high temperature short-time processing is recommended for maximizing the retention of anthocyanins and carotenoids (Lin and Chen, 2005) in foods. Although losses of naturally occurring antioxidant phytochemicals in fruits occur during sterilization, the formation of Maillard reaction products can result in elevated antioxidant capacity (Nicoli et al., 1999). The antioxidant activity of Maillard reaction products has been attributed to the high molecular weight brown melanoidin compounds formed in the advanced stages of the reaction (Anese et al., 1999).

Table 2. Potential Sites and Processing Variables Influencing the

Phytochemical Content of Processed Fruits

Process unit operation Variables influencing phytochemical content

Washing Spray vs immersion

Blanching Water vs steam Time and temperature

Peeling Amount of tissue removed Particle size reduction Degree of exposed surface area Screening Physical removal of skins and seeds

Brining Fruit/vegetable to brine ratio Brine composition

Thermal processing Static vs agitated Time and temperature

(adapted from Hager and Howard, 2006)


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6. THERMAL PROCESSING TECHNIQUES INFLUENCING PHYTOCHEMICAL CONTENT OF FRUIT

6.1 Survey of Industrial Processes

The greatest quantity of processed fruit is preserved by heat treatment. A

wide range of practical and theoretical knowledge is needed for an industrial process, as Figure 1 illustrates. Food processing involves the fields of microbiology, plant biology, thermophysics, food rheology and chemistry, packaging technique, unit operations, reactor techniques, construction and materials science, machinery, and electrophysics. The most important factors (besides the nature of the raw material and type of product) for constructing a plant are as follows:

type and size of the container (e.g., from small cans up to large tanks),

and mode of heat treatment (e.g., batch or continuous pasteurization of

closed containers; full aseptic process or some combination; temperature and pressure above or below 100°C and absolute pressure of 100 kPa).

In order to preserve the color of processed fruit, selection of fruit with

optimal pigmentation is recommended. Undesirable compounds such as melanoidins and melanins are formed in browning reactions. The major pigments in fruits are chlorophylls, carotenoids, and anthocyanins. Chlorophylls are lipid soluble pigments that disappear during fruit ripening. Carotenoids are subdivided into xanthophylls and carotenes. Xanthophylls are yellow (as in Golden Delicious apples and bananas), and carotenes are red (such as lycopene, the major pigment in watermelon and pink grapefruit flesh) and orange (the ß-carotene found in orange and apricots). Anthocyanins are water soluble pigments responsible for the red, blue, and purple of many fruits, flowers, and vegetables. All these pigments related to color are destroyed during thermal processing. The chlorophylls are degraded to brown pheophytins (Diane, 2005), the carotenoids are converted to epoxides, and the anthocyanins are rapidly degraded. During heat processing, anthocyanins react quite readily with the metal walls of nonlacquered cans. Thus, it is necessary to lacquer the cans to protect both the product and the can, as the color will usually pass out into the syrup during processing. There are two types of


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discoloration associated with anthocyanins. The first type occurs when leucoanthocyanins are converted to anthocyanins during canning. This causes the characteristic pink discoloration of pears and the excess red color in peaches. Other fruits that undergo pink discoloration include guava, lychee, and banana. The second type involves enzymic browning, which can be prevented by blanching and the addition of ascorbic acid, citric acid, and malic acid. Heat processing may also cause the formation of various zinc complexes such as in canned kiwifruit slices (Cano and Marin, 1992).

(adapted from Hui et al., 2006)

Figure 1. Co-operation between science and technology for achievements in heat treatment processes.


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6.2 Effects of Thermal on Carotenoids In most studies investigating processing effects on carotenoid retention

and isomerization the sterilization process has been delivered in a still or static mode. The high temperatures required to achieve commercial sterility of low acid canned fruits (>120°C) generally do not result in significant losses of carotenoids, but significant trans to cis-isomerization of carotenoids is evident The percent increase in total cis isomers on a percent basis as a result of thermal processing occurred in tomatoes (18%), and peaches (10%) (Lessin et al., 1997). In various commercial tomato products total cis isomers of lycopene increased 3.6% to 6% in response to thermal processing (Nguyen and Schwartz, 1998). In addition to process duration and temperature, food matrix components such as oil or fat have been shown to increase lycopene isomerization in tomatoes (Schierle et al., 1996). The major cis isomer of the provitamin A carotenoids in thermally processed red, yellow, and orange fruits was 13-cis, with much lower amounts of 9-cis and 15-cis isomers. In thermally processed tomato products the major cis isomer of lycopene is 5-cis lycopene followed by smaller quantities of 9-cis and 13-cis lycopene and other unidentified cis isomers (Schierle et al., 1996).

Carotenoids are well retained in fruit that receive a less severe thermal treatment (pasteurization), with negligible losses reported for mango slices and puree and papaya puree (Rodriguez Amaya, 1999). The increased levels of carotenoids in fruits after canning have generally been attributed to several factors including an unaccounted loss of soluble solids into the canning medium, inactivation of enzyme systems that oxidize carotenes during extraction of fresh samples, and increased extraction efficiency due to tissue softening and disruption of carotenoid–protein complexes and carotenoid–dietary fiber interactions. To assess the true retention of carotenoids in thermally processed fruits it is important to correct for weight changes during processing in order to account for losses of soluble solids and water. New extraction techniques for fresh samples are needed that result in rapid inactivation of carotene oxidizing enzymes and release of carotenoids bound to the sample matrices.

6.3 Effects of Thermal Processing on Flavonoids and Phenolic Contents

The flavonoid content of fruits is affected by unit operations such as

peeling, chopping, and blanching as well as heating and cooking method. In


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addition to processing factors, the retention of flavonoids and other phytochemicals is significantly influenced by leaching of compounds in the heating medium. Several studies have reported on flavonoid retention of fruits processed under commercial conditions and results indicate that thermal processing may enhance, reduce, or cause no change in flavonoids compared to those in fresh samples. The influence of phytonutrient solubility on retention during processing was demonstrated in a study on pasteurized yellow banana pepper rings (Lee and Howard, 1999). After processing, only 37% of ascorbic acid was retained, while 55% of quercetin and luteolin were retained, and capsaicinoids were fully retained. Hence, the polarity of the compounds (ascorbic acid > flavonoids > capsaicinoids) influenced their diffusion and solubility into the brine during processing. The effects of three different methods of processing tomatoes into sauce and paste on flavonoid content were reported by Re et al. (2002). Only 9% of naringenin was retained after processing tomatoes into sauce regardless of the processing method used, whereas 71% and 75% of rutin were retained by super cold break (65°C under vacuum) and hot break (90°C) methods, respectively, compared to only 48% retained by the cold break (65°C) method.

The effects of three different time–temperature process conditions (101°C for 40 min, 104°C for 10 min, and 110°C for 2.4 min) on the total phenolic and procyanidin contents in clingstone peaches were reported by Asami et al. (2003b). Peaches processed at 104°C for 10 min lost 21% of total phenolics, those processed at 110°C for 2.4 min lost 11% of total phenolics, while those processed at 101°C for 40 min retained comparable levels of total phenolics as the raw material. Peaches processed at 104°C for 10 min were analyzed for procyanidins with results compared to levels in frozen fruit. Marked reductions, 49% and 88% of procyanidin monomers and dimers, respectively, and a complete loss of procyanidin trimers through heptamers occurred in thermally processed peaches. In a follow-up study, the authors determined that loss of procyanidins in peaches during thermal processing was the result of leaching from the fruit into the syrup (Hong et al., 2004).

Several studies have reported changes in anthocyanins and other polyphenolics during canning of highly pigmented fruit. The total anthocyanin and total phenolic contents of pitted Bing cherries canned in light syrup did not change appreciably during canning, but approximately 50% of the anthocyanins and phenolics were transferred from the fruit into the syrup (Chaovanalikit and Wrolstad, 2004a). In another study involving canned Bing cherries increased levels of anthocyanins, total phenolics, hydroxycinnamates, epicatechin, and flavonol glycosides were observed after canning, which was


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attributed to increased extraction efficiency of the softened fruit (Chaovanalikit and Wrolstad, 2004b). Consistent with results from the first study, approximately 50% of the polyphenolics were leached into the syrup during canning. The anthocyanin content of fresh and frozen plums decreased 17% and 37%, respectively, during canning (Weinert et al., 1990a). During canning decreased levels of anthocyanins in the skin were accompanied by increased levels in the flesh and syrup, with uniformly distributed levels obtained in the skin, flesh, and syrup after one week of storage. In a follow-up study Weinert et al. (1990b) reported that anthocyanins losses during canning of plums were not associated with changes in polymer concentration, and suggested the losses were due to thermal destruction, irreversible binding, or oxidation.

The effects of processing unit operations on the total phenolic and total anthocyanin content of strawberry puree were studied by Klopotek et al. (2005). The mashing step resulted in a 15% loss in total phenolics, but an apparent 20% increase in total anthocyanins, which was thought to be the result of enhanced extractability. After pasteurization an additional 11% of total phenolics were lost, while total anthocyanin levels remained unchanged. Vacuum treated purees had similar levels of total phenolics and total anthocyanins than non-vacuum treated purees, indicating that the loss of phenolics was not related to oxidation. Strawberry processing to produce jams decreased the total ellagic acid content by 20% and the flavonoids by 15–20% (Hakkinnen et al., 2000). Aguilar-Rosas et al., (2007) who observed that, the high temperature sort time (HTST) treatment of apple juice caused a considerable lost of phenols (32.2%) when compared with the PEF treatment, which only caused a 14.49% reduction. Spanos and Wrolstad (1992) reported that total phenol concentration is reduced up to 50% in apple juice pasteurized thermally at 80°C for 15 min. Gardner et al., (2000) observed also considerable losses in phenolics in apple juice pasteurized by thermal means.

6.4 Effects of Drying Processing on Phytochemical Content of Fruit

6.4.1 Fruit Drying

Fruit drying has a long tradition. Inhabitants living close to the Mediterranean Sea and in the Near East traded fruits that had been dried in the open sun. Dried fruit is a delicacy, because of the nutritive value (66– 90% carbohydrate) and shelf life. For example, inhabitants of hillside villages


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isolated from the outside world by snow ate diets consisting primarily of seeds and dried fruits. Today, the production of dried fruits is widespread. Nearly half of the dried fruits in the international market are raisins, followed by dates, prunes, figs, apricots, peaches, apples, pears, and other fruits. Significant amounts of sour cherries, cherries, pineapples, and bananas are also dried. Fruit may be dried as a whole (e.g., grapes, various berries, apricot, plum, etc.), in sliced form (e.g., banana, mango, papaya, kiwi, etc.), in puree form (e.g., mango, apricot, etc.), as leather, or as a powder by spray or drum drying. Depending on the physical form of the fruit (e.g., whole, paste, slices), different types of dryers must be used for drying. Figure 2 illustrates the wide assortment of dryers that may be found in practice for drying of fruits. The selection of fruit for drying depends on local circumstances and customs. For example, in the Middle East, lemons with thin peel are dried whole. The taste and aroma are preserved in the brownish inner fleshy part, which remains soft. In the United States, blackberries, cowberries (ligonberries), and grapes are dried, while in Spain, red grapes are dried. Apricots, dates, plums, and tropical fruits are dried in the sun in several countries, while apples, pears, prunes, and peaches are dried by artificial means. Dryers with natural air ventilation were used in the 19th century in California for apple drying or to finish products that have previously been dried by the sun. Fruits dried in the sun or in dryers with natural air ventilation are referred to as “evaporated fruit,” while fruits

dried in dryers with artificial ventilation are described as “dehydrated fruits.”

The residual moisture content varies from small (3–8%) to large (16–18%) amounts, according to the type of fruit. Significant amounts are packaged in small portions (200–1000 g) in manufacturing plants. Often, countries importing dried fruit repackage it to meet the needs of consumers and large kitchens.

Figure 2. Various types of dryers for drying of fruit.


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Fruit mixtures are widely consumed both in the United States and Europe. Fruit is packed as a mixture or each component is packed separately in a transparent, appealing packaging. Well-known components are round slices of apples, apricots and peaches, pear halves, prunes, sour cherries, and dates. Often, walnuts and almonds are also added to the mixture. Dried fruit is widely used by the confectionery, baking, and sweets industries. Soup manufacturing plants use dried fruits in the various sauces, garnishments, puddings, and ice powders, and food for infants and children. Dried fruits are used in various teas, e.g., rose hips, and by the distilling industry (dried prunes, apricots). Applications include fruit powders processed from juices or pulps that dissolve quickly. The development of the fruit powders was possible through processing, which preserves color and flavor (vacuum drying, lyophilization, and swelling). Artificial drying made it economically possible to use raw materials at competitive prices and of high quality; examples are apples, prunes, and rose hips. Various milling procedures make it possible to dry highly valuable berries with soft flesh (strawberries, raspberries) and mature stone-fruits (apricots, peaches) (Hui et al., 2006).

6.4.2 Effects Of Dehydration On Flavonoids And Phenolic Compounds

Dehydration is one of the oldest methods for preserving foods and is still widely used in commercial manufacturing of dried fruit products. It is well known that drying methods employing high temperatures and long drying times result in thermal degradation of heat sensitive nutrients, including carotenoids and flavonoids, which can adversely affect the color of dehydrated products. Several different dehydration techniques available for preserving fruits vary significantly in both drying temperature and duration. Solar drying (SD) and conventional hot-air drying (HAD) methods require high temperatures and long durations, parameters that are detrimental to the texture, color, flavor, and nutritional quality of the product. Conversely, freeze drying (FD) of foods, although expensive compared to other dehydration methods, is a relatively benign method that minimizes thermal damage and generally results in excellent retention of color, flavor, texture, and nutritional quality. Vacuum microwave drying (VMD) has recently been promoted as an alternative method to improve the quality of dehydrated products. By combining the positive effects associated with vacuum (lower drying temperature and rapid mass transfer) with those of microwave heating (rapid energy transfer), products can be dried rapidly at lower temperatures. Additionally, VMD reduces the exposure of nutrients to oxygen, a critical step in minimizing oxidation of pigments responsible for acceptable product color.


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The effects of different dehydration methods on the retention of carotenoids and flavonoids in fruits have been studied. The studies indicate that phenolic compounds are much more susceptible to thermal degradation during dehydration than carotenoids, and that FD and VMD result in greater retention of the compounds than HAD. In a study of Saskatoon berries, the retention of total anthocyanins in FD, VMD, and HAD samples dried to similar water activity levels of 73%, 49%, and, 18%, respectively, as compared with levels found in fresh frozen berries (Kwok et al., 2004). Different classes of phenolic compounds show marked differences in their retention during dehydration, which appears to be related to their susceptibility to enzymatic oxidation. Changes in the phenolic composition of raisins subjected to three different dehydration treatments – sun-dried, dipped in hot water and dried (dipped), and dipped in hot water, treated with sulfur dioxide and dried (golden) – were compared with fresh and frozen Thompson seedless grapes (Karadeniz et al., 2000). Procyanidins and flavan-3-ols were completely degraded in all raisin samples, only 10% of the two major hydroxycinnamic acids (caftaric and coutaric acids) were retained, while approximately 38% of the major flavonols (quercetin and kaempferol glycosides) were retained. Golden raisins retained higher levels of caftaric and coutaric acids and had lower levels of oxidized cinnamic acids than the sun-dried and dipped samples, suggesting that the sulfur dioxide treatment ameliorated oxidation. The retention of flavonols among the three treatments varied, with golden raisins retaining higher levels of one quercetin glycoside but lower levels of rutin and two kaempferol glycosides than the sun-dried and dipped raisins. Ferreira et al. (2002) studied the effect of sun drying on the phenolic composition and content of a Portuguese pear (var. Bartolomeu). Compared to fresh fruit only 4%, 9%, and 32% of hydroxycinnamic acids, monomeric catechins, and procyanidins, respectively, were retained in the dried fruit. Arbutin was the only phenolic compound that was not degraded during sun drying presumably due to the low affinity of polyphenol oxidase (PPO) for the compound. Results from this study also indicated that the loss of large molecular weight procyanidins during sun drying was in part due to irreversible binding to cell wall polysaccharides, a phenomenon that may explain the sensorial loss of astringency in sun dried pears.

Shi et al. (1999) measured lycopene degradation and isomerization in tomatoes subjected to osmotic-vacuum drying, vacuum drying, and HAD. Osmotic-vacuum drying resulted in greater retention and less isomerization of lycopene than the vacuum and air drying methods. This was explained by the protective role of sugar present on the tomato surface in preventing oxygen


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from penetrating and oxidizing lycopene. HAD tomatoes retained the least amount of lycopene and contained the highest level of cis-isomers due to the adverse effects of heat and oxygen.

6.5 Effects of Microwave Heating on Carotenoids, Phenolic Acids and Flavonoids

Heating rate remains one of the major limitations for the optimization of

conventional thermal processes in which the heat is transferred through both conduction and convection, although advanced equipment such as rotary retorts and scraped-surface exchangers, etc., have been developed. Microwave heating is one of the volumetric heating methods that has the potential to lead to a quantum change in the ability of the food processor to achieve the heating rates necessary to deliver profiles that could improve current UHT process routes (Mullin, 1995). Microwaves used in the food industry for heating are of ISM (industrial, scientific and medical) frequencies (2450 or 900 MHz, corresponding to 12 or 34 cm in wavelength). In this frequency range the dielectric heating mechanism dominates up to moderated temperatures. Polar molecules, the dominant water try to align themselves with the rapidly changing direction of the electric field. The energy to achieve this alignment is taken from the electric field. When the field changes direction, the molecule “relaxes” and

the energy previously absorbed is dissipated into the surroundings, that is, directly inside the food. This means that the water content of the food is an important factor in the microwave heating performance of foods.

Microwave heating has been widely applied in industrial food applications such as defrosting or thawing of frozen foods, drying, blanching, and pasteurization. Sterilization using microwaves has been investigated for many years but the commercial introduction of this technique has only come about in the last few years in Europe and Japan. Microwave pasteurization and sterilization promise to give very quick heat processing that should lead to small quality changes due to the thermal treatment according to the HTST principle. However, it has turned out that very high requirements of heating uniformity must be met in order to fulfill these quality advantages (Ohlsson, 1991).

Microwave heating is reported to have varying effects on the retention of carotenoids and phenolics. De Ancos et al. (1999) studied the effects of microwave heating on carotenoid, chlorophyll, and anthocyanin contents of fruit purees. Generally, microwave heating produced minor modifications of the qualitative and quantitative composition of carotenoids in papaya and


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anthocyanins in strawberry purees, but resulted in extensive losses of chlorophyll a and b and xanthophylls in kiwi puree. In a study on apple mashes, juice from four heat treatments (40°C, 50°C, 60°C, and 70°C) in a 2450 MHz microwave oven at 1500 W of Fuji and McIntosh apple mashes were compared to juice from unheated mash. Microwave heat treatment of the mash increased extraction of phenolics and flavonoids from apple mash and resulted in juice with increased concentrations of total phenolics and flavonoids. Therefore, microwave heating of apple mash before juice extraction resulted in a high quality juice with increased phenolic and flavonoid content as well as increased juice yield (Gerard and Roberts 2004).

Chlorogenic acid concentration increased 2–3 times after the microwave heating of Idared apple puree, in comparison with control samples. Microwave energy has the advantage of heating solids rapidly and uniformly, thus inactivating the enzymes more quickly; it minimizes phenolic oxidation. The addition of ascorbic acid and microwave heating significantly increased not only chlorogenic acid concentrations, but also polymeric procyanidin concentration from 145 mg/kg to 404 mg/kg and 620 mg/kg, respectively. (Jan Oszmianski et al., 2008)

7. NON-THERMAL PROCESSING TECHNIQUES INFLUENCING PHYTOCHEMICAL CONTENT OF FRUIT Thermal processing is the most common method for extending the shelf

life of fruit products, by inactivating microorganisms and enzymes. However, thermal processing can diminish the sensory and nutritional qualities of juices (Pedro Elez-Martinez, 2007; Braddock, 1999).Consumer requirements for foods are constantly changing. Today consumers demand foods that are both fresh and natural. Therefore the steps used to process foods should be designed to preserve their natural quality. Hence non-thermal processing techniques such as high-pressure processing (HPP) and pulsed electric field (PEF) have been attracting more attention from food scientists and engineers in recent years not only because of their food preservation capabilities but also because of their potential to achieve some interesting functional effects.


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7.1 Effects of High-Pressure Processing on Carotenoids and Flavonoids

High-pressure processing (HPP) is an excellent alternative to thermal

processing of fruits as pressures commonly used affect primarily covalent bonds allowing for inactivation of microorganisms and enzymes, without adverse effects on flavor and nutritional quality. Homogeneous foods are most amenable to HPP, thus most of the research has focused on juices, purees, and soups. Most studies consistently show that HPP does not significantly alter levels of bioactive compounds or antioxidant activity of fruits. In a study of tomato puree HPP (400 MPa/25°C/15 min) treated purees retained much higher levels of individual and total carotenoids than purees subjected to low (70°C/30 sec) and high (90°C/1 min) pasteurization treatments (Sanchez-Moreno et al., 2006). In this as well as other studies of tomato (Sanchez-Moreno et al., 2004) and persimmon (De Ancos et al., 2000) purees HPP treatment increased the amount of extractable carotenoids compared with raw purees. The enhanced extraction of carotenoids by HPP may be due to several factors including membrane alteration, disruption of carotene–protein complexes, and alteration of macromolecular structures such as proteins and cell wall carbohydrates. Likewise, the carotenoid contents of a mixed juice (orange, lemon, and carrot) and carrot juice (Butz et al., 2003), tomato homogenate (Butz et al., 2002), and orange juice (Bull et al., 2004) were unaffected by ultra high pressure treatments ˃600 MPa. The effects of HPP

treatments on flavonoids have received little attention. Anthocyanins in strawberry jam are reported to be well retained after HPP processing compared with heat-processed jam, but anthocyanins in HPP-treated jam were more susceptible to degradation during storage (Gimenez et al., 2001). The greater instability of anthocyanins during storage of HPP-treated jams as opposed to heat-processed jams is most likely the result of residual enzymatic activities (peroxidase, polyphenol oxidase, and ß-glucosidase) that are readily destroyed by heat during traditional jam processing.

7.2 Effects Of Pulsed Electric Field Processing on Ascorbic Acid, Carotenoids and Flavonoids

The pulsed electric field (PEF) process is a new and innovative non-

thermal minimal processing technology that is used as an alternative preservation process for fruit juices. The aim of this technology is to inactivate


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microorganisms and to decrease the activity of enzymes in order to increase the shelf life of food products without undesirable heat and chemical effects. The theoretical basis of PEF technology is the use of an external electric field to destabilize cell membranes and form one or more pores in them. PEF technology applies high voltage pulses (generally 20– 80 kV/cm) for very short time (µs to ms), producing PEFs between two electrodes. This technique is very similar to electroporation, used in cell biology and genetic manipulation of cells. But, in the case of foods, the applied pulses are shorter and much more intense. The aim of the application of high voltage pulses to foods is not only to disrupt temporarily the cell membranes of microorganisms. However, in this process, the microorganisms are also killed or their numbers are drastically decreased by irreversible disruption of cell membranes. PEF has been mainly applied to preserve the quality of foods, such as to improve the shelf-life of bread, milk, orange juice, apple juice and liquid eggs (Hui et al., 2006).

A high retention of vitamin C content in orange juice was observed after PEF-processing with maximum values of 98.2% for PEF-treated orange juice. Retention of this vitamin after PEF treatment was always above 87.5% for orange juice, working with 35 kV/cm during 1000 µs at 200 Hz with monopolar pulses of 4 µs. Min, et al. (2003a) reported no differences between fresh orange juice and PEF-treated orange juice when they processed orange juice at 40 kV/cm for 97 µs at 2000 Hz with bipolar pulses of 2.6 µs and a maximum temperature of 45 °C. On the other hand, after processing orange juice by PEF with bipolar pulses of 4 µs at 800 Hz and 35 kV/cm during 750 µs (maximum temperature of 50 °C), a vitamin C retention of 93% was observed (Sanchez-Moreno et al., 2005). Evrendilek et al. (2000) reported that PEF-processing of apple juice did not alter the natural vitamin C of the juice (35 kV/cm, 94 µs, 952 Hz, monopolar pulses of 1.92 µs, maximum temperature 38 °C). Min et al. (2003b) did not observed differences in vitamin C retention between fresh and PEF-processed tomato juice at 40 kV/cm for 57 µs with bipolar pulses of 2 µs and a maximum temperature of 45 °C. When a thermal pasteurization (90 °C, 1 min) was applied to orange juice, the retention levels of vitamin C were 82.4%. Min, et al. (2003a, 2003b), and Sanchez-Moreno et al. (2005) also reported higher levels of vitamin C retention in orange and tomato juices treated by PEF compared with those processed by thermal treatment. High temperatures led to a loss of vitamin C because heat is known to speed the oxidation process of ascorbic acid (Gahler et al., 2003). Moreover, the depletion of vitamin C in fresh juices is also attributed to


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oxidative enzyme reactions promoted by ascorbate oxidase and peroxidase (Davey et al., 2000).

Gemma et al., (2009) who found that watermelon juices treated at 25 kV/cm for 50 µs at 50 Hz using mono- or bipolar 1-µs pulses exhibited the highest vitamin C retention (96.4–99.9%). On the other hand, vitamin C loss was higher than 50% when PEF treatment was set up at 35 kV/cm for 2050 µs at 250 Hz applying mono- or bipolar 7-µs pulses. Such severe conditions seem to greater affect vitamin C retention in watermelon juice than in other juices such as orange, orange–carrot or strawberry juices, which exhibited retention of vitamin C above 80% (Odriozola-Serrano et al., 2009). Applying the same PEF conditions, differences in vitamin C retention among PEF-treated juices could be due to their different pH, since more acidic conditions are known to stabilise vitamin C. Increased vitamin C retention of PEF-treated fruit juices in monopolar mode may be related to inactivation of enzymes that catalyse vitamin C oxidation. In PEF-treated orange juices, enzymes such as peroxidase were more inactivated with monopolar pulses than with bipolar pulses (Elez-Martinez et al., 2006). Loss of vitamin C in watermelon juice was accelerated when increasing severity of PEF treatments. In accordance, the lower the electric field strength, the treatment time, the pulse frequency or the pulse width, the higher the vitamin C retention in orange, tomato and strawberry juices. Lycopene retention in PEF-processed watermelon juice ranged from 87.6% to 121.2%. The content achieved with PEF treatments set up at 35 kV/cm with pulses of 250 Hz was slightly higher than that of untreated samples. The application of 35 kV/cm at low frequency led to a decrease in the lycopene content of treated watermelon juice of up to 10–12% compared to the fresh fruit juice. Maximal lycopene content of 114% in watermelon juice was achieved with 7-µs bipolar pulses for 1050 µs at 35 kV/cm and frequencies ranging from 200 to 250 Hz (Odriozola-Serrano et al., 2009). Cortés et al., (2006) observed that the carotenoid concentration in orange juice rose slightly after applying intense PEF treatments of 35 and 40 kV/ cm for 30–240 µs.

8. EFFECT OF FREEZING PROCESSING, FROZEN STORAGE AND THAWING ON BIOCHEMICAL CHANGES

Freezing is one of the best methods for long-term storage of fruits.

Freezing preserves the original color, flavor, and nutritive value of most fruits. Fresh fruits, when harvested, continue to undergo chemical, biochemical, and


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physical changes, which can cause deterioration reactions such as senescence, enzymatic decay, chemical decay, and microbial growth. The freezing process reduces the rate of these degradation reactions and inhibits the microbiological activity. However, it should be recognized that a number of physical, chemical, and biochemical reactions can still occur and many will be accentuated when recommended conditions of handling, production, and storage are not maintained. Although few microorganisms grow below −10°C, it should be recognized that freezing and frozen storage is not a reliable biocide. The production of safe frozen fruits requires the same maximum attention to good manufacturing practices (GMP) and hazard analysis critical control points (HACCP) principles as those used in fresh products. The quality of the frozen fruits is very dependent on other factors such as the type of fruit, varietal characteristics, stage of maturity, pretreatments, type of pack, and the rate of freezing. The freezing process reduces the fruit temperature to a storage level (−18°C) and maintaining this temperature allows the preservation of the frozen product for 1 year or more. Fruits are frozen in different shapes and styles: whole, halves, slices, cubes, in sugar syrup, with dry sugar, with no sugar added, or as juices, purees, or concentrates, depending on the industrial end-use (Hui et al., 2006).

8.1 Freezing Principles The freezing process reduces food temperature until its thermal center

(food location with the highest temperature at the end of freezing) reaches −18°C, with the consequent crystallization of water, the main component of plant tissues. Water in fruit and fruit products constitute 85–90% of their total composition. Crystallization of water during freezing reduces water activity (aw) in these tissues and consequently produces a decline in chemical and biochemical reactions and microbial growth. Freezing also involves the use of low temperatures and reactions take place at slower rates as temperature is reduced. The study of temperature changes during freezing is basic to an understanding of how products are processed. Figure 3 shows typical freezing curves at different freezing rates. When the product is cooling down to 0°C, ice begins to develop (see section A–S, Figure 3). The exact temperature for the formation of first ice crystal depends on the type of product and is a consequence of the constituents concentration independent of water content; for example, fruits with high water content (≈90%) have a freezing point

below −2°C or −3°C, while meat with less water content (≈70%) has a


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freezing point of −1°C; the main difference being the high sugar and organic acid concentration in fruits. Ice formation takes place after the product reaches a temperature below its freezing point (−5°C to −9°C) for only a few seconds. This process is known as super-cooling (position S in Fig. 3).

After that, due to heat release during the first ice formation, the temperature increases until the freezing point is reached (position B in Fig. 3). Section B–C in Fig. 3 corresponds to the freezing of most of the tissue water at a temperature that is practically constant, with a negative slope from a decline of the freezing point due to solute concentration. The increase of solute concentration as freezing progresses causes the unfrozen portion to undergo marked changes in such physical properties as ionic strength, pH, and viscosity. This increases the risk of enzymatic and chemical reactions, e.g., enzymatic browning or oxidation–reduction, with adverse effects on frozen fruit quality. A short B–C section increases the quality of frozen fruit. This means that a fast rate freezing produces a better quality frozen fruit (see curves b and c of Figure 3). Section C–D corresponds with the cooling of the product until the storage temperature, with an important increase of solute concentration in the unfrozen portion. Below −40°C, new ice formed is undetected. Up to 10% of the water can be unfrozen, mainly joined to protein or polysaccharide macromolecular structures that take part in the physical and biochemical reactions. In frozen foods the relationship between the frozen water and the residual solution is dependent on the temperature and the initial solute concentration. The presence of ice, and an increase in solute concentration, has a significant effect on the reactions and state of the fruit matrix. The concentration of the solute increases as freezing progresses; and thus, solute concentration of the unfrozen matrix can leach out of the cellular structures causing loss of turgor and internal damage. Solute-induced damage can occur whether freezing is fast or slow, and cryoprotectants, such as sugars, are usually added to aqueous solution to reduce the cell damage (Rahman, 1999).

8.2 Plant Cell Structure Understanding the effect of freezing on fruit requires a short review of

plant cell structure. Plant cells are surrounded by a membrane and interspersed with extensive membrane systems that structure the interior of the cell into numerous compartments. The plasmalemma or plasma membrane encloses the plasma of the cell and is the interface between the cell and the extracellular


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surroundings. Contrary to animal cells, plant cells are almost always surrounded by a cell wall and many of them contain a special group of organelles inside the plastids (chloroplasts, leucoplasts, amyloplasts, or chromoplasts). An important property of the plant cell is its extensive vacuole. It is located in the center of the cell and makes up the largest part of the cells volume and is responsible for the turgor. It helps to maintain the high osmotic pressure of the cell and the content of different compounds in the cell, among which are inorganic ions, organic acids, sugars, amino acids, lipids, oligosaccharides, tannins, anthocyanins, flavonoids, and more. Vacuoles are surrounded by a special type of membrane, the tonoplast. The cell wall of plants consists of several stacked cellulose microfibrils embedded in a polysaccharide matrix able to store water thereby increasing the cell volume (hydration and absorption). According to their capacity to bind or store water, the polysaccharides involved in the matrix can be classified as follows: pectin>hemicellulose>cellulose>lignin. Pectins are mainly polygalacturonic acids with differing degrees of G-galactosyl, L-arabinosyl or L-rhanmosyl residue and are predominant in the middle lamella, the layer between cells. The deesterification process of pectin is related to the softness of fruit tissues during ripening and processing (Hui et al., 2006).


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Figure 3. Typical freezing curves of foods at different rates: (a) very slow; (b) fast; and (c) very fast (Fennema, 1976).

8.3 Color Changes Color is the most important quality characteristic of fruits because it is the

first attribute perceived by the consumers and is the basis for judging the product acceptability. The most important color changes in fruits are related to chemical, biochemical, and physicochemical mechanisms: (a) breakdown of cellular chloroplasts and chromoplasts, (b) changes in natural pigments (chlorophylls, carotenoids, and anthocyanins), and (c) development of enzymatic browning. Mechanical damage (ice crystals and volume expansion) caused by the freezing process can disintegrate the fragile membrane of chloroplasts and chromoplasts, releasing chlorophylls and carotenoids, and facilitating their oxidative or enzymatic degradation. Also, volume expansion increases the loss of anthocyanins by lixiviation due to disruption of cell vacuoles.

(I) Chlorophylls

Chlorophylls are the green pigment of vegetables and fruits, and their structures are composed of tetrapyrroles with a magnesium ion at their center. Freezing and frozen storage of fruits cause a green color loss due to degradation of chlorophylls (a and b) and transformation in pheophytins, which transfers a brownish color to the plant product (Cano, 1996). One example is kiwi-fruit slices that show a decrease in chlorophyll concentration between 40% and 60%, depending on cultivar, after freezing and frozen storage at −20°C for 300 days (Cano et al., 1993).


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Figure 4. Pathways of chlorophyll degradation. (adapted from Heaton et al., 1996)

Different mechanisms can cause chlorophyll degradation; loss of Mg due to heat and/or acid, which transforms chlorophylls into pheophytins; or loss of the phytol group through the action of the enzyme chlorophyllase, which transforms chlorophyll into pheophorbide. Loss of the carbomethoxy group may also occur and pyropheophytin and pyropheophorbide can be formed (Figure 4) (Heaton et al., 1996). Acids, temperature, light, oxygen, and enzymes easily destroy the chlorophylls. Thus, blanching (temperature/time), storage (temperature/time), and acidity are the important factors to be controlled during processing in order to preserve chlorophylls. Other chlorophyll degradation mechanism can cause degradation by the action of peroxides, formed in the fruit tissue due to the oxidation reaction of polyunsaturated fatty acids catalyzed by the enzyme LOX. An important quality parameter employed to determine the shelf life of frozen green fruits is the formation of pheophytins from chlorophylls. As different types of enzymes can be involved in chlorophyll degradation (LOX, POD, and chlorophyllase), blanching and addition of inorganic salts such as sodium or potassium chloride and sodium or potassium sulphate are efficient treatments to preserve green color (Cano et al., 1993).

(II) Carotenoids

Carotenoids are among the most abundant pigment in plant products and are responsible for the yellow, orange, and red color of most of the fruits. All of them are tetraterpenes and contain 40 carbon atoms in eight isoprenes


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residues. ß-carotene and lutein are the carotenoids present in most of the fruits. Important sources of these pigments are as follows:

ß-cryptoxanthin: oranges lycopene: tomatoes, watermelon, papaya and persimmon α-carotene: banana and avocado zeaxanthin: orange and peach

Carotenoids are affected by pH, enzymatic activity, light, and oxidation

associated with the conjugated double bond system. The chemical changes occurring in carotenoids during processing have been reviewed by several authors (Rodriguez-Amaya, 1997). The main degradation reaction that damages carotenoid compounds is isomerization. Most plants appear to produce mainly trans forms of carotenoids but with increased temperature, the presence of light, and catalysts such as acids, isomerization to the cis forms increases, and the biological activity is dramatically reduced. However, heat treatments of products rich in carotenoids reduce the degradation of carotenoids because of the inactivation of enzymes LOX and POD. Blanching fruits before freezing could be efficient in the preservation of carotenoids due to enzyme inactivation. Although most carotenoids are heat resistant, some carotenoids, such as epoxycarotenoids, could be affected. Carotenoids are fat-soluble pigments and breakdown of chromoplasts, by heat treatment or mechanical damage, improves their extraction with organic solvents and bioavailability but not their loss by lixiviation (Hof et al., 2000). Freezing without protector pretreatment slightly decreases total carotenoid concentration (20%) of some fruits rich in carotenoids, such as mango and papaya. But after 12 months of frozen storage at −18°C, an important decrease of total carotenoid concentration (between 40% and 65%) occurred, although the carotenoid profilewas unchanged (Cano et al., 1996). Similar results have been found with frozen tomato cubes. A pronounced stability of total carotenoids, ß-carotene, and lycopene was recorded up to the 3rd month of storage. But after 12 months of storage at −20°C, the losses of carotenoids reached 36%, of ß-carotene 51%, and of lycopene 48% (Lisiewska and Kmiecik, 2000). Freezing and frozen storage could affect the carotenoid structure and concentration depending on the type of fruit and cultivar (pH, fats, antioxidants, etc.) and the processing conditions (temperature, time, light, oxygen, etc.) (Rodriguez-Amaya, 1997).


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(III) Anthocyanins

Anthocyanins are one class of flavonoid compounds, which are widely distributed plant polyphenols, and are responsible for the pink, red, purple, or blue hue of a great number of fruits (grape, plum, strawberry, raspberry, blackberry, cherry, and other types of berries). They are water-soluble flavonoid derivatives, which can be glycosylated and acylated. The effect of freezing, frozen storage, and thawing in different fruits rich in anthocyanins pigments have been reviewed by Skrede (1996). Anthocyanins in cherryfruit underwent pronounced degradation during storage at −23°C (87% after 6 months), but they are relatively stable at −70°C storage (Chaovanalikt and Wrolstad, 2004a). But in raspberry fruit, the stability of anthocyanins to freezing and frozen storage depends on the seasonal period of harvest. Spring cultivars were practically unaffected by freezing and frozen storage for 1 year at −20°C, but autumn cultivars showed a decreasing trend in total anthocyanin content (4–17%)(De Ancos et al., 2000b). In general, the freezing process does not affect the level of anthocyanins in raspberry fruit (Mullen et al., 2002). Authors explain degradation of anthocyanins during frozen storage by different chemical or biochemical mechanisms. Anthocyanins are water-soluble pigments located in the vacuoles of cell and are easily lost by lixiviation when the cell membranes break down. Also oxidation can play an important role in anthocyanin degradation catalyzed by light. PPO and POD enzymatic activities have been related to anthocyanin degradation. Thus, frozen–thawed cherry discoloration disappeared when the fruits were blanched before freezing. In slightly acidic aqueous solution at ambient temperatures, anthocyanins exist as essentially four species in equilibrium. These are the blue quinoidal base (A), the red flavylium cation (AH+), the colourless hemiacetal base (B), and the colourless chalcone form (C) (Figure 5). The changes in pH during processing can affect anthocyanin stability. Maintenance of red fruit requires an acid medium (pH < 3.5). The flavylium cation structure of anthocyanins transfers a red color to the fruit. But an increase in pH value produces a change from red to blue until the product is colorless, a consequence of transforming flavylium cation into a neutral structure (Figure 5). The loss of characteristic red color can also be produced by formation of the anthocyanin complex with different products present in the fruit matrix: ascorbic acid, acetaldehyde, proteins, leucoanthocyanins, phenols, quinones, metals (Fe3

+ and Al3+), hydrogen peroxide, etc. (Escribano-Bailon et al.,

1996).


27

Figure 5. Structural transformations of anthocyanins (anthocyanidin-3-glucosides). R3 and R5 are normally H, OH or OCH3 moieties; GL represents a glycosyl moiety (adapted from Dangles and Brouillard, 1992)

(IV) Enzymatic Browning

Browning usually occurs in certain fruits during handling, processing, and storage. Browning in fruit is caused by enzymatic oxidation of phenolic compounds by PPO (Martinez and Whitaker, 1995). PPO catalyzes either one or two reactions involving molecular oxygen. The first type of reaction is hydroxylation of monophenols, leading to formation of o-hydroxy compounds. The second type of reaction is oxidation of o-hydroxy compounds to quinones that are transformed into polymeric brown pigment. Freezing, frozen storage, and thawing of fruits, like mangoes, peaches, bananas, apples, apricots, etc., quickly develop color changes that result in nonreversible browning or darkening of the tissues. Freezing does not inactivate enzymes; however, some enzyme activity is slowed during frozen storage. Browning by PPO can be prevented by the addition of sulfites, ascorbic acid, citric acid, cysteine, and others. Selection of varieties with low PPO activity could help to control browning in frozen–thawed fruits (Cano et al., 1998).

8.4 Effect of Freezing Processing on Vitamin C, Carotenoids and Phenolic Compounds


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(I) Vitamin C

Freezing processes have only a slight effect on the initial vitamin C content of fruit (Cano and Marin, 1992). The destruction of vitamin C (ascorbic acid) occurs during freezing and frozen storage, and this parameter has been employed to limit the frozen storage period of frozen fruit. The main cause of loss of vitamin C is the action of the enzyme ascorbate oxidase. If pretreatments or freezing processes do not destroy this enzyme, it is continuously active during the frozen storage. Vitamin C degradation depends on different factors, such as time–temperature conditions, type of fruit, variety, pretreatments, type of package, freezing process, etc. (Skrede, 1996). Thus as the frozen storage temperature decreases, higher vitamin C retention is achieved for different fruits like berries, citrus, tomato, etc. (Skrede, 1996; Lisiewska and Kmiecik, 2000). Also, significantly different vitamin C retention values have been achieved between varieties of fruits such as raspberry (De Ancos et al., 2000c), mango, and kiwi (Cano and Marin, 1992), which were frozen and stored under the same conditions. Vitamin C stability in freezing and frozen storage of strawberries seems to be more dependent on storage temperature than on the type of freezing process. Nonstatistical differences were observed between strawberries processed by fast rate freezing (at −20°C) and quick rate freezing (at −50°C to –100°C), but great loss was shown between strawberries stored at −18°C and −24°C (Sahari et al., 2004).

(II) Provitamin A and Antioxidant Carotenoids

Some carotenoids, like ß-carotene, α-carotene, and ß-cryptoxanthin, are recognized as precursors of vitamin A. This provitamin A carotenoids, in addition to lycopene and lutein, constitute the group of antioxidant carotenoids. The prevailing opinion is that freezing and frozen storage do not prevent degradation of carotenoids. The content of ß-carotene, and consequently the provitamin A value, was decreased during frozen storage of mango, kiwi (Cano and Marin, 1992), papaya (Cano, 1996), and tomato (Lisiewska and Kmiecik, 2000). The losses were mainly due to the activity of enzymes (POD, LOX, and CAT), particularly during frozen storage in an oxygen environment. Lycopene, a characteristic carotenoid in tomato fruit, has been recognized as a powerful antioxidant (Lavelli et al., 2000). After 3 months of frozen storage (−20°C and−30°C), great stability of lycopene was recorded. After this period, slow losses occurred, the rate being faster at the higher storage temperature. After 12 months at −20°C and −30°C, the lycopene content was 48% and 26%, respectively, lower than that in the raw material (Lisiewska and Kmiecik, 2000). Other authors have reported an


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increase in the extraction of lycopene after 1 month of frozen storage, although after 3 and 6 months the loss of lycopene concentration was significantly higher than 40% (Urbanyi and Horti, 1989). Papaya fruit could be an important source of lycopene, but freezing and frozen storage at −20°C during 12 months produced a significant loss of lycopene concentration (34%) in frozen papaya slices (Cano, 1996).

(III) Phenolic Compounds

The freezing process does not modify either total phenolic content or ellagic acid concentration in raspberry fruit. There is an increasing interest in ellagic acid, a dimeric derivative of gallic acid, due to its anticarcinogenic and antioxidant effects. Although frozen storage produces a slight decrease in ellagic acid content because of PPO enzyme activity, frozen storage is a good methodology to preserve phenolic compounds during long term periods (De Ancos et al., 2000c). Asami et al. (2003b) evaluated the effects of storage at refrigeration and frozen temperatures on the concentration of total phenolics in clingstone peaches. Maturity stage III peaches of the Ross variety were peeled, pitted, sliced, and frozen at –12°C for a period of 3 months. There appeared to be a statistically significant increase (P < 0.05) in total phenolic content following freezing, and this higher content was retained after 2 and 3 months of frozen storage. It was postulated that the freezing process may have resulted in cellular disruption and more facilitated extraction of phenolics.

The effect of freezing and frozen storage on raspberry phytochemicals and volatiles was the subject of two manuscripts by De Ancos and colleagues (De Ancos et al., 2000a, 2000b). These authors compared two early-season and two late-season raspberry cultivars and found differential effects of freezing. In the early-season cultivars, freezing resulted in increased anthocyanin content, while in the late-season cultivars, which initially had higher concentrations of anthocyanins, freezing caused an overall reduction. The authors suggested that the preservation of anthocyanins during freezing depends on the pH of the fruit, organic acid content, sugar concentration, initial anthocyanin concentration, and initial cyaniding-3-glucoside content. They did not find a relationship between polyphenol oxidase activity and anthocyanin content. De Ancos et al., (2000b) also found that freezing had a slight effect on ellagic acid, vitamin C, and total phenolics, depending on the raspberry cultivar. Free radical scavenging capacity was decreased as a result of the freezing process, anywhere from 4 to 26%, again related to cultivar. Frozen storage of raspberries at –20°C for a 1-year period did not appear to affect total phenolics or free radical scavenging capacity, but did cause a


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decline in ellagic acid vitamin C. In another study of the effects of freezing on raspberry phenolics, ellagitannins, flavonoids, and antioxidant capacity (Mullen et al., 2002), these authors found that the antioxidant capacity of the fruit and vitamin C levels were not affected by freezing. The raspberry cultivar used in this study differed from those evaluated by de Ancos, however, and this may have affected the results. Freezing preservation of fruit is less destructive toward some antioxidant compounds, in particular total phenolics and ascorbic acid, than other means of preservation. One illustration of this is a recent publication (Asami et al., 2003a) in which Marionberries, strawberries, and corn were preserved using freezing, freeze-drying, and air-drying methods. The highest levels of both total phenolics and ascorbic acid (reduced form) were consistently found in the extractions of frozen samples, followed by those of freeze-dried and then air-dried samples. Freezing may cause some damage to cell structure, and application of a drying procedure following freezing, even though this is under vacuum at reduced temperatures, may result in even greater losses of beneficial nutrients. Air-drying at temperatures above 60°C may result in oxidative condensation or decomposition of thermolabile compounds, such as (+)-catechin and ascorbic acid. Therefore, the presence of total phenolics and ascorbic acid in the air-dried products was lower than that in either frozen or freeze-dried products.

8.5 Effect of Thawing on Phytochemical Content The quality of the original fruit, preserved by freezing, is retained by

quick thawing at low temperature in controlled conditions. During incorrect thawing, chemical and physical damage and microorganism contamination can also occur. Fruit products exhibit large losses of ascorbic acid (up to 40%) and color changes when thawed for an unusually long period, e.g., 24 h at room temperature. Good results in terms of vitamin C and anthocyanins retention (90%) were achieved by thawing small frozen fruits such as bilberry, raspberry, black currant, red currant, and strawberry at room temperature (18–

20°C /6–7 h), in a refrigerator (2–4°C /18 h), or in a microwave oven. Color and ascorbic acid retention of fruit was equally affected by thawing temperature and time. Thorough thawing must be determined by taking into account the size of the fruit and/or the type of packaging (Kmiecik et al., 1995).

In relation to the control samples (frozen and thawed by traditional methods) freezing in liquid nitrogen and thawing in microwave oven allowed


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an increase of the retention of polyphenols in all strawberry varieties (1.7–

25.7%). The difference between effects of the frozen strawberry preparation and thawing methods explained 68.9% of the total variation in principal component. The samples which were thawed in microwave oven had a much higher phenolic content (anthocyanins, proanthocyanins, (+)-catechin and ellagic acid) than samples which was thawed during 20 h at 20 °C. Probably enzymatic reaction could take place in destroyed strawberry tissues during long thawing process. To achieve a higher content of phenolic compounds in strawberries, they should be thawed by faster method in microwave oven (Jan Oszmianski et al., 2009).

Yurena et al.,( 2006) who found that, the relative to the content before freezing, differed depending on the thawing method (84 ± 7%, 91 ± 9% and 97 ± 6% when thawing at room temperature, in the refrigerator and in the microwave oven, respectively). There were no significant differences in the initial content and the values measured after microwave thawing. Thawing at room temperature resulted (for both standard solutions and extract) in lower AA content than thawing in the microwave oven. Microwave thawing was chosen on the basis of these results and because it was the most practical method for routine analysis.

9. PACKAGING, STORAGE AND HANDLING PROCEDURES INFLUENCING PHYTOCHEMICAL CONTENT OF FRUIT

9.1 Packaging Materials Used for Processed Fruits Thermally processed fruits have traditionally been packed in metal

containers and glass jars. Products stored in glass and metal containers generally maintain their nutritional and organoleptic quality for extended periods of time due to the durability and excellent oxygen and moisture barrier properties of the containers. Plastic packaging materials have recently become more popular due to their light weight, unbreakable nature, and convenience features. Driven by advances in aseptic processing, fruit purees (baby foods) are now available in reclosable, multilayer, barrier-plastic cups that are microwaveable. Plastic polyethylene terephthalate (PET) containers are also becoming more popular for high acid fruit products and many of the containers provide convenience for the consumer through easy dispensing designs. Although the convenience afforded by these products is unquestioned,


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information is lacking on how processing and storage of fruits in plastic containers affect the retention of phytochemicals. Storage stability of oxygen-sensitive phytochemicals may be an important issue since the plastic packaging materials are not completely oxygen impermeable.

9.2 Storage Factors Influencing Retention of Phytochemicals The stability of carotenoids and phenolics in processed fruits during

storage is influenced by temperature, light, oxygen, and chemical interactions, which may or may not be oxidative in nature. The package plays an integral role in protecting food constituents from the adverse effects of oxygen and light, which is especially important for dehydrated powders that are highly prone to oxidation. Although thermally processed fruits are typically stored at ambient temperature, storage at refrigerated temperatures can result in greater retention of color and phytochemicals.

(I) Cold Storage

At optimum cold storage conditions vitamin C content is decreasing; whereas most phenolics, carotenoids, glucosinolates and dietary fibers are relatively stable (Table 3). Deviations from optimum conditions may indeed affect the contents of health-related constituents. Suboptimum temperature and humidity usually give rise to enhanced rates of breakdown due to increased metabolism leading to faster maturation and senescence. Some constituents, such as phenolics, can increase their content under dehydration (without change in total amount) or when exposed to visible or UV radiation (with increased total amount). Except for vitamin C and phenolics, storage effects on nutrients and health-promoting phytochemicals have not been investigated to any great extent (Bengtsson, 2008). The biosynthesis of anthocyanins in red small fruits and berries tends to continue after harvest and during storage. Increased content of anthocyanins during cold storage has been recorded in strawberries, blueberries, grapes and pomegranates (Tomás-Barberán and Espín, 2001).

(II) Controlled Atmosphere (Ca) Storage

CA is a technologically advanced storage method for which temperature, humidity and the levels of oxygen and carbon dioxide are precisely controlled. Low oxygen concentration in the atmosphere during storage leads to reduced


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rate of respiration with a delayed onset of senescence. This has been exploited commercially for suitable species of fruit using special storage facilities.

In addition to typically 1–2 kPa concentration of O2, the CA contains CO2 at an elevated level, for instance 2–6 kPa. The exact concentrations used depend upon the product. CA storage is an extension of cold storage; thus an optimum temperature is also a prerequisite for optimum product quality. In general, CA storage retards the loss of health-related constituents compared to cold storage in air (Table 3). Some constituents can also increase their content during CA storage, for example some glucosinolates and phenolics. Controlled atmosphere (CA) storage of strawberry fruit did not affect anthocyanin content in external tissues but decreased anthocyanin content in internal tissues (Holcroft and Kader, 1999).

Table 3. General Effects of Storage on Contents of Health-Related Constituents of Fruits and Vegetables

Optimal temp.

Suboptimal temp.

Incident light

Reduced O2

Elevated CO2

Elevated O2

Dehydration

Vitamin C Decrease Decrease Decrease Slower decrease Decrease

Phenolics, fruits Stable Decrease Increase Stable or

increase Stable or increase Variable

Phenolics, berries Increase Increase Variable Stable or

decrease Stable or decrease Increase Increase

Carotenoids, fruits Variable Variable

Glucosinolates Stable or Decrease Decrease Variable Increase Decrease

Dietary fibre Stable Variable (adapted from Bengtsson et al., 2008)

Processing Techniques and Their Effect on Fruit Phytochemicals

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(III) Modified Atmosphere Packaging (Map)

Modified atmosphere packaging is an extension of CA to small packages for retail, the main difference being that the gas composition is not controlled by external systems. The atmosphere composition inside the closed package changes with time and is dependent on uptake and release of gases by the food, as well as on transmission of gases through the package. The effects of MAP on health-related constituents are very similar to those of CA storage, when similar atmosphere compositions are compared (Table 3).

In a study on fresh-cut jackfruit bulbs, it was observed that the total phenolic (TP) content decreased during storage of fresh-cut jackfruit bulbs in dip-pretreated as well as in untreated bulbs. Dip pretreatment coupled with MAP showed a significantly (p < 0.05) lower TP loss as compared to untreated samples which recorded a higher degree of degradation after just 7 days. The percentage phenolics loss in the pretreated samples was found to be 7–15%, as compared to 15–26% in the case of untreated samples kept under MA conditions (Alok Saxena, 2009). Alasalvar et al. (2005) reported that storage under low O2 atmosphere could reduce the accumulation of TP in shredded orange compared to those stored under air and high O2 conditions. Cocci et al., 2006), who reported a restricted degradation in TP, due to the reducing action of AA added in the dip pretreatment given to fresh-cut apple stored under MA conditions. The decrease in TP during storage of fresh-cut commodities could be attributed to enzymatic degradation by peroxidase (POD) and PPO activities. The loss in total flavonoids content was found to be 8–20% in the case of pretreated MA packaged samples as compared to a significantly higher loss of 20– 33% in the control samples under different MA conditions. Total carotenoids content was observed to decrease from the 7th day onwards in the control samples. The overall retention of total carotenoids was found to be in the range of 40–57%, in the case of pretreated samples, whilst the control ones showed a significantly lower retention (5–39%) under the various MA conditions during storage for 35 days (Alok Saxena, 2009). The pretreated samples kept under MA conditions showed a significantly (p < 0.05) higher retention of AA (56–69%), as against 10–49% in the case of untreated samples. Reports exist about higher retention of AA in fresh-cut commodities subjected to MAP (Odriozola-Serrano et al., 2008). Lower respiratory activity could be attributed to higher retention of AA content, due to restriction in enzymatic oxidation of AA into dehydroascorbic acid, through headspace oxygen in the MA-packaged samples during storage.


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9.3 Influnence of Storage and Handling on Health- Related Properties of Fruit

(I) Vitamin C

Vitamin C as L-ascorbic acid (AA) and its oxidation product L-dehydroascorbic acid (DHA) is present in all plants. The variation in content between various plant foods and within species is very large (Davey et al., 2000). L-dehydroascorbic acid is present in most fruits in small amounts, usually about 10% of the AA level, but it can increase during storage due to oxidation of AA. The storage stability of AA seems to be related to the initial level (Lee and Kader, 2000). The percentage losses are larger with a lower starting value. After cold storage for several months the AA level of apples is halved or more. Davey and Keulemans (2004) found among 31 Belgian apple cultivars that cultivars low in vitamin C had the largest losses during storage (both for three months at 1°C and for 10 days at room temperature) and these cultivars were also the ones with the worst storage outcome.

The effect of storage temperature (4°C) on the AA stability was studied by Silvia Tavarini et al., (2008) in two standard solutions (50 mg/l AA in water and 3% MPA–8% acetic acid, n = 3) and a ripe banana extract spiked with 50 mg/l AA (n = 3), which was selected as the extract model because it had the highest complexity among the extracts. After 24 h, the stability of AA kept at 4°C was 95 ± 5% from the initial AA content; after 4 days it was 75 ± 4% and after 8 days 51 ± 3% (Yurena et al., 2006). In kiwi fruit, the AA significantly decreased after 6 months of cool storage and slightly increased again after a week to ambient temperature.

(II) Phenolics

Apple is one of the most studied fruits with regard to phenolic content. The phenolic content in apple has been repeatedly reported to be relatively stable during long-term storage at low temperature (<4°C) in both normal air and CA with reduced O2 and increased CO2 levels (Awad and De Jager, 2003) and no changes in the phenolic content of „Aroma‟ apples were detected after

storage for 10 days in normal air at 10°C (Hagen et al., 2007). MacLean et al. (2006) observed a small decrease in anthocyanins and increase of chlorogenic acid, but no change in the overall content of phenolic compounds in „Red

Delicious‟ apples during 120 days of cold storage at 0–1°C and eight days of shelf-life at room temperature. In contrast, Leja et al. (2003) found a considerable increase in the total phenolic content of „Jonagold‟ and

„Sampion‟ apples stored in normal air or CA (2% O2 + 2% CO2) for 120 days


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at 1°C followed by an additional seven days at 16°C. A slight decrease in anthocyanin content was observed in the apples stored in normal air, but not in CA. In „Rocha‟ pears stored in air or CA (2 kPa O2 + 0–5 kPa CO2) at 2°C for four months, the content of hydroxycinnamic acid derivatives and flavonols were stable, the level of flavan-3-ols decreased and the concentration of arbutin increased as time elapsed. No differences in the effects of storage atmospheres were observed (Galvis-Sánchez et al., 2006). In a study about fresh-cut fruits versus whole fruits during storage, Gil et al., (2006) found that during 9 days of storage no significant changes occurred in phenol content in kiwifruits and no difference was determined between slices and whole fruits. Generally, phenol content may either increase or decrease in fruits and vegetables depend on the storage conditions (Kalt, 2005).

No changes in the levels of anthocyanins, flavonols or hydroxycinnamates, but a slight increase of total phenolics, were observed in red raspberries stored at 4°C for three days and then at 18°C for 24 hours, mimicking the route of fresh fruit after harvest to the supermarket and onto the consumer‟s table (Mullen et al., 2002). The effect of temperature on the rate of

anthocyanin formation during storage may depend on the maturity of the fruits at harvest (Kalt et al., 2003). CA storage, typically used with low O2 and high CO2 concentrations, may reduce the anthocyanin pigmentation in red fruits (Tomás-Barberán and Espín, 2001), but was found to have little or no effect on the phenolic content in blueberries (Schotsmans et al., 2007) and cranberries (Gunes et al., 2002). No changes in the content of phenolic compounds in „Superior seedless‟ table grapes were detected after storage under passive

MAP for seven days at 0°C followed by four days at 8°C, but slight decreases were registered after two days of simulated shelf-life at 20°C (Artés-Hernández et al., 2006). Elevated levels of O2 have been found to induce accumulation of phenolic compounds during the initial stage of storage of strawberries, but it may also promote the oxidation of phenolic compounds upon prolonged storage treatments (Zheng et al., 2007). In highbush blueberry, total phenolics and total anthocyanins as well as individual phenolic compounds were markedly increased by 60–100 kPa O2 treatments compared to 40 kPa O2 or normal air during 35 days of storage at 5°C (Zheng et al., 2003). During the 6 months storage the content of polyphenols in frozen control strawberries underwent decreases, which varied between 17.8% for cv. Senga Sengana and 36.6% for cv. Kent (Jan Oszmianski et al., 2009). Total phenolic compounds increased continuously in berries stored at 10°C and 5°C. However, strawberry fruit stored at 0°C maintained a constant value of total phenolic compounds during the storage period. Both temperature and storage


38

time had a significant effect (p 0˃.05) on total phenolic compounds of strawberry fruits (Fernando et al., 2004). A study involving the effects of blanching and processing on antioxidant capacity and phenolic content of peach puree reported that a major loss of antioxidant capacity occurred in pasteurized peach puree stored at 40°C for four weeks, which reflected major losses of chlorogenic acid (Talcott et al., 2000). Hydroxycinnamates, epicatechin, and flavonol glycosides are also subject to losses during storage. Bing cherries stored at 2°C for five months lost 8%, 20%, and 16% of hydroxycinnamates, epicatechin, and flavonol glycosides, respectively, while fruit stored at 22°C lost 30% and 16% of hydroxycinnamates and epicatechin, but an apparent 9% increase in flavonol glycosides was observed (Chaovanalikit and Wrolstad, 2004b).

(III) Carotenoids And Anthocyanins

Mature green mangoes ripened at 20°C will accumulate carotenoids after about one week and this is irrespective of prior hot water treatment (Talcott et al., 2005). When the mangoes were initially held at 5°C and then at 20°C, the carotene increase and the ripening were delayed. Wright and Kader (1997) studied changes in quality, retinol equivalents, and individual provitamin A carotenoids in fresh-cut Fay Elberta peaches held for 7 days at 5°C in air or CA. They concluded that the limit of shelf life was reached before major losses of carotenoids were observed. In a study about carotenoids changes after storage of Hayward kiwifruit, Silvia Tavarini et al., (2008) found that the carotenoid content in kiwifruit increased after 2 months of cool storage and after a week to ambient temperature, whereas after 6 months of cool storage, fruits harvested at showed a significant decrease in carotenoid content. Although carotenoid levels can be compromised during thermal processing due to oxidation and isomerization, they are generally well preserved during storage of canned products, primarily due to their non-polar nature and resistance to leaching into the canning medium. Provitamin A activity was also well retained in clingstone peaches stored for 18 months at ambient temperature. Storage temperature can significantly affect carotene retention in canned fruits. Canned peaches, peas, plums, spinach, and tomatoes stored at 10 and 18.3°C retained higher levels of carotenes over 24 months storage than products stored at 26.7°C. The type of packaging container is reported to influence retention of carotenoids during long-term storage of mango slices and puree (Godoy and Rodriguez-Amaya, 1987). No appreciable loss of β-carotene occurred in mango slices packed in lacquered (epoxy) or plain tin-plate cans during 10 months storage at ambient conditions, but significant


39

losses were observed after 14 months (50%) and 24 months (84%) of storage. Similar losses of β-carotene were observed in mango puree stored long-term in lacquered epoxy cans and bottles, but β- carotene showed a greater propensity to degrade when stored in bottles. Carotenoids in dehydrated fruit powders are prone to oxidation during long-term storage and require special packaging materials to exclude light and oxygen or gas flushing treatments completely to exclude oxygen. Low temperature during storage is, however, known to reduce the rate of anthocyanin formation. Increased levels of anthocyanins under storage at higher temperatures (15–25°C) have been reported for strawberries (Cordenunsi et al., 2005), blueberries (Kalt et al., 2003; Schotsmans et al., 2007), cherries (Gonçalves et al., 2004) and cranberries (Wang and Stretch, 2001).

During storage at 30 °C, significant changes were observed in the concentrations of procyanidins and cyanidin- 3-galactoside of all the apple purees. After 6 months of storage, cyanidin-3-galactoside was not detected in any samples. Anthocyanins are very sensitive to heat degradation (Garcia-Viguera et al., 1999). The procyanidins were more stable than were anthocyanins during puree storage. An increasing degree of procyanidin polymerization (DP) was observed during puree storage. Procyanidins in apple purees are better protected than are those in apple juice concentrate. Bengoechea et al. (1997) detected procyanidins in purees, but not in concentrates. During manufacturing of apple juice concentrate, they could polymerize to form insoluble forms and precipitate out, yielding lees or sediment. The procyanidins in purees are protected because of their ability to bind with cell-wall polysaccharides. (Guyot et al., 1998). Spanos et al. (1990) reported that apple juice stored for 9 months at 25 °C, of concentrates, showed 36% degradation of hydroxycinnamic acids, 60% degradation of quercetin and phloretin glycosides, and total loss of procyanidins. After 6 months of storage at 30°C, the apple purees had a maximal 26% degradation of chlorogenic acid and 18% degradation of phloretin-2 -glucoside. Anthocyanin content decreased in strawberry fruit stored at 0°C and 5°C during the first 5 days. Meanwhile, anthocyanin content in fruit stored at 10°C increased gradually during the storage period and reached its highest values near the end of the storage period (Fernando et al., 2004). Ochoa et al. (1999) reported that storage temperature markedly affected the color and anthocyanin content of raspberry pulp, with product stored at 4°C for 90 days retaining much higher levels of total anthocyanins than product stored at 20°C or 37°C. Fruits packed in liquid canning medium experience leaching of water-soluble phytochemicals during storage. In canned peaches stored for three months


40

procyanidin monomers, dimers, trimers, and tetramers decreased by 10, 16, 45, and 80%, respectively, with complete loss of pentamers through to octamers (Hong et al., 2004). Analysis of the canning syrup revealed that losses of procyanidins from the fruit during storage were mostly accounted for by migration into the syrup. Substantial losses of anthocyanins can also occur during storage of canned fruit, with losses being temperature dependent. Anthocyanin losses in canned Bing cherries stored for five months in syrup at 2°C and 22°C were 12% and 42%, respectively (Chaovanalikit and Wrolstad, 2004b), while anthocyanin losses in canned plums stored for 42 days at 4°C and 30°C were 13.5% and 46%, respectively (Weinert et al., 1990a).

10. FUTURE TRENDS

In a hungry and increasingly competitive world, reducing phytochemical food losses is a worthy agricultural goal. Most of the studies involving the effects of processing techniques on fruit phyochemicals have been conducted in pilot plants, which most likely do not accurately reflect actual losses that occur during commercial manufacturing. Due to the continuous nature of unit operations used in commercial production, the losses of phytochemicals may be ameliorated since the products are processed rapidly and steps are taken to minimize oxygen in the product prior to thermal treatments. Studies conducted in commercial settings are needed in order to assess the true retention of phytochemicals. Although kinetic data for time–temperature effects on carotenoid and anthocyanin degradation are available, this important information is lacking for other important phenolic compounds commonly found in fruit such as flavan-3-ols, procyanidins, flavonols, phenolic acids, and hydroxycinnamates. Generally, retention of maximum levels of antioxidants through application of appropriate food technology processes is an appropriate goal. However, few data exist on the nutritional value of specific non-nutrient antioxidants include flavonoids, polyphenols, and terpenes. Assuming such data are generated, there will be a need to adapt processes to maximise their retention. Innovative processing methods that minimize the amount of canning media or prevent the migration of phenolics into the canning media are needed. Information is also lacking about how new plastic packing materials (multilayer barrier-plastic cups and PET) impact the processing and storage stability of phytochemicals. Studies are needed to determine if greater losses of phytochemicals occur in these materials compared to products processed and stored in traditional glass and metal containers. Additional studies


41

involving optimization of non-thermal processes such as high pressure, pulsed electric field, and microwave processing or their combinations are needed to validate quality and phytochemical retention. PEF-processing has good prospects for use in the food industry as an alternative to thermal pasteurization, in order to achieve healthy foods. There is also a need for standardized methods to calculate the true retention of phytochemicals. These methods should include calculation of the phytochemical in a known weight of food before and after processing in order to account for solids losses that occur during processing. Almost all the studies published on minimally processed fruits and vegetables have been market quality studies. Except for irradiation studies, published data on nutrient content and nutrient retention of minimally processed foods are generally sparse and are needed. The potential safety considerations that may result from application of minimal processes also need to be understood.

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Zheng H., Wang C. Y., Wang S. Y. & Zheng W. (2003). Effect of high oxygen atmospheres on blueberry phenolics, anthocyanins, and antioxidant capacity. J Agric Food Chemistry, 51, 7162–7169.

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Foods, Diets and Disease Editors: Rakesh Sharma, Bharati D Shrinivas ©2009 Innovations And Solutions, Inc. ___________________________________________________________________________

Lecture 11

GRAFTING OF BIOFIBERS CARBOXYLIC ACID ACTION AT COLD PLASMA CONDITIONS


ABSTRACT

Physical and biochemical functionalisation of bast fibers are ways to improve thermo- and moisture regulation, anti-bacterial anti-allergies, hygiene, creating “smart”

textile. Enhancing natural properties of vegetable fibers is an intermediary step in the obtaining of new products with special applications. The vegetable fibers are biodegradable, can be recycled, and in natural state are highly polar and hydrophilic. To improve the properties of the cellulosic fibers, the chemical and/or physico – chemical modifications were applied. The surface esterification of the natural polymer with acids can be carried out to obtain biodegradable materials, novel fibres with tailored functionalities for special applications.

In this paper, starting from Spanish broom (Spartium junceum, syn. Genista juncea) fibers, under action of cold plasma discharges, and using different kinds of carboxylic acids, cellulose esters with short and long side chains have been synthesized.

The new grafted polymers were characterized by FT – IR spectroscopy (ATR), XPS and SEM in order to assess the existence of incorporated functional groups. The thermal characterization of the obtained fibres reveals their particular behaviour.

INTRODUCTION The development of synthetic materials (e.g. plastics) at the beginning of the 20th century

has caused the steady replacement of bio-based products. As a result of this change in raw material utilization, combined with an enormous increase in energy and chemical demand, the world is now facing an ecological crisis. This crisis will greatly intensify with the expected growth in demand for industrial products in developing countries. It has been estimated that global industrial output will be five to ten times that of world production in 1987, when the


world population stabilizes some time in the 21st century. Thus, the world community is facing a challenge of having to decrease pollution levels while at the same time, significantly increasing industrial output. Such predictions have led to a number of political initiatives, including support for enhanced industrial use of renewable resources (e.g. biomass) at the expense of non-renewable resources (plastic, glass fibres etc.). Plant fibres may therefore face a renaissance, not only for past uses, but also for the manufacture of three-dimensional products by hot-pressing of fibre mats or by extrusion or injection moulding of plant fibres in combination with plastic [1-4]. A classification of the plant fibres is given below – Scheme 1 [5].

Scheme 1. Classification of the plant fibres.

The applications of the plant fibres are very diverse such as: technical yarns, mechanical bonded nonwovens, various fields of application as reinforcing fibre, friction linings, paper production. Automotive components including natural fibres are currently being used by the following vehicle manufactures (as: Fiat, Ford, Mercedes Benz, Opel, Peugeot, Renault). Plant fibres have also found application in production of cement-based composites. A number of alternative crops have been identified particularly for southern Europe, while some progress has been made, as further production, processing and market development is required for large scale commercialization. A range of harvesting processes are being developed for each crop but harvesting cost and efficacy is still a limiting factor to economic production of high quality fibre. Much of the benefits of products derived from plant fibres are built on biodegradability. These benefits must be seen to be maintained in all new products.

Spanish Broom a member of the Pea family (Leguminosae), and known botanically as Spartium junceum (, syn. Genista juncea), also known as Weaver's Broom, is a perennial, leguminous shrub. It is a native of the Mediterranean region and the Canary Islands, in southern Europe, southwest Asia and northwest Africa, where it is found in sunny sites, usually on dry, sandy soils and is often cultivated. Spanish broom is a handsome shrub with long switch-like green few-leaved or leafless branches and large yellow sweet-scented papilionaceous flowers. Spanish Broom typically grows to 2-4 m tall, rarely 5 m, with main stems up to 5 cm thick, rarely 10 cm. It has thick, somewhat succulent grey-green rush-like shoots with very sparse small deciduous leaves 1-3 cm long and 2-4 mm broad. The whole plant, but especially the flower shoots and seeds have a bitter taste and tonic and diuretic

Grafting of Biofibres with Carboxylic Acids 3

properties, and were formerly used medicinally. The fibres of the young stems were used in making nets, carpets, mats, baskets, etc. The stem fibres are a hemp substitute being used mainly for coarse fabrics, cordage and paper. The stems are very pliable and can be used in basketry. It is also used for stuffing pillows etc., and for making paper. The smaller stems are used in basket making. The branches are often made into brooms. A yellow dye is obtained from the flowers. An essential oil is obtained from the flowers; it is used in perfumery [6].

During the last years, an increasing awareness of the public opinion about environmental and health problems pushed towards the utilization of natural raw materials, drawing the attention to industrial fibre crops. Industries all over the European Community are looking for raw material for replacing artificial fibres in composite materials to alleviate problems related with composite materials disposal at the end of the technical life. In Europe the use of natural fibres in the automotive industry in 1999 was about 21,300 tones and in 2000 about 28,300 tones. In 2005 the use of natural fibres was about 70,000 tones and in 2010 could increase to more than 100,000 tones of natural fibres for the effect of EU end-of-life vehicle directive than influence this development. The sources of raw material used in composites for automotive industry are mainly represented by flax, hemp, jute, kenaf, sisal and coconut fibres. Recently there has been a revival of interest in Spanish Broom as a possible source of natural fibre in automotive industry. Natural abundance, much higher strength per unit weight than most inorganic fillers, lower density and their biodegradable nature make natural fillers attractive as reinforcements of engineering polymer systems [7 – 9]. Spanish Broom cortical fibres are multiple elementary fibres (ultimates) arranged in bundles. The elementary fibres are bound together by lignin. A thick secondary cell wall indicates high cellulose content. The diameter of ultimates varies from 5-10 μm while the diameter of the whole bundle is about 50 μm. The values in tensile strength and elastic modulus are promising supporting the hypothesis that these fibres can be a potential replacement for man made fibres in composite materials. Spanish broom (Spartium junceum) fibres are new biofibres used as such or in composites [10]. The improvement of the properties is necessary in most purposes. Chemical processes of esterification with these acids are developed in special solvents for cellulose, in the presence of acid chlorides, pyridine and trifluoroacetic acid at very long reaction times. In these cases the pollution is high. [11] To avoid these drawbacks, a physical way of functionalization of vegetable fibre is proposed [12]. A highly hydrophobic product with retention of fibrous structure was obtained by the he reactions with higher saturated fatty acids (C10–C18) yielded lower DS values but still comparable hydrophobicity [13].

Possible ways of Spanish broom valorization are given in Scheme 2 [14, 15]. Enhanced fibre properties for improving the properties of new products and applications can be obtained by: new processing and production concepts include the development of environmentally friendly and energy-efficient processing and surface modification of fibres, yarns and fabrics. Creation of new natural polymer surfaces by plasma grafting and the deposition of thin film coatings by plasma assisted processes are applied in order to: improve: hydrophilisation, dyeing, reactive dyeing, printing, increase of the flame retardance and thermal insulation properties, increase abrasion resistance, to obtain higher conductivity, barrier layers to chemicals, UV protection, etc. use of various gases, mixtures of gases and monomers in vapor form and solutions, new softeners, to remove the impurities (reduction of chemical


pretreatments) which are summarized in the Scheme 3. Special applications are also for: specific protein attachment [16], biomolecule (e.g. heparin) immobilization [17], improved cell attachment and spreading [18, 19] reduction of calcium carbonate nucleation [20] siloxane coating [21] to create smart clothes / wearable computing [22] elaboration of technologies for nano and micro coatings on textiles, etc.

Scheme 2. Possible ways of Spanish broom (Spartium junceum, syn. Genista juncea) valorization.

Scheme 3. Low temperature plasma: effects on textile.

Several low temperature plasma systems are known as : low pressure plasma systems (glow discharge) and atmospheric plasma systems (corona discharge, dielectric barrier discharge, glow discharge) The last one can be integrated into the production line manufacturing processes and also offers some advantages such as sample size is unlimited,


are running at lower temperatures, [23, 24] and secondary reaction are avoided. Use of various gases, mixtures of gases and monomers in vapor form and solutions can be used [25 - 29]. Composites of natural fibers and thermo-plastics can be combined to form new enhanced materials. One of the problems involved in this type of composites is the formation of chemical bonds between the fibers and the polymers at the interface. Low energy glow discharge plasmas are used to functionalize cellulose fibers implanting polystyrene between the fibers and the matrix that improve the adhesion of both components, the adhesion in the fiber-matrix interface increasing with time in the first 4 min of treatment [30]. Methods of textile functionalization by plasma treatment include: a) plasma polymerization using different reactive gases, monomers or prepolymers, mixture of gases and monomers; b) plasma activation which mainly involves incorporation of new functional groups, which can be achieved by treatment with solutions and physical vapor deposition. Different procedures have been used to modify the cellulosic fibres from jute [31], wood fibres [32] cotton fabrics [33] for which the effect of corona discharge consists in the removal of impurities (reduction of chemical pretreatments), improvement of mercerisation process and dye uniformity in continuous dyeing, improvement of rubbing fastness of pigment printing and also it can influence the chemical finishing (softening, crease recovery, flame retardancy) and physical properties of linen fabrics and make raw cotton hydrophilic. The uniformity of dyeing is similar when the wetting agent in the recipe is replaced by a corona treatment. The authors claim that by corona treatment the wetting agents in different operations can be avoided, for example, in desizing, mercerization, bleaching and dyeing, higher number of barium is obtained in cotton mercerization, higher yield, penetration and rubbing fastness are obtained in pigment printing. In easy-care finishing, higher crease angle recovery, lower formaldehyde release, as well hydrophilisation is obtained.

This paper deals with plasma grafting of Spanish broom (Spartium junceum, syn. Genista

juncea) fibres with several acids in order to establish the optima conditions for their modification and obtaining of new improvements.

1. EXPERIMENTAL

Materials and Methods The composition of the Spanish broom (Spartium junceum) fibres undergone to plasma

treatment and those modified were globally characterized by FT-IR spectroscopy. The FT-IR spectra have been recorded by means of a DIGILAB Scimitar Series FT-IR

spectrometer (USA) at 4 cm-1 resolution. Five recordings were performed for each sample and the evaluations were made on the average spectrum obtained from these five recordings. FT-IR spectra are recorded in KBr pellets. Processing of the spectra was done by means of Grams/32 program (Galactic Industry Corp.).

The FT-IR spectra of the sample under study are given in Figure 1 and the assignment of the bands is presented in Table 1.


4000 3500 3000 2000 1500 1000

0.0

0.1

0.2

0.3A

bsor

banc

e, a

.u.

Wavenumber, cm-1

Figure 1. FT-IR spectra of the fibres from Spanish broom (Spartium junceum) fibres.

Table 1. Assignment of the FT-IR bands from the spectrum of Spanish broom (Spartium

junceum) fibres Band position (cm-1) Band assignment

3418 vs O(2)H…O(6) intramolecular H – bonds in cellulose, OH intermolecular, H – bond in the 10Ī plane

3277 sh O(6)H…O(3) intermolecular H bonds in cellulose 2913 s Asymmetric CH

2 valence vibration

2861 sh Symmetric CH2 valence vibration

1723 sh C=O stretch in unconjugated ketone, carbonyl and in ester groups (frequently of carbohydrate origin)

1639 s Protein impurity and water associated with lignin 1503 sh C=C stretching of the aromatic ring (G)CH deformation

1453 s CH

2 in pyran ring symmetric scissoring; OH in plane bending in cellulose I and cellulose

II 1429 s H-O-C in plane bending of alcohol groups 1373 s CH bending in cellulose I and cellulose II 1320 s CH

2 wagging in cellulose I and cellulose II

1277 m CH bending in cellulose I and cellulose II 1239 m OH in plane deformation, also COOH 1202 sh OH in plane bending in cellulose I and cellulose II 1160 s C – O – C asymmetric stretching in cellulose I and cellulose II 1114 s Ring asymmetric stretching in cellulose I and cellulose II 1057 s C – O valence vibration mainly from C(3) – O(3)H 1031 s Stretching C – O in cellulose I and cellulose II 896 w Anomere C – groups, C

1 – H deformation, ring valence vibration


In spectrum are identified all the bands of the main components mentioned in literature cellulose, lignin, pentosans, and extractives and also traces of amine groups [34].

A qualitative appreciation of the relative content of cellulose and lignin in these samples can be obtained from the ratios of the integral adsorption which are presented in Table 2. These ratios are proportional with lignin content which lied between 25 and 30 % [35, 36].

Table 2. Lignin/Carbohydrates ratio from FT-IR integral absorbances

Sample Relative intensities of aromatic skeletal vibration

I1505

/I1738

I1505

/I1375

I1505

/I1158

Height Area Height Area Height Area Spanish broom fibres 0.88 0.55 0.38 0.24 0.44 0.40

According to the literature data, the chemical composition of the whole stem outlined a

high content of cellulose (67-76 %) while lignin (13-22%), pentosans (4-5%) and extractives (6-7%) were low [37].

The following carboxylic acids have been used: butyric acid, oleic acid, olive oil, sunflower oil, lactic and polylactic acids.

Butyric acid was purchased from Merck, its purity is >99% being grade for synthesis; Lactic acid was also purchased from Merck, its purity was between 88 – 92 %; Olive oil contains between 55.0 - 83.0 oleic acid, 3.5 - 21.0 % linoleic acid and 7.5 - 20.0

palmitic acid, 0.3 - 3.5 % palmitoleic acid, 0.5 - 5.0 stearic acid and others in amounts less than 1%;

Oleic acid from sunflower oil which belongs to the vegetable oils group with a high content in mono and polyunsaturated acids of about 94 ,90% ( 23.7 % oleic acid and 59.8 % linoleic acid) and saturated acids 11,30% (mainly palmitic and stearic). It contains 99,90 % lipids, trigliceride 99,20%, moisture content 0,1% [38].

Polylactic acid was laboratory synthesized and it has a number average molecular weight of Mn = 2000.

Procedure of Grafting by Cold Plasma The experimental set - up for cold plasma fibers‟ grafting is presented in figure 2. In a

typical experiment, after several washing cycles with inert gas (nitrogen) from the gas metallic reservoir (6), into the cylindrical shaped vacuum plasma reactor (1), the working pressure was established (0.3 mm Hg) and then the R.F. power was transferred to the reactor through the semi cylindrical, external, silver-coated electrodes (8). The R.F. power was dissipated to the electrodes from a R.F. generator (11) with the possibility of generating 50 - 300 W. The samples were deposited on special glass support for fibres exposure to grafting (12). The Spanish broom fibres have been treated in plasma at a P=300 W, frequency 13.56 MHz, pressure 0.3 mm Hg for 5 or 10 minutes. Before treatment the samples were impregnated with solutions of acids as: butyric acid, sunflower oil and olive oil (solutions 20 % in acetone) or with lactic and polylactic (Mn = 2000) acids (solution 4 % in ethanol).


Figure 2: Experimental set-up for fibers‟ grafting: 1 – vacuum plasma reactor; 2 – close glass vacuum system; 3 – vacuum gauge; 4 – vacuum pump; 5 and 7 – glass valves; 6 – monomer flask (only for distilled monomers in plasma medium, not for impregnated samples); 8 – semicylindrical external silver coated electrodes; 9 – glass support for samples; 10 – central monomer‟s admission glass tube; 11

– HF generator (13.56 MHz); 12- special support for fibres exposure to grafting.

Unbounded acid from the treated samples was extracted for 6 h in a Soxhlet extractor. The treated fibres were dried and analysed.

After plasma treatment all modified fibres become much softer comparatively with untreated fibre.

2. INVESTIGATION METHODS: FT-IR spectroscopy – see above.

2.1. XPS Analysis of the Fibres Surface XPS spectra have been recorded by means of an Axis-Ultra de Kratos (UK) instrument

equipped with an electrostatic analyzer with a great radius and a detection system having 8 channels. Two X-rays sources are used one double of Al-Mg without monochromator and one of Al with monochromator. The system is also provided with a source of electrons of low energy in order to neutralize the electrostatic charge which will appear on the samples when they are exposed to the monochromatic X-rays beam. The spectrometer operates in a high vacuum of 5·10-9 mm Hg. The XPS chamber of analysis is connected with a


transport/preparation chamber having multiple uses allowing rapid introduction of the samples in the position suitable for analysis.

The samples in quartz crucibles have been introduced in XPS chamber and a pressure of 1-3·10-7 mm Hg was realized which is also necessary for the XDR source running.

The XPS spectra were acquired with an Al source of 300 watts power. For the elemental composition determination the recording of the total spectra was done with an energy of analyzer of 160 eV, with step size of 1 eV, lens act in hybrid mode which assures the maximum sensitivity. The spectra of high resolution for chemical analysis have been recorded using an energy of 20 eV, step size having 50 meV (lens in hybrid mode).

X-rays diffraction (XRD) analysis was done using a Bruker diffractometer equipped with a Kristalloflex 760 sealed-tube copper anode generator, operated at 40 kV and 40 mA, and a two-dimensional position-sensitive wire-grid detector (Bruker AXS) pressured with xenon gas. Collimation was effected by a graphite monochromator with a 0.8-mm pinhole sample-to-detector distance = 9 cm. Samples were placed in sealed Mark-Röhrchen glass capillaries (Charles Supper) of 1.0 mm inner diameter. Scans: 1200 s (1200 scans)

SEM images have been taken with a scanning electron microscope TESLA BS 301 (Acceleration potential 10 kV).

Differential scanning calorimetry (DSC).Thermal characterization was performed using a Mettler Toledo DSC 823e differential scanning calorimeter (DSC), calibrated with indium and flushed with nitrogen. The heating and cooling scans were performed at 10°C/min, on 5 to 15 mg of sample packed into standard aluminium pans. First-order transition temperatures are given by the peak values.

Thermogravimetry. Thermogravimetric analysis (TGA) was carried out under constant nitrogen flow (200 ml/min) at a heating rate of 10 °C/min using a Mettler Toledo TGA/SDTA 851 balance. The heating scans were performed on 10 to 15 mg of sample. The kinetic parameters have been evaluated by integral and differential methods. The kinetic parameters have been evaluated by integral or differential methods using VERSATILE commercial program which gives kinetic parameters by various methods (see below).

3. RESULTS AND DISCUSSION

3.1. FT-IR Spectra Results FT-IR spectra of the studied samples are given in Figures 3. The spectra of the treated

samples are different from that of untreated fibre and they are particular for each acid used for grafting. In all cases the evidence of the ester bonds in plasma grafted samples is clear at 1740 and 1610 cm-1. Bands shift and splits are also found at 2850 cm-1 and 2925 cm-1 (Figures 3 a and b).

A very different spectrum shows the sample grafted with polylactic acid. Supplementary bands being present at: 3020 and 1500 cm-1 and the band at 1750 cm-1 found in other samples is shifted to 1725 cm-1. On the basis of these results it can appreciate that the grafting took place with a high yield.


a) b)

c) d)

Figure 3. FTIR spectra of Spanish broom (Spartium junceum) fibres untreated and treated with different carboxylic acids.

XPS Results

The measurement of binding energy (BE) can be used to characterize materials. The observed BE depend on the specific environment where the functional groups are located, most changes being within 0.2 eV of variation [39, 40]. The C1s BE is observed to increase monotonically with the number of oxygen atoms bonded to carbon, that is C-C < C-O < C=O < O-C=O < O-(C=O)O- consistent with that the carbon becomes more positively charged with increasing number of oxygen atoms bonded to carbon.

The XPS intensity (integrated area under the photoelectron peak) is proportional to the atom quantity in the detected volume, therefore by integrating the area under a given peak and correcting for its ionization cross section, quantitative elemental analysis of the material can be made [41, 42]. This requires an algorithm for peak fitting, which should include all characteristic elements for a photoelectron peak, i.e. the peak height, width, shape. The relatively simple shape of a photoelectron peak, due to the one electron process involved, allows in most cases the deconvolution of complex experimental peaks. To extract


quantitative information from the XPS spectra, the area and the BE of each subpeak for a given orbital, e.g. C1s, must be determined. Typically, the spacing between subpeaks is similar or inferior to observed peak widths (1 eV). Thus, it is rare when individual subpeaks are completely separated in an experimental spectrum. This requires the use of a peak fitting procedure. Quantities in such procedures, performed using appropriate software, include the background, peak shape (Gaussian, Lorentzian, asymmetric, or mixtures thereof), peak position, and peak height and peak width.

In the XPS spectra of organic compounds, peaks corresponding to carbon atoms in different chemical environments every so often exhibit almost equal binding energies, and they cannot be correctly separated using a standard mathematical procedure, especially in substances with unknown chemical structures of the surface [43]. For example, in examining substances containing functional groups like those below:

C OH C O C

O

C O C

Peaks due to these groups in C1s spectra exhibit almost equal binding energies (about

286.5 eV) and, hence, cannot be separated using mathematical treatment. All the above is also true in the determination of functional groups by other XPS spectra such as O1s or N1s.

This problem became more severe once those modification techniques were developed to tailor the polymer surface properties, the chemical derivatization in XPS being firstly used in the fundamental research of phenomena that occur in surface polymer layers under exposure to plasma and other modifying agents.

XPS results for untreated and Spanish broom fibres grafted in cold plasma conditions are presented in Figures 4 and Tables 3 and 4.

The main bands in XPS spectra – Figure 4a are as it is expected those of carbon and oxygen. Each sample contains in traces some impurities.

The Si2p and Ca 2p bands are present in almost all spectra, this element being contained in raw material. Si might be from the holder of the samples. The unmodified fibres also contain traces of nitrogen and chlorine which are also find in plasma treated fibres with lactic acid and oleic acid. The Zn2p band was found in the fibres treated with butyric acid while the sample treated with polylactic acid contains manganese.


unmodified Butyric acid 10 min

Oleic acid 10 min Olive oil 10 min

Polylactic acid Lactic acid

Figure 4a. XPS spectra of the studied samples.


Untreated sample Butyric acid

Oleic acid 10 min Olive oil

Lactic Acid Polylactic acid

Figure 4b. Deconvoluted carbon bands of XPS spectra of the studied samples


Untreated sample Butyric acid 5 min

Oleic acid 5 min Olive oil

Lactic acid Polylactic acid

Figure 4c. Deconvoluted Oxygen bands of the XPS spectra of the studied sample.


Concerning the bands position, it can be observed a shift in C1s band for sample treated with lactic, oleic and olive oil, while O1s band is shifted to lower binding energy only in the spectra of sample treated with lactic acid – Table 3. The percentage area of the main elements takes values particular for each treated samples. The C1s area increases in the following order: untreated fibre < lactic acid < butyric acid < polylactic acid < oleic acid < olive oil cold plasma treated sample. The O1s area has an opposite variation.

This is an evident proof that the grafting reaction took place. The most evident change in the fibre structure at least at their surfaces is in the number and type of carbon atoms, as it appears from deconvoluted carbon bands in Figures 4b. The untreated fibres and butyric acid treated sample have four types of carbon atoms, while the other treated samples have at least six types of carbon atoms. The oxygen deconvoluted bands – Figure 4c – are much simpler being evidenced two types of oxygen atoms in untreated fibre and butyric acid or lactic acid treated fibres, while the oleic acid-, olive oil- and polylactic acid grafted fibres exhibit three kinds of oxygen atoms.

The assignment of bonds which belongs these atoms is given in Table 4 and it was made according to the literature data [44 – 47].

Table 3. Band position and percentage area of total area for untreated and cold plasma treated samples

Atom

Untreated Butyric acid Oleic acid Olive oil Lactic acid Polyactic acid Band position/% of total area

Band position/% of total area





C1s 282.91/67.6 282.91/74.08 281.91/87.7 281.91/89.90 281.91/70.43 282.91/ 82.36 O1s 529.91/31.3 529.91/24.58 529.91/11.5 529.91/9.32 528.91/28.10 529.91 /16.80 N1s 397.91/0.4 397.91/0.57 396.91/0.3 396.91/0.44 Si2p 99.91/0.4 99.91 /0.42 98.91/0.3 98.91/ 0.64 96.91/ 0.32 99.91/ 0.32 Cl2p 197.91/0.1 194.91/0.13 193.91/0.1 196/ 0.06 Ca2p 3442.9/0.2 344.91/0.21 342.91/0.08 343/ 0.04 344.91/0.29 Zn2p 1020.91/0.06

Table 4. Results of C1s and O1s high resolution spectral fitting (%)

Groups Untreated Butyric

acid Oleic acid Olive oil

Lactic acid

Polyactic acid

Carbon atom containing group

Positions of the carbon atoms in deconvoluted curves C-C (285.0eV) C-O-C (286.6 eV) O-C-O (288 eV) O-C=O (288.7

285.00 286.63 287.99 289.13

285.00 286.55 287.97 289.17

285.00 285.88 286.78 287.76 289.25

285.00 285.68 286.40 287.17 287.91 289.22

285.00 286.15 286.85 288.00 289.21

285.00 285.85 286.73 288.52 289.32


eV) O-CO-O (290.4 eV)

290.43 289.87 290.1 290.46

C1(C-C;C-H) 32.34 44.63 47.58 30.36 37.26 27.23 C2(C-O-) 51.57 41.62 17.73 17.29 26.32 20.87 C3 (O-C-O;C=O)

13.45 10.03 4.89 4.01 11.75 15.36

C4 (O-C=O) 2.65 3.72 4.42 4.40 7.01 5.28

Oxygen atom containing group Position of the oxygen containing groups in deconvoluted curves

532.93 533.55

532.89 533.79

532.61 533.82 535.02

532.81 535.55 534.16

533.02 534.10

532.79 534.10 535.26

O1 (OC=O) 78.41 90.04 45.18 39.36 94.00 35.87 O2 (C-O) 21.59 9.96 43.30 48.31 6.00 37.98 O3 - - 11.52 12.33 - 26.15

C1 is a carbon bonded only to another carbon (C-C) or a hydrogen atom (C-H); C2 – atom singly bonded to a oxygen atom (C-O-), other than a carbonyl atom; C3 – a carbon atom single bonded of two oxygen atoms (-O-C-O-) or to a single

carbonyl atom (-C=O); C4 – a carbon atom single bonded to an oxygen atom and to a carbonyl oxygen atom

(-O-C=O). C4 group (-O-C=O) is very low in untreated sample (2.65 %) and increases in treated sample as a result of ester bonds appearance. In the same time the content of C2 group (C-O- from C-O-H) decreases from the same reason. The results are in good agreement with FTIR measurements.

O1 is a oxygen atom linked to a carbon atom by a double bond, and also an oxygen single linked (O-C=O);

O2 – an oxygen atom linked to a carbon atom by single bond (C-O- ); O3 – oxygen atom from supplementary oxidation processes. Taking into account a content of hydroxyl groups in the superficial layers of the cellulose

fibers (up to ~ 10 nm depth) of 51,57 % and also the variation of the C2 groups (C-O- from C-OH) which decrease from 51.57 % to 16.13 % and the increase of the C4 (-O-C=O) groups from 2.65 % to 7.01 % can be estimate a degree of grafting of superficial layers which depends on the type of carboxylic acid employed as it appears in Table 5. . Some errors in XPS results for non-smooth topographies samples as in our case (see SEM results) can appear, but they do not affect the find results, because the modifications are very clear.


Table 5. Degree of grafting of the Spanish broom fibres with various carboxylic acids

Fibre grafted with: Degree of grafting (%) O/C atomic ratio Untreated 0 0.463 Butyric acid 19.3 0.332 Oleic acid 53.6 0.131 Olive oil 45.8 0.104 Lactic acid 68.8 0.399 Polilactic acid 62.8 0.204

The highest degree of grafting was achieved with lactic acid and this decreases in the

order: lactic acid > polilactic acid > oleic acid (sunflower oil) > olive oil > butyric acid. In cold plasma conditions the grafting can take place both with unsaturated and saturated

carboxylic acids, therefore the fibres grafted with sunflower and olive oil should have both chains with oleic acid, linoleic acid , palmitic and stearic.

The formation of other kinds of bonds is also possible, taking in the view the diversity of the active species possible to be formed in plasma conditions.

Comparing the values of the O/C atomic ratio it could appreciate that the grafting of the lactic, polylactic and butyric acids do not change significantly the surface composition while the other can be bonded by different kinds of bonds which lead to decrease of the oxygen percentage on the surface.

The morphological aspects of the grafted samples have been studied by XRD and SEM.

XRD Results

X-ray diffraction (XRD) techniques were used in the study of the effect of grafting on crystallinity. Generally XRD patterns of the cold plasma treated samples are similar with that of the untreated fibre – Figure 5.


Untreated

Butyric acid grafted

Oleic acid grafted


Olive oil grafted

Lactic acid grafted


Polylactic acid grafted

Figure 5. XDR patterns of the untreated and cold plasma treated Spanish broom (Spartium junceum, syn. Genista juncea) fibres.

The crystalline peaks are found in the untreated sample at 2 theta degree of: 3; 15.8; 22.5; 29 and 34 and they correspond to the Iα - cellulose crystalline structure [48, 49]. The same peaks characterize the crystalline fraction of all cold plasma grafted samples but they are shifted to higher 2 theta degree and they are much wider and the rings become much diffuse and for the fibres grafted with oleic acid, olive oil and lactic acid some rings can not be distinguish. This observation is in accordance with the data obtained for other cellulose esters [50]. It was shown that X-ray diffraction analysis indicates distinct crystal patterns for these crystalline cellulose esters, and differential thermal analysis shows strong melting peaks. X-ray diffraction analysis of secondary cellulose esters, that is, esters having a substantially lower degree of esterification, shows very diffuse patterns which are only slightly indicative of crystalline structure. On the other hand, differential thermal analysis, shows strong endothermic peaks which appear to indicate melting of crystalline material.

A small peak appears at 38 2 theta degree in the samples treated with butyric acid, oleic acid, lactic acid or polylactic acid, probably because of some changes in crystallinity network.

The crystallinity degree was evaluated as the ratio between the area under the crystalline

peaks and the area under the amorphous halo which is a broad hump in the XRD pattern –

Table 6. The decrease of the crystallinity degree after grafting with carboxylic acids is much important for the fibres grafted with oleic and lactic acid, therefore even the bulk morphology of the Spanish broom fibre is affected by grafting.


Table 6. XDR data for untreated and grafted Spanish Broom fibres with carboxylic acids

Spanish broom untreated

Treated butyric acid

Treated oleic acid

Treated olive oil

Treated lactic acid

Treated polylactic acid

Crystallinity index (%) 55.8 45.53 39.52 42.41 32.86 40.54

SEM Results

Untreated sample X 810 (1 cm = 10 μm)

Butyric acid grafted fibers (10 min) X 810 (1 cm = 10 μm)


Oleic acid grafted fibers (10 min) X 410

(1 cm = 20 μm)

Olive oil grafted fibers (10 min) X 430

(1 cm = 22 μm)


Acid polylactic grafted fibers (10 min) X 810

(1 cm = 10 μm)

Lactic acid grafted fibers (10 min) X 430

( 1 cm = 22 μm)

Figure 6. SEM micrographs of the untreated and grafted Spanish broom (Spartium junceum, syn. Genista juncea) fibres under cold plasma conditions.


The existence of the graft layers on the surfaces of the Spanish broom fibers is clearly evidenced by scanning electron microscopy (SEM) (Figures 6). Important morphological differences can be observed between the support and the grafted derivatives in all cases. It can be noticed that the aspect of the carboxylic acids grafted fibers are totally different from the surface of the control fibers (support). On the surfaces of the fibres grafted with oleic and olive oil some irregularities looking as depositions on particles are observed, while the fibres grafted with butyric acid, lactic and polylactic acid show a uniform aspect, the fibres becomes voluminous and fibrils seems to be expanded and distinct.

Thermal Properties

DSC Results

Two important aspects can be followed by DSC study of the cellulosic materials: influence of the substituents on thermal characteristics and because of their hydrophilicity the interaction with absorbed water.

Strong correlationship between the melting point and the length of substituents at the secondary hydroxyl groups at C2 and C3 positions, have been found for the cellulose fatty acid heteroesters (cellulose propanoate diacetate, cellulose butanoate diacetate, cellulose acetate dipropanoate, cellulose butanoate dipropanoate, cellulose acetate dibutanoate, and cellulose propanoate dibutanoate) [51].

Hatakeyama et al. found that vaporization peak is split into two peaks, one is at around 60°C and the other is at around 120°C. The high temperature vaporization peak is related with the structural change of amorphous chains of cellulose by desorption of bound water [52, 53]. Cieśla et al., established that the profiles of thermal effects depend on water content, time of conditioning, film pretreatment etc [54].

Both heating – Figure 7 - and cooling – Figure 8 - cycles have been applied from -50 – 250 oC to the untreated and grafted fibres. The DSC curves recorded by heating show two or three peaks. The process with peak temperature around 100 oC can be due to release of the absorbed water. A large amount of non-freezable strongly bounded water was also detected.

Cellulose absorbs water and the structural changes appear on ordering of polymer fraction. Due different strengths of water binding in the case of the grafted fibres the characteristic temperatures and the enthalpies vary with nature of grafts. Differences between interactions of particular cellulose fibres with water can be detected during the first, the second and the third heating. Because of water loss in the first run, in the next runs its quantity (and enthalpy) decreases and some temperatures are changed.

Comparing the data of Table 7 of the grafted fibres with those of untreated one the following conclusions can be draw: the first peak is shifted to lower temperature after grafting but its enthalpy is generally higher than of untreated fibre. Some peaks at negative temperatures are present in the DSC curve of fibres grafted with oleic and olive oil. The biggest difference between peak temperatures of the peak of absorbed water release is found for samples grafted with olive oil, lactic and polylactic acid. This should means that water is stronger bonded in grafted fibres than in untreated one but in the lower amount as was found also by TG/DTG (see below).


Temperature (°C)

-50 0 50 100 150 200

Exo

8

6

7

5

4

a)

3

3'

Figure 7. DSC thermograms of the treated and untreated samples obtained by heating at 10°C/min [with 8 = untreated; 7 = treated with oleic acid; 6 = treated with butyric acid; 5 = treated with lactic acid; 4 = treated with polylactic acid; 3 = treated with olive oil (first heating); 3‟ = treated with olive oil (second

heating)].

Similar values have been obtained for cellulose esters with linear aliphatic acyl substituents ranging in size from C[12] (lauric acid) to C[20] (eicosanoic acid). A series of transitions that represented motion by both ester substituents and cellulosic main chain. Broad crystallization and melting transitions attributed to side-chain crystallinity were observed in the range between -19 and +55°C. Melting temperature values of 96°C and 107°C [55].

As concerns the variation of the temperatures of the second peak which is probably a decomposition/melting step expecting fibres grafted with oleic acid, all other grafted fibres exhibit lower decomposition/melting temperatures and also lower enthalpy of this process, the most important decrease being found for fibres grafted with olive oil and lactic acid (which acts probably as plasticizer).


Table 7. Characteristic temperatures and enthalpies of the processes evidenced in DSC curves recorded by heating of the studied samples

Sample Peak 1 Peak 2 Tonset,

(oC) Tpeak,

(oC) Tendset,

(oC) H(J/g) Tonset,

(oC) Tpeak,

(oC) Tendset,

(oC) H(J/g)

Untreated 94.3 149.6 191.0 -59.63 179.5 186.4 198.5 -32.85 Butyric acid

-46.5 -18.2 17.7 -10.5 1.84 45.62 76.72 -3.48 144,7 164.4 172.7 -0.53 56.0 99.9 138.9 -107.2

94.06 130.9 178.1 -93.75 Oleic acid

-32.8 -10.6 3.9 -0.54

36.3 47.6 56.4 -1.14

99.05 132.8 172.4 -57.6 198.1 202.8 216.0 -1.32

Olive oil

-41.47 2.08 15.82 -4.81

44.32 106.49 153.1 -68.07 121.7 139.7 173.8 -3.28 Lactic acid 59.1 104.4 147.1 -79.9 154.2 160.1 168.1 -1.57 Polylactic acid

59.26 111.91 158.07 -92.76 163.58 169.47 179.68 -0.91

The DSC curves recorded by cooling are particular for each fibre studied and there are

big differences in respect with the untreated sample. The DSC curves of the untreated sample and that of the fibres grafted with butyric acid and polylactic acid show two exothermic processes with peak temperature at 38 and 181 oC, the temperatures of the grafted fibre being close to those of the untreated sample, that should means that this grafts do not influence these crystallization processes.

The crystallization process which occurs in the temperature range 50 – 200 oC is also present in the DSC curves of fibres grafted with oleic acid but it occurs at lower temperature. The fibres grafted with oleic acid, lactic acid and polylactic acid shows an exothermic process around 100 oC while the fibre grafted with olive oil and oleic acid show a crystallization peak at low temperatures at around -20 oC which could be due to the frozen bonded water.


Temperature (°C)

-50 0 50 100 150 200

Exo

-50 0 50 100 150 200 250

a) b)

8

7

3

Figure 8. DSC curves of the treated and untreated samples obtained by cooling at 10°C/min of a) untreated sample (8); treated with oleic acid (7) and b) treated with olive oil (3).

Table 8. Characteristic temperatures and enthalpies of the processes evidenced in DSC

curves recorded by cooling of the studied samples

Sample Peak 1 Peak 2 Tonset,

(oC) Tpeak,

(oC) Tendset,

(oC) H(J/g) Tonset,

(oC) Tpeak,

(oC) Tendset,

(oC) H(J/g)

Untreated 48.39 38.42 1.89 0.77 190.6 181.23 171.1 2.16 Butyric acid

55.1 33.3 21.9

0.54 198.4 193.6 182.9 0.27

Oleic acid -14.75 -24.12 27.6 1.46 202.3 195.2 184.8 0.32

39.5 29.3 11.5 0.35

116.4 110.97 98.7 0.43

Olive oil -3.4 -18.4 -43.87 4.22 220.8 215.2 201.94 2.08 Lactic acid 13.06 10.7 8.22 0.78 194.5 191.5 185.4 0.26

106.2 100.06 89.9 Polylactic acid

46.09 35.1 21.7 0.13 202.1 193.5 173.99 0.76 131.5 117.9 99.2 0.13

The sorbed water molecules are directly bound to the hydrophilic site to form non-

freezable water. Then, beyond a certain water content threshold, the sorbed water molecules


become freezable, but with a melting point lower than 0°C, due to their location in the second hydration layer [56, 57].

The peak at high temperature seems to be due to of a reversible process because it appears both during heating and cooling at close temperature. This is very difficult to be explained. It is possible as some substituents to act as plasticizer as was found by Aranishi et al., for a melt spun fiber comprising a thermoplastic cellulose mixed ester composition containing as plasticizer polylactic acid [58], and for cellulose oligomers with n>20: (cotton, wood, paper), a melting temperature > 250 oC is appreciated, but all time this process is accompanied by carbonization. For Eastman cellulose acetate a glass transition temperature is found at 180-186 oC [59] while the melting range lies between 230-250 oC. Therefore the first type of transition associated with other phenomena could be much probable in our case.

TG Results

There is a great number of studies thermal degradation of cellulose and cellulose esters such as cellulose acetates, nitrate, cellulose phosphate [60]; cellulose benzoate, cellulose succinate and cellulose cinnamate [61] esters with fluorine-containing substituents [62] cellulose fibers partially esterified with some long chain organic acids such as: undecylenic acid; undecanoic acid; oleic acid and stearic acid [63] and a particular behaviour was found in each case.

The TG/DTG curves of under study samples are given in Figure 9 and characteristics thermogravimetric data are summarized in Table 9.

Figure 9. TG/DTG curves of Spanish broom fibres untreated and grafted with different carboxylic acids in cold plasma conditions.

In the first thermogravimetric step, all characteristic temperatures for grafted fibres are superior those of untreated Spanish broom fibres and mass loss is lower. This process could attributed to water loss and should means that after grafting the bonding with water is stronger, but the absorbed amount is lower. In the second thermogravimetric step onset temperatures for all grafted fibres are higher than that of untreated one, while peak temperatures are almost unchanged. This step corresponds to decomposition and the shift of the onset temperatures to high temperature indicates an increase in thermal stability at least at

http://www.ingentaconnect.com/search;jsessionid=2nccrflq6jj9q.victoria?database=1&title=Undecylenic%20acid

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the fibres surface. This process is very complex showing many inflexions which appear at low temperatures for grafted fibres, indicating some changes in reaction mechanism as was previously shown [64].

Table 9. Thermogravimetric data of Spanish broom fibres untreated and functionalized

with different carboxylic acids

Thermogravimetric Characteristic

Spanish broom untreated


Treated oleic acid

Treated olive oil

Treated lactic acid


Peak I Tonset (oC) 22,5 24.9 25.1 23.5 23.9 23.6 Tpeak (oC) 49,0 49.0 53.8 49.0 49.0 55.2 Tendset (oC) 118,3 118.3 114.6 126.3 115.6 117.4 w (%) 5,4 5.0 3.5 3.9 3 2.7 Peak 2 Tonset (oC) 177,2 182.2 184.3 198.3 179.9 180.0

Tpeak (oC) 358,5 (i) 409,0 (i) 449,8

358.5 (i) 405.7 (i) 449.2

359.5 (i) 406.5 (i) 444.5

358.5 (i) 406.4

358.5 (i) 406.4 (i) 457.2

358.5 (i) 406.4 (i) 449.1

Tendset (oC) 550,6 547.9 578.2 529.2 534.5 542.6 w (%) 75,2 77.3 79.9 79.8 76.8 78.4

i – temperature at inflexion point of DTG curve. To have many information about the involved processes the overall kinetic parameters

have been evaluated – Table 10, using both differential and integral methods coupled in the commercial computing program VERSATILE.

Table 10. Kinetic parameters (E - overall activation energy and n - reaction order) of thermal decomposition (second step) of Spanish broom fibres untreated and grafted

with different carboxylic acids

Sample Spanish broom untreated


Treated oleic acid

Treated olive oil

Treated lactic acid


Methods of evaluation E, kJ/mol; n E, kJ/mol; n

E, kJ/mol; n

E, kJ/mol; n

E, kJ/mol; n

E, kJ/mol; n

Coats – Redfern [65] 89.18; 1.7 87.75; 1.6 85.52; 1.5 106.95; 1.7 88.99; 1.7 87.08; 1.6 Flynn-Wall [66] 94.38 ; 1.7 93.02; 1.6 90.92; 1.5 111.30; 1.7 91.06; 1.6 92.40; 1.6 van Krevelen [67] 131.09; 2.0 129.76; 1.9 121.88; 1.7 156.50; 2.0 127.70; 1.9 124.74; 1.8 Urbanovici-Segal [68] 90.53; 1.7 89.13; 1.6 86.89; 1.5 108.13; 1.7 90.25; 1.7 88.44; 1.6 Achar [69] 71.44; 1.3 72.98; 1.3 67.75; 1.1 91.46; 1.4 75.09; 1.4 77.48; 1.4 Freeman Caroll [70] 75.54; - 70.01; - 65.30; 90.91; - 66.98; - 64.44; - Piloyan 71] 62.97 - 64.25; - 66.25; 83.98; - 60.31; - 62.90; -

Significant changes in the kinetic parameters are obtained only for fibre grafted with

olive oil which contains 55.0 - 83.0 oleic acid, 3.5 - 21.0 % linoleic acid.


To explain the important changes in properties of the Spanish broom fibres after plasma treatment, the plasma action must be considered. Plasma aided synthesis (deposition and grafting reactions) involves fragmentation of plasma gases and reorganization of the resulting neutral and charged species, inside and outside the plasma area, into nonvolatile, high-molecular-weight structures. Recombination mechanisms developed in the absence of plasma (outside the plasma zone) usually lead to the incorporation of less fragmented building blocks into the nascent macromolecular structures [72-75].

Graft-polymerization reactions initiated from plasma-activated polymer surfaces involve two distinctive and consecutive processes: implantation onto polymer substrate of active sites, like free radicals or reactive functionalities followed by the initiation of conventional graft-polymerization reactions in situ or ex-situ conditions depending on the stability of the active sites. Supramolecular structure of polysaccharides controlled by intramolecular and intermolecular hydrogen-bonding (conformation of macromolecules), existence of crystalline and amorphous zones, exerts a significant influence on both their physical and chemical properties. Conventional modification techniques performed on lignocellulosics to make them compatible with other polymers or to enhance their surface properties by grafting with some additives (such fatty acids) alter significantly the supramolecular order of the macromolecular networks, diminishing their inherent physicochemical characteristics. Cold plasma environments offer a unique way for modifying these materials without altering the bulk structures and characteristics. Intrinsic properties can be preserved in this way, materials with advanced performances and tailored properties can be obtained. Peroxide functionalities are possible to be formed and they can strongly interact with the counterpart on surface.

The interaction of the active species of a plasma with polymer surfaces involves electron mediated processes and positive ion-induced reactions. The last leads through neutralization reactions. To energy concentrations localized on macromolecular chains (electronically excited states) which can promote hemolytic bond cleavages leding to the formation of free-radical sites. These reactive centers lead to a large variety of functionnalization mechanisms depending on the reaction environments under in situ or ex situ plasma conditions. Several possible ways of grafting are given in Scheme 4.


H

H

H

H

OH

O

CH2

OH

O

OHH

O

HH

N2

plasma1

23

4

5

6

C

C

C

C

C

OH

O

CH2

O

OHH

O

HH 13

4

5

6

C

C

C

C

C*

*

+ R-COOH

OOC-R

OH

OH

O

CH2

OH

O

OH

O

H1

3

4

5

6

C

C

C

C

Active sites at different carbon atoms

Fragmentation

*

* *

*

2

O

CH2

O

OHH

O

HH1

3

4

5

C

C

C

C

C

2R OH

OH

O

CH2

O

OHH

O

HH1

3

4

5

C

C

C

C

C

OH

2(OOCR)

(R)

R'COOH

or other different ways with various probabilities

*

Scheme 4. Suggested reaction mechanism for nitrogen plasma induced molecular activation, fragmentation and grafting of the Spanish broom (Spartium junceum, syn. Genista juncea) fibres.

The trapped free radicals can “survive” in the polymer matrix and their intensities vary

significantly with the nature of fibres and plasma gases. A good correlation has been found between free-radical concentrations and ex situ plasma surface oxidation reactions. As it was mentioned the characteristic cellulose peaks are: C-OH, C-O-C (286.6 eV) and O-C-O (288 eV) and also the existence of O-C=O (288.7 eV) and O-CO-O (290.4 eV) can be noticed. The formation of new functionalities (O-C=O and O-CO-O) are possible through the cleavage of C1-C2 bonds of the pyranosic ring.

It was demonstrated that all possible four hydroxyalkyl radicals (1,2, 3) are generated as primary structures, through the hydrogen abstraction mechanism, but with a preference at C2 and C5 carbon atoms. Plasma irradiation produces preferentially the alkoxy-alkyl radicals at C1 of the glucose units.

CONCLUSIONS The research was concentrated to obtain fibres with new or significantly improved

properties, with tailored functionalities for special applications starting from natural fibres based on concept of development of environmentally friendly and energy-efficient processing by surface modification of fibres, yarns and fabrics.

New fibres with enhanced properties based on existing natural compounds have been obtained by plasma grafting of the Spanish broom (Spartium junceum, syn. Genista juncea) fibres with carboxylic acids. The heterogeneous esterification reaction with five different acids was realized in mild conditions with a high yield. The characterization carried out with


X-ray photoelectron spectroscopy (XPS), XRD, SEM, differential scanning calorimetry and thermogravimetry showed that the individual fibers were covered with the corresponding esters by partial degrees of substitution of the cellulose and that the surface degree of substitution of the cellulose fiber was higher than for the bulk, showing that the esterification reaction was a surface phenomenon, mainly in cold plasma conditions.

New biofibres obtained could be processed in products which should improve comfort performance and enhance micro-climate of bed rooms. Develop additional properties enhancing natural properties of vegetable fibres lead to promotion of fibers with lower ecological impact, promotion of integrated European production chain

These fibers could be also useful as reinforcements in various composites (mainly containing polyolefins).

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In: Food, Diet and Disease Copyright 2009 Innovations And Solutions, Inc. Florida USA Editors: Rakesh Sharma, Bharati D Shrinivas

1

1876-3960/09 2009 Innovations And Solutions

Open Access

Bioactive Foods and Nutraceutical Supplementation Criteria in Cardiovascular Protection

Rakesh Sharma1,*, Bharati D Shrinivas*

1Department of Bioengineering, Florida State University & Tallahassee Community College, Tallahassee, FL 32304, USA

Abstract: Nutraceuticals are natural and commonly found in bioactive foods or whole plants to keep energy balance in the body and promise substantial therapeutic value in cardioprotection. Major cardioprotective bioactive foods and nutraceuticals are now part of nutrition supplements at nonprescription counters and their self-prescription is increased at large scale. The literature suggests the growing use of new bioactive foods and nutraceuticals in cardioprotection and management. The biochemical mechanisms of nutraceutical action in cardioprotection are poorly reported. The literature indicates the success of fish oils, nutaceuticals in vegetable fat-free diets and restricted life style to enhance cardioprotec-tion. The present paper highlights the need and benefits of newly introduced bioactive foods and revisits the cardioprotec-tive mechanisms of bioactive foods and nutraceuticals. Broadly cardioprotective nutraceuticals are polyunsaturated fatty acids, antioxidants, omega-3 fatty acids, vitamins, minerals and dietary fibers. Most of the nutraceuticals act as biochemi-cal metabolites by direct intervention in intermediary lipid metabolism or regulating proteins of vascular system responsi-ble of ‘cardiovascular incapability’.

Keywords: Cardioprotection, immunity, nutraceuticals, metabolites, diet, fat, nutraceutical supplementation.

INTRODUCTION

The term “bioactive foods” was first defined as “foods, food ingredients or dietary supplements that demonstrate specific health or medical benefits including the prevention and treatment of disease beyond basic nutritional functions”. Now bioactive foods and nutraceuticals have emerged as potential supplements in cardiovascular and cancer preven-tive natural sources from food [1]. Why new bioactive foods are important? Bioactive foods are fortified with nutrients to make them more usable within daily recommended allowences (RDA). These nutrients are rich in vitamins, minerals and nutraceuticals or any food or part of a food that provides health or disease prevention benefits with higher nutrition values. Bioactive foods are served as cuisine line of frozen foods on shelf such as Cam-den(balanced meal program) for hypertension, high choles-terol, or adult-onset diabetes; Tropicana Products, orange juice; Procter & Gamble's FruitCal® -calcium citrate malate etc. Growth in the bioactive food market has also rocked new combinatorial chemistry and profoundly accelerated the pace of discovery of new bioactive foods such as new high-oleic soybean with no trans fatty acids that reduces heart diseases. Both food industries and pharmaceutical industries have roped up to use bioactive food, pharmaceutical and nutrition products - from drinkable yogurt to mainstream designer bone, heart, and digestive health foods to calcium chews,

*Address correspondence to this author at the Department of Engineering and Technology, Florida State University, Tallahassee, Florida 32304 USA32304, USA;*Zoology Deptt. RTM University of Nagpur, Maharastra, India E-mails: [email protected] and [email protected]

from sports nutrition bar makers to soy burger manufacturers – bioactive foods are poised to undergo very rapid growth in the coming years. Bioactive foods are designed basically meeting four consumer demands: taste, convenience, simple proposition and price. A successful bioactive food product's bioactive role needs its perceptible health benefit. If a health benefit is clearly understandable, or if the health benefit is clearly perceptible - such as weight loss or stress reduction, or can be easily measured - such as a product that reduces cholesterol, then the product is much more likely to succeed. Now interest is growing for use of bioactive foods in cancer prevention. Recently JIVA™ a bioactive food made of res-veratrol combined with garlic has been advocated as poten-tial cardiovascular prevention formula [2]. Similar bioactive foods are in market by Kellogg Co.-cholesterol-reducing foods; Ensemble products - psyllium high fiber reducing cholesterol; Johnson & Johnson -cholesterol-lowering mar-garine, Benecol®; Balance Bar Company, Nestlé, Vevey, Switzerland – PowerBar. These bioactive foods work on principle that cancer and heart disease are concerns of fa-tigue/energy and stress. Tropicana Ultimate Smoothie com-bined with Galaxy's soy, rice and oats Veggie Milk® base with Tropicana's fruit juices. It was estimated that major benefactors were subjects with heart disease (75%), cancer (81%) including breast cancer (48%), colon cancer (37%) and prostate cancer (25%) using bioactive foods.[3]. The bioactive foods as part of daily diet and lifestyle guidelines for prevention of coronary artery disease (CAD) have been evidenced as a major interest during the last few decades. The concept of bioactive foods was initially considered as natural foods to provide energy as recommended daily requirement in the body to maintain health and lipid lower-ing till year 1990. Later the importance of nutraceuticals was

2 Sharma,Shrinivas

realized as beneficial in different cardiovascular disorders with growing use of the nutraceuticals as self prescription in cardiovascular and developmental conditions in the last dec-ade. In new era of 21st century showed enormous growing awareness of nutraceuticals as emerging potent therapeutic supplements with accepted concept of nutraceutical medicine as new branch of ‘complementary and alternative medi-cine’(CAM). In last three decades, national and federal bod-ies accepted nutraceuticals as possible neutraceutical therapy in main stream of medical education and health. The healthcare industry demonstrated the shift of growing popu-lation from medical treatment of dreaded cardiac arrest to-wards non-prescription nutraceuticals as self-medication in acute coronary syndrome and coronary heart disease man-agement and prevention of stroke. The best examples to mention are Atkin’s diet for lipid lowering and CoQ10 for reducing thrombosis. The growing awareness of nutraceuti-cal benefits and shift of healthcare economics in favor of nutraceuticals brought neutraceutical medicine in spotlight of government health policy on systematic use of nutraceuticals in cardiac protection and control of various coronary heart and cardiovascular diseases. In last sixteen years, National Heart Lung Institute (NHLI) and other global efforts have documented the fact sheets and several health documents on nutraceuticals in cardiovascular disease control. The major efforts were devoted in investigation of cardioprotective ef-fect of active nutraceutical component(s) on reduced athero-sclerosis plaque development, reduced coronary occlusion, cholesterol desaturation to result the reduced risk of cardiac arrest, heart failure and reduced risk of atherosclerosis and hypertension in initial stages. In last two decades the use of nutraceuticals in prevention and disease control has been extended further as protective nutrition supplementation pol-icy of center of disease control (CDC) under its independent supervision. However, mechanisms still remain unproven and unvalidated but practice of newly discovered nutraceuti-cals as food supplements in cardioprevention is acceptable.

What are Cardioprotective Nutraceuticals in Bioactive Foods?

Nutraceuticals are natural bioactive chemical compounds common in bioactive foods as products supplied from nutri-tion industries. Nutraceuticals have value in health promot-ing, disease preventing or semi-medicinal properties. Nu-traceuticals are found as natural products from (a) the food industry, (b) the herbal and dietary supplement, (c) pharma-ceutical industry, and (d) the newly emerged bioengineered microorganisms, agroproducts or active biomolecules. It may range from isolated nutrients, herbal products, dietary sup-plements and diets to genetically engineered “custom” foods and processed products such as cereals, soups and beverages. Chemically the nutraceuticals may be classified as isoprenoid derivatives (terpenoids, carotenoids, saponins, tocotrienols, tocopherols, terpenes), phenolic compounds (couramines, tannins, ligrins, anthrocynins, isoflavones, fla-vonones, flavanoids), carbohydrate derivatives (ascorbic acid, oligosaccharides, non-starch PS), fatty acid and struc-tural lipids (n-3 PUFA, CLA, MUFA, sphingolipids, lecithins), amino acid derivatives (amino acids, allyl-S compounds, capsaicnoids, isothiocyanates, indols, folate, choline), microbes (probiotics, prebiotics) and minerals ( Ca, Zn, Cu, K, Se) [4]. However, the nutraceuticals were

reported as active natural compounds. Majority of cardiovas-cular prevention evidence comes from clinical trials and animal studies [5, 6]. Self-described testimonies of nutraceu-tical medicine and its success accrued over years in favor of cardiovascular protection by lycopene, glucans(for cardio-vascular disease), noni Morinda citrifolia (for relief blood pressure, muscle pain) [7, 8]. Recently, the antiarrythmic effect of quercetin, lipid lowering effect of fish oils and beneficial effect of magnesium were reported including them as cardioprotective food supplements [9-11]. Dilemma of Nutraceuticals in Cardioprotection

Nutraceuticals may act as essential nutrient, as drug like, as regulatory biochemical metabolite and as phytohormone in the body as shown in Table 1. Most of the side effects of nutraceuticals remain undocumented and unnoticed. Re-cently, some prominent evidences are reported in favor of cardiovascular disease inhibitory metabolic activity of nu-traceuticals in the human body: 1. Nutraceuticals may act as essential amino acid drug like

essential nutrients. For example, tryptophan is needed for protein synthesis at low dose in humans [12].

2. The nutraceutical preparations containing phytosterols are effective in lowering LDL cholesterol [13].

3. Bovine milk fat globule act as anticardiovascular disease, anticholesterolemic, coronary heart disease [14].

4. The phytonutrients prevent myocardial cell proliferation and play significant role in the prevention of chronic de-generative diseases. Notable examples are ginseng, spi-rulina, gingko biloba, amino acids, glucosamine, chon-droitin and Aegle marmelos. Herbal and medicinal plants have shown significant inhibition of cell inflammation [15]. Phytoesterogens play role in reducing myocardial necrosis.

5. Vitamin C, vitamin E, β -Carotene, lycopene (caro-tenoids), lipoic Acid, glutathione(thiols) play role in car-diovascular disease prevention and inhibition of necrosis; Co-Enzyme Q-10, super oxide dismastase (enzyme), se-lenium, copper, manganese, zinc (minerals) act as anti-cardiovascular disease in cardiac cells[16].

7. Polyunsaturated fatty acids (PUFA) such as safflower oil, corn oil, soybean oil, mustard oil, evening primrose oil, flax oil, hemp seeds, borage seeds showed protective ef-fects in heart disease and stroke, inflammatory arthritis, inflammatory bowel disease, asthma, cardiovascular dis-ease [17].

8. Dietary fibers such as oats, dried beans, legumes, chicory as water soluble fibers, apple, orange, apricot, plum, pine apple contain 18-30% fiber contents. The fish oils and the vegetable sources such as cabbage, carrot, lettuce, on-ion, tomato containing 9 to 12 % fiber contents showed antioxidant and myocardial cell proliferation inhibitory properties [18].

9. Wild foods are other major source of nutraceuticals and phytoesterogens. Most of the wild plants, wild mush-rooms, wild fungi, wild vegetables, wild nuts, wild fruits and wild flowers as whole are considered as potential natural therapy alternatives rich in long chain omega 3 fatty acids [19, 20].

Bioactive Foods and Nutraceutical Supplementation Food, Diet and Disease 3

Table 1. Examples of Nutraceuticals are Shown with Their Benefits in Different Cardiovascular Diseases and Mechanism of Cardioprotective Action in the Body. The Structure of Active Nutraceuticals are Shown with Mechanism and Their Structure with Formula in Chemical Nomenclature

4 Innovations And Solutions, Inc. Sharma, Shrinivas


a-linolenic acid + antioxidant (20:3, n-3) or ALA Eicosapentanoic acid ++ antioxidant (20:5; n-3) or EPA Docosahexanoic acid ++ antioxidant (20:6; n-3) or DHA 10. Soy isoflavones, genistien, lycopene have emerged as

established cardioprotective nutraceuticals. The eicosap-entaenoic and docosahexaenoic acids reduce the oxidi-zability and thrombogenicity [21]

What Remains Still to Solve the Cardioprotection by Nutraceuticals?

The major issues that remain unsolved are the nutraceuti-cal side effects, dosage and mechanism, follow up conse-quences and mandatory guide lines of usage. The diet and lifestyle guidelines for prevention of coronary artery disease (CAD) have been evidenced as a major interest during the last few decades. Recommendations of the American heart Association (AHA 2007) have been reformed for better un-derstanding, based on new scientific evidences after publica-tion of guidelines in 2007 [22]. However, none of these guidelines emphasize the role of diet in patients with acute myocardial infarction (AMI) and stroke. Patients presenting with AMI are highly motivated to follow the advice of cardi-ologist due to serious AMI condition. AMI is associated with hyperglycemia, hyperinsulinemia, hypertriglyceridemia, free radical stress, rise in free fatty acid and pro-inflammatory cytokines, leading to endothelial dysfunction. There happens an acute generation of proinflammatory milieu among AMI patients which is known to cause disruption of atheroma plaque, resulting into re-infarction and death [24-29]. The synergy of these mechanisms in chronic disease is not clear in order to decide the intervention by nutraceuticals such as walnuts, ginko, vegetables [23-29]. Most American experts very deligently advise dietary patterns; including grains, vegetables, fruits, nuts, seeds and legumes, fat and oils based on research studies. Most of the times, the side effects of newly introduced products in market are not documented such as no recommendation for refined starches in the

prevention of endothelial dysfunction [24, 30]. However, there is no guideline about the type of oil and type of nuts depending upon the omega-3 fat and monounsaturated fatty acid (MUFA) content of these foods. While foods and bever-ages with added sugars and refined starches as well as excess of w-6, total and saturated fat and trans fatty acids, may be proinflammatory, increased intake of w-3 fatty acid and MUFA may be protective against surge of TNF-alpha,IL-6,IL-18 and adhesion molecules like VCAM-1(vascular cell adhsion molecule-1)and IVAM-1 caused by high glycemic, rapidly absorbed proinflammatory foods [27-32] (71-76). These foods are known to initiate a proinflammatory milieu in the body which is similar to that of AMI, causing further increase in complications among these patients.

In keeping these facts in mind, it is necessary to identify the concrete evidences of cardioprotective mechanism in both animals and clinical trials under controlled conditions with through investigations, careful nutrition formula design and success rate vs fallacies of earlier clinical experiences in favor of nutraceuticals in public use.

Animal Studies

A large volume of literature is available on nutraceutical inhibitory effect on cardiovascular disease cell growth based on observations of cultured cardiovascular disease cell pro-liferation, enhanced apoptosis, antioxidant action etc. Still attempts are in the direction of morphological, cytomorphic, histopathology evidences of nutraceutical induced lipid inhi-bition and thrombosis by using 3D localized molecular imag-ing techniques. Previous studies on micro-MRI and immu-nostaining suggested the reduced apoptosis in experimental rat, mice, rabbit, porcine, dogs experimental models [33]. Major evidence was the reduced oxidative stress, slowed

6 Innovations And Solutions, Inc Sharma,Shrinivas

down apoptosis, reduced proliferation, less plaque size, less necrosis and poor atherosclerosis growth in treated groups [34]. The mechanism of these nutraceuticals are still not established and it remains to investigate more scientifically diet controlled experimental methods [35-37]. Moreover the beneficial effects of nutraceuticals in experimental animals were reviewed and two third literature reports on nutraceuticals are documented on experimental animal cardiovascular disease studies as either reviews or animal bench experiments on cardiovascular disease prevention. The clinical evidence of nutraceutical cardiovascular disease prevention success is still based on biochemical mechanisms of nutrients in diets reported over several decades. Some mechanisms of nutraceutical action are reported as immune modulatory, induced apoptosis, removal of free radicals, inhibited cell proliferation, inhibited necrosis. New ayurved (Indian traditional medicine) concepts are also emerging as powerful nutraceuticals in cardiovascular disease prevention [38]. The growing literature on mechanism of nutraceutical action in the cardiovascular disease is supporting the extended benefits of nutraceuticals but it further needs more investigations as described in following separate section of new literature evidences [39-41].

Clinical Trials

Singh and coworkers used 400g/day of fruits, vegetables and legumes in conjunction with mustered oil to decrease the risk of hypertension, diabetes and CAD in 1090s similar with DART, DART II, GISSI [42]. This diet was re-examined by DASH investigators and subsequently by other group to ob-serve the reduced risk of hypertension in USA [43, 44]. In further randomized, controlled intervention trials, Singh et al. 2002, 2008 administered 400 g/day of fruits, vegetables and nuts(almonds and walnuts) and another 400g/day of whole grains including legumes in conjunction with 25-50g/day of mustered oil (ALA 2.9 g/day) in patients with high risk of vascular disease, which showed significant bene-fit [23, 45-47]. Other workers also found a beneficial effect of fruit, vegetables, nuts and ω-3 fatty acids (EPA+DHA 1.8 g/day) rich foods to patients on risk of coronary artery dis-ease [46, 47]. A randomized, double blind placebo controlled trial on 300 patients after MI supplemented with EPA+DHA 3.4-3.5 g/day or corn oil showed no change. Increased intake of monounsaturated fatty acid and ω-3 fatty acids have been suggested to be protective against diabetes and metabolic syndrome whereas increased consumption of trans fatty ac-ids, saturated fat and refined starches can predispose CVD. India has a rapid economic development causing increased consumption of salt, tobacco, fat, sugar, and energy in the last four decades. There is increase in per capita income, gross domestic product, food production and automobile production in the last four decades. This period from 1970 to 2008 has witnessed marked changes in nutraceutical rich diet and lifestyle, particularly in the urban populations among Indians. New bioactive factors have came in light of cardiovascular mechanisms likely af-fected by nutrients such as: 1. iodine induced T3 and nitric oxide decreases SVR by dilation of the arterioles protein kinase akt pathway via smooth muscle relaxation through nuclear transcription mechanisms [48]; 2. fish consumption

>300 gm/week reduced non-fatal coronary syndrome (CARDIO2000 study) [49]; 3. transcription of the positively regulated genes (alpha-myosin heavy chain (MHC) and cal-cium ATPase, SERCA2) both downregulate the expression of negatively regulated genes (beta-MHC and phospholam-ban) to increase cardiac contractile performance. There is possibility of nutraceutical protection to repair cardiac con-tractility and improved ejection time (LVET) [49, 50]; 4. improved cardiac output, reduced cardiac preload (low renin state and decreased erythropoietin secretion), increased vas-cular resistance, bradycardia, slightly depressed myocardial contractility and some increase in LV mass [51]; 5. IF chan-nel, L-type and T-type calcium channel, potassium channel and the ryanodine channel contribute to pacemaker functions and heart rate [52]; 6. dyslipidemia due to total cholesterol and low density lipoproteins (LDL) cholesterol, triglyc-erides, very low density lipoproteins (VLDL), intermediate-density lipoproteins, apoprotein A-1 and apoprotein B are observed as well [53, 54]; 7. cholesteryl ester transfer protein and hepatic lipase, increased levels of high-density lipopro-teins(HDL); 8. endothelial dysfunction, increased arterial stiffness, increased vascular resistance, and hypercoagulabil-ity with coronary artery disease [55]. However the effect of bioactive foods is not known if bioactive food affects cardiovascular morbidity or mortality. It might be beneficial to use bioactive food or nutraceuticals as supplements simultaneously with cardioprotective drug therapy. Recently reported noninvasive imaging methods such as Doppler echocardiography, carotid intima-media thickness, pulsed tissue Doppler imaging, cardiac MRI and radionuclide ventriculography to evaulate preejec-tion/ejection ratio systolic dysfunction may be more useful to establish the beneficial effect of nutraceuticals. Overall, tri-als evaluating cardiovascular mortality and mortality have yielded conflicting results [56].

Biochemical Basis of Nutraceuticals in Cardiac Prevention

Natural vegetables, herbs, plants, wild foods are complex in structural composition. The biochemical basis of individ-ual source of these foods is not explored due to their com-plex nature. Some of the evidences are in favor of the active food principles as nutraceuticals to show cardioprotective or preventive supplements. Some of nutraceuticals are in the phase of clinical trial or already available as food supple-ment. Complementary and Alternative Medicine is emerging in prevention of chronic coronary and heart diseases as safe practice because of the high risk of mortality and long-term morbidity associated with surgical procedures of coronary artery disease and high side effects of chemotherapy. Herbal medicines have shown reduced myocyte cell necrosis in cul-tured cells. The vitamins, minerals, dietary fat play a role in relation to cardioprevention and control. The mechanisms of nutraceutical action can be discussed broadly in following categories based on active metabolites present in nutraceuti-cals. 1. Niacin-bound chromium is reported to enhance myocar-

dial protection from ischemia-reperfusion injury [57]. 2. Mechanism of the antithrombotic effect was invented by

dietary diacylglycerol in atherogenic mice [58].


3. Protective effect of potassium against the hypertensive cardiac dysfunction was associated with reactive oxygen species reduction [59].

4. The atherogenic process is reduced by regulation of co-enzyme Q10 biosynthesis and breakdown.

5. The n-3 fatty acids reduce the risk of cardiovascular dis-ease. The evidence was explained and mechanisms was explored.

6. Mediterranean diet and optimal diets play role for pre-vention of coronary heart disease.

7. Alpha-tocopherol therapy was evidenced to reduce oxidative stress and atherosclerosis.

8. Genetic deficiency of inducible nitric oxide synthase re-duces atherosclerosis and lowers plasma lipid peroxides in apolipoprotein E-knockout mice.

9. Glutathione is the liver's most abundant protective con-stituent of antioxidant glutathione reductase enzyme. Glutathione functions as a substrate for the two key de-toxification processes in the liver: 1. transforming toxins into water soluble forms, 2. neutralizing and "conjugat-ing" with toxins for elimination through the gut or the kidneys. If either of these processes is impaired for any reason, toxins will accumulate in the body and lead to disease. The best nutrition with liver cardiovascular dis-ease focuses on improving the body's glutathione re-serves [60].

10. The Soy isoflavone Haelan951 (genistein and genistin) and garlic allicin were reported to have some role as a cardioprotective in humans [61]. Beta-glycoside conju-gate, genistin is abundant in fermented soybeans, soy-bean products such as soymilk and tofu. Beta-glycosyl bond of genistin is cleaved to produce genistein by mi-crobes during fermentation to yield miso and natto. Soy sauce has high isoflavone but low miso and natto con-tents. How much soy isoflavones needed? 1.5-4.1 mg/person miso isoflavone and 6.3-8.3 mg/person natto respectively [62].

11. Green tea has always been considered by the Chinese and Japanese peoples as a potent medicine for the mainte-nance of health, endowed with the power to prolong life [63].

12. The cardiovascular disease has been reported associated with vascular endothelial growth factor [64].

13. Some herbal plants act as cardioprotective medicine. The herbal extracts are known to reduce the circulating mark-ers of inflammation, including C-reactive protein (CRP), interleukine-6 (IL-6), tumor necrosis factor-α (TNF-α), serum amyloid A (SAA).

14. Combination of garlic, ginko biloba, herbs with rever-astrol inhibited a full 92 percent of age-related gene changes in the heart [2, 64].

Lipid Metabolism and Fatty Acid Modifiers as Basis of CVD and role of Nutraceuticals

Lipid metabolism is established a major factor in cardio-vascular protection by supplementing omega fatty acids as described with recent developments for interested readers.

The possible reversal of increased total cholesterol, increased LDL cholesterol, apolipoprotein B and decreased HDL con-centrations in cardiovascular patients on bioactive foods and nutraceuticals is controversial [65]. In several trials, total cholesterol levels, HDL, LDL-cholesterol, triglycerides, apolipoprotein A and B and lipoprotein A were not signifi-cantly improved with nutraceutical or vitamin-mineral treat-ment [65]. A trend was noted in favor of nutraceutical ther-apy with reduced total cholesterol TC level >240 mg/dL, LDL > 155 mg/dL TC levels (significant only for >240 mg/dL), and Body Mass Index > 25 kg/m2 was associated with better improvements [66].

Control of lipid metabolism and cholesterol desaturation in the blood has been cited as major factor in cardiovascular disease. The nutraceuticals have been reported as inhibitors of cholesterol synthesis and enhancing HDL lipoproteins in the body. To explain the effect of nutraceuticals, two major mechanisms play significant role in cholesterol saturation and lipoprotein synthesis. First, HMG CoA synthase enzyme controls the mevalonate to HMG CoA formation that subse-quently used in cholesterol formation while cholesterol oxi-dase enzyme oxidizes cholesterol to desaturate it. Second, cholesterol esterification by LCAT and ACAT enzymes and subsequently apoprotein binding controls the lipoprotein formation [67]. Mainly high density lipoprotein (HDL) plays significant role in scavenging cholesterol from blood as shown in Fig. (1). Low density lipoproteins (LDL) transport is controlled by LDL receptors in the cells. LDL lipoproteins get metabolized by lipo-oxygenase pathway as shown by Fig. (2).

The anti-inflammatory effects and antithrombogenic ef-fects of ω-3 fatty acids are eicosanoid–dependent process. More intake of EPA and DHA fatty acids increases these fatty acids in tissue, cellular and circulating lipids, along with a simultaneous reduction in ω-6 fatty acids. EPA acts as a substrate for both cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) enzymes to make derivatives from arachidonic acid (ΑΑ) such as leucotriene B5 (LTB5) is only about 10% as potent as LTB4 as a chemotactic agent and in promoting lysosomal enzyme release). The ω-3 fatty acids also result with reduced formation of thomboxane-2 (TxA2)and prostacyclin I2 (PGI2), as AA is a TxA2 and PGI2 precursor and inhibiting platelet aggregation (a less throm-bogenic state) as shown in Fig. 2). The fatty acids display three major beneficial effects: 1.lipid lowering in blood; 2.antiarrhythmic effect in CHD; 3. antithrombotic effects; 4. anti-atherosclerotic and anti-inflammatory effects; 4. improved endothelial function; and 5. lowering blood pressure. From biochemistry standpoint, the beneficial effect of ω-3 fatty acids on blood lipids is by the stimulation of the gene expression of lipoprotein lipase (LPL) enzyme in human adipose tissue with increase in the LPL mRNA. It results with post-heparin LPL activity, in conjunction with the low-ering effect of these fatty acids on the triglyceride levels, postprandial lipaemia and the levels of the highly athero-genic, small and dense LDL particles [24]. These fatty acids increase the expression of genes encoding enzymes critical to hepatic and skeletal muscle fatty acid β-oxidation while repressing genes encoding glycolytic, lipogenic and choles-

8 Innovations And Solutions, Inc. Sharma,Shrinivas

terolgenic enzymes. This twofold action results in the de-crease in lipid synthesis and a subsequent increase in lipid oxidation favorable for nutraceutical intervention. Despite the fact that the exact mode of action of ω-3 fatty acids is not fully understood, it is speculated that ω-3 fatty acids interact with three nuclear receptors–hepatic nuclear factor (HNF)-4α, liver X receptors (LXR) α and β and peroxisome prolif-

erators- activated receptors (PPARs) α, β and γ – and by regulating the transcription factor sterol regulatory element binding proteins (SREBPs) 1 and 2[25]. Ω-3 fatty acids also decrease excitability and cytosolic calcium fluctuations of ventricular myocytes via inhibition of Na+ and L-type Ca+2 channels. The mechanisms of action of ω-3 fatty acids have not been fully elucidated.

Fig. (1). Proposed role of LDL oxidation in the thrombogenesis and endothelial dysfunction. LDL crosses the endothelium in a concentra-tion-dependent manner and can become trapped in the extracellular matrix (1). The subendothelium is an oxidizing environment, and if the LDL remains trapped for a sufficiently long period of time, it undergoes oxidative changes (2). Mildly oxidized forms of LDL contain bio-logically active phospholipid oxidation products that affect the pattern of gene expression in endothelial cells (ECs), leading to, among other things, changes in the expression of monocyte binding molecules (designated X-CAM), monocyte chemoattractant protein (MCP-1), and macrophage colony stimulating factors (CSFs) (3). These factors in turn promote the recruitment of monocytes (4) and drive their phenotypic differentiation to macrophages (5). Further oxidation leads to alterations in apolipoprotein B such that LDL particles are recognized and in-ternalized by macrophages (6), progenitors of the lipid-laden foam cells. Marked increases in lipid and cholesterol oxidation products render the LDL particles cytotoxic, leading to further endothelial injury (7) and favoring further entry of LDL and circulating monocytes and thus a continuation of the disease process.

Fig. (2). The omega 3 and omega 6 fatty acids synthesize ecosanoids in the myocardial cells.


TREATMENT RECOMMENDATIONS FOR NEUTRACEUTICALS IN CARDIOVASCULAR PREVENTION

Who Need the Alternative Approaches of Nutraceuticals in CVD

Children below 18 years probably do not need nutraceu-ticals. Adults over 20-40 years need nutraceuticals and moni-toring CVD. Persons over sixty years in age, need CVD/ CHD watch and nutraceuticals as mandatory daily dietary supplements in practice. These senior persons may show the following major symptoms as causes of cardiovascular disorders and CVD development [68].

• Poor cytokines, inflammatory proteins gradually lead to apoptosis, loss of immunity

• Arteries and veins (and other tissues) become less elastic, as evidenced by our skin. Blood pressure may rise, as arteries lose their elasticity. (The amino acid taurine, found in fish, softens arteries and veins, as well as other connective tissue.)

• Inflammation and cholesterol-filled growths (plaques) in our blood vessels reduce their rates of flow. The loss of elasticity causes the heart to pump with less power and force.

• Insulin levels begin to rise as old cells become less re-sponsive to insulin, and the pancreas increases its output to compensate. This eventually leads to Type II diabetes and pancreatic cardiovascular disease in which old cells no longer respond to insulin and end up with heavy cardiovascular damage and cardiovascular disease.

• Kidneys lose reserve capacity, gradually fail to do normal function and develop cardiovascular disease.

• Reduced cell mediated immunity and humoral immunity leads to immune deficiency and cardiovascular disorders.

Present State of Art on Nutraceutical Medicine in Cardiovascular Prevention

FDA requires appropriate scientific evidence regarding safety of nutraceutical use as daily prescription. However, new recommendations suggested that daily diet must contain 6.25 grams of soy protein per serving, micro-compound al-licin (a small component of garlic) ad libitum amount, ecos-apentanoic acid/docosaheaxanoic acid as polyunsaturated fatty acids (PUFAs) from fish or fish oils. The complemen-tary medicine and alternative medicine approach is emerging as regulated tool to prescribe the norms of nutraceuticals as daily supplements in cardiovascular disease and other diseases [69].

Insurance and Prescription

National and federal agencies such as NCI and FDA need evidences and established data in large trials to approve nu-traceuticals in clinical practice. In lack of such evidences and database, still nutraceutical practice remains at the door steps as nonprescription self-prescription available on counter. As a result, insurance companies still shy to accept nutraceuti-cals as prescription.

Government Policy: Criteria of Suggested Practice of Nutraceuticals in Cardiovascular Prevention

The awareness of complementary and alternative medi-cine (CAM) is increasing rapidly among common public in developed countries [69]. Government agencies are actively participating in safe delivery of bioactive foods and dog-watch if any side effect. Several government reports have showed positive role to introduce new functional foods & nutraceuticals in CVD/CHD prevention in favor of guava, dietary fibers, soy, phystoesterogens, herbs, cruciferous vegetables [70]. Both bioactive food and nutraceuticals in diets were suggested as preventive in cardiovascular disease. Main causative factors of cardiovascular disease were free radicals, vitamin C,D,E deficiency, selenium deficiency and loss of cellular immunity in patients on daily diet [71].

Recently, National Heart and Lung Institute put forth the efforts on alternative ways of cardiovascular disease preven-tion as public awareness to main focus on life style, preven-tion and control care measures, eating habits, hazardous con-taminants with several successful attempts of antioxidants, garlic, vitamins [72]. Under supervision and dogwatch, most of the bioactive foods on counters and nutraceuticals are marketed as some of them listed in Table 2.

Bioactive Foods and Nutraceuticals in CVD/CHD: A Survey

In recent years during 2002-2008, the major focus was on more evidence based wider use of omega 3 fatty acids com-bined with multivitamin-multimineral and isolated bioactive components from plants and functional foods in various car-diovascular disease types. In last 4 years maximum efforts were devoted on reviews and compilation of evidenced ex-perimental results on vegetarianism in reducing cardiovascu-lar disease progress and identification of associations of ac-tive food components in diet with reduced lipids, myocardial necrosis and apoptosis. However, NHLI views that sequen-tial events during the nutraceutical treated cell growth or arrest cardiovascular disease are controversial [73]. The use of fish oils in elderly patients was revisited if any relation with arrhythmia and contractility. The literature during years 2002-2008 suggested major information for following: 1. direct link of vitamins, minerals in cardiovascular disease prevention; 2. new bioactive food components with new mechanism of lipid lowering; 3. more controlled trials and regulated studies under federal support; 4. new awareness of unpopular foods and common shelf food supplements in car-diovascular disease prevention; 5. new federal and statuary guidelines on nutraceutical recommended allowances and marketing.

The following information is grouped based on literature on nutraceuticals and nutraceuticals in cardiovascular disease management with major focus on controlled randomized trials in experimental cardiovascular diseases and clinical cardiovascular disease subjects. The description is divided into three sections. Bioactive foods and nutraceuticals in cardiovascular pre-vention during years 2000-2008: Nutraceuticals and local foods were suggested as readily available and their use with possibility of alternative pharmacotherapy to prevent cardio-


vascular diseases [73-75]. Less known bioactive foods con-taining ephedra and caffeine were reported to improve elec-trocardiograhic and hemodynamic effects [75]. Clear cardio-protective role of vitamin E and antioxidant supplements was reviewed in prevention of cardiovascular diseases [76-81]. Homocysteine, taurine, vitamins and omega 3-fatty acids were reinvestigated and confirmed their value in cardiovas-cular prevention [82-86]. Mechanism of cardiovascular prevention by nutraceuti-cals: Mainly cholesterol rich dietary fats enhances the risk of coronary heart disease while omega 3/omega 6 fatty acids reduce the risk of cardiovascular diseases and play cardio-protective role in primary, secondary and late- onset diseases [87-89]. Interestingly, author described the excessive linoleic acid is manifested as ‘linoleic acid syndrome’ in coronary heart disease [89]. Conjugated lineleic acid was reported as protective against cardiac hypertrophy [90]. Omega 3 fatty acids mainly lower the blood lipids.

The possible reasons of cardioprotection by ω-3 fatty acids in bioactive foods were: • Lipid lowering (reduction of fasting triglycerides,

attenuation of postprandial triglyceride response) • Antiarrhythmic effects • Antithrombotic and other effects on the haemostatic sys-

tems (i.e. reduced platelet reactivity, moderately longer bleeding times, reduced plasma viscosity)

• Inhibition of atherosclerosis and inflammation via inhibi-tion of smooth muscle cell proliferation, altered eicosa-noid synthesis, reduced expression of cell adhesion molecules and suppression of inflammatory cytokines production (IL’s, TNF-α) and mitogens

• Improvement of the endothelial function [through en-hancement of nitric oxide – dependent and nitric oxide independent vasodilatation]

Table 2. The Table Represents the FDA Approved Nutraceuticals with Recommended Quantity and Sources of Nutraceuticals on Shelf in Super Markets

Nutraceuticals Quantity Needed Common American Sources

Vitamin D 400 IU a day(2000 IU) Walmart's "OneSource" multivitamins

Multivitamin-minerals As 1 pill daily Centrum Silver A-Z with minerals

Natural Vitamin E (4 tocopherols + 4 tocotrienols) Two 400 IU capsules a week (800 mg) GNC natural Vitamin E

Selenium 200 mcgs. a day Walmart's "OneSource" multivitamins

Aspirin or ibuprofen* Baby aspirin a day nonprescription counter

Chocolate (best if fat-free) ?, 3 servings home made, Food emporium

Green tea ?, 3 servings home made, Foof emporium

Lycopene Cooked tomato sauces Domino's Pizza

Fish (tuna, salmon, mackerel)** or EHA+DHA Two servings a week Fresh phytosterols at Publix

Soy "meat", cheese, milk Ad libitum Publix, at the edge of the produce section. mozzarrela, sausage, burgers

Broccoli, cabbage, cauliflower Sulfhydrals ad libitum Piccadilly's tastes pretty good.

Blueberries A few tablespoons a day Publix' frozen foods (N. side, S. aisle)

Strawberries 4 or 5 large a day Publix' frozen foods (N. side, S. aisle)

Old-fashioned oatmeal One ounce? Publix, all supermarkets

Legumes (beans) Two servings a week Publix, all supermarkets

Low-fat blueberry yogurt 2 or 3 times a week Publix, all supermarkets

Yellow vegetables Ad libitum Publix(Piccadilly's tastes pretty good).

Purple grape juice, or red wine A glass a day Publix, for Welsh's grape juice

Turmeric roots Two capsules daily GNC natural body products

Herbs Two pills daily St John Warts natural source

Garlic, Soy products Ad libitum Walmart's "OneSource" ampoules

*aspirin and ibuprofen primarily act as anti inflammation. (Other agents such as fish also have anti-inflammatory properties.); **Tuna and mackerel contain mercury, dioxin, and PCB's, . The salmon fish is safe. Winn Dixie farm-raised salmon. canned salmon provides omega-3 fatty acids and , taurine which are vital to the nervous and cardiovascular systems (modified from the Source[Sharma 2009]).


• Improvement in blood pressure. III. CVD/CHD in the human body and nutraceutical pro-tection: Supplementation of fish oils dominate the scenario of lipid lowering in cardiovascular diseases [90]. New can-didates such as cinnamon, ginko biloba, bioactive peptides have been introduced in the list of nutraceuticals with cardioprotective properties [85, 91, 92]

Challenges, Hypes, Hopes and Futuristic role of Nutraceuticals in Cardioprotection

Most of the success of nutraceuticals is based on self-prescription and own individual experiences. Still it is far to realize the miraculous benefits of nutraceuticals unless con-trolled clinical trials support the evidences and facts of nu-traceutical preventive therapeutic efficacy. Major challenge is early detection of cardiovascular disease and timely effec-tive treatment. In spite of all tools available, cardiovascular disease is major health hazard. The major available data on nutraceutical benefits in cardiovascular disease comes from epidemiological health and population statistics. The reduced cardiovascular disease incidence due to nutraceuticals seems hype but greater hopes are anticipated with advancements in food science. However, still cardiovascular disease remains a major threat because of high mortality compounded with incomplete success of chemotherapy and surgery interven-tion. In future, bioengineered nutraceuticals will play signifi-cant role in CVD prevention as alternative therapeutics.

CONCLUSION

Bioactive foods with rich nutraceuticals still are growing in number as healthy food products introduced by companies and investigations suggest high hopes of nutraceuticals in cardiovascular disease prevention. The primary focus still remains on dyslipidemia and lipid lowering by fish oils and bioactive foods. The role of governments and globalization will certainly support the health risks and clinical trials on new bioactive foods and nutraceuticals. The nutraceuticals are becoming popular as they are harmless and natural food constituents. The nutraceuticals are still food supplements and last 5 years demonstrated enormous change in the per-ception of nutraceuticals as cardiovascular disease preven-tive and therapeutic supplements in cardiovascular diseases of different organs.

ACKNOWLEGEMENTS

The author acknowledges the opportunity of engineering and biotechnology internship under supervision of Dr Ching J. Chen at FAMU-FSU College of Engineering, Tallahassee, Florida.

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Received: July 29, 2009 Revised: September 3, 2009 Accepted: September 5, 2009 © Rakesh Sharma, Bharati D Shrinivas; Licensee.

This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

Comparison of Cholesterol Lowering Diets: Apple, Casein

Cytochrome P450 protein and Cholesterol 7α Hydroxylase Activities in Hamsters

Rakesh Sharma1, Rakesh K Tandon2

Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi 110029 India

Abstract

Lithogenic diet, casein and apple fiber diets were fed to hamsters for 3-5 weeks. For control group, animals were fed on normal Purina chow without any supplement. The cholesterol lowering effect of lithogenic diet, casein and apple diets were compared. After dietary regimen, animals were screened for any gall stone formation. The isolated liver microsomes were separated from animals and tested for the cholesterol-7α

Hydroxylase (CH) enzyme activity measurement in all three groups. The control animals did not show any gall stone formation and their CH enzyme activities were normal. The lithogenic diet showed significantly enhanced CH enzyme activities while animals fed on casein and apple diet regimen showed moderate increase in microsomal CH enzyme activity indicated cholesterol lowering in liver. In conclusion, cholesterol 7α hydroxylase may be a biomarker of cholesterol status in the body and microsomal CH enzyme may be lowered down after treatment of casein and apple diets. Key words: cholesterol 7α hydroxylase, apple, casein, hamster, gall stone, cholesterol lowering

Introduction Five million Americans suffer from some type of diagnosed symptomatic cholesterol saturation disorder while an even larger number are believed to be suffering from an undiagnosed cholelithiasis related disease. The cholesterol saturation in bile and blood is a common problem as hypercholesterolemia and it leads to gall stones mainly complicated with atherosclerosis, obesity and fatty liver sooner or later [1,2,3]. Dietary fibers, bran, pectins as bioactive foods have proven effective in serum cholesterol lowering or hypocholesterolemic effect. Apple dietary fiber and rice bran, a predominantly insoluble fiber source was reported to show hypocholesterolic effect in hamsters [4]. Apple fiber is among top five high fiber diets rich in cellulose, hemicellulose, lignin, pectin contents with potentials of hycholesterolemic effect[5,6]. Having evidences of their hypocholesterolemic effects, the mechanism of these dietary

1Present corresponding address: EB-5, Saket colony, New Delhi 110023 India 2 Present corresponding address: Department of Food and Nutrition, Florida State University, Tallahassee, Florida 32304 USA

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Foods,diets and disease (C)2009 Innovations And Solutions,Inc Editors:Rakesh Sharma,Bharati D Shrinivas _____________________________________________________________

fibers still is not clearly defined how cholesterol concentration in bile and liver is regulated by enzymes. Cholesterol is ultimate saturated lipid synthesized from precursor acetyl CoA (formed as end product of glycolysis) to mevalonate, sequalene and farnesyl phosphate intermediates through several biochemical reactions. Cholesterol serves as precursor of mobile lipids in the blood and bile emulsion made up of very low density (VLDL) low density (LDL), and high density (HDL) lipoproteins. In bile, cholesterol is synthesized from its precursor acetyl CoA to hydroxyethyl CoA and further to mevalonate intermediate precursor to synthesize cholesterol as shown in Figure 1. In catabolism, cholesterol breakdown takes place by cholesterol 7α hydroxylase enzyme[7]. The hydroxyl group on 7th carbon in cholesterol plays a major metabolic regulatory role in the action of cholesterol 7α hydroxylase enzyme activity. Dietary cholesterol controls the biosynthesis of cholesterol by inactivating the existing 3-hydroxymethylglutaryl CoA reductase (HMG-CoA Reductase) and suppressing the synthesis of additional reductase [8,9]. We showed the dietary fiber binds and removes the extra bile acids from the system and thereby causes cholesterol to be converted into replacement bile acids. However, postcholesystectomy develops symptoms despite of function gall bladder [10] In previous experiments, dietary supplemented bile acids and salts in combination showed cholesterol desaturation and reduced gallstone formation in animals and human both [11-17]. The major factor of cholesterol desaturation was the physical phase transformation of cholesterol into bile cholesterol esters and metabolic desaturation of cholesterol because of cholesterol conversion into different lipoproteins [18-23]. In present study, role of hepatic cholesterol 7α hydroxylase in cholesterol lowering and its regulation or reduced HMG-CoA: Cholesterol 7 α hydroxylase ratio is explored by apple diet supplementation to hamsters.

Materials and Methods Animals: Hamsters weighing 150-225 gm were locally raised at animal laboratory at All India Institute of Medical Sciences, and placed in plastic cages fed on cholesterol rich diet for the period of one-four weeks under constant supervision for any change in weight as per Institute Animal Care and Use Committee. Animals were grouped in four groups: control group, lithogenic or high cholesterol group, apple supplemented group, and casein supplemented group fed on normal Purina (Rodent Ralston Purina), high cholesterol diet, apple supplemented diet, casein supplemented diet respectively, with ad libitum water [24]. After different intervals, cytochrome P450 protein and CH enzyme were estimated as biomarker of time dependent cholesterol changes and cholesterol regulating CH enzyme in liver. The lithogenic high cholesterol dietary additive supplements* were used to fed animals and to develop cholesterol gall stones in animals as positive control animal group. Other two experimental animal groups included apple diet** and casein diet*** groups. The Purina diet contained sucrose (550 g/kg wt), vitamin mix (40 g/kg wt) *Cholesterol pure form 0.25% added in diet (w/w) **Apple pulp fiber was added in Purina 5% in diet (w/w) ***Casein powder (250 g/kg animal weight) was added in Purina 5% in diet (w/w)

At the end of each experiment, animals were killed by decapitation within 2-h span in an order determined to distribute time of death among different dietary treatments throughout that period. Livers were stored at -80 °C [24]. Isolation of hepatic microsomes: The hepatic microsomal preparations from hamster livers were done by liver cell isolation, density gradient ultrafast centrifuge at x105 g centrifugal force to isolate the microsomal fraction as source of cytochrome P450 and cholesterol 7α hydroxylase enzyme activity. The microsomal isolation was done by our modified method as described elsewhere [25]. Cholesterol enzyme estimation and enzyme activity: Cholesterol was delivered in Tween 80 as previously described and modified by our group [22]. Briefly, in a total volume of 500 μl, each assay tube contained 75 mmol/l phosphate buffer, pH 7.4, 1

mmol/l EDTA, 2.5 mmol/l DTT, 5 mmol/l MgCl2, 3 mmol/l NADPH, [4 14C]cholesterol (1.1×106 dpm, 100 000 pmol=0.2 mmol/l), 0.78 mg Tween 80 and 500 μg of liver

microsomal protein. The preincubation time during which NADPH was omitted was 2 min at 37°C. The incubation time was 20 min. The improved assay for cholesterol 7α-hydroxylase activity in hamster liver microsomes was performed as following [22]. In a total volume of 500 μl, each assay tube contained 75 mmol/l phosphate buffer, pH 7.4, 1 mmol/l EDTA, 0.5 mmol/l DTT, 5 mmol/l MgCl2, 1 mmol/l NADPH, [14C]cholesterol (1.1×106, 26 000 pmol, 52 μmol/l), 4.5 mg HPBCD,

10 mmol/l glucose-6-phosphate, 2 U glucose-6-phosphate dehydrogenase and 250 μg of

liver microsomal protein. Assay tubes were incubated at 37°C in a shaking water bath (Maxi-shake, Heto, OSI, France). The preincubation time during which only NADPH was omitted was 5 min and the incubation time following initiation of the assay with NADPH was 6 min. The reaction was terminated by adding 40 μl of 5 mol/l NaOH. Zero-controls were always run in parallel by the addition of 5 mol/l NaOH at the beginning of the preincubation. The sterols were extracted (after neutralization) with 4.3 ml of dichloromethane–ethanol (5:1, v/v, plus 1.2 ml of H2O). A mixture of unlabeled 7α- and 7β-hydroxycholesterol was added and [14

C]7α-hydroxycholesterol was separated from 7β-hydroxycholesterol by thin-layer chromatography (TLC) on silica gel G using a double migration technique with ethyl acetate–hexane (1:1, v/v). In all cases, activity was linear with respect to the amount of microsomal protein added and the incubation time. The amount of endogenous cholesterol present in assays ranged from 4.75–5.5 μg for 250

μg of microsomal protein. Enzyme cholesterol 7α hydroxylase reaction and effect of cytochrome P450 and cholesterol additives: In control and apple diet supplemented animal liver microsomal preparations, cholesterol 7α hydroxylase enzyme activity were mesaured at different concentrations of cytochrome P450 protein. In other set of experiment, cholesterol 7α

hydroxylase activity was measured at different cholesterol concentrations added in the reaction mixture.

Results The hamsters were active healthy and responsive throughout the span of study. The weight of animals varied in the range of 20%-40% after lithogenic diet. The animals did not show any symptoms of indigestion after dietary treatment of casein and apple fiber. The treatment of casein and apple fiber showed measurable difference in the CH enzyme activity in microsome. However, control animal CH activity remained constant through out study. The CH enzyme activity was linearly changed with the hydroxylation of cytochrome P450 protein. The cytochrome P450 hydroxylation was proportional to the cholesterol added in the enzyme reaction medium. The treatment of high casein and apple fiber contents in hamsters did not affect the enzyme activity in reaction medium and medium conditions. Table 1: Cholesterol 7α hydroxylase specific activities in animals fed on different diets. Diet Micrososomal Cytochrome P450/mg Cytochrome P450/mg protein Cholesterol 7α protein (total) (fraction A) Hydroxylase* _________________________ _________________________ Content 7α hydroxylase content 7α hydroxylase Control 2.20 + 0.25 18.4 + 0.2 0.70 + 0.1 18.5 + 2.2 0.85 + 0.15 Lithogenic 1.79 + 0.20 10.2 + 0.9 0.65 + 0.1 16.5 + 2.0 0.75 + 0.20 Apple diet 3.45 + 0.50 24.6 + 2.0 1.2 + 0.15 20.5 + 2.5 1.8 + 0.15 Casein diet 2.81 + 0.30 12.2 + 0.2 0.48 + 0.5 -- -- Specific activity of CH enzyme* is expressed as pmoles cytochrome P450 catalyzed per minute.

Figure 1: The cholesterol 7α hydroxylase enzyme activities are shown as histogram

graphs. The cholesterol 7 hydroxylase enzyme activity and cytochrome 450 concentration in liver cell microsomes: The purity of microsomes was 96% based on microscopy. In isolated microsomes the indicator enzymes showed the active state of microsomes without enzyme deactivation throughout the experiments. The enzyme activity was substrate concentration dependent and substrate specific. The end product of cholesterol conversion to hydroxycholesterol by CH enzyme indicated the nature of enzyme reaction as dependent on cytochrome P450 and NADP reduction at optimum pH 7.4 in Tris buffer medium. The addition of different cholesterol concentrations in enzyme reaction medium containing control microsome showed the linear change in CH enzyme specific activity. The cholesterol lowering effect of diets in different animal groups was in the increasing order of control group I > lithogenic diet II > casein group III > apple diet group IV as shown in Table 1 and Figure 1. The effect of each individual diet was distinct and CH activity was decreased in animals after diet treatment while enzyme reaction medium did

not show any change in cholesterol and cytochrome P 450 contents. It indicated further that enzyme activity changes were due to diet treatment.

Table 2: Effect of cytochrome P450 and cholesterol concentration on purified cholesterol 7α hydroxylase system (microsomal).

Diet (group) Concentration of Cholesterol 7α hydroxylase Cytochrome P450 concentration activity (in nanomoles)** added (picomoles/min) Control 0.250 25 n mol 36.5 Apple diet 0.250 25 n mol 35.0 Control 0.50 25 n mol 55.0 Apple diet 0.50 25 n mol 60.25 Control 1.0 25 n mol 57.0 Apple diet 1.0 25 n mol 65.5 Control + cholesterol 0.5 50 µM* 10.5 Control + cholesterol 0.5 100 µM* 28.5 Control + cholesterol 0.5 200 µM* 34.2 Control + cholesterol 0.5 400 µM* 55.0 Control: Group I; Apple diet: Group IV *Group I animal liver microsomes were added with different cholesterol concentrations. Animals on apple supplemented diets showed dependence of cholesterol 7α hydroxylase on cytochrome P450 protein content and cholesterol in the reaction mixture. With increasing cytochrome P450 protein in microsomes, enzyme elevated and apple diet enhanced further the enzyme activity. It indicated the apple diet effect independent from cytochrom P450 protein. The cytochrome P450 protein stimulated enzyme activity behavior was close to sigmoid pattern at constant cholesterol concentrations. Enzyme activity enhancement showed dependence with both cytochrome P450 protein concentrations and cholesterol concentrations. The cholesterol concentrations in reaction mixture and enzyme activities showed linear relationship as shown in Table 2. Discussion The cholesterol plays a major role in body as precursor of several vitamins, steroids, bile salts and synthesis of esters. The increased amount of cholesterol in the blood and tissues was discovered in the middle of past century and present time it is serious health hazard in stroke, cardiovascular and renal occlusion as cholesterol deposits around the walls with time [1,3,7,9,12,15]. However, in liver its role is more aggressive as cholesterol saturation during bile salt synthesis in bile formation. The balance between two enzymes for: mevalonate conversion in to HMG CoA by hydroxymethylglutaryl CoA reductase enzyme and cholesterol conversion to hydroxycholesterol by cholesterol 7α hydroxylase enzyme, is the key of cholesterol saturation or cholesterol desaturation in the body[8,9, 22]. The initial cholesterol saturation leads to effect bile saturation and slowly occludes vascular walls in renal, cardiovascular and cerebrovascular system[1,17,24].

An apple a day provides great phytonutrients (phyto=plant) and a good dose of fiber. One medium apple contains about 23 grams of carbs and 4 grams of fiber. Apple pulp processing and juice manufacturing is now a booming industry and recently apple products are identified with cholesterol lowering property [12,14,15,16]. The dietary fiber content in apple further adds up the double benefit of cholesterol lowering with renal dysfunction and enhanced fecal steroid excretion [12,14,15,16].

Table 3: Apple dietary composition is shown in whole apple and apple pulp in fiber content

Apple Pulp fiber Source Serving Content % energy

raw 1 small 55-60* 3.0 raw 1 med 70 4.0 raw 1 large 80-100* 4.5 baked 1 large 100 5.0 apple sauce 2/3 cup 182 3.6

Whole Apple raw 1 whole 17 0.8 dried 2 halves 36 1.7 canned in syrup 3 halves 86 2.5

*Important as dietary fiber is, laboratory technicians have not yet been able to ascertain the exact total content in many foods, especially vegetables and fruits, because of its complexity. Consequently, estimates vary from one source to another. Where differing estimates have been found, an approximation is given in the chart, as indicated by an asterisk. The same symbol following calorie content means the number of calories has been estimated, varying according to other added ingredients, especially fats and sugars, and to the size of the "average" fruit or vegetable unit. Reference: Bowel Function and Dietary Fiber: Warren Enker http://www.wehealny.org/healthinfo/dietaryfiber/index.html)

Lithogenic diets are advocated as inducing cholesterol saturation due to their action on cholesterol sterol to make cholesterol free as one of the major lithogenic effect. These lithogenic diets are considered to have negative effect on omega 3 and omega 6 fatty acids in the body and remain a major focus of their characteristic in plaque formation or atherosclerosis. The cholesterol lowering diets rich in omega 3 and omega 6 fatty acids have been investigated and reported to show major benefit in lipid lowering including cholesterol lowering [25]. The diets rich in fibers have been a major attention since last decade due to their double benefit to reduce lipids with cholesterol desaturation in the body and maintaining intestinal satiety free from any microvillus membrane damage [11,12,13,14,17].

The apple fiber was recently reported as major fruit content in the bulk of apple. The apple fiber was rich in cellulose, hemicellulose, lignin, and pectin contents and very compatible to the human digestive system free from any tropical sprue, pain or constipation[15,16,17]. The apple fiber has significant activity to lower down the cholesterol in the liver. The study focused on unique possibility of cholesterol lowering. The cause of cholesterol desaturation or cholesterol lowering could be either rapid cholesterol conversion to its hydroxylated product or it could be slowing down of cholesterol precursor HMG CoA formation to make less available cholesterol or to deprive the cholesterol formation. Here are several issues remain unanswered. First, the proposed cholesterol 7 hydroxylase activity is not solely cholesterol specific but includes other cholesterol derivatives or its analogues. Second, dependence of cytochrome P450 and NADP reduction is not solely represents hydroxylation reaction but other redox reactions active in the medium at optimal pH 7.4 and temperature 37 °C. Third, liver microsomes are very specific to intracellular conditions such as state of substrate, reaction medium composition, physiological variables etc. During liver cell fractionation and microsomal isolation there is every possibility of deactivation of enzyme and loss of enzyme protein. Fourth, the dietary effect on cholesterol lowering is not single biochemical or metabolic disorder but diet may also effect the other lipids and bile salts made from cholesterol. So, the dietary effect is a compound effect on group of lipids in both gall bladder and blood participating in lipid disorder. Fifth, cholesterol is not single lipid compound to represent the lipid disorder or bile disorder in the body as other lipid compounds also compete with cholesterol during bile formation or lipid lowering. Sixth, hypocholesterolemic effect is really increased by activation of fiber or pulp binding with alkaline earth metals, hydroxides, carbonates and phosphates or not. Recently, bioactive foods were highlighted to play major role in cholesterol desatutaion and lipid lowering such as guggulipid, garlic, fish fatty acids. Still fiber rich foods remain less known and there is paucity of information on the role fibers in lipid and cholesterol management. The study throws a possibility of the cholesteremic action of naturally available apple fibers and casein proteins in the body applicable to human. Casein is known to play a major role in cholesterol lowering and increasing HDL cholesterol in the body [27]. Still it remains to investigate if the biochemical action of both casein and apple diet is similar or analogous. Other available fiber rich foods do have potential of lowering lipids in the body such as soy, pectins, brans of cellulose, rice, oat [13,14,16,27]. Still main problem of isolated principle or fraction from complex fibers, diets and bran remains to solve while evaluation of cholesterol lowering based on enzyme activity is specific to cholesterol. How to enhance the hypocholesterolemic effect by apple diets? The method includes the steps of apple pulp processing by: (i) disrupting (rupturing) the cell structure of the pulp material, (ii) reacting the disrupted pulp material with a first reactant(s) capable of chemically modifying at least a portion of the pendant hydroxyl groups on the fiber material contained in the pulp to pendant groups capable of chemically coupling with alkaline earth metal ions, and then (ii) reacting the modified fiber material with a second reactant(s) capable of chemically coupling an alkaline earth metal ion to the modified pendant groups. An exemplary process includes the steps of

(aa) preconditioning the pulp by reacting the pulp with an aqueous solution of NaOH, (bb) reacting the preconditioned pulp with an aqueous solution of CH2 Cl-COOH to carboxylate the pendant hydroxyl groups on the fiber material contained in the preconditioned pulp, and then (cc) reacting the carboxylated fiber material with Ca(OH)2 so as to bond Ca.supplementation to the pendant carboxyl groups [13,14,16,27]. What present study highlights?

Present study is a preliminary report indicating an evidence of apple diet benefit in cholesterol lowering and a possible enzyme mechanism responsible of cholesterol lowering while cytochrome P450 protein may act as biomarker in serum to evaluate the cholesterol lowering. Current state of art is in the direction of investigation on dietary modification and effect on active or inactive forms of enzyme, better tracer techniques of enzyme estimation, better understanding of enzyme heterogeneity and physiological effects among species, apple processing advanced methods, and effective cholesterol lowering by dietary servings. The present study has limitation in predicting the effect of apple dietary fiber on cholesterol desaturation or lipid lowering as the study showed only evidence as preliminary data to support the possibility of cholesterol lowering by apple fiber supplementation to animals. It needs more investigations at different amounts of apple pulp supplementation in diet and estimation of cholesterol 7α hydroxylase in

optimized reaction mixture. Other issue is cytochrome P450 protein may not represent as true cholesterol 7α hydroxylase enzyme in liver. It needs additional experiments of isolation of apple dietary fiber principle(s) responsible of cholesterol lowering in blood or liver and additional benefits in renal dysfunction, fecal excretion etc. Present time, apple punch is FDA approved safe table drink with hope of cholesterol lowering effect but unconfirmed. Conclusion Apple diet is rich in pulp and apple fiber may have the cholesterol lowering effect on the lipid metabolism in body. Cholesterol 7 Hydroxylase is rate limiting enzyme of cholesterol degradation. Apple pulp fiber may have stimulatory effect on cholesterol 7α

hydroxylase enzyme while cholesterol also showed enhanced enzyme activities. Acknowledgements The financial grant from Indian Council of Medical Research to first author under supervision of Professor RK Tandon for this study is highly appreciated. The assistance of Mr Kishan Lal is highly appreciated to carry out daily lab work. References

1. Honda A, Yoshida T, Tanaka N, Matsuzaki Y, He B, Shoda J, Osuga T.Accumulation of 7 alpha-hydroxycholesterol in liver tissue of patients with cholesterol gallstones.J Gastroenterol. 1995 Oct;30(5):651-6. 2. Martin KO, Budai K, Javitt NB.Cholesterol and 27-hydroxycholesterol 7 alpha-hydroxylation: evidence for two different enzymes.J Lipid Res. 1993 Apr;34(4):581-8. 3. Einarsson K, Reihnér E, Björkhem I.On the saturation of the cholesterol 7 alpha-hydroxylase in human liver microsomes.J Lipid Res. 1989 Oct;30(10):1477-81. 4. Yoko Akazome Characteristics and physiological functions of polyphenols from applesVolume 22, Numbers 1-4/2004:311-314. 5. Ogino Y, Osada K, Nakamura S, Ohta Y, Kanda T, Sugano M. Absorption of dietary cholesterol oxidation products and their downstream metabolic effects are reduced by dietary apple polyphenols. Lipids. 2007 Mar;42(2):151-61. 6. Osada K, Suzuki T, Kawakami Y, Senda M, Kasai A, Sami M, Ohta Y, Kanda T, Ikeda M. Dose-dependent hypocholesterolemic actions of dietary apple polyphenol in rats fed cholesterol. Lipids. 2006 Feb;41(2):133-9. 7. Björkhem I, Eggertsen G, Andersson U.On the mechanism of stimulation of cholesterol 7 alpha-hydroxylase by dietary cholesterol.Biochim Biophys Acta. 1991 Oct 1;1085(3):329-35. 8. Tanaka S, Kinowaki M, Maeda Y, Nagatomo J, Kai MH, Kondo KH, Chijiiwa K.Species difference in cholesterol 7alpha-hydroxylase expression of rabbit and rat liver microsomes after bile duct ligation.J Surg Res. 2004 Jun 1;119(1):36-40. 9. Ndong-Akoume MY, Mignault D, Perwaiz S, Plaa GL, Yousef IM.Simultaneous evaluation of HMG-CoA reductase and cholesterol 7alpha-hydroxylase activities by electrospray tandem MS.Lipids. 2002 Nov;37(11):1101-7. 10. Anand AC, Sharma R, Kapur BM, Tandon RK. Analysis of symptomatic patients after cholesystectomy: is the term post-cholesystectomy syndrome an anachronism? Trop Gastroenterol. 1995;16(2):126-31. 11. Oakenfull DG, Fenwick DE.Adsorption of bile salts from aqueous solution by plant fibre and cholestyramine.Br J Nutr. 1978 Sep;40(2):299-309. 12. Dongowski G, Ehwald R.Binding of water, oil, and bile acids to dietary fibers of the cellan type.Biotechnol Prog. 1999 Mar-Apr;15(2):250-8. 13. Shimizu J, Yamada N, Nakamura K, Takita T, Innami S.Effects of different types of dietary fiber preparations isolated from bamboo shoots, edible burdock, apple and corn on fecal steroid profiles of rats.J Nutr Sci Vitaminol (Tokyo). 1996 Dec;42(6):527-39. 14. Ikegami S, Tsuchihashi F, Harada H, Tsuchihashi N, Nishide E, Innami S.Effect of viscous indigestible polysaccharides on pancreatic-biliary secretion and digestive organs in rats.J Nutr. 1990 Apr;120(4):353-60. 15. Aprikian O, Busserolles J, Manach C, Mazur A, Morand C, Davicco MJ, Besson C, Rayssiguier Y, Rémésy C, Demigné C.Lyophilized apple counteracts the development of hypercholesterolemia, oxidative stress, and renal dysfunction in obese Zucker rats.J Nutr. 2002 Jul;132(7):1969-76. 16. Sembries S, Dongowski G, Mehrländer K, Will F, Dietrich H.Physiological effects of extraction juices from apple, grape, and red beet pomaces in rats.J Agric Food Chem. 2006 Dec 27;54(26):10269-80. 17. Sembries S, Dongowski G, Mehrländer K, Will F, Dietrich H.Dietary fiber-rich colloids from apple pomace extraction juices do not affect food intake and blood serum lipid levels, but enhance fecal excretion of steroids in rats.J Nutr Biochem. 2004 May;15(5):296-302. 18. Maeda YR, Eggertsen G, Nyberg B, Setoguchi T, Okuda KI, Einarsson K, Björkhem I.Immunochemical determination of human cholesterol 7 alpha-hydroxylase.Eur J Biochem. 1995 Feb 15;228(1):144-8. 19. Kinowaki M, Tanaka S, Maeda Y, Higashi S, Okuda K, Setoguchi T.Half-life of cholesterol 7alpha-hydroxylase activity and enzyme mass differ in animals and humans when determined by a monoclonal antibody against human cholesterol 7alpha-hydroxylase.J Steroid Biochem Mol Biol. 2002 Aug;81(4-5):377-80. 20. Norlin M, Toll A, Björkhem I, Wikvall K.24-hydroxycholesterol is a substrate for hepatic cholesterol 7alpha-hydroxylase (CYP7A).J Lipid Res. 2000 Oct;41(10):1629-39.

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21. Souidi M, Parquet M, Dubrac S, Audas O, Bécue T, Lutton C.Assay of microsomal oxysterol 7alpha-hydroxylase activity in the hamster liver by a sensitive method: in vitro modulation by oxysterols.Biochim Biophys Acta. 2000 Aug 24;1487(1):74-81. 22. Boll M, Weber LW, Plana J, Stampfl A.In vivo and in vitro studies on the regulatory link between 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase in rat liver.Z Naturforsch C. 1999 May-Jun;54(5-6):371-82. 23. Souidi M, Parquet M, Lutton C.Improved assay of hepatic microsomal cholesterol 7 alpha-hydroxylase activity by the use of hydroxypropyl-beta-cyclodextrin and an NADPH-regenerating system.Clin Chim Acta. 1998 Jan 30;269(2):201-17. 24. Matheson HB, Colon IS, Story JA. Cholesterol 7α-hydroxylase activity is increased by dietary modification with psyllium hydrocolloid, pectin, cholesterol and cholestyramine in rats. J Nutr. 1995; 125: 454-458. 25. Nordstrom JL, Rodwell VW, Mitschelen JJ. Interconversion of active and inactive forms of rat liver hydroxymethyl-glutaryl-CoA reductase. J Biol Chem. 1977; 252:8924-8934. 26. Pella D, Dubnov G, Singh RB, Sharma R, Berry EM, Manor O. Effects of an Indo-Mediterranean diet on the omega-6/omega-3 ratio in patients at high risk of coronary artery disease: The Indian paradox. In: Omega-6/Omega-3 essential fatty acid ratio: The scientic evidence, editors: Simopoulos AP, Cleand LG. Karger AG. Switzerland 2003.pp 74-81. 27. Valhouny GV, Adamson I, Chalcarz W, Satchithanandam S, Muesing R, Klurfeld DM, Tepper SA, Sanghvi A, Kritchevsky D. Effects of casein and soy protein on hepatic and serum lipids and lipoprotein lipid distributions in the rat. Atherosclerosis. 1985;56(2):127-137.







APPENDIX:

EXTRA MATERIAL TO READ FROM LITERATURE 1. HUGH B. MATHESON, IVETTE S. COLON AHD JON A. STORY: Cholesterol 7

Hydroxylase Activity Is Increased by Dietary Modification with Psyllium Hydrocolloid, Pectin, Cholesterol and Cholestyramine in Rats.

2. LIEN B. NGUYEN, SARAH SHEFER, GERALD SALEN, JOHN Y. L. CHIANG, AND MUKUL PATEL: Cholesterol 7a-Hydroxylase Activities From Human and Rat Liver Are Modulated In Vitro Posttranslationally by Phosphorylation/Dephosphorylation

Biochemical and Molecular Roles of Nutrients

Cholesterol 7Â«-Hydroxylase Activity Is Increased byDietary Modification with Psyllium Hydrocolloid,Pectin, Cholesterol and Cholestyramine in Rats1Â«2Â«3

HUGH B. MATHESON,4 IVETTE S. COLON5 AHD JON A. STORY6

Department of Foods and Nutrition, Purdue University, West Lafayette, IN 47907

ABSTRACT Sources of dietary fiber known to altercholesterol metabolism and/or bile acid pool size werefed to rats, and activity of the rate-limiting step in bileacid synthesis, cholesterol 7Â«-hydroxylase, wasmeasured. In the first experiment, semipurified dietscontaining 5% cellulose, psyllium hydrocolloid, pectin oroat bran as dietary fiber sources or 2% Cholestyraminewere fed to groups of 10 male Wistar rats for 4 wk. Inthe second experiment, groups of six rats were fed dietscontaining 5% cellulose, rice bran, oat bran or psylliumwith and without 0.25% cholesterol. In the first experiment, the activity of cholesterol 7Â«-hydroxylase(prnol-mhr^mg protein"1) was highest in thecholestyramine-treated group (95.6 Â±3.6), followed bygroups fed psyllium (35.5 Â±3.5) or pectin (36.0 Â±4.5),which exhibited more than twice the enzyme activity ofgroups fed cellulose (16.9 Â±1.9) or oat bran (12.3 Â±2.0). In the second experiment, feeding cholesterolresulted in significantly higher enzyme activity when cellulose (65%), oat bran (118%) and rice bran (60%)were fed, but no difference in activity was observed whencholesterol was added to the psyllium-containing diet.Higher activity of cholesterol 7a-hydroxylase whenpectin or psyllium rather than cellulose was fed mayexplain the almost twofold higher bile acid pool sizespreviously reported in response to feeding either of thesefibers. These data support the hypothesis that thehypocholesterolemic effect of soluble fibers is modulatedthrough increased synthesis and therefore pool size ofbile acids. J. Nutr. 125: 454-458, 1995.

INDEXING KEY WORDS:

â€¢cholesterol-7a-hydroxylase â€¢ratsâ€¢dietary fiber â€¢cholesterol

There has been a great deal of interest in thehypocholesterolemic properties exhibited by some dietary fibers. Fibers with predominantly water-solublecomponents have proved to be more effective inreducing human serum cholesterol than have insoluble types of fiber (Anderson et al. 1988, Behall1990, Everson et al. 1992, Jenkins et al. 1993). It hasalso been reported that soluble fibers such as pectinand psyllium hydrocolloid significantly reduce serum

cholesterol concentrations in rats fed normal andcholesterol-supplemented diets (Anderson and Chen1979, Tsai et al. 1976). There is also evidence that oatbran, a fiber source with both soluble and insolublecomponents, has a hypocholesterolemic effect in ratsand humans (Anderson et al. 1984, Arjmandi et al.1992, Chen et al. 1981, Shinnick et al. 1988). Ricebran, a predominantly insoluble fiber source, has beenreported to reduce serum and liver cholesterol inhamsters (Kahlon et al. 1992). Despite considerableeffort, the hypocholesterolemic mechanism of thesefibers has not yet been clearly defined.

We have previously observed increases in the poolsizes of bile acids in rats fed pectin and psyllium(Matheson and Story 1994), suggesting an increase inthe catabolism of cholesterol through conversion tobile acids. The rate-controlling enzyme in the formation of bile acids is cholesterol 7a-hydroxylase (EC1.14.13.17) (Danielsson et al. 1967, Myant andMitropoulos 1977), which is expressed exclusively inthe liver and whose mRNA levels can be increased bydietary bile acid sÃ©questrants, cholesterol ormevalonate and decreased by dietary bile acids(Jelinek et al. 1990, Noshiro et al. 1990).

'These data were presented in part at Experimental Biology 93,

April 1, 1993, New Orleans, LA [Matheson, H. B. & Story, J. A.(1993) Changes in the activity of cholesterol 7a-hydroxylase bydietary modification using cellulose, psyllium, pectin, oat bran andCholestyramine. FASEB. J. 7: A722 (abs.|].

Supported in part by the Indiana Agricultural Research Programs (paper no. 14,339|, American Institute for Cancer Research(86A-25), and the Procter and Gamble Company.

3The costs of publication of this article were defrayed in part by

the payment of page charges. This article must therefore be herebymarked "advertisement" in accordance with 18 USC section 1734

solely to indicate this fact.4Current address: Department of Medical Nutrition, Huddinge

University Hospital F60, NOVUM, S-141 86 Huddinge, Sweden.5Current address: Nutrition Department, General Mills, Ine,

P.O. Box 1113, Minneapolis, MN 55440.6To whom correspondence and reprint requests should be ad

dressed.

0022-3166/95 $3.00 Â©1995 American Institute of Nutrition.Manuscript received 23 May 1994. Initial review completed 11 July 1994. Revision accepted 29 August 1994.

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FIBER MODIFIES CHOLESTEROL 7a-HYDROXYLASE 455

This study was designed to investigate whetherincreases in the enterohepatic circulation of bile acidsin rats, induced by dietary fiber, resulted from alteration in the activity of cholesterol 7a-hydroxylase andwhether there was any modulation of this effect bythe addition of low levels of dietary cholesterol.

MATERIALSAMD METHODS

Animals and diets. Male Wistar rats (HarÃanSprague Dawley, Indianapolis, IN), initially weighing62-88 g, were individually housed and adapted to areverse light cycle (dark 0400-1600 h), which optimized cholesterol 7a-hydroxylase activity at 1000 h(6 h after the beginning of the dark cycle) andminimized variation due to diurnal changes (Myantand Mitropoulos 1977). During a 7-d stabilizationperiod rats were given free access to ground nonpu-rified diet (Rodent Laboratory ChowÂ®, RalstonPurina, St. Louis, MO). In both experiments the basaldiet contained sucrose (550 g/kg), casein (250 g/kg),corn oil (100 g/kg), AIN-76 mineral mix (40 g/kg) andAIN-76A vitamin mix (10 g/kg) (Story et al. 1981).

In the first experiment, 50 rats were randomlyassigned to one of five treatment groups and fed for 28d the basal diet with cellulose, pectin, oat bran orpsyllium hydrocolloid added at 5% or cholestyramineadded at 2%; in each case fiber was added at theexpense of sucrose. In the second experiment, 48 ratswere randomly assigned to one of eight treatmentgroups and fed for 28 d a semipurified diet with 5%cellulose, oat bran, rice bran or psyllium, with andwithout addition of 0.25% cholesterol, added at theexpense of sucrose. Body weight and food intake weremonitored each week.

At the end of each experiment animals were killedby decapitation, beginning at 1000 h. Animals werekilled within a 2-h span, in an order determined todistribute time of death among the different dietarytreatments throughout that time. Serum and liversamples were stored at -80Â°C until analysis for

cholesterol (Rudel and Morris 1973) and insulin.Serum insulin was measured by RIA (Rat Insulin RIAKit, Lineo Research, St. Louis, MO). The experimental protocol was reviewed and approved bythe Purdue University Animal Care and Use Committee.

Cholesterol 7a-hydroxylase assay. Liver micro-somes were isolated by ultracentrifugation (Nordstrom et al. 1977) and stored in liquid nitrogen. Activity of cholesterol 7a-hydroxylase was measuredusing incorporation of liposome solubilizedcholesterol isotope into microsomal preparations(Junker and Story 1985). The results are expressed aspicomoles of 7a-hydroxycholesterol produced perminute per milligram of microsomal protein (Bio-RadProtein Assay, Bio-Rad Laboratories, Hercules, CA)(Bradford 1976).

Statistical analysis. Experimental values were analyzed using the Statistical Analysis System, version6.07 (SAS Institute, Gary, NC). Experiment 1 employed a one-way ANOVA and Experiment 2 a two-way ANOVA, with dietary cholesterol and dietaryfiber as main effects. In both experiments, ANOVAwas followed by Fisher's least significant differencetest (Snedecor and Cochran 1989) to compare meanvalues where appropriate. Data are presented as themean of 10 or 6 rats in Experiments 1 and 2, respectively, with pooled SEM.

RESULTS

Final body weights and average weight gain werenot significantly different among any of the groups, ineither experiment.

In the first experiment, when cellulose, oat bran,pectin, psyllium or cholestyramine was fed, serumand liver cholesterol concentrations were comparableamong the groups. Liver weight in animals fed pectin,psyllium or cholestyramine was significantly lowerthan in those fed cellulose or oat bran (Table 1). The

TABLE 1

Experiment 1: Serum and liver cholesterol concentrations in rats fed diets containing cellulose, psyllium,pectin, oat bran or cholestyramine1

GroupCellulose-fedOat

bran-fedPectin-fedPsyllium

-fedCholestyramine-fedPooled

errorLiver

weightg16.2a16.2a14.4b14.9b14.9b1.2Serumcholesterolmmol/L2.252.511.912.172.690.17Livercholesterol\im

ol/g7.507.248.287.246.470.54Total

livercholesterol\imol119a116a119a109a96b0.65Seruminsulinnmol/L0.155ab0.179a0.1

14b0.128ab0.148ab0.020

Values are means, n = 10. Within a column, values with different superscripts are significantly different (P < 0.05].

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456 MATHESON ET AL.

lower total liver cholesterol level in thecholestyramine-fed group would explain some of thedifference but does not account for differences ingroups fed pectin or psyllium.

Compared with cellulose-fed controls, rats fed 2%cholestyramine had a much higher activity ofcholesterol 7a-hydroxylase, which was of a magnitude similar to that previously reported (Hylemonet al. 1989). There was no difference in enzyme activity when oat bran was fed compared with cellulose,but both psyllium and pectin consumption resulted inactivities that were twice that of rats fed cellulose, adifference that was significant for both groups (Fig. 1).

In the second experiment, which was designed totest the modulation of the effects of cholesterolfeeding by cellulose, rice bran, oat bran and psyllium,serum cholesterol was significantly lower in rats fedthe diet containing psyllium without cholesterol thanany other dietary group. There was no difference inliver cholesterol concentration or total livercholesterol when the diets were fed without addedcholesterol, as was observed in Experiment 1. Whencholesterol was added to the cellulose diet, there wasapproximately a fivefold higher concentration of livercholesterol. When cholesterol was added to the otherdiets, the extent of liver cholesterol accumulationwas modulated by the dietary fiber sources. Oat branresulted in a cholesterol concentration that was significantly lower than that observed in rats fed cellulose with added cholesterol, whereas rice bran and

100-7

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Â±bTbTCTC

TO

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FIGURE 1 Hepatic cholesterol 7a-hydroxylase activity inrats fed semipurified diets containing 5% psylliumhydrocolloid, pectin, oat bran or cellulose or 2%cholestyramine. Values are means Â±SEM (n = 10). Bars withdifferent letters are significantly different (P < 0.05).

psyllium consumption resulted in the lowest livercholesterol concentration in rats fed cholesterol(Table 2). Total liver cholesterol level responded in asimilar fashion (Table 2).

There was no difference in the activity ofcholesterol 7a-hydroxylase in the cellulose-, oat bran-or rice bran-fed groups in the absence of dietary

cholesterol. When cholesterol was added to each ofthese diets, activity of the enzyme was significantlygreater but the values in the three groups did notdiffer (Fig. 2). This was not reflected in the psyllium-fed group, in which inclusion of cholesterol in thediet had no effect and enzyme activity was significantly higher than that observed in all other groupswith and without dietary cholesterol.

DISCUSSION

There have been several hypotheses proposed toexplain observed reductions in serum cholesterol concentrations in response to soluble components of dietary fiber. One of the most widely discussed is increased excretion of neutral and acidic biliary sterolsand diet-derived cholesterol, due to reduced intestinalabsorption and/or increased luminal binding of bileacids. This would lead to an increased conversion ofexogenous and endogenous cholesterol to bile acids inthe liver, causing an elevation in LDL receptor expression, thereby reducing serum cholesterol concentration. However, there have been conflicting reports

40 -,

FIGURE 2 Activity of hepatic cholesterol 7a-hydroxylasein rats fed semipurified diets containing 5% cellulose, oatbran, rice bran or psyllium hydrocolloid with (filled bars)and without (open bars) 0.25% dietary cholesterol. Valuesare means Â±SEM (n = 6). Bars with different letters aresignificantly different (P < 0.05).

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FIBER MODIFIES CHOLESTEROL 7a-HYDROXYLASE 457

TABLE 2

Experiment 2: Serum and liver cholesterol levels in rats fed cellulose, rice bran, oat bran or psyHium withand without added dietary cholesterol1

GroupCellulose-fedRice

bran-fedOat

bran-fedPsyllium-fedPooled

SEMDietary

LivercholesterolweightS16.9cd+

19.4a17.1cd+

20.0a172bcd+

19.0a15.5d+

18.4abc0.56Serum

cholesterolmmol/L2.09a2.15a2.08a2.26a2.24a2.30a1.77b2.07a3.72LivercholesterolÃimol/g7.83d38.0a7.66d22.6C6.96d30.0b9.02d20.2C0.80Totalliver

cholesterol\imol129d742a132d455bc119d579b140d370C18.2

^Values are means, n ==6. Within a column, values with different superscripts are significantly different (P < 0.05].

concerning fecal steroid excretion during dietary fiberintervention (Kritchevsky and Story 1988). Althoughsoluble fibers generally result in higher fecal bile acidexcretion in humans (Jenkins et al. 1993, Story 1985),the evidence is not consistent in rats, with bothhigher and lower levels of daily fecal acidic steroidexcretion having been reported for several solublefibers (Ide et al. 1990). The above hypothesis requiresan increased flux of cholesterol conversion to bileacids and therefore an increase in the biosyntheticcapacity of the bile acid pathway.

There is evidence that insulin stimulates activityof cholesterol 7a-hydroxylase at concentrations aslow as 1 nmol/L and that this response is dose dependent, occurring within 30 min (Skett 1990). Thesmall differences in serum insulin, which were significant only between rats fed oat bran and those fedpectin (difference between the two being 0.065 nmol/L) (Table 1), probably did not affect enzyme activity.This difference in insulin concentration may have hadmore effect on glycogen accumulation and, as aresult, liver weight.

Groups fed the soluble fibers pectin and psylliumhad greater than twice the activity of cholesterol7a-hydroxylase than those fed cellulose or oat bran(Fig. 1 and 2). This difference in enzyme activity is ofa similar magnitude to the previously observed bileacid pool size (2.7 to 3.1 times) in rats fed pectin orpsyllium compared with those fed cellulose(Matheson and Story 1994). This may have been sufficient to partially prevent liver cholesterol accumulation when dietary cholesterol was added because,although cholesterol feeding resulted in higherhepatic concentration of cholesterol in the group fedpsyllium, the increase was not as great as thatresulting from cholesterol feeding when cellulose oroat bran were fed (Table 2). Activity of cholesterol

7a-hydroxylase increased in response to cholesterolfeeding to approximately the same extent in thegroups fed insoluble fiber (cellulose or rice bran) andin the group fed the partially soluble oat bran. In thecase of psyllium, the high activity of the enzyme wasnot further increased by addition of cholesterol to thediet.

It has been shown that there are separate hepaticpools of cholesterol depending on its exogenous orendogenous origin (Balasubramaniam et al. 1973,Liscum and Dahl 1992) and that there is some stimulation of activity of cholesterol 7a-hydroxylase byincreased microsomal cholesterol availability (Strakaet al. 1990). Results from our experiments do notagree with the concept that the primary controllingfactor influencing cholesterol 7a-hydroxylase activityis total hepatic cholesterol concentration. These datasuggest that, in spite of differences in liver cholesterolconcentration and content in rats fed cellulose, ricebran or oat bran with added dietary cholesterol,enzyme activity is comparable for all three groups ofrats. The ability of rice bran and oat bran to preventcholesterol accumulation when cholesterol is addedto the diet does not seem to be mediated by changesin bile acid synthesis, as indicated by their similarcholesterol 7a-hydroxylase activities. Other components of these substances evidently are involved intheir hypocholesterolemic potential.

These results are consistent with the hypothesissuggested by our earlier work (Matheson and Story1994), indicating that soluble dietary fiber influencescholesterol metabolism by causing changes in thespectrum of circulating bile acids. It was suggestedthat these changes in bile acid composition and theconcomitant changes in hydrophobicity of bile wouldalleviate the bile acid feedback inhibition oncholesterol 7a-hydroxylase. These experiments substantiate this change in bile acid synthesis, further

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458 MATHESON ET AL.

supporting our hypothesis that the hypocholestero-lemic effect of soluble fibers is modulated throughincreased synthesis and therefore pool size of bileacids.

ACKNOWLEDGMENTS

The authors would like to express their thanks toEmily Furumoto for her excellent technical assistanceand to Penny Keller for help in preparation of themanuscript.

LITERATURE CITED

Anderson, J. W. & Chen, W.-J.L. (1979) Plant fiber carbohydrate andlipid metabolism. Am. J. Clin. Nutr. 32: 346-363.

Anderson, J. W., Story, L., Sieling, B., Chen, W.-J.L., Petro, M. S. &Story, J. (1984) Hypocholesterolemic effects of oat bran or beanintake for hypocholesterolemic men. Am. J. Clin. Nutr. 40:1146-1155.

Anderson, J. W., Zettwoch, N., Tietyen-Clark, J., Oeltgen, P. &Bichop, C. W. (1988) Cholesterol-lowering effects of psylliumhydrophilic mucilloid for hypercholesterolemic men. Arch.Intern. Med. 148: 292-296.

Arjmandi, B. H., Ahn, J., Nathani, S. & Reeves, R. D. (1992) Dietarysoluble fiber and cholesterol affect serum cholesterol concentration, hepatic portal venous short-chain fatty acid concentration and fecal sterol excretion in rats. J. Nutr. 122: 246-253.

Balasubramaniam, S., Mitropoulos, K. A. & Myant, N. B. (1973)Evidence for the compartmentation of cholesterol in rat-livermicrosomes. Eur. J. Biochem. 34: 77-83.

Behall, K. M. (1990) Effect of soluble fibers on plasma lipids,glucose tolerance and mineral balance. Adv. Exp. Med. Biol. 270:7-16.

Bradford, M. M. (1976) A rapid and sensitive method for quanti -

tation of microgram quantities of protein utilizing the principleof protein-dye binding. Anal. Biochem. 72: 248-254.

Chen, W.-J.L., Anderson, J. W. &. Gould, M. R. (1981) Effects of oatbran, oat gum and pectin on lipid metabolism of cholesterol-fedrats. Nutr. Rep. Int. 24: 1093-1098.

Danielsson, H., Einarsson, K. & Johansson, G. (1967) Effect ofbiliary drainage on individual reactions in the conversion ofcholesterol to taurocholic acid. Eur. J. Biochem. 2: 44â€”49.

Everson, G. T., Daggy, B. P., McKinley, C. &. Story, J. A. (1992)Effects of psyllium hydiophilic mucilloid on LDL-cholesteroland bile acid synthesis in hypercholesterolemic men. f. LipidRes. 33: 1183-1192.

Hylemon, P. B., Struder, E. J., Pandak, W. M., Heuman, D. M.,Vlahcevic, Z. R. & Chiang, J.Y.L. (1989) Simultaneous measurement of cholesterol 7a-hydroxylase activity by reverse-phase

high performance liquid chromatography using both endogenousand exogenous [4-14C)cholesterol as substrate. Anal. Biochem.

182: 212-216.

Ide, T., Horii, M., Yamamoto, T. & Kawashima, K. (1990) Contrasting effects of water-soluble and water-insoluble dietaryfibers on bile acid conjugation and taurine metabolism in therat. Lipids 25: 335^340.

Jelinek, D. F., Andersson, S., Slaughter, C. A. & Russell, D. W.(19901 Cloning and regulation of cholesterol 7a-hydroxylase, therate-limiting enzyme in bile acid synthesis. J. Biol. Chem. 265:8190-8197.

Jenkins, D.J.A., Wolever, T.M.S., Rao, V., Hegele, R. A., Mitchell, S.J., Ransom, T.P.P., Boctor, D. L., Spadafora, P. J., Jenkins, A. L.,Mehling, C., Relie, L. K., Connelly, P. W., Story, J. A., Furumoto,E. J., Corey, P. & Wiirsch, P. (1993) Effect on blood lipids of veryhigh intakes of fiber in diets low in saturated fat andcholesterol. N. Eng. J. Med. 329: 21-26.

Junker, L. H. & Story, J. A. (1985) An improved assay for cholesterol7a-hydroxylase activity using phospholipid liposome solubilizedsubstrate. Lipids 20: 712-718.

Kahlon, T. S., Choq, F. L, Sayre, R. N. & Betschart, A. A. (1992)Cholesterol lowering in hamsters fed rice bran at various levels,defatted rice bran and rice bran oil. J. Nutr. 122: 513-519.

Kritchevsky, D. & Story, J. A. (1988) The influence of dietary fiberon cholesterol metabolism in experimental animals. In: CRCHandbook of Dietary Fiber in Human Nutrition (Spiller, G. A.,ed.), pp. 129-142. CRC Press, Boca Raton, FL.

Liscum, L. & Dahl, N. K. (1992) Intracellular cholesterol transport.J. Lipid Res. 33: 1239-1254.

Matheson, H. B. & Story, J. A. (1994) Dietary psyllium hydrocolloidand pectin increase bile acid pool size and change bile acidcomposition in rats. J. Nutr. 124: 1161-1165.

Myant, N. B. & Mitropoulos, K. A. (1977) Cholesterol 7a-hydroxy-lase J. Lipid Res. 18: 135-153.

Nordstrom, J. L., Rodwell, V. W. & Mitschelen, J. J. (1977) Interconversion of active and inactive forms of rat liver hydroxymethyl-glutaryl-CoA reducÃase. J. Biol. Chem. 252: 8924-8934.

Noshiro, M., Nishimoto, M. & Okuda, K. (1990) Rat livercholesterol 7a-hydroxylase pretranslational regulation for cir-cadian rhythm. J. Biol. Chem. 265: 10036-10041.

Rudel, L. L. & Morris, M. D. (1973) Determination of cholesterolusing o-phthaldialdehyde. J. Lipid Res. 14: 364-366.

Shinnick, F. L., Longacre, M. J., Ink, S. L. &.Marlett, J. A. (1988) Oatfiber: composition versus physiological function in rats. J. Nutr.118: 144-151.

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Cholesterol 7a-Hydroxylase Activities From Human and RatLiver Are Modulated In Vitro Posttranslationally by

Phosphorylation/Dephosphorylation

LIEN B. NGUYEN,1 SARAH SHEFER,1 GERALD SALEN,2 JOHN Y. L. CHIANG,3 AND MUKUL PATEL1

ciency of C7aH (activity per protein mass unit) is modu-Purified cholesterol 7a-hydroxylases (C7aH) from hu-lated, in vitro, posttranslationally by a phosphorylation/man and rat liver microsomes, and from transformeddephosphorylation mechanism in both the human andEscherichia coli expression systems, were incubatedthe rat enzymes. (HEPATOLOGY 1996;24:1468-1474.)with 0.3 mmol/L [g-32P] adenosine triphosphate (ATP) in

the presence and absence of bacterial alkaline phospha-tase (AP) or rabbit muscle adenosine 3*,5*-cyclic mono- Cholesterol 7a-hydroxylase (C7aH) (EC 1.14.13.17) is thephosphate (cAMP)-dependent protein kinase. The first step and rate-limiting enzyme in the conversion of cho-amounts of 32P incorporation after separation of human lesterol to bile acids in the liver.1,2 This enzyme has beenand rat C7aH proteins by sodium dodecyl sulfate–poly- purified by various laboratories,3-6 its gene cloned, sequenced,acrylamide gel electrophoresis (SDS-PAGE) were re- localized to chromosome 8q11-q12, and partially character-lated to C7aH catalytic activities (determined by a radio- ized.7-11 The catalytic activity of C7aH is regulated by a diur-isotope incorporation method) and enzyme protein mass nal rhythm and various dietary, drug, and hormonal fac-(determined by Western blotting and laser densitome- tors.12-17 The preferred substrate pool for C7aH is newlytry). Both human and rat C7aH activities significantly synthesized cholesterol.18 Therefore, bile acid synthesis isdecreased after dephosphorylation by AP (057%–072%) regulated by the activity of both C7aH and 3-hydroxy-3-and increased up to twofold with phosphorylation by methylglutaryl coenzyme A reductase, the rate-limiting en-rabbit muscle cAMP-dependent protein kinase. The in- zyme that controls the formation of endogenous choles-creases in C7aH activities were proportional to the terol.15-17 The supply of cholesterol up-regulates C7aH activ-amounts of cAMP-dependent protein kinase used, and ity in the rat,15,19 but inhibits it in the rabbit,20 the hamster,19

were coupled to 32P incorporation into the purified en- and the African Green monkey.21 The reasons for such specieszymes. Both the activation of C7aH and the amounts of differences have not been established. There is still a contro-32P incorporation were time-dependent and reached a versy about mechanisms by which C7aH activity is controlledmaximum after 1 hour of incubation with 5 U of cAMP- by the enterohepatic flux of bile acids.15,16 Earlier studiesdependent protein kinase. In a second set of experi- have suggested that C7aH activity could be regulated post-ments, purified human and rat liver C7aH were dephos- translationally by mechanisms involving cytosolic factors,22

phorylated by 30-minute incubation with AP, followed disulfide bonds in the enzyme structure,23 and phosphoryla-by inactivation of the phosphatase by the inhibitor NaF, tion/dephosphorylation of the enzyme protein.24-28 In contrastand rephosphorylation of C7aH by 30-minute incubation to 3-hydroxy-3-methylglutaryl coenzyme A reductase, whichwith rabbit muscle cAMP-dependent protein kinase or is deactivated by phosphorylation and stimulated by dephos-bovine heart cAMP-independent protein kinase. Re- phorylation,29 it was earlier suggested that C7aH was deacti-phosphorylation of the dephosphorylated C7aH pro- vated by phosphatase-mediated dephosphorylation and stim-teins by cAMP-dependent protein kinase increased ulated by a number of protein kinases.24-26 However,C7aH catalytic activities up to fourfold, and the stimula- phosphorylation/dephosphorylation as a mechanism for post-tion in catalytic activities paralleled the increases in 32P translational regulation of C7aH activity is still disputed byincorporation into the purified enzymes. Bovine heart some investigators.29-31

protein kinase was as potent as rabbit muscle cAMP- The objectives of this study are to (1) examine the effectsdependent protein kinase in stimulating catalytic activ- of phosphorylation/dephosphorylation on catalytic activitiesity and 32P incorporation into the human C7aH protein. of various preparations of purified C7aH from human andBecause the protein mass of these purified enzymes did rat liver; and (2) relate changes in catalytic activities undernot change, the short-term regulation or catalytic effi- varying phosphorylation/dephosphorylation conditions to theamounts of 32P incorporated into the purified enzyme pro-teins.

Abbreviations: C7aH, cholesterol 7a-hydroxylase; AP, alkaline phosphatase; cAMP,MATERIALS AND METHODSadenosine 3*,5*-cyclic monophosphate; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide

gel electrophoresis; ATP, adenosine triphosphate.Purification of Human and Rat C7aH. Four preparations of puri-From the 1Department of Medicine and the Liver Center, University of Medicine and

fied rat C7aH were used: two were isolated as previously described5Dentistry of New Jersey–New Jersey Medical School, Newark, NJ; 2Gastroenterology Sec-tion, Veterans Administration Medical Center, East Orange, NJ; and 3Department of Bio- from cholestyramine-fed rats and two were obtained from Esche-chemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, richia coli that had been transformed with an expression vector con-Rootstown, OH. taining a C7aH complimentary DNA (cDNA).9 Human C7aH protein

Received June 28, 1996; accepted July 25, 1996. was expressed similarly and purified from transformed E. coli.10 TheSupported in part by U.S. Public Health Service Grants DK 26756, HL 17818, DK 44442, C7aH proteins isolated from E. coli expression systems were trun-

and the Research Service Veterans Administration Medical Center.cated enzymes that lacked the N-terminal 24-amino acid residuesAddress reprint requests to: Lien B. Nguyen, Ph.D., UMDNJ-NJ Medical School, Depart-but were catalytically similar to the full-length enzymes purifiedment of Medicine, 185 South Orange Ave., MSB H-532, Newark, NJ 07103.from human and rat liver microsomes.9,10 All purified enzyme prepa-Copyright q 1996 by the American Association for the Study of Liver Diseases.

0270-9139/96/2406-0028$3.00/0 rations showed single bands upon sodium dodecyl sulfate–polyacryl-

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AID Hepa 0022 / 5p19$$$421 11-02-96 00:39:59 hpta WBS: Hepatology

HEPATOLOGY Vol. 24, No. 6, 1996 NGUYEN ET AL. 1469

amide gel electrophoresis (SDS-PAGE)32 and had a molecular weight prestained molecular-weight standards (Bio-Rad Laboratories, Mel-ville, NY) and purified C7aH proteins as markers, and separatedof 51,000. Polyclonal antibodies against rat and human enzymes

were raised in rabbits as described previously.5 These specific anti- by electrophoresis32 using a Hoeffer system (Model SE600, HoefferScientific Instruments, San Francisco, CA). The gels were stainedbodies could cross-react with heterologous antigens of both full-

length and truncated enzymes.5 with Coomassie Brilliant Blue, and 32P radioactivity in the gel slicescontaining the C7aH bands were determined by liquid scintillationPhosphorylation/Dephosphorylation of C7aH. Aliquots (0.5-4 mg) of

human- and rat-purified C7aH proteins were incubated at 377C for spectroscopy.Assay of C7aH Activity, Mass, and Catalytic Activity. The phosphor-30 minutes with varying amounts of E. coli–type III alkaline phos-

ylation/dephosphorylation of the microsomal and purified C7aH pro-phatase (AP) or rabbit muscle adenosine 3*,5*-cyclic monophosphateteins, or rephosphorylation of AP-treated C7aH, were performed as(cAMP)-dependent protein kinase (Sigma Chemical Co., St. Louis,described above, except that unlabeled ATP was used. After inactiva-MO) in buffer (50 mmol/L Tris, 100 mmol/L NaCl, 5 mmol/L MgCl2,tion of the AP/protein kinase with 50 mmol/L NaF and 10 mmol/LpH 7.4) containing phospholipids (5 mg dilauryl phosphatidylcholinedisodium ethylenediamine tetraacetate, cofactors needed for thedissolved in 5 mg deoxycholic acid) in a final volume of 50 mL. Theassay for C7aH catalytic activity (5 mmol/L dithiothreitol, 30 mmol/LAP was solubilized in 20 mmol/L imidazole buffer, pH 7.4, dilutednicotinamide, and 2 U of reduced nicotinamide adenine dinucleotideto 100 U/mL, and added in increasing amounts (0.2, 0.5, and 1 U)phosphate cytochrome P450 reductase in 100 mmol/L K2HPO4, pHas active or heat-inactivated phosphatase (boiled for 5 minutes for7.4) were added with the labeled substrate (200 nmol [4-14C]cholest-control samples). The cAMP-dependent protein kinase was dissolvederol, from Amersham Life Science, Arlington Heights, IL, dissolvedat a concentration of 800 U/mL in 100 mmol/L K2HPO4, pH 7.4, andin 10 mL of 50% b-cyclodextrin,33 from Cyclodextrin Technologies,added in aliquots containing 1, 2, and 5 U, together with 5 mCi [g-Inc., Gainesville, FL) in a total volume of 0.48 mL. The mixtures32P] adenosine triphosphate (ATP) (Amersham Life Science, Arling-were preincubated for 2 minutes at 377C, and the 7a-hydroxylationton, IL; diluted with unlabeled ATP for a final concentration of 0.3reactions were started with the addition of 20 mL of reduced nicotin-mmol/L) and 50 mmol/L cAMP. In a time-course study of the phos-amide adenine dinucleotide phosphate (1.2 mmol/L final concentra-phorylation reaction, 5 U of cAMP-dependent protein kinase wastion). The products were extracted, separated by thin-layer chroma-added to 2 mg C7aH with [g-32P]ATP and cAMP (as above) and thetography, and quantitated by liquid scintillation counting asmixtures were incubated for various intervals (15, 30, 60, and 120previously reported.34

minutes).The C7aH protein mass was measured by immunoblotting andIn a second set of experiments, the purified human and rat C7aH

densitometry after SDS-PAGE separation. The proteins andproteins (2-4 mg) were incubated with AP (0.5 U) for 30 minutes atprestained markers were transferred electrophoretically from the377C in a total volume of 30 mL (50 mmol/L Tris, 100 mmol/L NaCl,SDS-PAGE gels to nitrocellulose membranes (Amersham Life Sci-5 mmol/L MgCl2, 5 mg dilauryl phosphatidylcholine/deoxycholic acid,ence) at 100 V for 2 hours in a Trans-Blot Cell, equipped with apH 7.4). The phosphatase was then inactivated with 5 mL of 500Model 200/2.0 power supply (Bio-Rad Laboratories) and a refriger-mmol/L NaF, then increasing amounts (0, 1, 2, 5 U) of rabbit muscleated circulating bath (Haake, Inc., Saddle Brook, NJ). The mem-cAMP-dependent protein kinase or bovine heart cAMP-independentbranes were blocked at room temperature for 2 hours (20 mmol/Lprotein kinase (Sigma Chemical Co., St. Louis, MO) were added withTris, 150 mmol/L NaCl, 0.02% NaN3, 5% nonfat dry milk, 0.2%5 mCi [g-32P]ATP with or without cAMP, to bring the total volumeTween-20, pH 7.6), and incubated overnight in heat-sealed bags atto 50 mL. The mixtures were incubated for an additional 30 minutes47C with polyclonal rabbit anti-human C7aH IgG at a concentrationto rephosphorylate the dephosphorylated (AP-treated) C7aH pro-of 5 mg/mL in Tris-buffered saline (20 mmol/L Tris, 500 mmol/L NaCl,teins, before the measurements of 32P incorporation, C7aH catalyticpH 7.5) containing 5% nonfat dry milk. The membranes were washedactivity, and protein mass.three times (10 minutes each) with Tris-buffered saline containingTo investigate the role of endogenous hepatic protein kinase/phos-0.05% Tween-20 and incubated with biotinylated anti-rabbit IgGphatase in the activation/deactivation of C7aH, livers from adult(Vector Laboratories, Inc., Burlingame, CA) at a concentration of 5Sprague Dawley rats were used. Two rat liver microsomes (P andmg/mL in Tween-20 Tris-buffered saline for 1 hour at room tempera-dP) were prepared with different buffers. The homogenizing bufferture. After washing in Tween-20/Tris-buffered saline the membranesfor microsomes P is a phosphate buffer (50 mmol/L K2HPO4, 154were incubated for 1 hour at room temperature with horseradishmmol/L KCl, 1 mmol/L disodium ethylenediamine tetraacetate, 1peroxidase conjugated to strepavidin (Amersham Life Science). Themmol/L dithiotreitol, 75 mmol/L nicotinamide, pH 7.4) that containsconjugated C7aH protein bands were detected by color development50 mmol/L of the phosphatase inhibitor NaF; the homogenizingusing 4-chloro-1-naphthol–containing reagents (Bio-Rad Labora-buffer for microsomes dP is the same, except 50 mmol/L imidazoletories) according the the manufacturer’s instructions. The proteinand 50 mmol/L KCl replace K2HPO4 and NaF, respectively. The mi- mass of C7aH under various experimental conditions was quanti-crosomal fractions, separated by ultracentrifugation between tated by densitometric scanning of the immunoblots with a LKB10,000g and 100,000g, were resuspended in buffers (microsomes P: 2202 laser densitometer equipped with a LKB 2220 recording inte-100 mmol/L K2HPO4, 50 mmol/L NaF, 1 mmol/L disodium ethylene- grator, and C7aH relative peak area used as protein mass units.diamine tetraacetate, 5 mmol/L dithiotreitol, 75 mmol/L nicotin- The catalytic efficiency (catalytic activity per protein mass unit)amide, 20% glycerol, pH 7.4; microsomes dP: same buffer with imid- of phosphorylated and dephosphorylated C7aH was calculated byazole and KCL replacing K2HPO4, and NaF) and stored at 0807C dividing the C7aH catalytic activity (pmol/mg protein/min) by the

until used for enzymatic assays. Dephosphorylation of microsome P enzyme protein mass (relative peak area/mg protein).was carried out by incubating 100 mg microsomal proteins with 5 U Statistical Analysis. Data were analyzed statistically by the one-of AP at 377C for 30 minutes in a buffer (50 mmol/L Tris, 100 mmol/ way ANOVA, comparison of confidence intervals of the means, orL NaCl, 5 mmol/L MgCl2, pH 7.4). Phosphorylation of microsomal the unpaired t test.35

protein dP was carried out by incubating 100-mg microsomal proteinswith 100 U of rabbit muscle cAMP-dependent protein kinase, 2 mmol/ RESULTSL ATP, and 50 mmol/L cAMP at 377C for 30 minutes in the same

Table 1 shows C7aH catalytic activities in rat liver micro-Tris buffer. After the phosphorylation/dephosphorylation reactions,somes P (prepared from liver homogenized with a phosphate50 mmol/L NaF and 10 mmol/L disodium ethylenediamine tetraace-buffer in the presence of the phosphatase inhibitor NaF) andtate were added to deactivate phosphatase/protein kinase prior to the

measurements of C7aH catalytic activities. Preliminary experiments microsomes dP (prepared in the absence of phosphate andwith the same microsomal preparations showed that varying the NaF), as well as the effects of AP and cAMP-dependent pro-composition of the C7aH assay buffer (phosphate, imidazole, or Tris) tein kinase on microsomal C7aH activities. Microsomes dPused during the incubation with the [14C]cholesterol substrate did showed significantly lower C7aH catalytic activities (060%,not change C7aH catalytic activities. P õ .01) than microsomes P, where the phosphorylated state

Measurements of 32P Incorporation. At the end of the phosphoryla- of C7aH was protected. Dephosphorylation of microsomes Ption, dephosphorylation or rephosphorylation reactions as describedwith AP significantly reduced C7aH catalytic activities,above, the incubation mixtures were ultrafiltrated (Centricon-30;whereas phosphorylation of microsomes dP with cAMP-de-Amicon, Beverly, MA) to remove unreacted [32P]ATP, and dissolvedpendent protein kinase raised C7aH catalytic activities toin 50 mL of gel-loading buffer (100 mmol/L Tris, 200 mmol/L dithio-the high level found in microsomes P.threitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol, pH 6.8). The

samples were loaded on 1.5-mm-thick 10% polyacrylamide gel with When human and rat purified C7aH proteins were incu-


1470 NGUYEN ET AL. HEPATOLOGY December 1996

TABLE 1. Effects of AP and cAMP-Dependent Protein Kinase onC7aH Activities in Rat Liver Microsomes

C7aH Activities†Treatment* pmol/mg/min

Microsomes P 107.2 { 6.3 (100)Microsomes dP 43.1 { 8.6‡ (40)Microsomes P { AP 36.2 { 7.6‡ (34)Microsomes dP { cAMP-dependent protein kinase 108.4 { 26.8 (101)

* Microsomes were prepared from liver homogenized with either a phos-phate buffer containing 50 mmol/L NaF (microsomes P) or an imidazole bufferwith 50 mmol/L KCl replacing NaF (microsomes dP). Treatment with 5 U ofAP or 100 U of cAMP-dependent protein kinase, 2 mmol/L ATP, and 50 mmol/L cAMP was for 30 minutes, followed by deactivation of the phosphatase andprotein kinase, before cofactors and substrate were added for the assay ofC7aH activities.

† Means { SEM from three to six animals, with percentage in parentheses.‡ Significantly lower than activities in microsomes P, P õ .01.

bated for 30 minutes with increasing amounts of AP, C7aHcatalytic activities significantly decreased and reached maxi-mum inhibition with 0.5 U AP (Põ .01, Table 2). In contrast,rabbit muscle cAMP-dependent protein kinase increasedC7aH catalytic activities with the increasing amounts of pro-tein kinase (Table 2). C7aH proteins purified from livers ofcholestyramine-fed rats and isolated from E. coli expressionsystems gave similar results, and have been combined in thepresentation of data in all tables and figures. When humanand rat purified C7aH proteins were incubated with 5 Uof rabbit muscle cAMP-dependent protein kinase for longerperiods (up to 2 hours), C7aH catalytic activities increasedlinearly with incubation times and reached a maximum (atwofold increase relative to baseline) after 1 hour of incuba-tion (Fig. 1). Thus, C7aH catalytic activities significantlydecreased with AP-mediated dephosphorylation and substan-tially increased with protein kinase–mediated phosphoryla-tion (Fig. 1). The increases in catalytic activities of both hu- FIG. 1. Effects of AP and rabbit muscle cAMP-dependent protein kinase on

human and rat C7aH activities. Purified C7aH proteins (2 mg) were incubatedman and rat purified C7aH after phosphorylation werewithout (C, control), and with AP (0.5 U) or cAMP-dependent protein kinase(KN, 5 U) for 1 hour before the assay for C7aH catalytic activities. Means {SEM from three measurements of E. coli-expressed human C7aH, and multiplemeasurements of four purified rat C7aH proteins (two from livers of cholestyra-TABLE 2. Effects of AP and cAMP-Dependent Protein Kinase onmine-fed rats and two isolated from E. coli expression systems, which gavePurified C7aH Activitiessimilar results). Catalytic activities representing 100% for human and ratC7aH were 1.96 { 0.31 and 2.13 { 0.31 nmol/mg/min, respectively. *Signifi-C7aH Activities†cantly different from control, P õ .01.Source Treatment* nmol/mg/min

Human Control, 0 U 1.96 { 0.31 (100)AP coupled to increases in amounts of 32P incorporated into the

0.2 U 1.04 { 0.39 (53) C7aH proteins. As with C7aH catalytic activities, amounts0.5 U 0.55 { 0.21‡ (28)of 32P incorporation increased linearly with C7aH protein1.0 U 0.77 { 0.22§ (39)concentrations (Fig. 2A) and incubation times (Fig. 2B), andcAMP-dependent protein kinase32P incorporation/mg C7aH protein reached a maximum after1 U 1.85 (94)

2 U 2.29 (117) incubation with [g-32P]ATP and 5 U of cAMP-dependent pro-5 U 2.94 { 0.09\ (150) tein kinase for 1 hour (Fig. 2B).

Rat Control, 0 U 2.13 { 0.31 (100) Figure 3 presents the catalytic activities of human and ratAP C7aH proteins after dephosphorylation by 30-minute incuba-

0.2 U 1.18 { 0.68 (55) tion with 0.5 U of AP, inactivation of the phosphatase with0.5 U 0.91 { 0.19‡ (43) NaF, then rephosphorylation of the C7aH proteins by 30-1.0 U 0.92 { 0.19‡ (43)

minute incubation with increasing amounts of two differentcAMP-dependent protein kinaseprotein kinases. Both human and rat C7aH catalytic activi-1 U 2.20 { 0.20 (103)ties significantly increased with rephosphorylation by cAMP-2 U 2.60 { 0.32 (122)

5 U 3.15 { 0.45 (148) dependent protein kinase, and these increases were proteinkinase concentration-dependent. With 5 U of cAMP-depen-

* Incubation for 30 minutes with 2-4 mg C7aH. dent protein kinase, human and rat C7aH catalytic activities† Means { SEM from two to four measurements of purified human C7aH were stimulated fourfold and twofold, respectively, relative

and at least two measurements of each of four different preparations of purified to baseline activities of the dephosphorylated enzymes. Therat C7aH (two from livers of cholestyramine-fed rats and two from E. colibovine heart cAMP-independent protein kinase was as potentexpression systems). Values in parentheses are percentages.as the rabbit muscle cAMP-dependent protein kinase in up-‡ Significantly lower than control, P õ .01.regulating the human C7aH catalytic activities. It should be§ Significantly lower than control, P õ .05.

\ Significantly higher than control, P õ .05. noted that the rephosphorylation reactions and up-regulation



of catalytic activities shown in Fig. 3 were not complete be-cause the rephosphorylation was performed for only 30 min-utes; yet, the up-regulation of enzyme catalytic activities wasalready significant. The stimulation of human and rat C7aHcatalytic activities by rephosphorylation with increasingamounts of rabbit muscle cAMP-dependent protein kinaseand bovine heart protein kinase paralleled the increases in32P incorporation into the C7aH proteins (Fig. 4). Both C7aHcatalytic activities and 32P incorporation into the human andrat C7aH proteins increased linearly with the incrementsin amounts of cAMP-dependent protein kinase. The humanC7aH protein was rephosphorylated (32P incorporated intothe enzyme protein) and its catalytic activity was stimulatedas efficiently by the bovine heart protein kinase as by theFIG. 2. Effects of C7aH concentrations and incubation times on the

amounts of 32P incorporated into the purified C7aH proteins. (A) 0.2 to 4 mg cAMP-dependent protein kinase (Fig. 4).C7aH was incubated in a total volume of 50 mL with 0.3 mmol/L [g-32P]ATP, Figure 5 shows the SDS-PAGE separation of human and50 mmol/L cAMP, and 5 U of cAMP-dependent protein kinase for 30 minutes. rat C7aH proteins after incubation with cAMP-dependent(B) 2 mg C7aH was incubated with 0.3 mmol/L [g-32P]ATP, 50 mmol/L cAMP,

protein kinase without (Fig. 5A) and with (Fig. 5B) priorand 5 U of cAMP-dependent protein kinase for 15-120 minutes. Means { SEMfrom three to four measurements of E. coli–expressed human C7aH, and dephosphorylation by bacterial AP. In the absence of proteinmultiple measurements of three purified rat C7aH proteins (one from livers kinase and AP (Fig. 5A lanes 4 and 9, Fig. 5B lane 7), oneof cholestyramine-fed rats and two isolated from E. coli expression systems). major band was detected slightly above the 50,000 molecular-

weight marker. Neither the addition of varying amounts ofthe rabbit muscle cAMP-dependent protein kinase (Fig. 5A),nor the incubation with AP before addition of cAMP-depen-dent protein kinase (Fig. 5B) changed the apparent mass ofthe purified C7aH proteins. The constant relative proteinmass of human and rat C7aH proteins under various experi-mental conditions was substantiated by densitometric mea-surements of the C7aH protein bands in the immunoblots,which remained unchanged. The catalytic efficiencies (nmol/min/protein mass unit) of both human and rat C7aH proteins(Table 3), markedly decreased after dephosphorylation by APand increased after rephosphorylation by cAMP-dependentprotein kinase.

DISCUSSION

Both human and rat purified C7aH activities significantlydecreased with AP-mediated dephosphorylation and in-creased two fold to fourfold by cAMP-dependent protein ki-nase–mediated phosphorylation. When purified C7aH pro-teins were phosphorylated by cAMP-dependent proteinkinase, the amounts of 32P incorporated into both humanand rat C7aH proteins increased linearly with C7aH protein

FIG. 4. The amounts of 32P incorporation (solid and open circles) and C7aHFIG. 3. Effects of rephosphorylation by rabbit muscle cAMP-dependentprotein kinase (solid bars) and bovine heart protein kinase (hatched bars) on catalytic activities (solid and open triangles) after rephosphorylation of C7aH

with rabbit muscle cAMP-dependent protein kinase and bovine heart proteinthe catalytic activities of dephosphorylated C7aH. The broken lines representactivities of C7aH after dephosphorylation by 30-minute exposure to 0.5 U kinase. After dephosphorylation of C7aH by 30-minute exposure to AP, then

inactivation of the phosphatase with NaF, the C7aH proteins were rephosphor-of AP (Human Å 550 { 207 pmol/mg/min; Rat Å 907 { 187 pmol/mg/min).Rephosphorylation was performed by incubation for 30 minutes with 0.3 mmol/ ylated for 30 minutes with 0.3 mmol/L ATP and 5 U of rabbit muscle cAMP-

dependent protein kinase (solid lines and filled symbols) or bovine heart cAMP-L ATP, 5 U of protein kinase with or without cAMP, after inactivation of APwith NaF. Averages from two measurements of the E. coli–expressed human independent protein kinase (broken lines and open symbols). Averages of two

measurements of the E. coli–expressed human C7aH protein and means {C7aH protein and means{ SEM from measurements of four purified rat C7aHproteins (two from livers of cholestyramine-fed rats and two isolated from E. SEM from measurements of four purified rat C7aH proteins (two from livers

of cholestyramine-fed rats and two isolated from E. coli expression systems).coli expression systems).



TABLE 3. Effects of Dephosphorylation by AP andconcentrations and incubation times, and occurred in parallelRephosphorylation by cAMP-Dependent Protein Kinase on Catalyticto the stimulation in catalytic activities. With 5 U of cAMP-

Efficiency of C7aHdependent protein kinase, both the amounts of 32P incorpo-rated into human and rat C7aH proteins and C7aH catalytic Source Treatment* Catalytic Efficiency†activities reached maximum levels after 1 hour of incubation.

nmol/min/protein mass unitThese observations demonstrated that, in in vitro experi-Human Control, 0 U 3.9 { 0.6ments, both human and rat C7aH are regulated posttransla-

APtionally by a phosphorylation/dephosphorylation mechanism.0.2 U 1.9 { 0.3‡In contrast to 3-hydroxy-3-methylglutaryl coenzyme A reduc- 0.5 U 1.1 { 0.2§

tase, which is inactivated with phosphorylation, C7aH is 1.0 U 1.6 { 0.1‡stimulated by the incorporation of phosphate groups and de- AP (0.5 U) / cAMP-dependentactivated with their removal. protein kinase

0 U 1.1 { 0.2The C7aH cDNA used to obtain the E. coli–expressed rat1 U 3.3 { 1.0C7aH protein was isolated with antibodies against C7aH2 U 4.3 { 1.4purified from liver of rats that were fed the bile acid seques-5 U 5.0 { 0.2\trant cholestyramine to enhance C7aH formation.7,9 C7aH

Rat Control, 0 U 4.9 { 0.8purified from cholestyramine-fed rats and preparations fromAPE. coli were similar in catalytic activities, responses to AP 0.2 U 2.7 { 1.0

and protein kinase, as well as 32P incorporation. The similar- 0.5 U 1.6 { 0.6‡ity in the data from the two sources of rat enzymes is evi- 1.0 U 1.7 { 0.6‡denced by the coefficients of variation, which were less than AP (0.5 U) / cAMP-dependent

protein kinase14% when C7aH activities in untreated or protein kinase–0 U 1.6 { 0.6treated rat enzymes from both sources were pooled and ana-1 U 2.8 { 0.8lyzed. The coefficient of variation of 32P incorporation mea-2 U 3.4 { 0.5surements in both the liver and the E. coli-expressed enzymes5 U 4.5 { 0.8Øis also small (less than 8%).

Diven et al.24,25 showed that the inactivation of C7aH by * Incubation of 2-4 mg C7aH with or without AP for 30 minutes, inactivationdephosphorylation could be effected by phosphatases of he- of AP with 50 mmol/L NaF, then incubation for an additional 30 minutes withpatic source. Our results with microsomal C7aH, which or without rabbit muscle cAMP-dependent protein kinase.

† Means { SEM from four measurements of purified human C7aH and twoshowed high C7aH activity in microsomes P, where the phos-to four measurements of each of three different preparations of purified ratphorylated form of enzyme was protected during preparation,C7aH (one from cholestyramine-fed rat liver and two from E. coli expressionand loss of activity when this microsomal fraction was ex-systems).posed to AP, suggest a role of hepatic phosphatase/protein

‡ Significantly lower than control, P õ .05.kinase in the regulation of microsomal C7aH in the liver. It§ Significantly lower than control, P õ .01.is interesting to find that the E. coli–expressed C7aH protein\ Significantly higher than samples incubated with only 0.5 U of AP, P õ

is phosphorylated partially, because it could be deactivated .01.by AP-mediated dephosphorylation and its activity restored Ø Significantly higher than samples incubated with only 0.5 U of AP, P õand enhanced above baseline values with protein kinase– .05.mediated phosphorylation. Apparently, C7aH can be phos-phorylated/dephosphorylated by protein kinase/phosphatasewith broad substrate specificity. and possibly other endogenous kinases, could effectively up-

regulate C7aH in humans. The rabbit muscle cAMP-depen-In our in vitro experiments, the stimulation of C7aH cata-lytic activities by phosphorylation is not limited to cAMP- dent kinase used in this study is commercially prepared ac-

cording to a published method.36 One unit is defined as thedependent protein kinase. The bovine heart protein kinase,

FIG. 5. SDS-PAGE separation of human and rat C7aH after phosphorylation/dephosphorylation and rephosphorylation reactions. (A) Lanes 5 and 10,molecular weight markers (top to bottom: b-galactosidase, 121,000; bovine serum albumin, 86,000; ovalbumin, 50,700; carbonic anhydrase, 33,600; soybeantrypsin inhibitor, 27,800; lysosyme, 19,400), lanes 1-4, 4 mg purified C7aH from rat liver with 5, 2, 1, 0 U cAMP-dependent protein kinase (KN); lanes 6-9, 4mg purified human C7aH protein with 5, 2, 1, 0 U cAMP-dependent protein kinase. (B) Lane 2, molecular-weight markers as in (A); lanes 1, and 3-5, 4 mgpurified human C7aH dephosphorylated by AP prior to rephosphorylation by 0, 5, 2, 1 U cAMP-dependent protein kinase; lanes 6 and 7, incubations of humanC7aH in the absence of AP and absence of both AP and protein kinase, respectively, in the dephosphorylation/rephosphorylation experiment.



amount that will transfer 1 pmol of phosphate from [g-32P]- Acknowledgment: The dedicated technical assistance ofEva Paroulek is acknowledged.ATP to hydrolyzed and partially dephosphorylated casein per

minute at pH 6.5 at 307C. It should be noted that the specificREFERENCESphosphorylating activity of this holoenzyme preparation was

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