Tipologia: Original Article

IN VITRO A PRELIMINARY STUDY OF THE INTERACTION SALIVA-PECTIN- TANNIN: POSSIBLE APPLICATIONS IN THE PREVENTION OF THE LIP AND ORAL CANCER INDUCED BY CONSUMPTION OF BETEL

Menicagli Roberto1, Duca Massimo2, Marotta Ortensio3, Rancoita Paola Maria Vittoria4

1 F.A.C (Biomedical) Mediglia ItalyVia Martiri Libertà 6° 20060 Mediglia

2 Inst. Clin. Perf. Milan Italy.Via Castelvetro 22 20154 Milano

3 Unit of Otorhinolaryngology, “Sant’Anna e San Sebastiano” Hospital, Via Ferdinando Palasciano – 81100, Caserta, Italy

4 “San Raffaele” Hospital Milan Italy Via Olgettina 60 20124 Milano

Corrisponding Author: Menicagli Roberto

Menicagli Roberto

mail: menicagli@libero.it

ABSTRACT

Introduction: The aim of this study is to verify if the pectin can prevent precipitation of proteins induced by tannic substances present in betel. The polyphenols precipitate proteins ,depriving the mouth of his main defense ,and it is possible to have a highest indices of incidence for cancers of the oral cavity.

Materials and Methods: In saliva samples supplied by four volunteers were determined values of total protein, pH, and thereafter added variable mixtures of pectin and tannin; the results are examined by statistical fisher exact test. For a 1: 1 ratio (w / w), pectin tannin has for two saliva samples with initial pH acid, the partial precipitation of the protein phase, while with a ratio of 2: 1 regardless of the initial value of the same, there is no protein in the solution and form a complex that fleet towards the surface salivary.

Results: The results indicate that the adsorption of salivary proteins has a trend of depending in the first place from the initial pH value of the saliva when the concentration of pectin is lowest (rho=1 and p-value = 0.083, with 6 g/l addition pectin, rho=0.77 and p-value = 0.225, with 9 g/l addition pectin). With the tannin a 1: 1 ratio tannin / pectin , for two samples at lowest acid pH proteins remain in solution (rho=0.94 and p-value = 0.057), while if the ratio is pectin/ tannin (2: 1), we obtain the total adsorption of salivary.

Discussion: The pectin is a effective component to avoid the salivary proteins precipitation by tannins interactions ,and improvement the floatation adduct with the consequence that it’s possible the hypothesis that this same adduct, Pectin –Protein :tannin , just for the stabilization mechanism in vitro, indicating, the probability, that in vivo the addition of pectin allowing the accession of the protein layer to the oral mucosa, preserving the natural defenses.

Key words: pectin, salivary protein, tannin, mucin

INTRODUCTION

The incidence of cancer of the lips and oral cavity in the world has very different values depending on the different geographical areas. For countries like India, Sri Lanka, Bangladesh, Pakistan and New Guinea, indexes ASR (Age Standardization Rate)

are much higher than, for example, two other Asian nations such as China and Japan In the first countries is extremely important then the number of cases that affects the population below the age of 45, a number which among other things increases every year. The overall data are published by the IARC and reported in "Globocan 2012" (1). Because of the high incidence of the disease and the increasing number of new cases in the youngest age group of the population, Health Institutions, in many countries, have launched the alarm on the consumption of the seeds of areca catechu (betel), which seems to be, according to many researchers, the main cause of this phenomenon. In New Guinea was even passed a law that prohibits the consumption (1), but with little success among the population. In this country it is estimated that each year 25,000 people die from oral cancer and 10,000 new cases are expected. A Taiwan also carcinoma of the oral cavity is the leading cause of death among young males aged between 25 and 44 years (2). Not speaks of 'lifestyle habit as a real ritual which has its roots in more than two thousand years ago. It is estimated that in these areas are roughly between 200 and 300 million regular consumers who chew betel nut (3) and over a billion worldwide. The betel is a mixture of products, essentially formed by a leaf of the plant of pepper (betel), from which, improperly, the name of the food bolus that covers the main component, or the seed of a palm called Areca catechu. This seed is cut into slivers and put inside the leaf with the addition or absence of other plant and/or minerals but have , between the components of which are outstandingly high concentration tannins, (20-35%) (4). These chemical compounds have a structure of the type polyphenolic, whose reactivity, against salivary proteins, can be expressed, sometimes, with their precipitation, and with the feeling of well-known "astringency" - One speaks of astringency when, the cavity is exposed to oral astringent molecules (5, 6). The importance of saliva as a lubricant oral is well demonstrated by the people who suffer from xerostomia with marked reduction in salivary flow below the value of 0,1 mL/min (7). The xerostomia or even just the feeling of dry mouth can result from alterations in the physicochemical properties of saliva. Although saliva is constituted for 99% of water, are the salivary proteins and ions which confer such properties. Among all the salivary proteins, MUC5B and MUC7, produced by sublingual and submandibular glands, and that represent about 20-30% on the total are essential to the lubricating properties, depending on their structure (8-10), which derive from their glycosylation. It is believed that these mucins are adsorbed in a protein layer at the interface liquid of the saliva-cell, to form the so-called salivary pellicle, which layer has an average thickness of 70-100 microns (11) such as to guarantee in all the oral cavity and larynx lubrication and protection. This function can only be guaranteed under certain conditions of rheology that prevents

the film salivary mucin, in the process of sliding and renewal, the phenomena of abrasion and laceration (12). Also note that if the saliva increases very its viscosity (9) inhibits its same lubricating ability, preparing also in this case the oral cavity to various diseases. Ultimately mucins are essential in maintaining the lubricity, adhering strongly to the surface in layers, formed with the repulsive areas, by a the steric-electrostatic bounds (13). The concept of astringency, therefore indicating a phenomenon which results in the precipitation of all salivary protein with also mucins, and is induced primarily by polyphenolic compounds, according to the proposed scheme (14). In practice, the seizure of the proteins by the tannins is because the presence of aminoacid groups and this involves the formation of an aggregate very stable and insoluble in water due to the formation of bonds between the OH groups present in tannins with oxygen ketoimidic bond (-CO-NH-) peptide of aminoacids. It' s clear that, the concentration of polyphenols contained in most of some foods, tea, apples, etc., can cause the entire precipitation of salivary mucins. This occurs, however, as has been demonstrated (14), during the chewing of betel. In this case it has, for the entire period of mastication, the total deprivation of the oral mucosa of the protective film, and it is in contact with products carcinogens. In nature, the main food of vegetable origin that is high in tannins, such as apples and persimmons, the sensation of astringency is changed during the process of fruit ripening. At the end of this step significantly increases the concentration of pectin, which by binding to the tannin, it inhibits its efficacy, neutralizing it. Recent work (15, 16), aimed at changing the structure of certain foods and their attractiveness, have studied, in fact, the interaction of some carbohydrates used in the food industry in the process of precipitation of salivary proteins to work of tannins; between the various compounds employed as arabic gum, pectin and polygalacturonic acid, the latter proved to be the most efficient in inhibiting this phenomenon. Similarly, however, the same pectin can interact with proteins, as demonstrated in the case of poly-L-lysine (17), through processes of cross-linking, in the presence of pH values close to neutrality, forming colloids gel type, which can flocculate, if the proteins are in excess. Proteins, being surface active can play major role in the formation and stabilization of emulsions in the presence of polysaccharide, while interacting through electrostatic or hydrophobic-hydrophobic interactions. On the other hand, polysaccharides being hydrophilic in nature generally remain in aqueous phase thus help in controlling the aqueous phase rheology like thickening, gelling and acting as stabilizing agents. The formation and deformation of polysaccharide-protein complexes and their solubility depend on various factors like charge and nature of biopolymers, pH, ionic strength and temperature of the medium. For the above, the aim of this study, was to investigate

whether these interactions, pectin-tannin, pectin–protein, tannin-protein, possible separately, may occur simultaneously. The goal is to have in the certain conditions, an adduct in which the pectin binds all tannic matter ,binds the proteins ,making but free protein sites, which in turn become fixed to the oral mucosa.

MATERIALS AND METHODS Four volunteers, two male and two female all not smokers, were asked to provide a saliva sample, obtained from the collection of the same, at various times of the day. The volunteers were chosen from all non-smoking and previous anamnesis, showed no diseases that could affect the value of salivary proteins concentration, such as for Sjogreen syndrome, nor that presented examination target oral disease such as gingivitis. The sampling was performed within two hours in the morning and also in the afternoon, without stimulators processes, taking care to provide saliva about an hour away from meals. The volunteers gave each a sample of whole saliva of approximately 50 cc ,and for every test are employed 5 ml; of these samples was carried out biochemical analysis for total protein concentration (mg/dl), and after for pH value. The concentration of total proteins was performed by photometric Biureto test while the pH was determinate by the use of sticks for urinalysis, type Uriscan Roche, reading the values with automatic analyzer URIYSIS 2400 ROCHE. The pectin is made by BIOLINE: AGLU PET: LA-S20. The tannin is of the type used in moderate reactivity UVO TAN BO; MILANO DAL CIN. The measure of concentrations of pectins in salivary medium was determinate by the use of a refractometer DR 10, with double scale Brix (Maselli Misure co). The experimental approach of this work, namely the determination of the amount and concentration of tannin and pectin to be added to whole human saliva samples were identified based on the results obtained in vitro in a previous work (14), which simulates, in vitro, the protein-tannin interaction during chewing of betel. In this work, the total precipitation of the proteins was insured for a minimum concentration of tannin in the volume of saliva employees equal to 0.4 g/l. The ratio p/p, tannin/salivary proteins, was defined in the range from 1: 4 to 1: 1 with a maximum quantity of tannin of 1 g/l, a concentration approximately 8 -15 times lower than that which is liberated during the chewing of betel. The relationships tannin/pectin employed in these experiments have been identified, in line entirely theoretical, based on the interactions made possible and probable by the molecular structures of the polyphenols with compounds of the class of polysaccharides, in a range of experimentation in (w/w), tannin/pectin, which varies from 2:1 to 1:2. The amount of pectin in the first time employed, and the pectin–tannin mixtures (in equal part), after, to be added to the saliva samples as powders under agitation.

Spearman’s rho correlation coefficient was used to asses correlation between pH and Protein % absorption. P-values less than 0.05 were considered significant and due to the small sample size, p-values between 0.05 and 0.1, were discussed as trend.

RESULTSThe main parameters considered for testing interaction saliva or saliva more pectin and pectin and tannin, are total salivary proteins and pH salivary medium: the results for the interaction, protein-pectin are showed in table 1. N ° sample pH Proteins mg/dl

1 6 310

2 8 290

3 6.5 320

4 7 300

Table 1. Samples of whole saliva.

For the samples in question have been added to the preparation of pectin at different concentrations; results are shown in Table 2.

sample

6 g/l addition pectin 9 g/l addition pectin 12 g/l addition/pectin

pH Protein mg/dl

Protein% absorption

Pectin in solution g/l

pH

Protein mg/dl

Protein % absorption

Pectin in solution g/l

pH

Protein mg/dl

Protein % absorption

Pectin in solutiong/l

1

6 155 53% 1.2 g/l

6 90 73% 3.4g/l

6 0 100% 6.8g/l

2

8 0 100% 1.5g/l

8 0 100% 3.8g/l

8 0 100% 6.5g/l

3

6,5 120 64% 1.1g/l

6,5

0 100% 3.5g/l

6,5

0 100% 7.0g/l

4

7 50 82% .,4g/l 7 0 100% .,0g/l 7 0 100% 6.0g/l

Table 2. Interaction pectin/salivary protein.

These ratios, between total protein and pectin, expecially with the polysaccharide in excess, has allowed that the gel formation of the complex pectin protein not to collapse and after are flocculated , as experienced in another work (17), but to be formed, in all tests, an opalescent colloid instead fleet towards the surface of saliva, as in photo 1 attached. For the analytical determination of the various indices examined were taken from samples of saliva underlying colloid solution floated.An examination of table 2 it can be seen how the adsorption of salivary proteins has a trend of depending in the first place from the initial pH value of the saliva when the concentration of pectin is lowest (rho=1 and p-value = 0.083, with 6 g/l addition pectin, rho=0.77 and p-value = 0.225, with 9 g/l addition pectin). With the lowest value of pH (pH 6), when the concentration of pectin is 6 g/l, it also has the lowest amount of proteins that interact with the pectin to form a complex floating in saliva.With the value instead of the initial pH equal to eight and the same concentration of pectin, the value of the adsorbed protein is 100%.Even the concentration of the pectin the ratio p/p on proteins is important. In test with a concentration of pectin equal to 12 g/l, one has the complete absorption of the protein in all samples. In the second set of trials have examined the effects of the addition to the saliva of mixtures of tannin and pectin in variable ratios. In the tables 3, 4, 5 you can instead highlight as the concentration of tannin added to the saliva has the highest weight important. With a 1:1 ratio tannin/pectin, for two samples at lowest acid pH proteins remain in solution (rho=0.94 and p-value = 0.057), while if the ratio is pectin/tannin (2:1), we obtain the total adsorption of salivary proteins to form an floating adduct .sample

pH proteinsmg/dL

proteinsadsorption%

pectin insolution

notes

1 6.0 0 100 0 flocculation

2 7 0 100 0 flocculation

3 6 0 100 0 flocculation

4 6.5 0 100 0 flocculation

Table 3. Saliva + Tannin (6gr/L) + Pectin (3gr/L)

sample

pH

proteins mg/dL

proteinsadsorption %

pectin in solution

notes

1 6 90 69 0 floc+float

2 7 0 100 0 floc+float

3 6 45 86 0 floc+float

4 7 0 100 0 floc+float

Table 4. Saliva + Tannin (6gr/L) + Pectin (6gr/L)

sample

pH

proteins mg/dL

proteinsadsorption %

pectin in solution

notes

1 6.5 0 100 0.2 floating

2 7.5 0 100 0.2 floating

3 6.5 0 100 0.9 floating

4 7 0 100 1.0 floating

Table 5. Saliva + Tannin (6gr/L) + Pectin (12gr/L)

DISCUSSION The results obtained and presented in tables 2, 3, 4 are in full agreement with the studies and the assumptions, reported in literature (18) as the mechanisms of formation of pectin complex protein.Polysaccharide and protein complex formation is mainly driven by various non-covalent interactions, like electrostatic, H-bonding, hydrophobic, and steric interactions .Protein carries +ve or –ve zeta potential based

on the pH of the medium (+ve at pH lower than pI and vice-versa). This +ve or –ve electrical charge on the protein chain point towards the presence of different amino acids in the protein molecules and their mode of ionization at different pH ranges. Carboxyl ate polysaccharides get deprotonated (become anionic) at a pH range higher than its pKa. This electrical charge on the back bone of protein or polysaccharide chain is responsible for electrostatic attraction or repulsion between them. Again, presence of -COOH group on the polysaccharide and -NH3, -COOH groups on the protein chain are the sources of hydrogen bonding between these two bio-polymers. Extent of both of this hydrogen bonding and electrostatic interaction depends on the solution parameters such as pH, ionic strength, temperature etc. Except these ionic patches on the bio-polymers, few non-polar segments are also present on the bio-polymers, which are responsible for the hydrophobic staking with each other. Even though solution parameters are important factors to control the different mode of interactions between protein and polysaccharide, type of proteins/polysaccharides, molecular weight, charge density, and hydrophobicity of the bio-polymers are also play significant role towards the extent the linkage between two bio-polymers at a fixed condition. How can 'easily inferred from the observation, two parameters, pH and the concentration of pectin, but not the concentration of the total protein present in saliva, affect, significantly, the percentage values of the proteins responsible for the formation of colloidal complexes. The most relevant data for sample 1 (initial pH 6), for which with the addition of pectin in a concentration of 3g/l, a significant amount (47%) of total salivary proteins remains in solution; this value drops to 27% when the concentration of pectin added salt to 6 g/l. The main parameter that still seems to be what the conditions the process of interaction and the linkage of pectin with salivary proteins is the pH value, as is clear to the observation of the results expressed in the tables 2 and 3.Whe the pH values increase, the molecular configuration of pectin extends spatially, offering more interaction sites to the proteins. This phenomena is really expressed with the results of the tables 2 and 3. Another, relevant data, complete reaction of the protein to form a colloid, already in pectin concentration of 3 g/l, one has to sample 4 where the initial pH 7 is also it important to emphasize that below a certain concentration of pectin, at least in the range of the initial pH of the saliva samples, analyzed, does not occur the total precipitation of the protein fraction. This value as one can easily derive from the results of tables 1 and 2, is related to a ratio pectin/protein greater than 5.However, is very important to indicate that the formation of a stable colloid depends of the value of pH also to the pectin concentrations, and in fact this chemical-physical phenomena stops when the amount of pectin is in large excess as 12 g/l. Analyzing the

data, but more importantly, seeing the picture, we can speculate how such a phenomenon observed in vitro, can be translated in vivo in the formation of a colloid laminated and bound to the oral mucosa through the groups protein, thereby preserving the protective function of the protein fraction of the saliva. After these experiments, were added to samples of human saliva, in order next time, prepared aqueous solutions of tannin abs well be seen from the tables, and from photos regardless of the data obtained in all tests have been formed a ternary complexes, protein-pectin-tannin. Practically, the process of their formation can be hypothesized in two phases:A-pectin bound protein;B-excess of pectin not bound with protein interacts with tannin.This second phase, hypothesized with a mechanism shown in figure 1, is determinate by various parameters, principally substrates concentration and value of pH medium, and is more favored by electrostatic force than tannins on the proteins interactions, see figure 2.

Figure 1. Pectin–tannin interaction, a model of possible linkage

Figure 2. Excess of pectin not bound with protein interacts with tannin

In table 5 we can see how with a concentration of tannin of 6 g/l, twice the pectin, the predominance of phenolic groups, totally inhibit the adsorbing power of the pectin to the proteins, precipitating the same and the polysaccharide (see figure 3).

Figure 3. Excess of tannin precipitate the proteins and the pectin

The salivary pH of the solution in all the four samples is lowered for samples 1- 3-4 up-acidity and for the sample 2 to neutrality. Bringing the ratio tannin/pectin to equal, see table 6, can be observed in all four samples, the formation of three distinct phases, (see figure 4), with the separation of a flocculated based mainly tannins, an intermediate solution, which in the samples 1-3 still contains protein, and a colloid floating, pectin based.

Figure 4. Formation of three distinct phases

The final pH value in these samples is acidic, lower than one unit, compared to the samples 2 and 4.

sample cc saliva cc tannincc pectin

WBC RBC µL

pH proteins mg/dL

proteinsadsorption %

pectin in solution

notes

1 5 2.5+2.5 0 0 6 35 69 0 floc+float

2 5 2.5+2.5 0 0 7 0 100 0 floc+float

3 5 2.5+2.5 0 0 6 15 90 0 floc+float

4 5 2.5+2.5 0 0 7 0 100 0 floc+float

Table 6. Saliva + Tannin (6 g/l) + Pectin (6 g/l)

Probably the initial pH more acidic in these samples, facilitates the partial hydrolysis of polysaccharide, inhibiting its sequestering ability for a better interaction of tannins with salivary proteins. There is also the possibility that the presence of ions, such as sodium and chlorine in the saliva, can also destabilize the colloids of pectin and proteins formed, as demonstrated in a recent article (19), even if the pH of the solution in this case was considerably more acid "5" and the solution of sodium chloride addition had a molarity of 0.15 M ie with concentrations of Na and Cl much higher than those normally present in human saliva (20). The main hypothesis remains that is due to the influence of pH, which, however, requires further experimentation with the examination of the process of interaction tannin-pectin in the absence of protein. In table 7 are finally reported the data related to testing in which the relationship tannin pectin has been reversed or 1:2, in this case in all the samples there is the disappearance of salivary proteins entirely sequestered to form a "colloid multilayer" that fleet to the surface, see figure 5.

sample cc saliva

cc tannincc pectin

WBC RBC µL

pH proteins mg/dL

proteinsadsorption %

pectin in solution

notes

1 5 2.5+2.5 0 0

6.5 0 100 0.2 floating

2 5 2.5+2.5 0 0

7.5 0 100 0.2 floating

3 5 2.5+2.5 0 0

6.5 0 100 0.9 floating

4 5 2.5+2.5 0 0

7 0 100 1.0 floating

Table 7. Saliva + Tannin (6 g/l) + Pectin (12 g/l)

Figure 5. Colloid multilayer

The hypothesis most likely, also confirmed by other authors in a recent article (21), is that the formation of a ternary complex, protein -pectin- tannin, as determined by us from both hydrophilic and hydrophobic interactions, which in vivo by its nature should form a multi-layer adhering to the oral mucosa. The experimental aspects used in this work, egg type of saliva, its value of pH, and proteins concentration, cover wide range of real-life situations. Also the extreme experimental conditions have been obtained in these tests with the addition of tannin. With these assumption it can be said as the data obtained can be sufficient to demonstrate how at least, in vitro, the ability to form a stable colloid that incorporated the proteins to form a multilayer structure which does not precipitate, it is possible when the concentration of pectin is at least twice of the tannin. The consequence of these results is that it can be assumed with sufficient probability the multilayer colloid remains bound to oral mucosa. If fact we eat unripe fruit, we experience the phenomenon of astringency because the tannins precipitate mucins, leaving the oral mucosa discovery in some places where the nerve receptors are stimulated by the same tannin. When the fruit is ripe or when there is a sufficient amount of pectin, this does not happen, because the polysaccharide neutralizes the tannins and mucin remains bound to the oral mucus. So in the test 7, in which there is the flotation in the presence of an excess of pectin, it is permissible hypothesize that the colloid remains bound to oral epithelium. The work described here highlights the importance in the control of all those factors and not food, that produce the phenomenon of astringency, a feeling that may signal a biochemical imbalance in mouth parts, when perceived in a chronic, especially for the use and/or abuse of substances such as betel. This indicates that sensation in the oral cavity occurs even if partially and/or limited in time, a process of precipitation of salivary proteins, and between those of mucin, the most important defense against the chemical, physical and biological (22) attacks. In this work was again demonstrated such as pectin, in the chemical and physical conditions of the saliva of the various samples examined to form stable colloids with proteins, which in vitro-floating, thus being able, speculate that in vivo, such structures can remain adhering to the oral mucosa, protecting it. Certainly the introduction of tannin in samples of human saliva, in an amount double that of the double pectin may inhibit this protective effect, at least in certain samples of saliva, where the initial condition of pH are low or when the adsorbing capacity of the same can be prevented by the considerable presence of salivary calcium ions as in Sjogren syndrome and in others conditions. Based on the results of this work could be considered the use of betel replacing the lime dose of pectin in an amount at least double those of the tannin present in the seed of Areca catechu. In this case, obtaining in vitro formation in salivary solution, of a ternary

stable colloid, which is also of the floating type, it can be assumed that in vivo it should ensure the defense of the oral mucosa. Certainly, these data need to not only further evidence, with others, and different physical and chemical characteristics for biological samples of human whole saliva, but also and above all, an audit in vivo.

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