bioefficacy assessment of licorice
TRANSCRIPT
BIOEFFICACY ASSESSMENT OF LICORICE
NUTRACEUTICS AGAINST METABOLIC
DISORDERS
By
Muhammad Sohail M.Sc. (Hons.) Food Technology
A dissertation submitted in partial fulfillment of the
requirements for the degree of
DOCTOR OF PHILOSOPHY
IN
FOOD TECHNOLOGY
NATIONAL INSTITUTE OF FOOD SCIENCE & TECHNOLOGY
FACULTY OF FOOD, NUTRITION AND HOME SCIENCES
UNIVERSITY OF AGRICULTURE
FAISALABAD
2017
DEDICATED
To
Holy Prophet Muhammad
(Peace be upon him)
3.7.3. Sensory evaluation 33
3.7.4 Selection of best treatments 33
3.8. Bioefficacy trial 33
3.8.1. Hepatoprotective perspectives 35
3.8.1.1 Oxidative stress biomarkers in liver 35
3.8.1.1.1. Superoxide dismutase (SOD) 35
3.8.1.1.2. Catalase 35
3.8.1.1.3. Melandialdehyde (MDA) 36
3.8.1.2. Serum specific biomarkers 36
3.8.2. Serum lipid profile and glucose & insulin levels 36
3.8.2.1. Cholesterol 36
3.8.2.2. High density lipoprotein 36
3.8.2.3. Low density lipoprotein 36
3.8.2.4. Triglycerides 36
3.8.2.5. Serum glucose and insulin levels 36
3.8.3. Safety assessment studies 37
3.8.3.1. Renal functioning tests 37
3.8.3.2. Hematological analyses 37
3.9. Statistical analysis 37
4. RESULTS AND DISCUSSION 38
4.1. Phytochemical screening and antioxidant activity assays for CSE 38
4.1.1. Total phenolic content (TPC) 38
4.1.2. Total flavonoids (TF) 41
4.1.3. Free radical scavenging activity (DPPH assay) 43
4.1.4. Ferrous reducing antioxidant power (FRAP) assay 44
4.1.5. ABTS assay 46
4.2. Phytochemical screening and antioxidant activity assays for SFE 48
4.3. Quantification of active ingredients 50
4.4. Selection of best treatments 54
4.5. Development of licorice based drink 54
4.5.1. Physicochemical analysis of licorice drinks 54
4.5.2. Antioxidant potential of licorice drinks 62
4.5.3. Sensory Evaluation 62
4.6. Selection of best treatments 74
4.7. Bioefficacy trial 74
4.7.1 Hepatoprotective perspective 75
4.7.1.1. Alanine Transaminase (ALT) 75
4.7.1.2. Aspartate Transaminase (AST) 78
4.7.1.3. Alkaline Phosphatase (ALP) 80
4.7.1.4. Superoxide dismutase (SOD) 82
4.7.1.5 Catalase 83
4.7.1.6. Malondialdehyde (MDA) 87
4.7.1.7. Bilirubin 89
4.7.2. Hypocholesterolemic perspective 91
4.7.2.1. Total cholesterol 91
4.7.2.2. High density lipoproteins (HDL) 94
4.7.2.3. Low density lipoproteins (LDL) 97
4.7.2.4. Serum triglycerides 99
4.7.2.5. Glucose 102
4.7.2.6. Insulin 105
4.7.3. Safety assessment studies 107
4.7.3.1 Renal functioning tests 107
4.7.3.1.1. Urea 107
4.7.3.1.2. Creatinine 107
4.7.3.2. Hematological analyses 109
5. SUMMARY 115
RECOMMENDATIONS 122
LITERATURE CITED 123
APPENDIX 140
i
AACCKKNNOOWWLLEEDDGGEEMMEENNTTSS
All praises for Almighty Allah, who creates everything by saying ‘Kun Fayakoon’. All respect
and reverence for Holy Prophet Muhammad (P.B.U.H.) whose teachings are complete
guidance for humanity.
I pay cordial gratitude to my worthy and zealot supervisor Dr. Allah Rakha, Assistant
Professor, National institute of Food Science & Technology, Faculty of Food Nutrition &
Home Sciences, University of Agriculture, Faisalabad, for his invaluable help, thought-
provoking guidance, immense intellectual input, sympathetic and kind attitude throughout the
study. He supervised the whole work critically and gave tremendous constructive comments for
the preparation of this manuscript.
It is my utmost pleasure to avail this opportunity to extend my heartiest gratitude to Prof. Dr.
Masood Sadiq Butt, Dean, Faculty of Food, Nutrition and Home Sciences, University of
Agriculture, Faisalabad, for his inspiring guidance and ever encouraging attitude during my
research work. With due respect, I am deeply and strongly obliged to Prof. Dr. Muhammad
Asghar, Department of Biochemistry, University of Agriculture, Faisalabad, for his counseling,
patronizing and scholarly knowledge.
It is imperative to mention my lab mates for their assistance and good company. I would like to
offer my heartiest graditude to my beloved friends, Jawad Iqbal and Iahtisham-ul-Haq for their
support and cooperation at every step of my study. I express my deep sense of gratitude to Faiza
Ashfaq and Kanza Aziz Awan for their dexterous & untiring cooperation and encouragement
for the completion of research. Special thanks to Muhammad Rizwan for his company during
research and thesis work. No acknowledgements would ever adequately express my obligation
to my parents who always wished to see me glittering high on the skies of success. Whatever, I
am today, is because of their love and prayers.
Muhammad Sohail
ii
LIST OF TABLES
Sr. No. Title Page No.
1 Treatments for solvent extraction 28
2 Treatments for supercritical fluid extraction 29
3 Treatments plan for licorice drink development 32
4 Experimental plan for bioefficacy study 35
5 Mean squares for antioxidant indices of licorice solvent extracts 39
6 Means for TPC (mg GAE/100g) of licorice solvent extracts 42
7 Means for flavonoids (mg/100g) of licorice solvent extracts 42
8 Means of DPPH activity (%) of licorice solvent extracts 45
9 Means for FRAP assay (μM Fe2+/g) of licorice solvent extracts 45
10 Means for ABTS assay (µM TE/g) of licorice solvent extracts 47
11 Mean squares for antioxidant indices of licorice supercritical fluid
extracts 49
12 Mean for antioxidant indices of licorice supercritical fluid extracts 49
13 Mean squares for HPLC quantification of bioactive components 51
14 HPLC quantification of bioactive components of licorice 53
15 Mean squares for color tonality of licorice drinks 57
16 Effect of treatments and storage on L* value of licorice drinks 57
17 Effect of treatments and storage on a* value of licorice drinks 58
18 Effect of treatments and storage on b* value of licorice drinks 58
19 Effect of treatments and storage on chroma of licorice drinks 59
20 Effect of treatments and storage on hue angle of licorice drinks 59
21 Mean squares for pH, acidity and TSS of licorice drinks 61
22 Effect of treatments and storage on pH of licorice drinks 61
23 Effect of treatments and storage on acidity of licorice drinks 63
24 Effect of treatments and storage on TSS/brix of licorice drinks 63
25 Mean squares for antioxidant indices of licorice drinks 64
26 Mean squares for sensory evaluation of licorice drinks 69
27 Effect of treatments and storage on color of licorice drinks 70
28 Effect of treatments and storage on flavor of licorice drinks 70
29 Effect of treatments and storage on taste of licorice drinks 72
30 Effect of treatments and storage on mouthfeel of licorice drinks 72
31 Effect of treatments and storage on sweetness of licorice drinks 73
32 Effect of treatments and storage on overall acceptability of licorice
drinks 73
33 Effect of licorice drinks on ALT levels (IU/L) of rats in different 76
iii
studies
34 Effect of licorice drinks on AST levels (IU/L) of rats in different
studies 79
35 Effect of licorice drinks on ALP levels (IU/L) of rats in different
studies 81
36 Effect of licorice drinks on SOD (IU/mg protein) of rats in different
studies 84
37 Effect of licorice drinks on catalase activity (IU/mg protein) of rats in
different studies 86
38 Effect of licorice drinks on MDA (nM/mg) level of rats in different
studies 88
39 Effect of licorice drinks on bilirubin level (mg/dL) of rats in different
studies 90
40 Effect of licorice drinks on cholesterol (mg/dL) of rats in different
studies 92
41 Effect of licorice drinks on HDL (mg/dL) of rats in different studies 95
42 Effect of licorice drinks on LDL (mg/dL) of rats in different studies 98
43 Effect of licorice drinks on triglycerides (mg/dL) of rats in different
studies 101
44 Effect of licorice drinks on glucose (mg/dL) of rats in different studies 103
45 Effect of licorice drinks on insulin (µU/mL) of rats in different studies 106
46 Effect of licorice drinks on urea level (mg/dL) of rats in different
studies 108
47 Effect of licorice drinks on creatinine level (mg/dL) of rats in
different studies 110
48 Effect of licorice drinks on RBC (cells/pL) of rats in different studies 112
49 Effect of licorice drinks on WBC (cells/nL) of rats in different studies 113
50 Effect of licorice drinks on Platelets (103/µL) of rats in different
studies 114
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LIST OF FIGURES
Sr. No. Title Page No.
1 Effect of treatments on antioxidant indices of licorice nutraceutical
drink 66
2 Effect of storage on antioxidant indices of licorice nutraceutical
drink 67
3 Percent reduction in ALT levels as compared to control drink 76
4 Percent reduction in AST levels as compared to control drink 79
5 Percent reduction in ALP levels as compared to control drink 81
6 Percent increase in SOD levels as compared to control drink 84
7 Percent increase in catalase levels as compared to control drink 86
8 Percent reduction in MDA levels as compared to control drink 88
9 Percent reduction in bilirubin levels as compared to control drink 90
10 Percent reduction in cholesterol levels as compared to control drink 92
11 Percent increase in HDL levels as compared to control drink 95
12 Percent reduction in LDL levels as compared to control drink 98
13 Percent reduction in triglycerides levels as compared to control 101
14 Percent reduction in glucose levels as compared to control drink 103
15 Percent increase in insulin levels as compared to control drink 106
v
LIST OF APPENDICES
Sr. No. Title Page No.
I Sensory evaluation performa for licorice drink 140
vi
ABSTRACT
Recent research in the field of food and nutrition has extensively focused on the dietary
approaches, as an effective tool for healthy lifestyle. Functional foods and nutraceutics have
successfully coined as therapeutic interventions against various metabolic disorders. The main
objective of this study was to explore the role of licorice bioactive components against
dyslipidemia and hepatic malfunctions. In current project, extraction and characterization of
licorice bioactive moieties was carried out followed by product development and bioefficacy
assessment using rat modeling. For optimum recovery of nutraceutics, three solvents (ethanol,
methanol and ethyl acetate) were employed at different ratios with water (25:75, 50:50 and
75:25) whereas supercritical fluid extracts were obtained at varying pressures (3500, 4500 and
5500 psi). . The resultant conventional extracts were tested for total phenolic content (TPC),
total flavonoids (TF) 2,2-diphenyl 1-picrylhydrazyl (DPPH),ferric reducing antioxidant power
(FRAP) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) ABTS assays. Afterwards,
two best treatments, one from each conventional solvent and supercritical fluid extracts were
selected on account of promising phytochemistry and maximum antioxidant potential. Results
indicated that 75% ethanolic extract showed maximum antioxidant activity among solvent
extracts; TPC 897.24±31.49 mg GAE/100g, TF 286.17±9.85 mg CE/100g, DPPH
72.65±2.45%, RFAP 451.52±15.73 μM Fe2+/g and ABTS 11.02±0.46 μM Trolox/g. Among
supercritical fluid extracts, 5500 psi extract (TSC3) exhibited best results; TPC 1532.75±36.84
mg GAE/100g, TF 576.13±23.51 mg CE/100g, DPPH 88.26±3.255%, FRAP 743.45±19.38 μM
Fe2+/g and ABTS 17.85±0.55 μM Trolox/g. Afterwards, all the conventional solvent and
supercritical fluid extracts were analyzed for their glycyrrhizin and glabridin content via HPLC
quantification. Results depicted that highest concentrations of glycyrrhizin and glabridin were
detected in TSC3 as 5.02±0.031 and 2.97±0.012 mg/g, respectively. Amongst conventional
solvent extracts, the highest glycyrrhizin content was detected in 25% methanolic extract as
2.41±0.027 mg/g whereas, highest glabridin content was observed in 75% ethanolic extract i.e.
1.13±0.010 mg/g. . On the basis of HPLC analysis, one selected treatment from each extraction
mode; 75% ethanolic extract and supercritical carbon dioxide extracts at 5500 psi pressure were
further proceeded for the development of drink by adding 0.4% nutraceuticalCSE (T1), 0.2%
nutraceuticalCSE (T2), 0.1% nutraceuticalSFE (T3), 0.2% nutraceuticalSFE (T4) and control (T0)
treatment without any extract. Physicochemical analysis revealed significant effect of
treatments on color parameters (L*, a*, b*, chroma) whereas pH, acidity and brix were non-
significantly changed. Likewise, treatments and storage interval significantly affected the
antioxidant potential of drinks. Based on sensory evaluation scores, T1 (drink containing 0.2%
CSE) and T4 (drink containing 0.2% SFE) were selected for bio-evaluation. During
bioevaluation trial, three simultaneous studies namely study I (normal rats), study II
(hypercholesterolemic rats) and study III (hepatotoxic rats) were carried out for 90 days. Each
study was further splitted into three groups based on diets; control (D0), nutraceuticalCSE drink
(D1) and nutraceuticalSFE (D2). Results indicated that serum ALT, AST, ALP, bilirubin and
MDA levels were significantly decreased by 20.51 & 31.19%, 17.91 & 28.62%, 16.11 &
23.75%, 15.53 & 26.21% and 20.76 & 38.33% in D1 and D2 groups, respectively in hepatotoxic
rats. Whereas, SOD and catalase activities were significantly enhanced by 19.26 & 31.95% and
17.32 & 25.78%, respectively in D1 and D2 groups. Likewise, provision of nutraceuticalCSE and
nutraceuticalSFE drinks resulted in significant decrement in serum total cholesterol, LDL,
triglycerides and glucose by 11.24 & 18.52%, 17.56 & 24.37%, 9.57 & 15.74% and 5.17 &
7.28%, respectively in hypercholesterolemic rats. Nevertheless, HDL and insulin levels were
improved significantly. Moreover, kidney functioning biomarkers and hematological aspects
were within normal ranges. Conclusively, licorice nutraceutics have potential to mitigate
hepatotoxicity and dyslipidemia through different mechanism.
1
CHAPTER 1
INTRODUCTION
Scientific research on the relationship between dietary habits and disease risk has shown a
direct impact of food on human health. Changing dietary patterns; shift towards refined
and processed foods along with less consumption of plant based diet set the seed of many
metabolic disorders including hepatotoxicity, hypercholesterolemia, diabetes,
cardiovascular diseases and several types of cancer (Espin et al., 2007). Recently, an
increasing trend has been observed in the use of processed and junk foods in Pakistan
(Shamoon et al., 2012). Novel nutritional approaches have successfully been employed as
therapeutic interventions against these maladies. Scientific evidences have supported
dietary intervention as an effective tool for health promotion (Shahidi, 2000). In this
context, plant derived non-nutritive secondary metabolites (referred as phytochemicals)
have shown promising capacity to be utilized as therapeutic agents. Recently, the
philosophy “Let food be your medicine and medicine be your food” is gaining special
attention forming the basis of functional foods and nutraceutics (Mollet and Rowland,
2002).
Nutraceuticals are nutritional supplements that are isolated from natural sources with the
intent to prevent a particular disease or group of diseases and has no regulatory concerns.
Thus, nutraceutics are considered as part of food that provide health benefits and have
some therapeutic properties to address different metabolic diseases such as
hypercholesterolemia, cardiovascular diseases, liver malfunctions and diabetes
(Rajasekaran et al., 2008). Nutraceutics in the form of antioxidants, phytochemicals,
dietary fiber, prebiotics, probiotics and polyunsaturated fatty acids have been investigated
and are being used for better health (Whitman, 2001). Natural commodities are the main
source of bioactive components to develop functional foods and traditional medicines
against specific ailments. Nearly one half of the medicinal components have their origin
from natural sources. Plants have a promising future as a source of bioactive moieties with
disease modulating potential. So far, only 6% of the total estimated plant species (250,000-
400,000) have been investigated for their biological activity (Lakshmi and Geetha, 2011).
2
Licorice is the root of Glycyrrhiza uralensis Fisch., G. inflata Bat., and G. glabra L. plants
belongs to the Leguminosae family. It is a small shrub with flat pods, purplish or white
flower clusters, oval shaped leaflets, main taproot and numerous runners. It is widely
cultivated in India, Spain, Persia, Afghanistan, Kazakhstan, Russia, Tajikistan, China and
some areas of Pakistan. In Pakistan, licorice is native to Punjab, Baluchistan and some
areas of Jammu Kashmir region. In Chinese Pharmacopoeia, roots of G. glabra, G. inflate
and G. uralensis are all regarded as licorice (Yang et al., 2014). The first documented
literature on the medicinal use of licorice dated back to 2100 BC in Shennong’s Classic of
Materia Medica, the first Chinese dispensary. Licorice has several food applications in
industry as flavoring and sweetening agent and got the status of GRAS by United States
Food and Drug Administration (FDA) (Zhou et al., 2013. Numerous studies have reported
pharmacological and nutraceutical potential of licorice such as antiviral (Baltinar et al.,
2012), anti-inflammatory, hepatoprotective, (Zhang et al, 2012; Sun et al, 2010), antitumor
(Tao et al, 2013; Wang et al., 2013) and immune stimulating activities (Kim et al., 2013b).
However, some studies have reported that the over consumption of glycyrrhizin (the major
bioactive component of licorice) is associated with hypermineralocorticoid condition (Isbrucker
and Burdock, 2006).
Fresh licorice root encompasses about 20% extractable substances. The main extractable
moiety is glycyrrhizin (3-5%) which occurs in the form of calcium and potassium salts.
Flavonoids are the second major extractives (1-1.5%) and give bright yellow color to
licorice root. Root extract also contain starch, essential oils, resins, reducing and non-
reducing sugars, gums, inorganic salts and minute quantities of nitrogenous components
like nucleic acids, proteins and individual amino acids (Isbrucker and Burdock, 2006).
More than 400 compounds have been reported in licorice. Flavonoids (mainly glabridin)
and triterpenoid saponins (predominantly glycyrrhizic acid) are the main bioactive moieties
present in licorice (Zhang and Ye, 2009).
Earlier studies on active ingredients of licorice mainly focused on glycyrrhizic acid and its
derivatives. These components are reported to be responsible for antiulcer and
hepatoprotective effects and their pharmacokinetics has been well studied (Fiore et al.,
2004). Glycyrrhizin, also known as glycyrrhizic acid or glycyrrhizinate, makes up about
10-25% of licorice extract. Glycyrrhizic acid is a triterpenoid saponin compound composed
3
of a triterpenoid aglycone, glycyrrhetic acid (glycyrrhetinic acid; enoxolone) conjugated to
a disaccharide of glucuronic acid. Glycyrrhizin can form a number of salts, potassium and
calcium salts are the notable ones. Ammoniated salt of glycyrrhizin is prepared
commercially from licorice root extract and is used as flavor in confectionary industry
(Isbrucker and Burdock, 2006). Recently, licorice flavonoids have gained immense interest
owing to their structural and functional diversity and pharmacological potential. The
flavonoids isolated from licorice root include isoflavans, isoflavones, flavonones,
chalcones, flavanonols, arylcoumarins and isoflavenes (Lee el al., 2007; Xie et al., 2009).
Glabridin, the chief isoflavan derived flavonoid in licorice, possesses promising anti-
atherosclerotic, anti-inflammatory, hypolipidemic and anti-tumor potential (La et al., 2010;
Vaya et al., 2003; Kang et al., 2006). Being a potential antioxidant, it also protects low
density lipoprotein (LDL) against oxidation by scavenging reactive oxygen species (ROS)
(Rosenblat et al., 2002). Isoliquiritigenin and licochalcone are flavonoids with chalcone
structure and possesses high anti-tumor, antiradical and anti-inflammatory potential (Park et
al., 2009; Fu et al., 2004). Glycyrol, another important flavonoid present in licorice with
arylcoumarins structure has also been reported to possess anti-inflammatory response (Shin
et al., 2008).
The bioactive components from licorice can be recovered using different conventional and
novel extraction methods. These extraction techniques carries their own merits and demerits.
Differential solubility of bioactive components in different solvents make it possible to use
a variety of solvents for efficient extraction of the desired component. Conventional
solvent extraction has long been used for separation and isolation of active moieties by
using water, ethanol, methanol, n-hexane, ethyl acetate and other solvents (Pan et al.,
2000). However, various disadvantages of traditional solvent extraction have been reported
like low yield of desired component, long extraction time, more solvent requirement, high
cost and safety concerns linked with their use in food applications. In this context, novel
extraction methods such as microwave assisted extraction, ultrasonic extraction, subcritical
and supercritical fluid extraction have emerged as effective alternates. (Sun et al., 2007).
Supercritical fluid extraction (SFE) is one of the novel technique being employed for the
recovery of biologically active components with potential safety and high extraction rate
(Kim et al., 2004). This technique offers numerous advantages such as non-toxicity,
4
provision of non-residual extract along with environment friendly extraction. This
technique can effectively be used as a replacement for solvent extraction (Klejdus et al.,
2005). Moreover, SFE has been documented as an efficient method to prepare antioxidant
rich extracts from herbal plants (Marongiu et al., 2004), so it is a promising extraction
process to be employed in designer foods and drug preparation industry. Liver is the main
metabolic center of the body and plays a key part in excretion of toxins. It is frequently and
consistently exposed to an array of xenobiotics that can cause acute or chronic liver
dysfunctions with complex pathology and pathogenesis. Hepatotoxicity and associated
problems are among most common afflictions in medical practice. Currently available
drugs for the treatment of liver disorders are often less effective and have several side
effects. Therefore, efforts are being made to explore new and more potent therapeutic
agents from natural products by virtue of their fewer side effects and little or no toxicity
(El-Tawil et al., 2013).
Glycyrrhizic acid (glycyrrhizin) from licorice root has demonstrated promising role to
address liver ailments. It breakdown in vivo to form glycyrrhetinic acid which is the main
active form and responsible for therapeutic effects. Both glycyrrhizic acid and
glycyrrhetinic acid have shown liver-protective effects against CCl4-induced liver damage
and retrorsine-induced hepatotoxicity. Moreover, glycyrrhetinic acid has also been
reported to be an effective inhibitor of bile acid-induced necrosis and apoptosis in liver
(Asl and Hosseinzadeh, 2008; Gumpricht et al., 2005).
Cardiovascular diseases (CVDs) are a major health problem in both developed as well as
developing counties. Dyslipidemic condition is a major indicator of CVDs. Overweight
and obesity are also positively related with increase in dyslipidemia. Restoration of normal
serum lipid patterns is an important concern in this regard. LDL oxidation and higher
serum cholesterol are directly related with atherosclerosis and related discrepancies
(Abeywickrama et al., 2011). Owing to its rich phytochemistry, licorice can successfully
be used as a diet based strategy to regulate blood lipid levels. Many recent studies have
suggested that bioactive components from licorice root are effective in modulating
abdominal fat and overall lipid profile. Licorice has shown the potential to reduce total
cholesterol and low density lipoprotein (LDL) in moderately hypercholesterolemic patients
(Mirtaheri et al., 2015). Earlier research has advocated that flavonoids are the main
5
components responsible for lipid modulating potential of licorice root, glabridin bring the
major one (Tominaga et al., 2009).
Diabetes mellitus is one of the major cause of illness and deaths worldwide. It is
characterized by disturbance in glucose metabolism coupled with complete or relative
deficit insulin secretion and sensitivity. When unnoticed or uncontrolled, diabetes can lead
to serious health complications including neuropathy, nephropathy and cardiovascular
disorders (Sen et al., 2011). Strict dietary restrictions, synthetic drugs and insulin
injections are major modes to curtail the peril. A number of hypoglycaemic medicines have
been developed and used to treat diabetes but all are linked with several side effects such as
liver disorders, lactic acidosis and diarrhea, in addition to the development of drug
resistance (Inzucchi, 2002). Recently, the research is focusing on effective antidiabetic
moieties from natural sources. Licorice and its components have been reported to show
significant hypoglycemic activity which is mainly attributed to glycyrrhizin. Current
investigations regarding the effect of glycyrrhizin on diabetes have delineated significant
reduction in streptozotocin-induced diabetic changes and associated oxidative damage by
the administration of glycyrrhizin (Sen et al., 2011).
The current research work is an endeavor to explore the bioefficacy potential of licorice
nutraceutics against hypercholesterolemia, hyperglycemia and liver dysfunctions. The
objectives of designed project are mentioned herein:
1. Extraction of nutraceutics from licorice root through solvent and supercritical fluid
extraction techniques
2. Development and characterization of licorice based drink
3. Bioevaluation of licorice drink to attenuate lipidemic and hepatic malfunctions
using rodent modeling
6
CHAPTER 2
REVIEW OF LITERATURE
Dietary choices coupled with lifestyle are regarded as the major determinants of health;
owing to their direct relationship with lifestyle related metabolic disorders. Caloric dense
diet, refined and junk foods along with sedentary lifestyle are the key risk factors involved
in the incidence of metabolic ailments including dyslipidemia, diabetes, insulin resistance,
coronary heart diseases, hepatic and renal disorders. Over the years, diet based therapies
have gained special momentum as a promising tool to combat various health discrepancies.
Plant derived phytochemicals have been reported to hold numerous health benefits and are
effective in tailoring the diet for specific health use against targeted disorders. These
phytonutrients are often termed as “nutraceuticals or nutraceutics” and are in limelight
owing to their disease modulating effect, safety, natural origin, cost effectiveness and ease
in availability, processing and consumption for the masses. Herbal plants have a rich
history of use for the treatment of various diseases and recent research has revealed the
presence of hundreds of phytochemicals in these plants. Licorice (Glycyrrhiza Glabra) is
one of most widely used medicinal plant in various ancient schools of medicines including
Ayurveda and Chinese pharmacopia. Licorice root is rich in phytochemicals with strong
nutraceutical potential. Current research project is an endeavor to ascertain the role of
licorice nutraceutics against hepatotoxicity, dyslipidemia and hyperglycemic conditions.
Literature regarding this project has been reviewed and discussed comprehensively herein.
2.1. Nutraceuticals; an overview
2.2. Licorice; at a glance
2.3. Nutraceuticals from licorice
2.4. Antioxidant potential of licorice nutraceutics
2.5. Extraction of bioactive components of licorice
2.5.1. Conventional solvent extraction
2.5.2. Supercritical fluid extraction
2.6. Licorice nutraceutics against metabolic disorders
2.6.1. Hepatoprotective perspectives
2.6.2. Hypolipidemic activity
2.6.3. Antidiabetic potential
7
2.1. Nutraceuticals; an overview:
Epidemiological and scientific studies have suggested a strong relationship between health
status, well-being and dietary habits. It is generally accepted that populations utilizing a
greater quantity of plant-based diet including nuts, vegetables, cereals, fruits, legumes,
whole grains, spices and herbs are at lower risk of metabolic disorders (Shahidi, 2009).
Diet related chronic health maladies such as type II diabetes, neurodegenerative diseases,
cardiovascular diseases, liver disorders, renal dysfunctions and several types of cancer (for
example gastrointestinal and lungs cancer) continue to inflate with age. An increase in
plant based diet has been recommended by the global health organizations in order to
improve health and delay the onset of such ailments (Espin et al., 2007).
The ability of plant-based foods to curtail the risk factors associated with certain diseases
has been, in part, associated with the presence of secondary metabolites, generally referred
as phytochemicals. These secondary metabolites are non-nutritive and has been reported to
put forth a number of physiological benefits. The bioactivity of phytochemicals is low as
compared to synthetic drugs, but since they are utilized in considerable quantity on regular
basis as a part of diet, they may cause noteworthy long term biological effect (Mannarino
et al., 2014). During last decades, many bioactive components from different natural
sources has been extracted and commercialized in the form of capsules, pills, gels,
powders, liquors, granulates and nutritional supplements. Furthermore, food product
enriched with bioactive components have attained special interest of researchers and
consumers. These products cannot be accurately categorized as ‘food’ or ‘pharmaceutical
drugs’ hence, a new hybrid term “nutraceuticals” has been coined to designate these
products (Espin et al., 2007).
The concept of nutraceuticals was given by Dr. Stephen DeFelice in 1989 who used this
term first time. In marketing, the term nutraceuticals is used for different types of
nutritional supplements which are marketed with a claim to treat or prevent certain
disorders hence there is no regulatory definition available. Generally, a nutraceutical or
nutraceutic may be defined as any substance that is food or part of food and is associated
with certain physiological functions including treatment or prevention of certain disorders.
Nutraceuticals may range from isolated natural ingredients and diet formulations to herbal
8
products, processed foods and genetically engineered “pharma” or “designer” foods (Eskin
and Tamir, 2005). Whereas, functional foods are food items in their natural form which
provide some health benefits along with basic nutrition. The process of developing
enriched foods is termed “nitrification”. In order to be included in functional foods
category, a food must be an essential component of daily diet, it should be in its natural for
and should have a desease modulating potential (Chaturvedi, 2011).
Functional foods and nutraceuticals have gained significant interest from consumers owing
to their potential therapeutic and nutritional benefits along with their presumed safety. This
shift in consumer’s interest is an advantage for the industry involved in the business of
functional foods and nutraceutics. According to market statistics, the growth rate of global
functional foods and nutraceuticals market is overtaking traditional processed food’s
market (Espin et al., 2007). Generally, the use of nutritional supplements is safe but the
overdose of any specific nutrient can cause some health issues. Studies on the vitamin and
mineral supplementation have reported that there is no clear evidence of beneficial effects
of these supplements in individuals with no nutritional deficiencies. However, the overdose
may cause serious health issues like photosensitivity and neurotoxicity (Ronis et al., 2017).
Prevention of the disease risks and improvement of health status are short term goal of
functional foods and nutraceutics. These products are also recommended for their long-
term goals which include increase in life expectancy and overall quality of life. Lifestyle
related disorders and metabolic syndromes are the key targets of functional foods and
nutraceutics. Changing dietary habits, consumption of junk foods coupled with sedentary
lifestyle may lead to obesity, hypertension, hypercholesterolemia, hyperglycemia,
cardiovascular diseases and related complications (Moebus and Stang, 2007). A diet rich in
phytochemicals or bioactive components has potential to mitigate all the aforementioned
medical complications. Research has favored the use of such products as it has been
reported that individuals consuming functional food and nutraceuticals are at lower risk of
illnesses (Bjelakovic et al., 2007; Jenkins et al., 2008).
2.2. Licorice; at a glance
Plant roots have been reported to possess various valuable bioactive components. These
can be effective to curtail several lifestyle related metabolic disorders. (Shabani et al.,
9
2009). Licorice (Glycyrrhiza glabra) is a perennial herbaceous plant. Its origin is
Mediterranean region however, it is widely grown in Middle East, Asia and Europe
(Blumenthal et al., 2000). Licorice is an ancient medicinal plant as its roots have been a
history of use since 500 BC, hence maned as “the grandfather of herbs” (Ody, 2000).
Licorice is also known as sweet root, liquorice, yashtimadhu and gancao (Nomura et al.,
2002). In Pakistan, it is commonly known as “mulathi”. Subtropical climate is best suited
for its cultivation. The roots of licorice can grow up to five feet deep and consists of
fibrous wood (Khanzadi and Simpson, 2010).
Licorice has an ancient history of use as folk medicine in both Western and Eastern
civilizations. Historically, Greeks were first to use this herb for medicinal purposes where
it was recommended to treat peptic and gastric ulcers. Licorice was prescribed to treat
fever, asthma and cough in Chinese Pharmacopeia and appeared as a herbal component in
about 60% of all traditional Chinese medicines (Fu et al., 2013). Likewise, licorice is one
of the oldest and widely used herbal plant from ancient times in Ayurveda. It has been used
as medicine, ingredient in medicine and as flavoring agent to mask the undesirable flavor of
other medicines. In Indian Ayurveda, currently it is being used to treat eye diseases, throat
infection, peptic ulcers, liver disorders and different types of inflammations. Other
medicinal uses of licorice includes the treatment of bronchitis, tuberculosis, dyspepsia and
as a laxative, antiviral, antibacterial, antioxidative, antiallergic, expectorant and antitussive
(Biondi et al., 2005).
Research on the chemical composition of licorice have reported that licorice roots and
stolons are rich in valuable phytochemicals. A study on the proximate composition of
licorice root has reported that it contain 1.95% fat, 4.58% ash, 5.30% protein and 10.00%
moisture content (Karami et al., 2013). Additionally, a significant quantity of biologically
active components has also been reported. Among these, flavonoids and triterpene
saponins are most important compounds with greater bioactivity. Flavonoids (for example
glabridin and hispaglabridins) impart yellowish color to the root and are considered as
potential antioxidants (Shabani et al., 2009).
The genus name “Glycyrrhiza” excellently reflects the major features of this plant as this
word is derived from Greek words “glykos” meaning sweet and “rhiza” meaning root.
10
Licorice is highly nutritious and beneficial plant which is extensively used in food and
drug industry. The sweet taste of root is attributed to “glycyrrhizin” which is the main
bioactive component of root. Studies have reported that glycyrrhizin is 50 times sweeter
than sucrose (Isbrucker and Burdock, 2006). Due to its intense sweetness, licorice is
widely utilized as sweetener and flavoring compound in various food products like
candies, toothpaste, tobacco, chewing gums and beverages. In United States, tobacco
industry is the major sink of licorice and its components whereas remaining licorice is
equally shared among pharmaceutical and food industries (Fu et al., 2013).
Commercial products of licorice are derived from root extract of the plant. Licorice root is
harvested in autumn after 3-4 years of growth. Roots are dug up followed by washing,
transportation to warehouse, bailing, grading and dehydrating for further processing.
Dehydrated roots are milled with millstones to make pulp which is subsequently boiled to
obtain root extract. Solids are removed and the extract is dried under vacuum to make thick
paste which is filled in the blocks or can further dehydrated to make powder. Licorice
powder is ideal for pharmaceutics and in confectionary making. Licorice powder is
preferred as flavoring compound in tobacco industry (Carmines et al., 2005).
2.3. Nutraceuticals from licorice
A number of moieties have been extracted from licorice root with biological activity
against different metabolic syndromes. Licorice root contain about 45-50% water soluble,
biologically active substances on dry weight basis. Polysaccharides, pectin, gums, amino
acids, simple sugars, flavonoids, saponins, sterols, mineral salts, resins, proteins, tannins,
essential oils, glycosides, asparagines and several other substances have been extracted
from licorice root (Saxena, 2005). Glycyrrhizin (GL) is the primary bioactive component
of licorice and it constitute about 10-25% of licorice root extract. Chemically glycyrrhizin
is composed of a triterpenoid aglycone, glycyrrhetic acid (glycyrrhetinic acid; enoxolone)
conjugated to a disaccharide of glucuronic acid. Both glycyrrhizin and glycyrrhetic acid
can exist in two sterioisomeric forms (18α and 18β).
Glycyrrhizin has been reported to be effective against lipid peroxidation reactions by acting
as a blocking agent. Bioefficacy trails have revealed the in vivo antiproliferative,
chemopreventive and antioxidant activities of glycyrrhizin (Rahman and Sultana, 2007).
11
Glycyrrhizin or glycyrrhizic acid is the most studied and one of the most important
constituent of licorice root. It’s about 3.63–13.06% of the dried roots depending upon
variety, harvesting time, climatic and soil conditions (Wang and Nixo, 2001). It is often
used as a tool to recognize this species (Glycyrrhiza). Glycyrrhizin is about 170 times
sweeter than sucrose with a more persistent sweetness (Shibata, 2000).
Apart from triterpenoids, 1-5% of dried toots consist of 300 other polyphenols which have
successfully been isolated from Glycyrrhiza species. These includes flavans, flavones,
isoflavonoids and chalcones. Licorice extract is rich is phenolic acids, especially
flavonoids, which are responsible for most of the antioxidant potential of licorice root.
These compounds act as powerful antioxidants through free radical scavenging, metal
chelating, hydrogen donation, reducing potential and anti-peroxidative mechanisms
(Visavadiya et al., 2009). Flavonoids from licorice roots possess remarkable antioxidant
activity. Licorice flavonoids are reported to be 100 times more potent antioxidants as
compared to vitamin E. According to a study, licorice flavonoids can scavenge more
reactive species (20.6% inhibition) at a dose of 2.58 mg/mL than vitamin E at a dose of
258 mg/mL (11.2% inhibition). Licorice flavonoids are considered as strongest
antioxidants from natural origin. For this reason, licorice extract is frequently used in
cosmetics to protect the hairs and skin against oxidative damage (Cronin and Draelos,
2010).
Flavonoid rich fractions of licorice contain glabridin and its derivatives, liquirtin,
isoliquertin, glucoliquiritin, liquiritigenin, rhamnoliquirilin, shinpterocarpin,
hispaglabridins A and B, apioside, shinflavanone, 1-methoxyphaseolin and
prenyllicoflavone A. One of the major flavonoids is glabridin which is about 11.6% (wt/wt)
of the licorice root extract. Glabridin and its isoflavan derivatives are potential antioxidants
and have shown significant activity. Isoflavans is a subclass of flavonoids, these
compounds possess a peculiar chemical structure in which ring A is fused to ring C which
is further connected to ring B through carbon 3. Glabridin has antiradical, hypoglycemic,
cardiovascular protective, hypolipidemic, anti-inflammatory, antimicrobial,
antiatherosclerotic, antinephritic and estrogen-like activities. The hydroxal group on B ring
of glabridin is mainly responsible for its antioxidant and free radical scavenging activity
(Kang et al., 2005). Glabridin, methylglabridin compounds and hispaglabridin A & B are
12
reported to have liver protecting function by preventing oxidative stress on liver
mitochondria (Haraguchi et al., 2000). Glabridin treated mice demonstrated less 80% LDL
oxidation as compared to placebo treated mice at a dose of 20 µg/mouse/day for 6 weeks
(Wang and Nixo, 2001).
2.4. Antioxidant potential of licorice nutraceutics
Extraction and utilization of natural components with antioxidant properties has been a
major area of research in food sciences and pharmacology since last two decades. Natural
antioxidant compounds with strong free radical scavenging activates are in demand owing
to their presumed safety and effectiveness against many types of free radicals (Tohma and
Gulcin, 2010). All aerobic organisms have antioxidant defense mechanism including
antioxidant enzymes (e.g. superoxide dismutase, catalase) and antioxidants from food that
helps in the removal of free radicals and tissue repair. These antioxidants help the body to
fight against oxidative stress mediated dysfunctions and certain chronic disorders including
hypercholesterolemia, diabetes, hypertension, liver & kidney problems, neurodegenerative
disorders and cancer (Koksal and Gulcin, 2008). Apart from their use in maintaining
human health, antioxidants are also used in food industry due to their potential to retard
lipid peroxidation and hence increasing shelf life of food products during processing and
storage. Therefore, there is a growing interest among scientific community to explore more
components with antioxidant like properties from natural sources (Gulçin, 2010).
Licorice encompasses excellent complex of phytochemicals with promising antioxidant
activities in both in vivo and in vitro systems. A number of earlier research investigations
have focused on isolation of bioactive moieties from licorice root followed by the
determination of antioxidant activities of these phytocomplexes. Licorice flavonoids are
among the extensively studied biologically active natural compounds in this regard (Lee et
al., 2007). Licorice flavonoids have gained significant interest due to their potential
antioxidant & physiological activities and structural diversity. The important classes of
flavonoid compounds includes chalcones, flavanonols, isoflavans and arylcoumarins. (Xie
et al., 2009). Glabridin, isoliquiritigenin, Licochalcone A, licoricidin, licorisoflavan A and
glycyrol has been reported as major flavonoid compounds responsible for most of the
antioxidant capacity of licorice root.
13
Licorice root extract holds significant free radical scavenging activity owing to its rich
phytochemistry. Varsha et al. (2013) undertake a study to explore the phytochemistry and
antioxidant potential of aqueous methanolic (50% v/v) extract of licorice root using in vitro
models. Their results demonstrated the presence of different secondary metabolites in
aqueous methanolic extract including tannins, saponins, terpenoids, glycosides and
flavonoids. The extract showed significant OH radical binding potential with an IC50 value
of 80µg/ml (52.5±0.79) as compared to standard Ascorbic acid (positive control) having
IC50 value 50µg/ml (51.11±0.66). Earlier, Mekseepralard et al., (2010) evaluated the
antioxidant potential of four therapeutic plants used in traditional Thai treatment including
licorice. ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assay was
employed for the purpose. They reported highest ABTS scavenging activity (169.18
μg/mL) for licorice as compared to other herbs.
Murcia et al. (2004) carried out a comparative study to explore the antioxidant properties
of commonly used dessert spices including licorice compared to commonly used synthetic
antioxidants. The effect of radiation treatment on antioxidant properties was also evaluated.
Results indicated that licorice extract exhibited 66.21±2-71.04±3% inhibition of
peroxidation in the lipid system, 56.39-57.90% inhibition of OH- radicals and 42.3-52.2%
scavenging of superoxide radicals. Total antioxidant activity was determined through
Trolox equivalent antioxidant capacity (TEAC) assay using 2,2′- azinobis(3-
ethylbenzthiazoline-6-sulfonate) (ABTS-) free radicals. Licorice extract showed greater
TEAC activity as compared to butylated hydroxyanisole (BHA). Moreover, oxidative
stability of fats and oils (olive, sunflower and corn) was also reported (110 °C Rancimat).
No significant difference was noted for the antioxidant activities of irradiated and non-
irradiated samples as tested through different in vitro models.
Several studies have focused on licorice flavonoids for their antioxidant activities against
hydroxyl, superoxide and peroxyl radicals. Di-Mambro and Fonseca (2004) evaluated the
stability of topical formulations following supplementation of many plant extracts with high
flavonoid contents (Glycyrrhiza glabra, Ginkgo biloba, Nelumbium speciosum, Symphytum
officinale L and Arctium majus root). Results exhibited that Glycyrrhiza glabra (licorice)
had highest polyphenols and flavonoids content. Licorice extract resulted in significant
inhibition of lipid peroxidation and chemiluminescence intensity (99% inhibition at 0.5
14
µL/mL). Similarly, DPPH (2,2-diphenyl 1-picrylhydrazyl) assay also revealed that licorice
extract has higher antioxidant capacity as it resulted in 88% reduction in absorbance at 517
nm. This reduction in absorbance indicate H donation ability of licorice which is a
mechanism of action as an antioxidant.
Glabridin show good antioxidant potential and associated physiological benefits. It has
been reported to possess protective effect against LDL oxidation (Armeli and Ogelman,
2009), adipogenesis and obesity (Ahn et al., 2013). Armeli and Ogelman (2009)
determined effect of glabridin on oxidation of LDL by estimating the development of
Thiobarbituric acid reactive substances (TBARS). They reported 20% reduced level of
LDL oxidation and oxidative stress in healthy subjects after 6 month oral supplementation
of licorice ethanolic extract. Later, Ahn et al., (2013) reported that glabridin rich
supercritical extract of licorice caused significant weight loss in high fat-fed rodents. They
also observed that glabridin rich extract inhibited hepatic stress markers developed due to
high fat diet.
2.5. Extraction of Bioactive components from licorice
Selection of extraction technique is one of the most important factor considered in the
quantitative and qualitative studies of bioactive moieties (Sasidharan et al., 2011).
Extraction is the first and foremost step of any study concerned with medicinal plants and
their health benefits. Selection of proper extraction methods plays an important and crucial
role on final results. Extraction techniques are also denoted as “sample preparation
method”. The sample preparation and extraction of desired components is of paramount
importance in any analytical study and roughly two-third effort of a researcher account for
sample preparation techniques (Azmir et al., 2013). Extraction of target bioactive
components can be done through several conventional and non-conventional extraction
techniques, each with associated merits and demerits. A number of non-conventional
extraction techniques has been developed to increase yield and selectivity of bioactive
compounds from target materials. However, conventional methods like solvent extraction,
maceration and hydrodistillation are still in use to extract bioactive moieties and are used
as reference techniques to compare the effectiveness of newly developed extraction
techniques (Wang and Weller, 2006). Some commonly employed extraction techniques
15
used for licorice bioactive components are described as under:
2.5.1. Conventional Solvent Extraction (CSE)
The bioactive component of licorice are readily soluble in different solvents hence can be
extracted effectively using conventional solvent extraction. Previously, water, ethanol,
methanol, ethyl acetate and hexane have been used to extract licorice nutraceutics, each
solvent with different degree of selectivity and solubilizing power. Glycyrrhizin is readily
soluble in water and give highest extraction rate in aqueous medium. Whereas, glabridin can
be extracted more efficiently with ethanol as compared to other solvents (Tian et al., 2008).
Methanol has also been used by various researchers as a preferred solvent to isolate target
bioactive components from licorice. Moreover, most of the research studies have focused
on binary solvents to attain selectivity and fractionation. Additionally, a number of organic
solvents based partitioning techniques are being employed to isolate and purify specific
bioactive components from crude extracts (Shen et al., 2007).
Ahn et al. (2013) investigated different extraction solvents to extract glycyrrhizin and
glabridin from licorice root. Dried licorice was subjected to solvent extraction using water
and ethanol. Distilled water was used with 10 times boiling for 4 hours whereas ethanolic
extract was obtained using 70% ethanol at 25 oC. Resultant extracts were concentrated
using rotary evaporator at 50 oC following by freeze drying for further analysis. Extraction
yields for water and ethanolic extracts were 16.3% and 10.9% (w/w), respectively. Results
showed that hot water extract contain 56.65±2.8 mg/g glycyrrhizin and 3.66±0.11 mg/g
glabridin whereas ethanolic extract exhibited higher concentration of glabridin (4.19±7.74
mg/g) and very less concentration of glycyrrhizin (2.18±3.75 mg/g licorice extract).
Tian et al. (2008) investigated the impact of different extraction conditions on the
extraction rate of glycyrrhizic acid (glycyrrhizin) and glabridin from licorice. A
comparative study was carried out using ethanol, methanol, water, acetonitrile and
chloroform as extraction solvents. Results indicated that water was the most efficient
solvent to extract glycyrrhizic acid (2.44 mg/g) whereas maximum glabridin recovery
(0.93 mg/g) was noted in ethanolic extract. Very low quantity of glabridin (0.006 mg/g)
was extracted using acetonitrile whereas glycyrrhizic acid was not found in this extract.
Different ratios of ethanol to water (90:10, 70:30, 50:50, 30:70, 10:90 v/v) were also tested
16
to investigate comparative abundance of both the active components. It was reported that
aqueous ethanol at a ratio of 70:30 exhibited optimum recovery of conditions with 2.39
mg/g of glycyrrhizic acid and 0.92 mg/g of glabridin providing 89.7% and 72.5%
recoveries, respectively. A recent study has reported 1% and 3.24% glabridin in
methanolic and chloroform extracts of licorice (Rebhun et al., 2015).
Various research trails have delineated beneficial effects of licorice flavonoids other than
glabridin such as glabrene, liquiritin, isoliquiritigenin and liquiritigenin and focused on the
extraction of these valuable components. Fu et al. (2005) carried out an experimentation to
extract licorice flavonoids using solvent extraction to test their inhibitory effect
against tyrosinase enzyme which is reported to be responsible for browning and
melanization in in plants and animals, respectively. Purposely, powdered licorice was
extracted twice with ethanol (70% v/v) followed by sonication at 25 oC for 30 min.
Resultant extract was centrifuged at 6000 rpm for 10 min followed by rotary evaporation at
40 oC. The concentrated extract obtained in this way was analyzed through nuclear
magnetic resonance (NMR) spectrometer to identify different flavonoids. Results indicated
that four flavonoids, liquiritin, licuraside, isoliquiritin, and liquiritigenin were efficiently
extracted and isolated from licorice root using ethanol as extraction medium.
Similarly, Račková et al. (2007) conducted an experiment to extract polyphenols and
flavonoids from licorice roots and rhizomes using solvent extraction. 1400 g of dried,
ground licorice was extracted with 60% methanol at a root solvent ratio of 15:150 (m/v)
for 30 min. Extract was filtered and remaining residues were extracted twice with 60%
methanol. Furthermore, extracts were mixed and concentrated at 40 oC followed by freeze
drying to yield crude extract. The extraction yield was 8.57%. HPLC quantification of
active components revealed that methanolic extract of licorice contain 117.63±0.95 mg
epicatechin equivalents/g polyphenols, 32.39±0.41 mg quercetin equivalents/g total
flavonoids and 19.10±1.68% glycyrrhizin. The resultant extract also exhibited significant
antioxidant potential as estimated through DPPH assay and inhibition of lipid peroxidation.
2.5.2. Supercritical Fluid Extraction (SFE)
An increase in public concern has been noted regarding the use of organic solvents in food
industry for the extraction of different desirable components in relation with its safety,
17
effect on human health and environment. Additionally, the residual limits and the
contamination of finished products with traces of solvents is also a major concern. Food
industry is always looking for alternates of conventional solvent extraction with better
recovery, high purity and ease in the extraction of bioactive components (Kompella and
Koushik, 2001). Other factors triggering this shift included high cost of solvents, need for
high purity of extracts and strict environmental regulations. In this regard, supercritical
fluid extraction (SFE) has been in limelight as a competent alternate to conventional
extraction techniques which use organic solvent (Norulaini et al., 2009; Zaidul et al., 2006,
2007a).
The application of SEF for the recovery of bioactive compounds from plant material is
advantageous and environmentally safe as compared to solvent extraction methods. This
technique showed greater prospective in food and other industries as it ensures better
extraction as well as fractionation of desired components, which is otherwise not properly
achieved in the case of solvent extraction. Supercritical fluid extraction technique uses a
supercritical fluid as solvent and extraction medium. A number of supercritical fluids are
available, each distinguished by its characteristic critical pressure and temperature (Herrero
et al., 2010). Irrespective to the pressure applied, a gas cannot be converted to liquid above
its critical temperature. Above its critical temperature, a gas is converted to a state which is
closed to liquid. When a gas is at above its critical temperature and pressure, it is in
supercritical stat hence named as “supercritical fluid”. For instance, the critical temperature
and pressure of CO2 are 31.1 oC and 7.38 MPa, respectively (Mendiola et al., 2007).
Supercritical fluids possess some desirable characteristics which make them ideal solvents
for the extraction of target compounds. These characteristics includes their density, thermal
conductivity, viscosity and diffusivity. Supercritical fluids have a smooth flow and greater
penetration in the matrices owing to their low viscosities. Likewise, they have a greater
solublisation power due to high densities. All the aforementioned characteristics are greatly
affected by temperature and pressure. Carbon dioxide is considered as one of the best
supercritical fluids due to its low critical pressure and temperature, inflammability, low
cost and greater extraction yields. Due to its non-polar nature, CO2 is considered as ideal
medium for the extraction of fat soluble components (Dunford et al., 2003).
18
Ahn et al. (2013) conducted a comparative study to extract bioactive components of
licorice using supercritical fluid extraction and conventional solvent extraction.
Supercritical fluid extraction was performed at 30 MPa extraction pressure, 40 oC
temperature and 150 g/min CO2 flow rate for 1 hour using 154 g dried licorice sample in a
300 mL extraction vessel. Results showed an extraction yield of 3.57% (w/w). It was
reported that supercritical fluid extract of licorice showed higher glabridin content
(45.12±0.14 mg/g licorice extract) as compared to both hot water (3.66±0.11 mg/g) and
ethanolic (4.19±7.74 mg/g) extracts. Highest Isoliquiritigenin content (2.62±0.11 mg/g)
was also noted in supercritical fluid extract. However, hot water was a better extraction
medium for glycyrrhizic acid.
Earlier, Kim et al. (2004) investigated optimum conditions for the recovery of glycyrrhizin
from licorice using SFE technique. The morphological characteristics of residue tissues left
after extraction was also studied through scanning electron microscopy. It was observed
that glycyrrhizin could not extracted efficiently while using only CO2 as a solvent however,
the use of water as a modifier was effective to extract significant quantity of glycyrrhizin.
Highest recovery (~97%) of glycyrrhizin was obtained using 70% aqueous methanol as
modifier. Optimum temperature and pressures for the extraction of glycyrrhizin were 60 oC
and 30 MPa respectively. Additionally, licorice tissues left after extraction with 70%
methanol as modifier were found to be highly damaged by swelling as compared to other
extraction conditions. This suggest that maximum damage to tissue structure was caused
by these extraction conditions resulted in maximum recovery of glycyrrhizin.
Recently, Hedayati and Ghoreishi (2015) compared soxhlet and supercritical carbon
dioxide extraction techniques for the recovery of glycyrrhizin from licorice and
investigated the optimum conditions for maximum recovery through Response Surface
Methodology (RSM). Temperature (45-85°C)), extraction time (40-120 min), pressure (10-
34 MPa), CO2 flow rate (0.8-2 ml/min) and modifier concentration (0-100% methanol)
were the variables studied in this regard. Results explicated that supercritical fluid
extraction was much better when compared with conventional solvent extraction
considering yield, recovery, extraction time and process efficiency. It was evident from
RSM modeling that maximum glycyrrhizin can be recovered at 29.6 MPa pressure, 68 oC
19
temperature, 108 min time and 2 ml/min CO2 flow rate.
2.6. Licorice nutraceutics against metabolic disorders
Dietary patterns and choice of lifestyle are two major determinants of health status.
Consumption of foods closed to their natural forms along with active lifestyle has been
reported to be associated with good health, less morbidity and low mortality rates.
However, increased use of refined foods, less consumption of natural foods and sedentary
lifestyle is resulted in several metabolic disorders such as diabetes, cardiovascular diseases
(CVDs), dyslipidemic conditions, hypertension, liver and kidney problems,
neurodegenerative disorders and many type of cancer. Novel nutritional approaches have
successfully been employed to curtail all the aforementioned maladies. Scientific and
epidemiological studies have supported the use of nutritional interventions as an effective
tool to address these ailments. Licorice has a history of traditional use in Chinese folk and
Indian Ayurveda medicine to treat several metabolic disorders. Recent research on
therapeutic effect of licorice has revealed its potential to alleviate different lifestyle related
health discrepancies owing to its excellent phytochemistry.
2.6.1. Hepatoprotective perspectives
Liver is the major metabolic and excretion center of the human body. During its metabolic
activities, it is often exposed to a number of xenobiotics that have shown hepatotoxic effect.
Metabolism of xenobiotics resulted in free radical generation that can react with major
cellular constituents including lipids, proteins, DNA and RNA (Ajith et al., 2007). Carbon
tetrachloride (CCl4) is a potential hepatotoxic compound that has been used as a model
compound to study the mechanisms of different food formulations and drugs against toxin
induced liver damage. CCl4 mediated liver damage is caused by the production of reactive
degradation products, trichloromethyl or trichloromethyl peroxyl, as a result of CCl4
metabolism by cytochrome P450 2E1. Lipid peroxidation of endoplasmic reticulum and cell
membranes is initiated by these free radicals. These processes in turn cause DNA damage,
decline in protein synthesis and increase in membrane permeability, resulted in necrosis and
degeneration of liver cells (Prasenjit et al., 2006).
The use of natural components from different medicinal plants are presumed as safe and
20
effective alternates to synthetic drugs for the treatment of hepatotoxicity. Various studies
has explored the use of natural antioxidant compounds against CCl4 induced liver injury
through numerous mechanisms including restoration of anti-oxidant enzyme activities,
reduction in the expression of pro-inflammatory mediators and inhibition of lipid
peroxidation. These components restore normal liver functions to different extent depending
upon their mode of action and active dose. Therefore, bioactive components from various
food materials can be used efficiently to design model foods aimed at lowering hepatic
disease burden. Licorice has exhibited promising potential to address CCl4 induced
hepatotoxicity and associated health complications in various animal trails (Huo et al.,
2011).
Various studies have supported that licorice and its extracts are effective in alleviating the
symptoms of liver ailments. Huo et al., 2011 assessed the liver-protective potential of
licorice extract on CCl4-induced hepatic damage in rodents. They observed significant
impact of licorice administration on different parameters. Licorice extract effectively
controlled the elevation in serum aspartate aminotransferase (AST), alanine
aminotransferase (ALT), alkaline phosphatase (ALP) and also addressed reduced levels of
different proteins which was caused by CCl4 administration. Licorice water extract also
elevated liver catalase, glutathione reductase, super oxide dismutase and Glutathione S-
transferase activities. Tekla et al., (2001) reported a considerable reduction in ALT levels of
subjects with chronic hepatitis C as a result of glycyrrhizin administration.
Abdelrahman et al. (2012) carried out animal modeling to study the ameliorative potential
of licorice and dates aqueous extracts on CCl4-induced hepatotoxicity. Three groups of test
animals were made. Group I test animals were administrated with a mixture of CCl4 and
olive oil (1:1 v/v) at a dose of 0.6 mL/kg for 4 consecutive days. Group II animals orally
received both extracts for 24 days and were administrated with CCl4 on 4, 10, 11 and 12th
day. Likewise, group III animals were fed on both extracts for 14 successive days and
received intra peritoneal injection of CCl4 on 1st, 2nd and 3rd day of the experiment. Extent of
liver damage was measured through histology, liver morphology and estimation of plasma
levels of liver enzymes including aspartate aminotransferases (AST), alanine
aminotransferase (ALT) and alkaline phospatase (ALP). Results exhibited a significant
21
reduction in elevated liver enzymes concentration in plasma as a result of CCl4
administration in licorice and dates extract fed groups. Histopathology of liver tissue also
revealed less damage as a result of licorice and date extract administration. Additionally, it
was also reported that licorice and date extract stop fibrosis and edema of hepatic
parenchyma.
Numerous clinical studies have provided convincing evidences regarding the efficacy of
licorice nutraceutics against toxins induced liver injury in in vivo and in vitro models. In a
trails, aqueous extract of licorice root was tested for its potential to curtail hepatotoxicity and
oxidative stress using isolated primary hepatocytes of rats. It was evident from results that
oxidative stress and cytotoxicity was caused when isolated hepatocytes were exposed to 5
mM CCl4. This condition eventually resulted in a considerable increase in ALT, AST &
LDH leakage and caused loss of cell viability. However, a significant reduction in ALT,
AST, LDH, oxidative degeneration and cell damage was observed when hepatocytes were
pre-incubated with 25 μM/mL licorice solution. Moreover, the depletion of glutathione and
formation of thiobarbituric acid reactive substances (TBARS) was also prevented. It is clear
from the results that licorice extract is effective in modulating hepatotoxicity condition
caused by CCl4 (El-Tawil et al., 2013).
Likewise, Al-Razzuqi et al. (2012) evaluated the potential of licorice extract to ameliorate
CCl4 induced acute liver damage. Liver injury was induced in experimental rabbits by 1.25
mL/kg dose of CCl4 and olive oil mixture. Aqueous extract of licorice was orally given to
rabbits for 7 days at a dose of 2 g/kg body weight. Histopathology and liver functioning
tests were performed to assess the shielding effect of licorice against CCl4 induced
abnormalities. Results delineated significant reduction in serum ALT, AST and ALP and
bilirubin levels in rabbits consuming licorice extract. Moreover, serum proteins were
improved significantly and hepatocellular architecture was restored towards normal in
rabbits fed on licorice extract. Absence of necrosis in hepatic cells also gave a clear image
of the hepatoprotective effect of licorice. Conclusively, aqueous extract of licorice can be
effective in restoring normal liver functions and tissue morphology in acute liver injury.
Recently, Zhao et al. (2015) documented the hepatoprotective effect of Isoliquiritigenin
(isoLQ), a bioactive component of licorice, against CCl4 induced oxidative damage of liver.
22
Purposely, 0.5 mL/kg body weight CCl4 was given twice to induce severe liver injury
characterized by increased ALT, AST levels, hepatic tissues degeneration and necrosis.
Administration of isoLQ at a dose of 20 mg/kg for three days markedly protected the liver
from these adverse changes. Serum ALT levels exhibited significant reduction from
265.50±20.07 U/L to 185.00±19.67 and 135.58±14.40 U/L with the provision of 5 mg and
20 mg/kg of isoLQ, respectively. Likewise, serum AST levels were reduced from
174.09±25.30 U/L to 112.67±7.31 and 79.52±5.19 U/L respectively with aforementioned
doses of Isoliquiritigenin. Additionally, treatment with isoLQ also reversed the decline in
hepatic antioxidant status caused by CCl4 and curbed the expression of tumor necrosis
factor-alpha in liver. These results strongly advocated the hepatoprotective and anti-
inflammatory effect of isoLQ.
2.6.2. Hypolipidemic activity
Epidemiological and clinical studies have reported a strong relationship among dietary
habits and health status of individuals. Unhealthy diet along with physical inactivity is the
root cause of illness and resultant deaths around the globe. Cardiovascular diseases (CVDs)
are leading cause of morbidity and mortality around the globe. They cause more deaths per
year as compared to any other single cause. According to the statistics, about 17.3 million
people die annually due to VCDs which accounts for 31% of all the worldwide deaths. It
was reported that middle and low income countries, like Pakistan, are more affected and
75% of CVDs deaths are reported in these countries. In Pakistan, CVDs are the major reason
for 30-40% of all deaths. It represents that about 200,000 deaths are caused by CVDs every
year. Adherence to strict dietary guidelines and positive changes in lifestyle has been
recommended to mitigate CVDs and allied health complications.
The key risk factors for the occurrence and development of CVDs includes elevated low-
density lipoprotein (LDL), triglycerides and total cholesterol levels and a subsequent
decrease in high density lipoprotein (HDL) levels. Therefore, a decrease in total cholesterol
level is important to prevent or cure CVDs. Lipoproteins (HDL, IDL, LDL and VLDL) are
the carriers of plasma cholesterol and are used for its transportation in the body (Roberts et
al. 2007).
Different bioevaluation studies have narrated the positive role of licorice nutraceutics in
23
modulating serum lipid profile. Previous research has advocated that licorice has potential
to address dyslipidemia through different mechanisms including reduction in abdominal fat
deposition, serum total cholesterol, LDL cholesterol and triglycerides levels whereas
improving serum HDL cholesterol and overall fat metabolism (Mirtaheri et al., 2015).
Numerous clinical studies provide convincing evidences regarding licorice bioactive
components in modulating coronary disorders and their root causes. Mirtaheri et al. (2015)
determined the impact of licorice extract on lipid patterns and atherogenic indices of
overweight subjects. For the purpose, sixty four overweight subjects were recruited and
divided in two equal groups. One group was given with 1.5g/day licorice ethanolic dried
extract for a period of 8 weeks along with low caloric diet whereas, other group received
corn starch as a positive control. There was not a significant difference in lipid profile of the
subject at baseline however at the end of trail, significant decrement in total cholesterol
(TC), LDL cholesterol, TC:HDL and LDL:HDL was observed. Moreover, no effect was
evident for triglycerides and HDL levels. Body mass index and weight was also changed
non-significantly among the groups.
Licorice flavonoids are the main bioactive components in licorice extract with cholesterol
lowering potential. Beneficial effect of licorice flavonoids (mainly glabridin) has been
reported in preventing LDL oxidation and also against atherosclerotic lesions development.
Fuhrman et al. (2002) reported that licorice extract supplementation increased the resistance
towards LDL oxidation along with normalizing serum lipid profile in hypercholesterolemic
subjects. In their experiment, hypercholesterolemic subjects (with serum cholesterol level of
220-260 mg/dL) were given with ethanolic dried extract of licorice at a dose of 0.1 g/day for
30 days. Serum lipid profile and LDL oxidation levels were measured at the termination of
trail. It was evident from the results that licorice consumption reduced the oxidation of
plasma by 19% and increased the resistance of LDL towards oxidation by 55%.
Additionally, blood chemistry analysis showed a momentous decrement in TC (5%), LDL-c
(9%), VLDL (13.8%) and triglycerides (14%) was observed. One month consumption of
licorice extract reversed the risk factors of hypercholesterolemia towards normal values.
Flavonoids and saponins are the major compounds of interest in licorice but several other
classes of compounds have also shown considerable bioactivity. Chalcones or chalconoids
24
are aromatic ketones which constitute the central ring of many bioactive moieties.
Isoliquiritigenin is an important chalcone compound found in licorice with considerable
hypocholesterolemic, hepatoprotective, antitumor and antidiabetic potential. In a recent
study, isoliquiritigenin and liquiritigenin (flavonoid) were isolated from licorice root to
study their bioactivity. It was reported that Isoliquiritigenin administration at a dose of 100
mg/kg of body weight reduced serum triglyceride level by 38.41% and increased the serum
HDL by 55.65%. Likewise, liquiritigenin-7,4-dibenzoate reduced serum triglyceride level
from 177.33 mg% to 105.75 mg% (40.37) and increased the HDL cholesterol by 61.74% at a
dose of 50 mg/kg (Gaur et al., 2014).
Recently, many novel extraction techniques are being employed to obtain more pure and
bioactive component rich fractions of plant materials to study their in vitro and in vivo
characteristics. Ahn et al. (2013), for example, studied the effect of glabridin rich
supercritical fluid extract (SFE) of licorice on obesity indicators and serum lipid patterns of
high fat-fed rats. Supercritical CO2 extract comprising of 45.12 mg/g of glabridin was fed at
a dose of 0.1% and 0.25% in diet. Results indicated 15% and 35% reduction in weight gain
as a result of 0.1% and 0.25% addition of supercritical fluid extract in diet. Similarly, diet
containing 0.1% SFE reduced serum total cholesterol by 32% and triglycerides by 7.79%.
Whereas, diet supplementation with 0.25% SFE caused 20.57% reduction in total
cholesterol and 19.48% decline in serum triglycerides level. Results of the study showed
that glabridin possess high anti-adipogenic activity and can effectively use for weight
reduction and to modulate blood lipid profile.
2.6.3. Antidiabetic potential
Diabetes is a widespread metabolic syndrome characterized by increased serum glucose
levels and absolute or relative deficiency in secretion or action of insulin. Type II diabetes
mellitus is the most encountered form of diabetes, accountable for more than 80% of the
total cases and is predicted to increase by 5.4% till 2025 (Kim et al., 2006) If unchecked,
diabetes can lead to serious allied health problems including nephropathy, cardiovascular
disorders, retinopathy and neuropathy. Additionally, this condition may affect peripheral
nerves, vascular system and skin thus can prove extremely injurious to health (Maritim et
al., 2003).
25
Risk factors for the initiation and progression of this menace includes genetic factors,
autoimmune disorders, viral infection, lifestyle and dietary abuses. When it comes to the
treatment, strict dietary restrictions, lifestyle modifications, insulin injections and oral
medication are the major modes. In last two decades, a number of synthetic drugs has been
formulated and marketed for the management of diabetes mellitus but most of these drugs
are linked with some side effects and may lead to the development of drug resistance.
Therefore, recent research is focusing on the use of natural resources to develop herbal
formulations with less or no side effects (Sen et al., 2010).
Among various therapeutic herbs, licorice has attained forefront position to combat against
hyperglycemia, hyperinsulinemia and autoimmune dysfunctions. Substantial evidences have
revealed the role of licorice as an anti-diabetic agent due to rich phytochemistry.
Modifications in the glucose metabolism, affirmative influence on insulin secretion and
absorption through the ß-cells are the major mode of action of licorice nutraceutics (Ko et
al., 2010). Glycyrrhizin has been reported to possess hypoglycemic potential by stimulating
glucose-induced secretion of insulin in pancreatic islet cells. Elevated plasma insulin levels
has been observed as a result of glycyrrhizin treatment in diabetic animal models (Kalaiarasi
and Pugalendi, 2009). Similarly, other bioactive components of licorice (glabridin,
Isoliquiritigenin, liquiritigenin) have also shown considerable antidiabetic potential through
different mechanisms (Gaur et al., 2014; Yehuda et al., 2011).
Licorice extracts and its isolated bioactive components has been successfully administrated
to animal models against hyperglycemic conditions. Sen et al. (2010) delineated the
potential of glycyrrhizin, the major water soluble bioactive constituent of licorice root, in
attenuating streptozotocin (STZ) induced diabetes and oxidative stress markers in rat
models. Male rats were grouped in to normal control, normal rats treated with glycyrrhizin,
STZ induced diabetic control, STZ-induced diabetic rats administrated with glycyrrhizin
and diabetic rats given with glibenclamide, a standard antidiabetic drug. It was evident from
their results that glycyrrhizin treatment ameliorated STZ-induced diabetogenic markers
including increased serum glucose level, decreased insulin level, glucose intolerance and
elevated serum cholesterol & triglyceride level. Additionally, serum concentrations of
oxidative stress markers including SOD, ctalayse, MDA and fructosamine were also
restored towards their normal values in diabetic rats. Moreover, the antidiabetic potential of
26
glycyrrhizin was comparable with that of reference drug, glibenclamide.
Studies have shown that there are a number of bioactive components in licorice with
antidiabetic prospective. Gaur et al. (2014) reported that some derivatives of licorice
derived isoliquiritigenin and liquiritigenin are potential anti-diabetic and hypoglycemic
agents. STZ- induced diabetic rats were given with 200 mg/kg body weight of
isoliquiritigenin and 50 mg/kg body weight of liquiritigenin. Serum glucose level was
significantly lowered in rats administrated with both bioactive components as compared to
the control. Hyperglycemic condition also caused hepatic and renal abnormalities such as
increased liver enzymes levels elevated serum urea and creatinine. These malfunctions
were ameliorated upon the treatment with isoliquiritigenin and liquiritigenin. Likewise, both
bioactive moieties were found helpful in restoring serum lipid levels which were disturbed
as a result of hyperglycemia in STZ- induced diabetic rats.
Earlier, Mae et al. (2003) documented that non-aqueous extract of licorice root is effective
against metabolic syndrome and its complications including type II diabetes, obesity, insulin
and resistance. Results of their study exhibited that non-aqueous extract of licorice showed
significant PPAR- γ ligand binding potential and was found to be helpful in alleviating
health complications caused by metabolic syndrome. It was reported that 0.1-0.3 g/100 diet
of licorice ethanolic extract significantly lowered down serum glucose level. Two type of
experiments were conducted to examine preventive as well as ameliorative effect of licorice
extract. In preventive experiment, 38% reduction was observed at a dose of 0.1 g/100g diet
of licorice extract whereas 39.5% reduction was noted at a dose of 0.2g/100g diet of extract.
Likewise, in ameliorative experiment, 34.07% and 30.09% decline in serum glucose level
were observed at 0.1 g/100g and 0.2g/100g of licorice extracts, respectively. Serum insulin
levels were also affected significantly as a result of licorice extract administration.
27
CHAPTER 3
MATERIALS AND METHODS
The current research was conducted in the Functional and Nutraceutical Food Research
Section, National Institute of Food Science and Technology (NIFSAT), University of
Agriculture, Faisalabad (UAF). In the present study, bioactive components of licorice
(Glycyrrhiza Glabra) were evaluated for their disease modulating potential. Purposely,
bioactive moieties of licorice were extracted using conventional solvent (CSE) and
supercritical fluid extraction (SFE). Resultant extracts were evaluated for their total
phenolic and total flavonoid content followed by assessment of their antioxidant potential.
Major bioactive compounds (glycyrrhizin and glabridin) were quantified through HPLC
system and one treatment was selected from each extraction mode, based on their
phytochemical contents, for further study. Licorice based drinks were prepared using
different levels of selected extracts and were evaluated for their physicochemical,
antioxidant and sensorial attributes. Animal trail was carried out to evaluate the
hepatoprotective and hypercholesterolemic potential of licorice based nutraceutical drink.
The materials and methods followed are as under.
3.1. Procurement of raw materials
Licorice was purchased from local market of Faisalabad. . It was cleaned to remove any
foreign particles and dust followed by grinding and storage for further analysis. The
standards and reagents (HPLC and analytical grade) were procured from Sigma-Aldrich
(Tokyo, Japan) and Merck (Darmstadt, Germany). For biochemical assays, kits were
purchased from Cayman Chemicals (Cayman Europe, Estonia), Bioassay (Bioassays
Chemical Co. Germany) and Sigma-Aldrich. The test animals (male Sprague Dawley rats)
were purchased from National Institute of Health (NIH) Islamabad and were kept in the
animal room of NIFSAT, UAF.
3.2. Preparation of licorice extracts
3.2.1. Preparation of solvent extracts
The solvent extracts (Table 1) were prepared using three binary solvent including aqueous
28
ethanol, methanol and ethyl acetate (25:75, 50:50 and 75:25 v/v) following the prescribed
methods (Tian et al., 2008). Afterwards, all extracts were filtered and concentrated using
rotary evaporator (Eyela, Japan) followed by freeze drying to make powder. Extraction
yield of respective samples were calculated and stored at refrigeration tempeature for
further analysis.
3.2.2. Preparation of supercritical fluid extracts
Supercritical fluid extracts (SFEs) of licorice were obtained using SFE-150 system
(Supercritical Fluid Technologies, Inc. Delaware, USA) following the method as outlined
by Ahn et al. (2013). The treatment plan is mentioned in Table 2. For extraction purpose,
100 g licorice powder was filled in 150 mL tubular extraction vessel. CO2 gas was used as
extraction medium. Gas was passed through a chiller at 4 oC followed by compression using
high pressure pump and heating to convert in supercritical fluid. The supercritical CO2 was
allowed to enter the extraction vessel adjusted to 40 oC and varied pressure, as per
treatment. A stay time of 3 hours was given and extracts were collected through a metering
valve.
Table 1: Treatments for solvent extraction
Treatment Solvent Solvent:water
T
1
Ethanol 25:75
T
2
Ethanol 50:50
T
3
Ethanol 75:25
T
4
Methanol 25:75
T
5
Methanol 50:50
T
6
Methanol 75:25
T
7
Ethyl acetate 25:75
T
8
Ethyl acetate 50:50
T
9
Ethyl acetate 75:25
29
Table 2: Treatments for supercritical fluid extraction
Treatment Pressure
(psi)
Temperature
(oC)
TSC1 3500 40
TSC2 4500 40
TSC3 5500 40
3.3. Phytochemical screening assays
The powdered extracts of licorice were dissolved in their respective solvents and SFEs
were dissolved in ethanol at 200 µg/mL concentration. Resultant solutions were used for in
vitro analysis including phytochemical screening and antioxidant activity assays.
3.3.1. Total Phenolic Contents (TPC)
TPC of licorice extracts were assessed following the method of Sengul et al., 2010). The
method was based on Folin-Ciocalteu reagent. Purposely, 0.1 mL licorice extract was
taken in flask trailed by the mixing of Folin-Ciocalteu reagent (1 mL) and distilled water
(46 mL). The mixture was shaken continuously for 3 min. Afterwards, 3 mL, 2% Na2CO3
was mixed and flask was intermittently shaken for 2 hrs. After stay time, absorbance of the
mixture as taken at 760 nm. Standard solution of gallic acid (0-1000 mg/0.1 mL) were
made and absorbance was measured using same procedure and standard curve was
obtained. The results were presented as mg GAE/100g of licorice.
3.3.2. Total Flavonoids (TF)
The total flavonoid contents were evaluated by following spectrophotometric method as
described by Ghasemzadeh and Jaafar (2013). Briefly, 1 mL extract solution of licorice
was diluted by adding 4 mL water in a flask. Afterwards, 0.3 mL NaNO2 (5%) was added
and mixed for 5 min trailed by the mixing of 10% AlCl3 and 2 mL 1.0 M NaOH after 6
min. Absorbance of this solution was noted at 430 nm. Results for total flavonoids were
presented as mg catechin equivalent /100g of licorice.
30
3.4 Antioxidant activity assays
3.4.1. Free radical scavenging ability (DPPH assay)
The capacity of licorice extracts to scavenge DPPH radicals was assessed using protocol
outlined by Cheel et al. (2007). Principally, this method measures the potential of H-
donors to react with DPPH radicals. DPPH is reduced during the reaction and change in
absorbance takes place which is used to measure antioxidant activity of test sample.
Absorbance at 571 nm was measured to calculate scavenging activity. For each sample, the
values of absorbance was taken in triplicate were presented as means ± SD. The % values
were calculated by using following formula:
DPPH-scavenging activity (%) = [(E – S) / (E)] x 100
Where E = A - B and S = C – D, A is absorbance of the control; B is absorbance of the
control blank; C is absorbance of the sample; D is absorbance of the sample blank.
3.4.2. FRAP assay
Licorice extracts were subjected to FRAP assay to assess their reducing power as per
procedure outlined by Baek et al. (2008). 1 mL extract solution, 1 mL potassium
ferricyanide (1%) and 1 mL sodium phosphate buffer (200 mM, 6.6 pH) were mixed and
20 min stay time was given at a temperature of 50 ◦C. Afterwards, 1 mL Trichloroacetic
Acid (10%) was mixed and the solution was subjected to centrifugation for 5 min at
13,400×g. The supernatant layer was collected and mixed with 0.1 mL ferric chloride
(0.1%) and 1 mL distilled water. Absorbance of this solution was taken at 700 nm.
Aqueous solutions of FeSO4.7H2O (100-1000 µM) were used for standardization and
values were presented as micromoles Fe (II) per gram.
3.4.3. ABTS assay
ABTS free radical scavenging activity of licorice extracts was estimated according to the
method outlined by Hossain et al. (2008). For the preparation of ABTS radicals, 5 mL
freshly prepared ABTS solution (7 mM) was mixed with 5 mL potassium persulfate
solution (2.45 mM) to make 10 mL total volume. The mixture was transferred to opaque
bottle and allowed to for 16 hrs in a dark place to reach a stable oxidized state. The mixture
was diluted with ethanol and was adjusted to give 0.7 absorbance at 734 nm. Additionally,
31
10 µL licorice extract was added 1 mL ABTS solution, mixed thoroughly and subjected to
spectrophotometer to measure the absorbance at 734 nm after 30 min stay time. The
antioxidant activity was compared with trolox, used as standard. Values obtained from
calibration curve were reported in µmol Trolox/g sample extract.
3.5 Quantification of active ingredients
All the extracts of licorice were analyzed for their glycyrrhizic acid and glabridin contents
using HPLC system (PerkinElmer, Series 200, USA) according to the protocols of Tian et
al. (2008). All the extracts were vortexed using gyromixer and filtered to remove any undesirable
substance before HPLC analysis. All the sample solutions were filtered through 0.2 μm
disposable syringe filters before HPLC analysis. UV/Vis detector was used and adjusted at
252 nm. Mobile phase comprised of methanol-water binary solvent (70:30, v/v, containing
1% acetic acid) with a flow rate of 1.0 mL/min. Glycyrrhizic acid and glabridin were
analyzed through shim-pack C18 column (15 cm x 4.6 mm, 5.0 μm particle size).
3.6 Selection of best treatments
One best treatment from solvent as well as supercritical fluid extracts will be selected for
further analysis based on comparative abundance of phytochemicals (glycyrrhizic acid and
glabridin) as analyzed through HPLC.
3.7 Development of nutraceutical drink
In the product development module, four treatments of licorice drink were developed by
incorporating different levels of selected conventional (T1 & T2) and supercritical extracts
(T3 & T4). A control (T0) without extracts was also be formulated for comparison purpose
(Table 3). Raw materials used for drink preparation were table sugar, aspartame, citric
acid, carboxy methyl cellulose, sodium benzoate, food grade color and flavor. All the
materials were purchased from local market and accurately weighed. All drinks were
prepared by mixing sugar, aspartame, citric acid and carboxy methyl cellulose in water
followed by heating for 1 minute at 90 oC. Afterwards, sodium benzoate, food grade color
and flavor was added and thoroughly mixed. Additionally, solvent and supercritical
extracts were also added in respective drinks at a dose descried in Table 3. All the drinks
were cooled up to 15 oC instantly by using iced water bath. Citric acid was added in all the
32
treatments to maintain the pH at 4.5 for uniformity. Moreover, food grade lime yellow
color and mango flavor were added to increase sensory response and to impart appealing
and similar look. All the drinks were filled in transparent bottles and stored at 4 oC.
Table 3. Treatments plan for licorice drink development
Product Treatment Percentage
Control T0 -
Drink with conventional solvent extract (NutraceuticalCSE drink) T1 0.2
T2 0.4
Drink with supercritical fluid extract (NutraceuticalSFE drink) T3 0.1
T4 0.2
3.7.1 Product analysis
The developed drinks were analyzed for their physicochemical, antioxidant and sensorial
attributes at 0, 30th and 60th day during two month storage study.
3.7.1.2 Color
Color of licorice based drinks was assessed through digital Color Meter (CIELAB SPACE,
Color Tech-PCM, USA) employing the protocol as described by Lara et al. (2010). The
results were obtained in the form of L* a* and b* values which were further used for the
calculation of chroma value and hue angle.
Chroma = [(a*)2+(b*)2]1/2
Hue angle = tan-1 (b*/a*)
3.7.1.1 pH
pH of all licorice based drinks was determined through digital pH meter (InoLab 720,
Germany) following AOAC (2006) method.
3.7.1.2 Acidity
Total acidity of licorice based nutraceutical drinks was determined by titration method.
33
Samples were titrated against 0.1N NaOH as per guidelines of AOAC (2006).
3.7.1.3 Brix
The brix of licorice drinks was recorded by using Digital Refractometer following the
guidelines of AOAC (2006).
3.7.2 Antioxidant potential
All the prepared drinks were subjected to in vitro phytochemical screening (TPC, TF) and
antioxidant activity assays using DPPH, FRAP and ABTS methods as per protocols
described in section 3.3 and 3.4.
3.7.3 Sensory evaluation
All the developed drinks were evaluated for different sensorial attributes as described in
Appendix-I according to the protocols of Meilgaard et al. (2007). For the purpose, scores
for different sensory attributes including color, flavor, taste, sweetness, mouthfeel and
overall acceptability were recorded at 0, 30 and 60 days. For evaluation, panelists (age 25-
40, all male) were provided with licorice drinks (chilled at 4 oC. Drinks were labelled with
codes and were served I transparent glasses. Judges were given with unsalted crackers and
mineral water to neutralize the receptors for accurate results. Samples were presented
randomly to the panelists and were requested to assign scores for given characteristics.
3.7.4. Selection of best treatments
One best nutraceuticalCES as well as nutraceuticalSFE drink was selected on the basis of
physicochemical and sensorial properties for rodent modeling.
3.8 Bioefficacy trial
Bioefficacy trial was conducted to assess the disease modulating potential of licorice based
nutraceutical drinks against hepatotoxicity and dyslipidemia. Perposly, 90 rats were
acquired and kept in animal room under controlled feeding and environmental conditions. A
basil diet was provided for 7 days to acclimatize the test animals. During complete trial
period, the animal housing facility was kept at constant relative humidity (55±5%) and
temperature (23±2°C) with 12:12 hr light: dark cycle. Three studies were conducted
independently (Table 3) involving normal (Study I), hypercholesterolemic (Study II) and
34
hepatotoxic (Study III) rats. Three groups of rats having 10 rats in each group were formed
under each study based on type of licorice drink provided. During 12 weeks trial, control,
nutraceuticalCSE and nutraceuticalSFE drinks were given to respective groups to evaluate their
therapeutic effects.
Study I: Normal rats
Study I was comprised of normal rats administered on standard laboratory diet throughout
the trial period. Rats were divided into three groups depending type of nutraceutical drink
administrated; control, nutraeuticalCSE and nutraceuticalSFE. For first week, the rates were
provided with water and basil diet to acclimatize them to the environment. The
experimental diet for normal rats was composed of 82% wheat flour, 10% corn oil, 3%
minerals mix, 4% casein and 1% vitamin mix. Rats were given with nutraceutical drinks
and experimental diet for 12 weeks. At the termination of study, fasted rats were sacrificed
and blood samples were taken to assess the effect of licorice drinks on dyslipidemia and
hepatotoxicity biomarkers. Liver tissues were also to study tissue specific biomarkers of
liver toxicity.
Study II: Hypercholesterolemic rats
The diet for study II consisted of same components in addition to 1.5% cholesterol and
0.5% cholic acid. This diet was given to all three groups to develop hypercholesterolemia
with simultaneous provision of licorice drinks to respective groups. Blood samples were
taken at the termination of the study to check the hypocholesterolemic and hypoglycemic
potential of licorice drinks.
Study III: Hepatotoxic rats
In study III, hepatotoxicity was induced at the end of the study by intra-peritoneal injection
of CCl4 (2 mg/Kg body weight) followed by slaughtering within 24 hours. Blood and liver
tissues were collected to assess hepatoprotective effect of respective licorice drinks. Blood
samples were centrifuged @ 4000 rpm for 6 min ((5804 R, Eppendorf, Germany) to
separate serum for biochemical tests. The respective sera samples were stored for
biochemical assessment.
35
Table 4. Experimental plan for bioefficacy study
Study I
(Normal Rats)
Study II
(Hypercholesterolemic
rats)
Study III
(Hepatotoxic rats)
D0 D1 D2 D0 D1 D2 D0 D1 D2
D0 = Control drink D1 = NutraceuticalCSE drink D2 = NutraceuticalSFE drink
3.8.1 Hepatoprotective perspectives
The blood serum and hepatic tissues collected from normal (Study I) and hepatotoxic rats
(Study III) were analyzed for hepatic stress markers.
3.8.1.1 Oxidative stress biomarkers in liver
Oxidative stress specific markers in liver tissues including superoxide dismutase (SOD)
and catalase (CAT) were assessed as per protocols of Jodynis-Liebert et al., (2000). The
liver tissue were expurgated and homogenized in phosphate buffer (pH 7.4) followed by
differential centrifugation to prepare microsomal and cytosol fractions.
3.8.1.1.1. Superoxide dismutase (SOD)
SOD activity was measured by following spectrophotometric method. Purposely, 0.1 mM
EDTA was mixed in 50 mM carbonate buffer and pH was adjusted to 10.2 at room temperature. 10
mM HCl was used to prepare epinephrine solution. Cytosolic fraction (0.5 mg protein),
epinephrine solution (10 mM) and carbonate buffer were mixed to give 1.5 mL total
volume. Epinephrine oxidation was determined at 320 nm wavelength and 25 oC. SOD
activity was calculated by using the standard curve.
3.8.1.1.2. Catalase
Catalase activity was measured spectrophotometrically in terms of reduction of H2O2. The
reaction mixture included potassium phosphate buffer (50 mM, pH 7.0) and H2O2 (54 mH)
to 3 mL final volume. The assay was initiated with the inclusion of cytosol fraction.
Reduction of H2O2 was determined spectrophotometrically at 240 nm. The results were
expressed in units per mg protein.
36
3.8.1.1.3 Malondialdehyde (MDA)
Serum malondialdehyde level of normal and hepatotoxic rats was evaluated according to
the procedure as outlined by Zhao et al. (2015) following thiobarbituric acid methods using
MDA kit. Results were presented as nmol/mg protein.
3.8.1.2 Serum specific biomarkers
Serum specific oxidative stress markers like ALT, AST and ALP were investigated
following the respective procedures by using commercial kits (Bio-Merieux Laboratory
Reagent and Products, France).
3.8.2 Serum lipid profile and glucose & insulin levels
The collected sera from normal (Study I) and hypercholesterolemic (Study II) rats was
analyzed for lipidemic and glycemic biomarkers. Serum specific biomarkers including
total cholesterol, LDL, HDL and triglycerides were analyzed as per their respective
protocols. Further detail is as under:
3.8.2.1 Cholesterol
Total cholesterol was determined using CHOD–PAP method as described by Kim et al.
(2011).
3.8.2.2 High density lipoprotein
High density lipoprotein (HDL) was assessed according to the protocol as described by
Alshatwi et al. (2010).
3.8.2.3 Low density lipoprotein
Low density lipoproteins (LDL) of serum samples was evaluated by using method of
Alshatwi et al. (2010).
3.8.2.4. Triglycerides
Triglycerides level of serum samples was estimated following the method of Demonty et al.
(2010).
3.8.2.5. Serum glucose and insulin levels
Serum glucose level in all samples was evaluated as per guidelines of Kim et al. (2011)
37
whereas, insulin level was analyzed using the protocols described by Ahn et al. (2011).
3.8.3 Safety assessment studies
Renal functioning indicators and hematological aspects were determined in all three studies
to assess the impact of licorice drinks on respective parameters.
3.8.3.1 Renal functioning tests
Creatinine and urea levels were determined spectrophotometrically according to the
protocols of Salah et al. (2012) using manual commercial reagent kits.
3.8.3.2 Hematological analyses
The blood biochemistry with respect to red blood cells, white blood cells and platelets will
be investigated as per the guidelines of AlHaj et al. (2011).
3.9. Statistical analysis
The data for each parameter was analyzed statistically to check the level of significance
(Montgomery, 2008). Analysis of variance was performed by using ANOVA test and means
were interpreted by Tukey’s HSD test.
38
CHAPTER 4
RESULTS AND DISCUSSION
Plant based nutraceutics provide protection against various health maladies thereby, improving
overall health status of the body. Herbal plants are a rich pool of biologically active
components with a history of use against serval diseases. Licorice is one of the commonly used
herb in various formulations and possess numerous health benefits. In this context, current
study was planned to explore the disease modulating potential of licorice bioactive moieties
with special reference to hepatic and lipidemic malfunctions. The study was divided into three
parts; firstly, licorice was subjected to conventional solvent (CSE) and supercritical fluid
extraction (SFE) followed by phytochemical profiling. Further, licorice based drink was
developed using different levels of two selected extracts, one from each extraction mode. In
last phase of the study, hepatoprotective and hypocholesterolemic perspectives of developed
drink were assessed using rat modeling. The results with discussion regarding all parameters
are as under.
4.1. Phytochemical screening and antioxidant activity assays for CSE
4.1.1 Total phenolic content (TPC)
Mean squares in Table 5 exhibited significant effect of solvents and their concentration on the
TPC of licorice extracts however, their interaction was non-significant. It is evident from mean
values for TPC that highest recovery of phenolic compounds was noted in ethanolic extracts
of licorice (897.24±31.49 mg GAE/100g) followed by methanol (673.38±24.51 mg
GAE/100g) and ethyl acetate (555.07±17.35 mg GAE/100g). The values for total phenolic
content increased with increasing the concentration of solvent. Maximum value of
859.47±21.26 mg GAE/100g was observed at a solvent:water of 75:25. However,
686.40±19.58 mg GAE/100g and 579.82±16.23 mg GAE/100g values were observed for 50:50
and 25:75, respectively (Table 6).
Polyphenols are very important components of plant extracts due to their free radical
scavenging capacity which is mainly attributed to their hydroxyl groups. Hence, the phenolic
content of plant extracts is directly related with their antioxidant potential (Karami et al.,
2013). In current study, different solvents and their concentrations were compared for their
TPC, determined through Folin-Ciocalteau method. The results of this study are in close
39
Table 5. Mean squares for antioxidant indices of licorice solvent extracts
SOV df TPC TF DPPH FRAP ABTS
Solvent (A) 2 271784 ** 7154.7**
478.47** 44621.5**
11.17**
Ratio (B) 2 179274** 11107.4**
478.71** 5621.6**
13.00*
A x B 4 1953 NS 365.7NS
10.48NS 309.6NS
0.14NS
Error 18 1339 155.6 10.32 140.6 1.58
* = Significant **= Highly significant NS= Non significant
40
agreement with the outcomes of Gabriele et al. (2012). They investigated TPC and antioxidant
activity of licorice cortex and inner part extracts obtained through different solvents using
soxhlet extraction. Their results showed 763 and 644 mg GAE/100g total phenolics in ethanolic
extract of licorice root inner part and cortex, respectively. Moreover, methanol was reported
as less efficient for the extraction of phenolic components with 419 and 122 mg GAE/100g
values of TPC for inner part and cortex, accordingly. Earlier, Di-Mambro et al. (2005)
compared different medicinal plants, including licorice, for their antioxidant potential and
reported 724 mg GAE/100g total phenolics in licorice extract.
The extraction of phenolics is greatly affected by the type of solvent and its concentration
being employed for extraction. In a study, Tohma and Gulçin (2010) compared different
solvents for the extraction of phytochemicals from licorice root and antioxidant activity of
resultant extracts. For the purpose, ethanolic and aqueous extracts of licorice root were
obtained and subjected to different in vitro antioxidant activity assays. Results exhibited that
ethanol was better solvent for the recovery of phenolic components as compared to water. In
current study, an increase in TPC was observed by increasing the concentration of ethanol
which is well supported by aforementioned study explaining that ethanol possess greater
potential to extract phenolic compounds as compared to water.
The phytochemical content of plants is significantly influenced by a number of factors during
pre and post-harvest time. These factors include genotype, climatic conditions, harvesting time,
cultivation techniques and storage practices (Gao et al ., 2011). All of these factors are crucial
in determining chemical structure of plants in general and may also effect the phytochemical
content and bioactivity of these components in particular. In a study, Karami and coworkers
(2013) determined the effect of harvesting time on antioxidant activity of licorice root extracts
and reported that the TPC of licorice solvent extracts varied significantly during different
harvesting times.
In a similar study, Cheel et al. (2013) observed a significant variation in TPC of licorice when
determined at different harvesting times (February to November). Their results showed that
the TPC of licorice root was positively associated with the maturity stage of the plant. The
observed value for TPC was 72.01±0.51 mg/g in February
41
which then progressively increased to 88.43±0.49, 99.86±0.72 and 107.93±0.74 mg/g in May,
August and November, respectively. Apart from the harvesting time and maturity stage,
difference in licorice verities, climatic conditions, soil type and sample preparation methods
are the major contributing factors in the fluctuation of the results as reported in various studies.
Cheel et al. (2010), for example, prepared licorice infusion by adding 1.50g licorice powder to
150 mL distilled water followed by brewing (20 min), filtration and lyphilization. The water
extract thus prepared exhibited 1750 mg GAE/100g total phenolic content.
4.1.2 Total flavonoids (TF)
It is evident from the mean squares for TF of licorice extracts that both solvents and their
concentrations significantly affected the TF of different extracts. Whilst, their interaction
remained non-significant (Table 5). The mean values for effect of solvents showed highest
value of TF (286.17±9.85 mg CE/100g) in ethanol extracts trailed by methanol (255.41±8.34
mg CE/100g) and ethyl acetate (229.86±9.81 mg CE/100g). Considering solvent to water ratio,
maximum flavonoids (289.02±7.24 mg CE/100g) were recovered at 75% solvent concentration
and a decreasing trend was observed as we decrease the concentration of solvents (Table 7).
Flavonoids are important secondary metabolites of plants serving many vital functions
including floral pigmentation (yellow or red/blue coloration), UV filtration, regulation of
physiological functions and cell cycle inhibition. Licorice flavonoids are among most potent
natural antioxidant components. They follow different mechanisms including hydrogen
donation, free radical scavenging and metal chelating (Visavadiya et al., 2009). The
extraction of flavonoids can be modulated by using different solvents and extraction
techniques. In current investigation, the efficiency of different solvents and their
concentrations was compared for the recovery of flavonoids from licorice root.
The results of current investigation are in agreement with the outcomes of Asan-zusaglam
and Karakoca (2014) who investigated the antioxidant capacity of Turkish licorice root and
reported that licorice flavonoids are potential antioxidants. In their study, dried licorice root
was extracted with n- hexane using soxhlet apparatus for 24 hours. The TF contents of
resultant extract was 392 mg QE/100g. In another study, Tohma and Gulçin (2010) compared
the antioxidant and radical
42
Table 6. Means for total phenolic contents (mg GAE/100g) of licorice solvent extracts
Solvent:Water
Solvents Means
25:75 50:50 75:25
Ethanol 753.06±17.31 881.36±30.84 1057.29±27.53 897.24±31.49a
Methanol 527.25±21.62 667.18±18.68 825.72±26.42 673.38±24.51b
Ethyl Acetate 459.14±17.44 510.67±16.85 695.39±18.41 555.07±17.35c
Mean 579.82±16.23c 686.40±19.58b
859.47±21.26a
Table 7. Means for flavonoids (mg CE/100g) of licorice solvent extracts
Solvent:Water
Solvents Means
25:75 50:50 75:25
Ethanol 235.03±8.22 298.97±8.76 324.50±9.08 286.17±9.85a
Methanol 219.59±5.25 261.47±9.32 285.16±7.16 255.41±8.34b
Ethyl Acetate 203.82±7.48 228.34±6.59 257.41±7.93 229.86±9.81c
Mean 219.48±6.32c 262.93±7.65b
289.02±7.24a
43
scavenging activity of root and areal parts of licorice. According to their results, the TF
contents of licorice root and areal parts were 420 and 440 mg QE/100g, respectively. Earlier,
Di-Mambro et al. (2005) investigated antioxidant potential and flavonoid content of different
medicinal plants and found that licorice root extract exhibited 88 mg QE/100g total flavonoid
content, highest among all the plant extracts tested.
Total flavonoid content of licorice root is highly influenced by maturity stage, harvesting time
and climatic conditions. In a similar study, Cheel et al. (2013) reported significant variations
in total flavonoid contents of licorice harvested at different times. According to their results,
total flavonoids increased with the passage of time from February (18.42±0.49 mg/g extract)
to August (44.20±0.64 mg/g extract). However, further maturity of licorice adversely affected
the total flavonoid content as the value for this trait decreased significantly after August to
November (35.03±0.65 mg/g extract).
4.1.3 Free radical scavenging activity (DPPH assay)
Mean squares for DPPH free radical scavenging activity showed significant differences for
solvents and their concentration however, the interaction among these factors was non-
significant (Table 5). Means for DPPH activity delineated maximum value for ethanolic
extracts 72.65±2.45% followed by methanolic 66.22±2.84% and ethyl acetate extracts
58.10±2.11% (Table 8). DPPH free radicals inhibition activity was also affected by solvent to
water ratio and maximum activity (71.97±2.81%) was observed at 75% solvent
concentration. Whereas, 67.33±2.76% and 57.68±2.15% inhibition was noted at 50 and 25%
solvent concentration, respectively.
DPPH assay is widely used in food science and nutrition, phytochemistry and pharmacology
to determine free radical scavenging potential. DPPH is a free radical that is easily converted
to stable molecule by accepting a hydrogen radical or an electron. This method is sensitive
enough to determine the antioxidant activity in samples with low analyte concentration and
also can handle comparatively large number of samples within short time (Yokozawa et al.,
1998). The results of this study are in close agreement with the results of Di-Mambro et al.
(2005) who evaluated the antioxidant capacity of several medicinal plants including licorice,
using DPPH free radical assay. Licorice extract exhibited significant inhibition of DPPH
44
radicals as evident from 88% decrease in the absorbance at 517 nm even at lower concentration
(1 µL/mL).
The existing results of DPPH free radical scavenging activities of licorice solvent extracts are
also well supported by the findings of Gabriele et al. (2012). They assessed radical
scavenging activities of licorice cortex and inner yellowish part extracts obtained through
different solvents and observed that cortex extracts delineated greater scavenging potential
than the inner portion of the root. The reported values of free radical scavenging activity of
cortex extracts ranged from 90 to 98% in ethanolic extracts. However, the methanolic
extracts exhibited comparatively less free radical scavenging activities (67-92%). Besides,
ethanolic extracts of inner yellowish part of licorice root showed 75-86% radical scavenging
activities in contrast to methanolic extracts, exhibiting 28-81% free radical scavenging
potential.
Likewise, Lateef et al. (2012) evaluated the antiradical activity of licorice methanolic extract
and its sub fractions prepared in n-butanol, ethyl acetate and chloroform. Results of their
study exhibited that methanolic extract showed highest free radical scavenging potential
(91.3%) and it increased in a dose-dependent way. Among sub fractions, chloroform extract
delineated maximum DPPH free radical scavenging capacity with 87.7% inhibition. Earlier, Jo
et al. (2003) investigated the electron donating potential of licorice ethanolic extract through
DPPH free radical scavenging method. They observed 70.44% free radical scavenging ability
for licorice extract.
4.1.4 Ferrous reducing antioxidant power (FRAP) assay
The mean square values for FRAP assay of licorice solvent extracts presented significant effect
of solvents and their concentrations on the ferrous reducing power of different extracts whilst,
their interaction remained non-significant. Mean values, as presented in Table 9, elucidated
that ethanol was the best solvent among all the three solvents tested for the extraction of
phytochemicals from licorice with highest FRAP value followed by methanol and ethyl
acetate. Mean values for FRAP assay were 451.52±15.73, 369.91±10.64 and 311.32±9.12 μM
Fe2+/g for ethanol, methanol and ethyl acetate, respectively. As a function of solvent
concentration, values for FRAP increased by increasing the concentration of solvents and
maximum value (404.07±13.51 μM Fe2+/g) was observed at 75% concentration.
45
Table 8. Means of DPPH activity (%) of licorice solvent extracts
Solvent:Water
Solvents Means
25:75 50:50 75:25
Ethanol 64.38±2.61 75.43±2.86 78.15±3.04 72.65±2.45a
Methanol 56.47±2.27 68.24±3.15 73.96±2.67 66.22±2.84b
Ethyl Acetate 52.18±1.68 58.32±2.37 63.81±1.78 58.10±2.11c
Mean 57.68±2.15c 67.33±2.76b
71.97±2.81a
Table 9. Means for FRAP assay (μM Fe2+/g) of licorice solvent extracts
Solvent:Water
Solvents Means
25:75 50:50 75:25
Ethanol 416.28±14.97 452.91±10.86 485.36±17.52 451.52±15.73a
Methanol 348.31±13.22 364.26±12.39 397.15±11.85 369.91±10.64b
Ethyl Acetate 298.68±8.34 305.57±10.67 329.71±8.88 311.32±9.12c
Mean 354.42±11.62c 374.25±12.04b
404.07±13.51a
46
Whereas, 374.25±12.04 and 354.42±11.62 μM Fe2+/g values were exhibited at 50% and 25%
solvent concentration, respectively.
Antioxidant species with ferric ion reducing potential are effective electron donor entities
which form stable products by neutralizing free radicals. Many earlier research studies reported
that licorice bioactive components possess significant reducing potential with special reference
to ferric and cupric ions. Current findings are in agreement with Tohma and Gulçin (2010)
who evaluated the antioxidant potential and radical scavenging activity of ethanolic and
aqueous extracts of licorice root and areal parts of the plant. They noticed significant variations
in Fe3+ reducing ability of ethanolic and water extracts of licorice root and areal parts. Their
results showed that ethanolic extract of licorice areal parts exhibited 0.808±0.019 absorbance
whereas, the observed value for aqueous extract was 0.597±0.045. Similarly, 0.759±0.028
absorbance was noted for ethanolic extract of root in contrast to 0.453±0.011 value for aqueous
extract of root. Moreover, 1.097±0.074 and 1.414±0.97 absorbance values were observed for
α-tocopherol and trolox, respectively which were used as standards. They concluded that
ethanolic extract of licorice has greater potential to reduce Fe3+ ions as compared to aqueous
extract.
4.1.5 ABTS assay
Statistical analysis for ABTS assay of licorice solvent extracts depicted significant
differences for different solvents and their concentration on the reducing ability of ABTS
radicals (Table 5). Considering the effect of solvents, maximum value for ABTS assay was
observed for ethanolic extracts (11.02±0.46 µM TE/g) trailed by methanol (9.58±0.29 µM
TE/g) and ethyl acetate (8.66±0.22 µM TE/g). As a function of solvent concentration, 75%
was noted as the optimum solvent concentration with maximum ABTS value of 10.98±0.29
µM TE/g whereas, 9.85±0.38 µM TE/g and 8.42±0.026 µM TE/g values were observed at
50% and 25% solvent concentration, respectively (Table 10).
ABTS assay determine the antioxidant potential of biologically active moieties by following
different mechanism than DPPH assay. ABTS•+ radicals exhibits more reactivity as compared
to DPPH radicals and the reaction involves electron transfer mechanism whereas DPPH
radicals follow hydrogen atom transfer mechanism. The results of current study are in
harmony with the findings of Tohma and Gulçin (2010), determined the anti-radical
47
Table 10. Means for ABTS assay (µM TE/g) of licorice solvent extracts
Solvent:Water
Solvents Means
25:75 50:50 75:25
Ethanol 9.54±0.36 11.05±0.42 12.46±0.39 11.02±0.46a
Methanol 8.13±0.28 9.86±0.25 10.75±0.31 9.58±0.29b
Ethyl Acetate 7.59±0.31 8.65±0.27 9.73±0.35 8.66±0.22c
Mean 8.42±0.026c 9.85±0.38b
10.98±0.29a
48
potential of aqueous and ethanolic extracts of licorice root and areal parts. According to their
results, ethanolic extract of areal parts of licorice exhibited 98.7±3.2% ABTS radical
scavenging activity whereas aqueous extract showed 98.7±3.2% inhibition. Likewise,
ethanolic extract of licorice root delineated 95.8±4.0% free radical scavenging activity in
contrast to the aqueous extract that showed 81.7±11.3% inhibition of ABTS radicals.
It is concluded from the discussion that licorice is a rich source of phytochemicals with high
antioxidant potential. The antioxidant activity of licorice extracts varies with solvents and their
concentrations. Generally, the antioxidant potential of all extracts increased with increasing
the solvent concentration. Moreover, ethanol was most suitable solvent for the extraction of
flavonoids and phenolic compounds from licorice, resulted in greater anti-radical potential of
the extract.
4.2. Phytochemical screening and antioxidant activity assays for SFE
Mean squares regarding phytochemical screening and antioxidant activity assays for
supercritical fluid extracts of licorice exhibited significant effect of pressure on total phenolic
contents, total flavonoids, DPPH, FRAP and ABTS assay values (Table 11). Generally,
increasing the pressure favored the recovery of total phenolics and flavonoids which resulted
in increased antioxidant activity as determined through different in vitro assays.
The mean values of all parameters as affected by varying pressure are presented in Table 12.
It is evident from the mean table that maximum TPC was observed in Tsc3 i.e. 1532.75±36.84
mg GAE/100g followed by Tsc2 (1475.28±47.62 mg GAE/100g) and Tsc1 (1286.51±41.15 mg
GAE/100g). Likewise, highest total flavonoid content was delineated by Tsc3 (576.13±23.51
mg CE/100g) trailed by Tsc2 (531.64±21.46 mg QE/g) and Tsc1 (462.87±17.59 mg CE/g).
Means for DPPH free radical scavenging activity of SFE explicated highest value
(88.26±3.25%) for Tsc3 whilst, Tsc1 showed lowest value (82.49±2.27%). The FRAP values
for Tsc1, Tsc2 and Tsc3 were 610.88±17.08, 698.71±23.74 and 743.45±19.38 μM Fe2+/g,
respectively. A similar trend was observed for ABTS values which was highest in Tsc3
(17.85±0.55 µM TE/g) followed by Tsc2 (16.09±0.47 µM TE/g) and Tsc1 (14.62±0.62 µM
TE/g).
SFE is a novel extraction technique for the recovery of biomolecules from pant matrices. The
solvation potential of supercritical fluid can be effectively increased by changing pressure
49
Table 11. Mean squares for antioxidant indices of licorice supercritical fluid extracts
SOV df TPC FT DPPH FRAP ABTS
Treatment 2 49785.5** 9768.25**
26.39* 13645.3**
7.84**
Error 6 1854.3 464.41 2.94 294.7 0.10
* = Significant **= Highly significant
Table 12. Mean for antioxidant indices of licorice supercritical fluid extracts
Parameters TSC1 TSC2 TSC3
TPC
(mg GAE /100g)
1286.51±41.15c
1475.28±47.62b
1532.75±36.84a
Total Flavonoids
(mg CE/100g)
462.87±17.59c
531.64±21.46b
576.13±23.51a
DPPH
(%) 82.49±2.27c
86.57±3.04b 88.26±3.25a
FRAP
(µM Fe2+/g) 610.88±17.08c
698.71±23.74b 743.45±19.38a
ABTS
(µM Trolox/g) 14.62±0.62c
16.09±0.47b 17.85±0.55a
TSC1 = Supercritical fluid extract at 3500 psi, 40 oC
TSC2 = Supercritical fluid extract at 4500 psi, 40 oC
TSC3 = Supercritical fluid extract at 5500 psi, 40 oC
50
and/or temperature, therefore a remarkable selectivity can be achieved. The process is carried
out at low temperature which favor the extraction of thermo-labile components and undesirable
processes like oxidation, hydrolysis, rearrangement and degradation are also avoided (Lang
and Wai, 2001). Pressure is the most important parameters and the recovery of desired
components can be modulated by merely changing the pressure, keeping other parameters
constant. In current investigation, an increase in recovery of phenolic acids and flavonoids was
observed by increasing the pressure which in turn increased the antioxidant activity of resultant
extracts. The reason behind this phenomena is an increase in the recovery of major bioactive
moieties (glycyrrhizin and glabridin) of licorice with the increase in pressure, as supported by
previous studies (Hedayati and Ghoreishi, 2015; Wei et al., 2004).
Conclusively, the recovery of phytochemicals from licorice is significantly improved in
supercritical fluid extraction as compared to conventional solvents.
4.3. Quantification of active ingredients
High performance liquid chromatography (HPLC) is an advanced analytical tool employed
for the quantification and characterization of biologically active components. Glycyrrhizin
and glabridin are the major bioactive moieties of licorice accounting for its antioxidant
potential and other therapeutic attributes. Precise determination of exact quantity of these
biomolecules in licorice extracts is important to assess its effective dose. For the purpose, all
the solvent and supercritical fluid extracts were subjected to HPLC analysis for accurate
quantification of glycyrrhizin and glabridin.
Statistical analysis regarding HPLC quantification of licorice bioactive components delineated
significant differences in glycyrrhizin and glabridin content of conventional solvent and
supercritical fluid extract as a function of treatments (Table 13). Means concerning the effect
of different solvents and their ratios elucidated highest recovery of glycyrrhizin (2.41±0.027
mg/g licorice) in 25% methanolic extract whereas, highest concentration of glabridin
(1.13±0.010 mg/g licorice) was observed in 75% ethanolic extract (Table 14). Generally,
ethanolic extracts showed higher glabridin recovery which increased with the increase in
solvent concentration. However, methanol was proved as better solvent for the recovery of
glycyrrhizin. It is evident from the results that increasing water concentration favored the
recovery of glycyrrhizin. Ethyl acetate on the other hand was least effective for the recovery
51
Table 13. Mean squares for HPLC quantification of bioactive components
Conventional Solvent Extracts
SOV df Glycyrrhizin Glabridin
Treatment 11 0.147** 0.0493**
Error 25 0.00064 0.00008
Supercritical Fluid Extracts
SOV df Glycyrrhizin Glabridin
Treatment 11 1.026* 1.368**
Error 25 0.003 0.001
52
of either component. Among supercritical fluid extracts, the recovery of both bioactive
moieties increased with the increase in pressure and highest recovery of glycyrrhizin
(5.02±0.031 mg/g licorice) and glabridin (2.97±0.012 mg/g licorice) was detected in TSC3
(5500 psi, 40 oC) followed by TSC2 and TSC1.
The current results regarding the effect of different solvents on the extraction rate of licorice
bioactive components are in harmony with the results of Tian et al. (2008) who investigated
the effect of different solvents on the recovery of glycyrrhizin and glabridin from licorice root.
Their results confirmed that ethanolic extract exhibited highest glabridin content (0.93 mg/g
of licorice) followed by methanol and water. Whilst, highest recovery of glycyrrhizin (2.44
mg/g of licorice) was obtained in water extracts followed by methanol and ethanol.
Furthermore, increasing ethanol concentration favored the recovery of glabridin whereas
glycyrrhizin concentration gradually decreased from 2.44 mg/g to 1.09 mg/g when the
concentration of ethanol was improved from 10 to 90%, respectively.
Recently, Deyab (2015) evaluated the effect of solvent concentration on the recovery of
glycyrrhizin and glabridin from licorice. Purposely, licorice was extracted with 10-90%
ethanol. Their results showed that glycyrrhizin content of licorice extracts increased from 0.75
mg/g to 2.25 mg/g by decreasing the concentration of ethanol from 90% to 10% and increasing
the water concentration in the same manner. However, an increase in glabridin content was
evident from 0.79 mg/g to 0.90 mg/g by increasing the ethanol concentration from 10% to
90%. The outcomes of current study are also in accordance to the work of Ahn et al. (2013)
who compared the recovery of licorice bioactive moieties through conventional solvent and
supercritical fluid extraction. HPLC analysis depicted that supercritical fluid extract of licorice
exhibited significantly higher content of both bioactive components. Earlier, Wang and Yang
(2007) reported 1.212±0.054 to 7.881±0.141 mg/g glycyrrhizin content in one year old licorice
root. They documented that glycyrrhizin content of licorice increases with the age of the plant.
Recently, Hedayati and Ghoreishi (2015) evaluated the impact of different process variables
on the recovery of glycyrrhizin from licorice. Their results showed that increasing pressure
favored the recovery of glycyrrhizin by increasing the density of supercritical CO2. The
solubility of glycyrrhizin increased as a function of density resulted in better extraction yield.
Moreover, it was reported that methanol provided better recovery of glycyrrhizin as compared
53
Table 14. HPLC quantification of bioactive components of licorice
Treatment Glycyrrhizin
(mg/g licorice)
Glabridin
(mg/g licorice)
Conventional Solvent Extracts
T1 2.24±0.024 0.87±0.024
T2 2.13±0.015 0.95±0.013
T3 2.05±0.018 1.13±0.010
T4 2.41±0.027 0.78±0.013
T5 2.32±0.014 0.85±0.015
T6 2.19±0.019 0.92±0.021
T7 1.95±0.011 0.72±0.011
T8 1.82±0.015 0.74±0.028
T9 1.76±0.016 0.78±0.012
Supercritical Fluid Extracts
TSC1 3.87±0.034 1.64±0.014
TSC2 4.26±0.027 2.51±0.017
TSC3 5.02±0.031 2.97±0.012
TSC1 = Supercritical fluid extract at 3500 psi, 40 oC
TSC2 = Supercritical fluid extract at 4500 psi, 40 oC
TSC3 = Supercritical fluid extract at 5500 psi, 40 oC
T1= 25% Ethanol T2= 50% Ethanol T3= 75% Ethanol T4= 25% Methanol T5= 50% Methanol T6= 75% Methanol T7= 25% Ethyl acetate T8= 50% Ethyl acetate T9= 75% Ethyl acetate
54
to ethanol and use of 50:50 v/v methanol with water further enhances the recovery rates.
Earlier, Wei et al. (2004) also documented that the extraction of phenolics and flavonoids from
licorice increased at elevated pressure due to their polar nature.
In the nutshell, supercritical fluid extraction improved the recovery of glycyrrhizin and
glabridin as compared to the conventional solvent extraction. Among solvent extracts, ethanol
provided highest recovery of glabridin whereas, methanol was the most suitable solvent for the
recovery of glycyrrhizin.
4.4. Selection of best treatments
On the basis of relatively higher content of glabridin and glycyrrhizin, T3 (75% ethanolic
extract) was selected among solvent extracts and TSC3 was selected among supercritical fluid
extracts for product development and bioefficacy trial.
4.5. Development of licorice based drink
In the product development module, four treatments of licorice drink were developed by
incorporating different levels of selected conventional (T1 & T2) and supercritical extracts (T3
& T4). The levels of both extracts were selected on the basis of their active dose and relative
content of bioactive components. A control (T0) without extracts was also formulated for
comparison purpose. The resultant drinks were analyzed for physicochemical attributes,
antioxidant potential and sensory evaluation during 60 days storage study at refrigeration
temperature.
4.5.1. Physicochemical analysis of licorice drinks
The physicochemical attributes of licorice based drinks were analyzed including color, pH,
acidity and brix. The results regarding these parameters are discussed below.
For color analysis, CIELB color system was used which is based on L*, a* and b* values. L*
value is indication of lightness and darkness, a* value represents greenish and reddish tone,
b* value indicates yellowish and bluish color.
Statistical analysis regarding color of licorice drinks exhibited significant differences in L*,
a*, b* and chroma values whereas, hue angle was affected non- significantly as a function of
storage intervals (Table 15).
55
56
Means pertaining L* values (Table 16) of licorice drinks explicated that control treatment (T0)
showed maximum L* value (79.65±2.84) whereas, minimum L* value (52.42±1.49) was noted
for licorice drink containing 0.4% solvent extract (T2). This suggests that T2 had darker color
among all the drinks whilst, T0 has brighter color. The L* values for T1, T3 and T4 were
63.64±2.73, 70.67±2.84 and 69.49±2.31 respectively. Drinks containing supercritical fluid
extracts (T3, T4) had brighter color as compared to the drinks with solvent extracts (T1, T2). A
significant reduction was observed in L* value from 68.98±2.42 at 0 day to 65.71±2.06 at 60th
day proposing that the color of licorice drinks became dull with the time.
Means regarding a* value explicated that the color tonality shifted towards reddishness with
the addition of licorice extracts in all treatments (Table 17). The mean values of a* were
5.15±0.13, 7.24±0.18, 8.41±0.35, 6.86±0.18 and 7.42±0.37 for T0, T1, T2, T3 and T4,
respectively. As a function of time, a* values decreased from 7.53±0.34 to 6.53±0.14 during
60 days storage study.
Likewise, The observed values for b* were 63.54±2.17, 58.24±2.36, 46.79±1.41, 61.37±2.25
and 61.16±2.01 for T0, T1, T2, T3 and T4, respectively (Table 18). More the b* value greater is
the yellowish tone in the color. It is evident from mean values that control (T0) treatment has
more yellowish color pattern as compared to other ones and minimum yellowish tone was
noted in licorice drink containing 0.4% solvent extract (T2). A significant reduction in b* value
was observed during storage study from 59.92±1.74 to 56.51±1.88.
Chroma value represents color saturation, more the chroma value more will be the intensity of
the color. Means concerning chroma values (Table 19) exhibited highest chroma value
(63.75±1.92) for control whereas, minimum value (47.54±1.76) for this character was noted in
licorice drink containing 0.4% supercritical fluid extract (T2). Moreover, a significant decline
in chroma values was observed during storage from 60.42±2.33 to 56.91±1.58. Similarly,
means regarding hue angle (Table 20) were 85.36±3.04, 82.91±2.85, 79.81±2.44, 83.62±3.28
and 83.09±3.37 for T0, T1, T2, T3 and T4, respectively. Whilst as a function of storage interval,
the recorded values at 0,30th and 60th day were 82.66±2.93, 82.99±3.11 and 83.22±3.42,
respectively.
Conclusively, the color tone of licorice drinks changed from yellowish towards brownish
during two months of storage. The results of current study are well supported by the
57
Table 15. Mean squares for color tonality of licorice drinks
SOV df L* a* b* Chroma Hue
Treatments (A) 4 907.40** 12.74**
399.45** 377.43**
36.35*
Storage (B) 2 41.55* 3.76**
43.60* 46.20*
1.19NS
A x B 8 0.76 NS 0.18NS
2.68NS 2.72NS
0.12NS
Error 30 9.30 0.12 8.56 8.69 8.43
Table 16. Effect of treatments and storage on L* value of licorice drinks
Storage
Interval
(Days)
Treatments
Mean
T0 T1 T2 T3 T4
0 80.92±3.07 65.73±2.56 54.98±1.48 72.34±2.96 70.95±2.55 68.98±2.42a
30 79.39±2.31 63.02±2.27 51.83±1.61 70.76±2.46 69.17±2.17 66.83±2.23ab
60 78.64±2.67 62.18±2.35 50.46±1.26 68.92±2.19 68.34±2.48 65.71±2.06b
Mean 79.65±2.84a 63.64±2.73c
52.42±1.49d 70.67±2.84b
69.49±2.31bc
T0= Control T1= Drink with 0.2% CSE
T2= Drink with 0.4% CSE
T3= Drink with 0.1% SFE
T3= Drink with 0.2% SFE
58
Table 17. Effect of treatments and storage on a* value of licorice drinks
Storage
Interval
(Days)
Treatments
Mean
T0 T1 T2 T3 T4
0 5.24±0.15 7.78±0.32 9.08±0.39 7.45±0.35 8.12±0.25 7.53±0.34a
30 5.17±0.19 7.10±0.27 8.36±0.31 6.94±0.22 7.33±0.19 6.98±0.16b
60 5.04±0.11 6.85±0.34 7.79±0.26 6.18±0.27 6.81±0.32 6.53±0.14c
Mean 5.15±0.13d 7.24±0.18b
8.41±0.35a 6.86±0.18c
7.42±0.37b
T0= Control T1= Drink with 0.2% CSE
T2= Drink with 0.4% CSE
T3= Drink with 0.1% SFE
T3= Drink with 0.2% SFE
Table 18. Effect of treatments and storage on b* value of licorice drinks
Storage
Interval
(Days)
Treatments
Mean
T0 T1 T2 T3 T4
0 64.45±2.44 60.72±2.29 48.32±1.94 62.25±2.67 63.85±2.31 59.92±1.74a
30 63.21±1.76 58.17±2.43 46.77±1.36 61.53±1.85 61.49±2.64 58.23±2.28ab
60 62.96±2.23 55.82±1.85 45.28±1.72 60.34±2.13 58.14±1.97 56.51±1.88b
Mean 63.54±2.17a 58.24±2.36b
46.79±1.41c 61.37±2.25ab
61.16±2.01ab
T0= Control T1= Drink with 0.2% CSE
T2= Drink with 0.4% CSE
T3= Drink with 0.1% SFE
T3= Drink with 0.2% SFE
59
Table 19. Effect of treatments and storage on chroma of licorice drinks
Storage
Interval
(Days)
Treatments
Mean
T0 T1 T2 T3 T4
0 64.66±2.13 61.22±2.38 49.17±1.62 62.69±1.56 64.36±2.25 60.42±2.33a
30 63.42±2.85 58.60±1.76 47.51±1.88 61.92±2.31 61.93±2.10 58.68±1.74ab
60 63.16±2.39 56.24±2.05 45.95±1.59 60.66±2.08 58.54±1.94 56.91±1.58b
Mean 63.75±1.92a 58.69±2.24b
47.54±1.76c 61.76±1.52ab
61.61±1.81ab
T0= Control T1= Drink with 0.2% CSE
T2= Drink with 0.4% CSE
T3= Drink with 0.1% SFE
T3= Drink with 0.2% SFE
Table 20. Effect of treatments and storage on hue angle of licorice drinks
Storage
Interval
(Days)
Treatments
Mean
T0 T1 T2 T3 T4
0 85.35±3.21 82.69±2.99 79.35±2.52 83.17±3.15 82.74±3.18 82.66±2.93
30 85.32±2.58 83.03±3.28 79.85±3.06 83.56±2.94 83.20±3.26 82.99±3.11
60 85.42±3.15 83.00±2.74 80.23±3.17 84.14±3.13 83.32±3.51 83.22±3.42
Mean 85.36±3.04a 82.91±2.85b
79.81±2.44c 83.62±3.28ab
83.09±3.37ab
T0= Control T1= Drink with 0.2% CSE
T2= Drink with 0.4% CSE
T3= Drink with 0.1% SFE
T3= Drink with 0.2% SFE
60
early work of Jo et al., (2003) who estimated the effect of storage and irradiation on color
parameters and functional properties of licorice root extract at different temperatures. They
reported a decrease in L* value of irradiated licorice extract during two weeks storage. This
change was more pronounced in extracts stored at 4 oC as compared to their counterparts
stored at -20 oC. Moreover, a decrease in a* and b* value of licorice extracts was also
observed during storage. They concluded that the yellow color of licorice extracts gradually
changed to brown during storage. Likewise, the results regarding color of licorice drinks are
also in close agreement with the research outcomes of Alighourchi and Barzegar (2009) who
investigated the effect of storage on the physicochemical attributes of pomegranate juice.
They reported a considerable decrease in L*, a* and b* values of juice during 210 days storage
study. They inferred that the decrease in L* and a* values indicate a fading of color and the
juice turned brownish during storage. Earlier, Marti et al. (2002) noticed a similar trend and
reported a decrease in L* value and increase in hue angle during storage of pomegranate
juice at room temperature resulted in darker color.
Statistical analysis pertaining to the effect of storage intervals and treatments on pH of licorice
drinks exhibited significant effect of storage for this parameter however, treatments imparted
non-significant effect (Table 21). Means related to the pH of licorice drinks depicted a decline
in values from 4.48±0.02 at the initial day to 4.22±0.08 at 60th day (Table 22). Addition of
licorice extracts slightly lowered the pH value of drink from 4.44±0.02 in control treatment to
4.23±0.04 in drink with 0.4% solvent extract whilst, the values for T1, T3 and T4 were
4.35±0.03, 4.41±0.05 and 4.37±0.03, accordingly.
Mean squares regarding acidity of licorice drinks exhibited non-significant effect of treatments
while significant variation was observed during storage study. Means values of acidity for T0,
T1, T2, T3 and T4 were 0.14±0.01, 0.15±0.01, 0.15±0.02, 0.14±0.01 and 0.15±0.01,
respectively (Table 23). Moreover, a significant elevation in acidity was noted during 60
days storage (0.14±0.01 to 0.16±0.01).
The results regarding the change in pH and acidity of licorice drink are in hormony with the
earlier work of Kausar et al. (2012). They investigated the storage stability of cucumber-melon
based functional drink and reported a decline in pH value from 4.89 to 4.77 whereas an increase
in acidity was noted from 0.44 to 0.51%. Likewise, El-Faki and Eisa (2010) evaluated the
effect of storage
61
Table 21. Mean squares for pH, acidity and TSS of licorice drinks
SOV df pH Acidity TSS
Treatments (A) 4 0.053NS 0.0006NS
0.204NS
Storage (B) 2 0.247* 0.0002*
0.330NS
A x B 8 0.001NS 0.00002NS
0.027NS
Error 30 0.023 0.00009 0.276
Table 22. Effect of treatments and storage on pH of licorice drinks
Storage
Interval
(Days)
Treatments
Mean
T0 T1 T2 T3 T4
0 4.56±0.04 4.45±0.03 4.38±0.02 4.52±0.06 4.47±0.04 4.48±0.02a
30 4.46±0.05 4.39±0.01 4.29±0.06 4.45±0.04 4.41±0.03 4.40±0.05b
60 4.31±0.06 4.23±0.04 4.06±0.05 4.28±0.07 4.25±0.07 4.22±0.08c
Mean 4.44±0.02 4.35±0.03 4.23±0.04 4.41±0.05 4.37±0.03
T0= Control T1= Drink with 0.2% CSE
T2= Drink with 0.4% CSE
T3= Drink with 0.1% SFE
T3= Drink with 0.2% SFE
62
on physicochemical attribute of soft drinks and noticed a decreasing trend of pH during six
month storage study. They reported that the pH of lemon lime, apricot and cherry drinks
decreased from 2.91 to 2.84, 3.35 to 3.12 and 3.16 to 2.90, respectively. Similarly, results of
Fasoyiro et al. (2005) were also in close agreement with current study who reported an
increase in acidity with a subsequent decline in pH during storage of fruit based drinks.
Likewise, Ahmed et al. (2008) also observed the same trend during refrigerated storage of
mandarin based dink during 60 days study. Lately, González-Molina et al. (2009) prepared
polyphenols rich beverage using pomegranate and lemon juices in varying concentrations.
They documented a similar trend in pH and acidity as in the present case however, the
differences were non-significant.
Mean squares concerning brix of licorice drinks elucidated non-significant effect of storage
intervals and treatments on brix/TSS value. The observed mean values for brix were
12.93±0.51, 13.23±0.49, 13.33±0.57, 13.09±0.39 and 13.15±0.32 for T0, T1, T2, T3 and T4,
respectively (Table 24). Moreover, a minor increase in brix value was observed during 60
days storage study from 12.98±0.38 to 13.27±0.42.
These results pertaining to increase in TSS during storage study are in line with the research
outcomes of Kausar et al. (2012) who prepared cucumber-melon based functional beverage.
They reported an increase in TSS of functional drink from 15.49 to 16.09% during 120 days
storage. Earlier, Alighourchi and Barzegar (2009) also noticed similar trend and reported an
increase in soluble solids content of pomegranate juice from 13.7 to 14.1 during 210 days
storage study.
4.5.2. Antioxidant potential of licorice drinks
Mean squares concerning the phytochemical screening assays and antioxidant potential of
licorice drinks (Table 25) explicated significant effect of treatments and storage intervals
however, an insignificant effect was noted for their interaction.
Means regarding effect of treatments on TPC, TF, DPPH, FRAP and ABTS assays are shown
in Figure 1. For treatments, the observed values for TPC in licorice drinks were 5.81±0.14
(T0), 15.93±0.54 (T1). 30.71±0.84 (T2), 18.17±0.66 (T3) and 35.28±1.22 mg GAE/100g (T4).
Similarly, the values for total flavonoids of licorice drink ranged from 2.37±0.10 mg
CE/100g (T0) to 8.95±0.21 mg CE/100g (T4). For DPPH free
63
Table 23. Effect of treatments and storage on acidity of licorice drinks
Storage
Interval
(Days)
Treatments
Mean
T0 T1 T2 T3 T4
0 0.14±0.01 0.15±0.01 0.15±0.02 0.14±0.01 0.14±0.01 0.14±0.01b
30 0.14±0.01 0.15±0.01 0.15±0.01 0.14±0.01 0.15±0.02 0.15±0.01ab
60 0.15±0.01 0.16±0.02 0.16±0.01 0.15±0.01 0.16±0.01 0.16±0.01a
Mean 0.14±0.01 0.15±0.01 0.15±0.02 0.14±0.01 0.15±0.01
T0= Control T1= Drink with 0.2% CSE
T2= Drink with 0.4% CSE
T3= Drink with 0.1% SFE
T3= Drink with 0.2% SFE
Table 24. Effect of treatments and storage on TSS/brix of licorice drinks
Storage
Interval
(Days)
Treatments
Mean
T0 T1 T2 T3 T4
0 12.81±0.37 12.87±0.36 13.25±0.42 12.96±0.27 13.01±0.45 12.98±0.38
30 12.96±0.46 13.38±0.41 13.36±0.58 13.10±0.63 13.16±0.35 13.19±0.61
60 13.02±0.54 13.43±0.24 13.39±0.35 13.22±0.46 13.27±0.52 13.27±0.42
Mean 12.93±0.51 13.23±0.57 13.33±0.49 13.09±0.39 13.15±0.32
T0= Control T1= Drink with 0.2% CSE
T2= Drink with 0.4% CSE
T3= Drink with 0.1% SFE
T3= Drink with 0.2% SFE
64
Table 25. Mean squares for antioxidant indices of licorice drinks
SOV df TPC TF DPPH FRAP ABTS
Treatments
(A)
4
1265.55**
66.28**
3086.78**
6774.46**
29.51**
Storage (B) 2 58.81** 4.38**
105.45** 762.22**
1.94**
A x B 8 4.75NS 0.30NS
2.62NS 22.23NS
0.13NS
Error 30 3.18 0.21 4.29 17.68 0.06
* = Significant **= Highly significant NS= Non significant
65
radical scavenging activity, maximum activity was noted in T4 (55.01±1.27%) followed by T2
(48.17±0.59%), T3 (43.89±1.12), T1 (34.97±085%) and T0 (7.42±0.15%). Likewise, same
trend was observed for FRAP values with maximum value observed for T4 (87.05±2.42 μM
Fe2+/g) followed by T2 (77.04±2.38 μM Fe2+/g) whilst the noted values for T0, T1 and T3 were
16.09±0.56, 53.31±2.12 and 64.17±2.68 μM Fe2+/g, respectively. Moreover, a significant
difference in ABTS values was detected as 1.23±0.04 µM TE/g (T0), 4.22±0.15 µM TE/g (T1),
5.34±0.17 µM TE/g (T2), 4.97±0.12 µM TE/g (T3) and 5.84±0.19 µM TE/g (T4).
Means regarding effect of storage interval on TPC, total flavonoids, DPPH, FRAP and ABTS
assays are presented in Figure 2. A significant decline in TPC was observed during 60 day
storage study from 23.15±0.69 to 19.19±0.68 mg GAE/100g. The observed values for total
flavonoids at 0, 30th and 60th day of storage were 6.16±0.24, 5.53±0.21 and 5.09±0.18 mg
CE/100g. Similarly, the DPPH free radical scavenging capacity of licorice extracts
supplemented drinks also exhibited a decreasing trend from 40.47±1.29% at initiation of study
to 35.18±1.09% at the end of 60 days storage trial. Moreover, a significant decline in FRAP
and ABTS values was observed from 66.88±2.14 to 52.65±1.56 μM Fe2+/g and 4.65±0.16 to
3.93±0.18 µM TE/g, respectively.
Polyphenols are prone to degradation with the course of time due to certain factors including
oxidation, pH change, enzymatic degradation and reactions with other substances.
Polymerization is another major contributing factor in the loss of bioactive moieties during
storage. Resultantly, the total phenolic content and antioxidant potential of extract product
decrease with time (Choi et al., 2002). In such a study, Alighourchi and Barzegar (2009)
evaluated the effect of storage on degradation kinetics of anthocyanin in pomegranate juice.
They reported a substantial decrease in total anthocyanin content of juice during 210 days
storage study i.e. 96.9±0.9, 91.3±0.6 and 71.8±0.5% degradation at 37, 20 and 4 oC,
respectively. They inferred that the loss of anthocyanin was attributed to oxidation and
condensation with ascorbic acid. The breakdown products of ascorbic acid and
monosaccharides were identified as major components that accelerated the degradation of
anthocyanins. Later, Fang and Bhandari (2011) assessed the storage stability of bayberry
polyphenols at different temperatures during 6 months study. They reported a significant
5.81
15.93
30.71
18.17
35.28
2.37
4.30
7.83
4.52
8.95
7.42
34.97
48.17
43.89
55.01
16.09
53.31
77.04
64.17
87.05
1.23
4.22
5.34
4.79
5.84
T0
T1
T2
T3
T4
TP
C
F L A V
O N
O I D
S D
P P
H
F R A
P
A B
T S
Fig
ure 1
. Effec
t of trea
tmen
ts on
an
tiox
ida
nt in
dices o
f licorice n
utra
ceu
tical d
rink
66
67
Figure 2. Effect of storage on antioxidant indices of licorice nutraceutical drink
0 Day 30 Day 60 Day
TPC F L A V O N O I D S D P P H F R A P A B T S
23
.15
21
.20
4
19
.19
6.1
6
5.5
3
5.0
9
40
.47
38
.02
35
.18
66
.88
59
.06
52
.65
4.6
5
4.2
8
3.9
3
68
decrease in TPC and anthocyanins by 6-8% and 7-27%, respectively at 4 oC. Moreover, this
decrease was more pronounced at higher temperatures.
The results obtained in current investigation are in close agreement with the earlier work of Jo
et al. (2003). They investigated the effect of storage on the electron donating capacity of
licorice extract through DPPH assay and reported a significant decrease in free radical
scavenging potential from 70.44 to 66.05% during two weeks storage at refrigeration
temperature. Likewise, Chen et al. (2003) reported the antioxidant activities of different
herbal drinks prepared from Chinese medicinal herbs including licorice root. They noticed
that licorice extract exhibited highest DPPH radical inhibition (90.93%) among all the 29
herbs selected for the preparation of drink. Furthermore, the DPPH free radical scavenging
activity of prepared herbal drinks ranged from 38.85 to 50.19%. Conclusively, licorice based
drinks have considerable antioxidant activity owing to the rich phytochemistry of licorice
extracts. Moreover, drinks containing SFE had greater phytochemical content and antioxidant
capacity as compared to drinks with CSE, even at lower concentration.
4.5.3. Sensory Evaluation
Sensory evaluation is an important tool to study the acceptability and marketability of food
products being formulated. Licorice based drinks were assessed for sensory properties
including color, flavor, taste, mouthfeel, sweetness and overall acceptability. Mean squares
regarding sensorial attributes of licorice drinks exhibited significant effect of treatment on
color, taste, flavor, mouthfeel and overall acceptability whereas, sweetness was changed non-
significantly as a function of treatment (Table 26). Regarding storage interval, color and overall
acceptability explicated significant variations in sensory evaluation scores while rest of the
parameters were non-significantly affected. Moreover, interaction showed insignificant effect
for all sensorial attributes of licorice drinks.
Means for color (Table 27) delineated that maximum score for this parameter was observed in
T4 (7.81±0.24) followed by T0 (7.74±0.13), T1 (7.70±0.17), T3 (7.53±0.24) and T2 (7.35±0.23).
Storage imparted significant decline in score for color from 7.80±0.24 at initiation to 7.42±0.21
at the termination of storage study. For flavor, highest score was assigned to T4 (7.77±0.21)
whereas, minimum score was given to T0 (7.36±0.16). For storage interval, sensory score
showed a decreasing trend from 7.70±0.16 to 7.46±0.15 during 60 days.
69
Table 26. Mean squares for sensory evaluation of licorice drinks
SOV
df
Color
Flavor
Taste
Mouthfeel
Sweetness Overall
acceptability
Treatments
(A)
4
0.714*
0.522*
0.863*
0.260*
0.135NS
0.538*
Storage (B) 2 1.30* 0.495NS
0.498NS 0.495NS
0.198NS 0.506*
A x B 8 0.048NS 0.003NS
0.012NS 0.0009NS
0.005NS 0.003
Error 90 0.104 0.164 0.208 0.210 0.213 0.110
* = Significant **= Highly significant NS= Non significant
70
Table 27. Effect of treatments and storage on color of licorice drinks
Storage
Interval
(Days)
Treatments
Mean
T0 T1 T2 T3 T4
0 7.87±0.15 7.84±0.19 7.62±0.24 7.71±0.18 7.95±0.23 7.80±0.24a
30 7.76±0.22 7.72±0.16 7.46±0.26 7.56±0.21 7.82±0.22 7.66±0.18ab
60 7.60±0.18 7.53±0.23 6.98±0.22 7.32±0.26 7.66±0.19 7.42±0.21c
Mean 7.74±0.13ab 7.70±0.17b
7.35±0.23c 7.53±0.24bc
7.81±0.24a
T0= Control T1= Drink with 0.2% CSE
T2= Drink with 0.4% CSE
T3= Drink with 0.1% SFE
T3= Drink with 0.2% SFE
Table 28. Effect of treatments and storage on flavor of licorice drinks
Treatments
Storage Interval
Mean
T0 T1 T2 T3 T4
0 7.51±0.14 7.76±0.22 7.64±0.16 7.70±0.24 7.88±0.26 7.70±0.16
30 7.35±0.11 7.68±0.15 7.52±0.23 7.58±0.17 7.79±0.24 7.58±0.18
60 7.22±0.18 7.59±0.16 7.38±0.19 7.46±0.21 7.65±0.19 7.46±0.15
Mean 7.36±0.16c 7.68±0.21a
7.51±0.24b 7.58±0.18ab
7.77±0.21a
T0= Control T1= Drink with 0.2% CSE
T2= Drink with 0.4% CSE
T3= Drink with 0.1% SFE
T3= Drink with 0.2% SFE
71
It is evident from the means for taste (Table 29) that T0, T1, T2, T3 and T4 were assigned with
sensory scores of 7.27±0.17, 7.60±0.15, 7.43±0.18, 7.48±0.24 and 7.66±0.19, respectively.
Moreover, a non-significant drop in sensory scores was noted during 60 days storage study
showing that no off taste was observed by the consumers. The means for mouthfeel (Table
30) were 7.38±0.24 (T0), 7.61±0.16 (T1), 7.46±0.21 (T2), 7.53±0.25 (T3) and 7.66±0.16 (T4).
Storage resulted in minor change in score from beginning to termination of the study i.e.
7.65±0.24 to 7.41±0.23. Similarly, for sweetness, sensory evaluation scores for T0, T1, T2, T3
and T4 were 7.68±0.22, 7.59±0.19, 7.47±0.21, 7.54±0.19 and 7.63±0.23, respectively.
Whereas, the scores for sweetness at different storage intervals were 7.66±0.23, 7.57±0.25
and 7.51±0.19 at 0, 30th and 60th day. For overall acceptability, maximum score was observed
for T4 (7.75±0.23) followed by T1 (7.71±0.17), T3 (7.65±0.19), T2 (7.47±0.22) and T0
(7.38±0.21). A decline was noted for overall acceptability scores during storage study from
7.71±0.24 to 7.47±0.17.
In current study, drinks containing supercritical fluid extract got higher scores as compared to
their counterparts with conventional solvent extract, showing better sensory profile of extract
obtained through SFE. Change in color of licorice drinks from bright yellowish to dull
brownish tonality was evident by their L*, a*, b*, chroma and hue values during storage study.
This change in the color is further confirmed by the sensory scores for this trait which changed
negatively as a function of time. Shabani et al. (2009) reported that the yellowish color of
licorice and its extract is mainly attributed to the flavonoids, mainly glabridin and
hispaglabridins. It is evident from HPLC quantification and in vitro testing of licorice extracts
that supercritical fluid extracts have higher total flavonoids and glabridin. This explains the
reason behind brighter yellowish color of drinks with supercritical extracts as compared to
brownish color of drinks with conventional solvents extracts as they are deficient in total
flavonoids and glabridin. Moreover, conventional solvent extraction is not much selective for
the extraction of only desired components and a number of undesirable coloring substances are
also present in crude extract. Furthermore, the change in pH and acidity of drinks affected the
taste, flavor, sweetness and mouthfeel of licorice drinks and scores for these attributes
decreased with time.
The results of this project pertaining to the sensorial attributes of licorice based functional
drinks are in line with the findings of Kausar et al. (2012). The research group assessed the
72
Table 29. Effect of treatments and storage on taste of licorice drinks
Treatments
Storage Interval
Mean
T0 T1 T2 T3 T4
0 7.48±0.22 7.72±0.13 7.61±0.19 7.66±0.22 7.78±0.21 7.65±0.24
30 7.27±0.16 7.61±0.18 7.43±0.24 7.46±0.27 7.65±0.23 7.48±0.21
60 7.06±0.25 7.48±0.21 7.25±0.23 7.33±0.16 7.56±0.14 7.34±0.22
Mean 7.27±0.17d 7.60±0.15a
7.43±0.18c 7.48±0.24b
7.66±0.19a
T0= Control T1= Drink with 0.2% CSE
T2= Drink with 0.4% CSE
T3= Drink with 0.1% SFE
T3= Drink with 0.2% SFE
Table 30. Effect of treatments and storage on mouthfeel of licorice drinks
Treatments
Storage Interval
Mean
T0 T1 T2 T3 T4
0 7.52±0.26 7.72±0.25 7.57±0.18 7.64±0.26 7.78±0.19 7.65±0.24
30 7.38±0.21 7.61±0.17 7.48±0.23 7.53±0.24 7.66±0.21 7.53±0.21
60 7.25±0.25 7.49±0.14 7.34±0.26 7.41±0.27 7.55±0.25 7.41±0.23
Mean 7.38±0.24c 7.61±0.16a
7.46±0.21b 7.53±0.25ab
7.66±0.16a
T0= Control T1= Drink with 0.2% CSE
T2= Drink with 0.4% CSE
T3= Drink with 0.1% SFE
T3= Drink with 0.2% SFE
73
Table 31. Effect of treatments and storage on sweetness of licorice drinks
Treatments
Storage Interval
Mean
T0 T1 T2 T3 T4
0 7.75±0.17 7.67±0.26 7.56±0.24 7.62±0.18 7.70±0.27 7.66±0.23
30 7.68±0.25 7.58±0.18 7.45±0.15 7.53±0.17 7.62±0.26 7.57±0.25
60 7.60±0.24 7.52±0.23 7.39±0.26 7.48±0.22 7.56±0.18 7.51±0.19
Mean 7.68±0.22 7.59±0.19 7.47±0.21 7.54±0.19 7.63±0.23
T0= Control T1= Drink with 0.2% CSE
T2= Drink with 0.4% CSE
T3= Drink with 0.1% SFE
T3= Drink with 0.2% SFE
Table 32. Effect of treatments and storage on overall acceptability of licorice drinks
Treatments
Storage Interval
Mean
T0 T1 T2 T3 T4
0 7.53±0.23 7.82±0.19 7.58±0.25 7.75±0.26 7.85±0.21 7.71±0.24a
30 7.39±0.14 7.71±0.24 7.49±0.16 7.66±0.24 7.76±0.17 7.60±0.18ab
60 7.22±0.18 7.61±0.26 7.33±0.17 7.54±0.21 7.63±0.22 7.47±0.17b
Mean 7.38±0.21c 7.71±0.17a
7.47±0.22b 7.65±0.19ab
7.75±0.23a
T0= Control T1= Drink with 0.2% CSE
T2= Drink with 0.4% CSE
T3= Drink with 0.1% SFE
T3= Drink with 0.2% SFE
74
sensorial characteristics of cucumber-melon based functional drink during four month storage
study at 15 days interval. Their results exhibited a decrease in sensory evaluation score for
color from 7.52 to 6.52 whereas the score for flavor decreased from 7.68 to 6.68 during 120
days. Moreover, the scores for taste and overall acceptability decreased in a similar manner
from 7.40 to 6.44 and 7.48 to 6.48, respectively. The results of present study are further
supported by the work of Murtaza et al. (2004) who evaluated the storage stability and sensory
attributes of strawberry juice stored at different temperatures for three months. The results of
their study exhibited a variation in scores for different sensorial attributes including taste,
color and flavor during 90 days storage study. They documented that amino acids and
reducing sugars reacted at elevated temperature and caused non-enzymatic browning which
in turn resulted in color differences. Moreover, increase in acidity was also reported as a
main reason for decrease in sensory scores for flavor and color.
Conclusively, the addition of licorice extracts had no deleterious effect on the storage and
sensory evaluation of drinks. Moreover, all the scores for different traits were in acceptable
range showing a good sensory response from the panelists. The addition of licorice extracts
also resulted in a significant increase in the phytochemical content and antioxidant capacity.
4.6 Selection of best treatments
On the basis of antioxidant potential and sensory evaluation scores, T1 (0.2% CSE) and T4
(0.2% SFE) were selected for bioevaluation trial from drinks containing conventional and
supercritical fluid extracts, respectively.
4.7. Bioefficacy trial
Bioefficacy trial was conducted to assess the therapeutic effect of licorice based drink against
hepatotoxicity and hypercholesterolemic condition. Animal modeling was selected due to its
ease in controlling the environmental conditions and provision of drinks at planned intervals
in predefined quantity. Three studies were conducted independently involving normal (study
I), hypercholesterolemic (study II) and hepatotoxic (study III) rats. Three groups of rats were
formed under each study based on type of licorice drink. During 12 weeks trial, control,
nutraceuticalCSE and nutraceuticalSFE drinks were given to respective groups to evaluate their
75
therapeutic effects. At the termination of bioefficacy study, rats were sacrificed for the
collection of serum and tissues to evaluate lipidemic, glycemic and hepatic biomarkers.
4.7.1. Hepatoprotective perspective
The effect of licorice based functional drinks in normal and hepatotoxic rats was assessed with
special reference to liver enzymes including Aspartate Transaminase (AST), Alanine
Transaminase (ALT), Alkaline Phosphatase (ALP) and endogenous antioxidant compounds
(superoxide dismutase, catalase and malondialdehyde). In the current study, CCl4 was used to
induce acute liver injury in the test animals. CCl4 is a renowned liver toxin which is widely
used to evaluate the effect of hepatoprotective agents against toxin-induced liver damage.
CCl4 mediated liver damage takes place to the generation of reactive substances,
trichloromethyl or trichloromethyl peroxyl, as a result of CCl4 metabolism by cytochrome
P450 2E1. Lipid peroxidation of endoplasmic reticulum and cell membranes is initiated by
these free radicals. These processes in turn cause DNA damage, decline in protein synthesis
and increase in membrane permeability, resulted in necrosis and degeneration of liver cells.
Resultantly, the serum and liver tissue specific biomarkers are changed significantly. The
effect of licorice based nutraceutical drinks on these biomarkers is discussed as under.
4.7.1.1. Alanine Transaminase (ALT)
Statistical analysis showed non-significant effect of treatments on serum ALT levels in study
I (normal rats) however, significant differences were observed in study III (hepatotoxic rats)
(Table 33). Means for study I exhibited maximum ALT level 42.64±1.73 IU/L in D0 (control
drink) followed by D1 (41.75±1.67 IU/L) and D2 (41.08±1.63 IU/L). In study III, a noticeable
increase in serum ALT was detected in D0 group (154.29±6.87 IU/L) that significantly
reduced to 122.65±4.58 and 106.17±3.71 IU/L as a result of nutraceuticalCSE (D1) and
nutraceuticalSFE (D2) drinks, respectively. It is evident from the graphical representation that
licorice drink containing supercritical fluid extract (D2) showed more reduction in serum ALT
levels than drink with conventional solvent extract (D1) (Figure 3). In study III, provision of
nutraceuticalSFE drink (D2) resulted in 31.19% decline in serum ALT whereas, 20.51%
reduction was noted as a result of nutraceuticalCSE drink (D1).
76
Table 33. Effect of licorice drinks on ALT levels (IU/L) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 42.64±1.73 41.75±1.67 41.08±1.63 1.19NS
Study III 154.29±6.87a 122.65±4.58b 106.17±3.71c 29.6**
NSNon-significant
**Highly significant
Study I : Normal rats
Study III: Hepatotoxic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
35
30
25
20
15
10
5
0
Study I Study III
D1: Nutraceutical CSE Drink D2: Nutraceutical SFE Drink
Figure 3. Percent reduction in ALT levels as compared to control drink
3.66 2.09
31.19
20.51
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The results of this study are in line with the work of Huo et al. (2011), revealed the
hepatoprotective effect of licorice aqueous extract through rat modeling. Purposely,
hepatotoxicity was induced by 3 mL/kg body weight dose of CCl4. Rats were divided in six
groups; normal control, model control (CCl4 only), CCl4+100mg/kg extract, CCl4+150mg/kg
extract, CCl4+300mg/kg extract and positive control (100mg/kg bifendate) group. Results
exhibited that serum ALT level increased about 2.5 folds in CCl4 group as compared to normal
control. However, provision of 100, 150 and 300 mg/kg body weight of licorice extract for 15
days significantly lowered the ALT concentration by 61.69, 59.27 and 82.99%, respectively.
Reportedly, the major bioactive component of licorice, glycyrrhizin along with other
triterpene and saponins are responsible for liver protecting effect of licorice, either alone or in
combination with other biologically active compounds. The mechanisms followed for this
effect possibly includes free radical scavenging, stimulation of endogenous enzymes activity
and reducing the formation of inflammatory cytokines which ultimately protects the liver
against acute injury.
The current data pertaining to the effect of licorice supplementation on serum ALT levels are
in close agreement with the results of Al-Razzuqi et al. (2012). They carried out an
experiment to assess the hepatoprotective effect of licorice aqueous extract in rodent models
with acute liver damage induced by CCl4. A substantial increase in serum ALT level
(140.3±1.80 IU/L) was observed resulted in CCl4 administration in comparison to the control
(38.31±1.71 IU/L). However, dietary inclusion of licorice aqueous extract effectively reduced
serum ALT concentration by 78.2%.
Later, Zhao et al. (2015) reported ALT modulating effect of licorice bioactive component,
isoliquiritigenin, at different concentrations (5mg and 10 mg/kg body weight) in CCl4 induced
hepatotoxicity in rats. Results demonstrated a substantial increase in serum ALT levels from
41.46±2.07 U/L in control group to 265.50±20.07 U/L in CCl4 treated group within 12 hours.
Provision of licorice derived isoliquiritigenin at a dose of 5mg and 10mg/kg body weight for
three consecutive days significantly lowered down the increased serum ALT levels to
185.00±19.67 and 135.58±14.40 U/L, respectively in comparison with CCl4 treated negative
control group.
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4.7.1.2. Aspartate Transaminase (AST)
The F value regarding serum AST expounded non-significant difference for study I however,
effect of treatments was significant in study III (Table 34). Means pertaining serum AST in
study I were 93.87±4.01 IU/L, 90.92±3.82 and 89.75±3.74 IU/L in D0, D1 and D2 groups,
respectively. However, mean AST level for D0 in study III was 182.45±8.68 IU/L which then
reduced to 149.77±6.59 and 130.23±5.33 as a result of D1 and D2, respectively. It is clear
from the Figure 4. that nutraceuticalSFE drink showed greater reduction in serum AST levels.
In study III, nutraceuticalSFE drink reduced the serum AST level by 28.62% whereas, 17.91%
reduction was observed in AST level of rats fed on nutraceuticalCSE drink.
The results of current investigation are in agreement with the work of Zhao et al. (2015),
declared a significant drop in serum AST levels of CCl4 induced hepatotoxic rats as a result
of licorice derived isoliquiritigenin administration in a dose-dependent way. According to their
results, treatment with CCl4 drastically elevated the serum AST levels from 31.12±2.28 U/L
to 174.09±25.30 U/L. Inclusion of isoliquiritigenin in diet at a dose of 5mg and 10mg/kg body
weight effectively reduced serum AST levels by 35.28% and 54.32%, respectively. Likewise,
the serum AST lowering effect of licorice extracts is also in accordance with the work of Al-
Razzuqi et al. (2012). They reported 77.8% reduction in serum AST level in CCl4 induced
hepatotoxic rabbits fed on aqueous extract of licorice at a dose of 2gm/kg body weight.
Previously, Huo et al. (2011) also documented that water extract of licorice can efficiently
mitigate CCl4 induced elevation in serum AST levels in wister rats. In their experimental trial,
different groups of rats received 100, 150 and 300 mg/kg body weight licorice extract during
15 days study. The result exhibited that CCl4 treatment caused a marked increase in serum
AST concentration from 171.82±13.54 U/L to 401.45±32.07 U/L. Conversely, the dietary
supplementation of 100, 150 and 300mg/kg body weight of licorice extract lowered down the
elevated AST levels by 27.93, 25.98 and 51.88%, accordingly.
Afterwards, Abdelrahman et al. (2012) evaluated the combined effect of licorice and dates on
CCl4-induced hepatic injury in dogs. Purposely, three groups of test animal were formed i.e
CCl4-treated group (received 0.6 mL/kg CCl4 on day 1, 2 and 3), prophylactic group (received
1g/kg date, 0.4g/kg licorice + CCl4 injection at days 10, 11 and 12) and curative group (licorice
+ date + CCl4 on days 1, 2 and 3). Serum biochemistry was analyzed after 6 and 15 days of
79
Table 34. Effect of licorice drinks on AST levels (IU/L) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 93.87±4.01 90.92±3.82 89.75±3.74 1.72NS
Study III 182.45±8.68a 149.77±6.59b 130.23±5.33c 21.0**
NSNon-significant
**Highly significant
Study I : Normal rats
Study III: Hepatotoxic rats
Do : Control drink D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
35
30
25
20
15
10
5
0
Study I Study III
D1: Nutraceutical CSE Drink D2: Nutraceutical SFE Drink
Figure 4. Percent reduction in AST levels as compared to control drink
28.62
17.91
3.14 4.39
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CCl4 intervention to assess the prophylactic & curative role of licorice and dates. Their results
exhibited a significant reduction in CCl4-induced elevation in serum AST levels in both
treatment groups. Provision of licorice and dates in combination resulted in 26.80 and 61.70%
decline in AST levels of curative and prophylactic group, respectively after six days of CCl4
injection. Moreover, a greater decrement i.e. 52.79 and 62.95% was noticed in aforementioned
groups after 15 days of CCl4 intervention. Histopathological analysis revealed absence of
necrosis and edema in addition to less inflammatory changes in groups provided with
combined extract of licorice and dates. They elaborated that licorice flavonoids are potent
antioxidants with strong potential to halt the activity of free radicals produced as a result of
CCl4 metabolism. Additionally, glycyrrhizin and its major metabolite 18β-glycyrrhetinic acid
have significant potential to restore hepatocellular architecture by maintaining the structural
integrity of hepatocytes cell membrane.
4.7.1.3. Alkaline Phosphatase (ALP)
Statistical analysis showed that treatments brought non-significant variation in serum ALP in
study I whereas, significant reduction was observed in study III (Table 35). An abrupt
increase in serum ALP level was noted as a result of CCl4 treatment which was effectively
addressed by licorice based drinks. Means regarding this parameter showed that in study I,
highest ALP level (165.48±7.59 IU/L) was observed in D0 followed by D1 (162.14±6.98 IU/L)
and D2 (160.57±7.05 IU/L). Likewise, means for study III reflected maximum value for D0
(841.25±38.84 IU/L) that significantly reduced to 705.83±33.17 and 641.49±29.06 IU/L in D1
and D2, respectively. It is obvious from Figure 5. that maximum reduction in serum ALP level
was exhibited as a result of D2 (nutraceuticalSFE drink) in both studies. In study III, treatments
D1 and D2 resulted in 16.11 and 23.75% decline in serum ALP, respectively.
The current trend for the reduction in CCl4 induced elevated serum ALP levels as a result of
licorice supplementation is supported by the findings of Al-Razzuqi et al. (2012). The results
of their investigation delineated a momentous increase in serum ALP concentration upon CCl4
administration at a dose of 1.25mg/kg body weight from 49.66±2.53 to 291.73±7.99 U/L.
Nevertheless, provision of licorice extract at a dose of 2gm/kg body weight significantly
reduced the serum ALP level to 46.83±0.59 U/L. One of the mechanistic approaches for
hepatoprotective effect of licorice is the strong free radical scavenging potential of licorice
81
Table 35. Effect of licorice drinks on ALP levels (IU/L) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 165.48±7.59 162.14±6.98 160.57±7.05 0.45NS
Study III 841.25±38.84a 705.83±33.17b 641.49±29.06c 32.8**
NSNon-significant
**Highly significant
Study I : Normal rats
Study III: Hepatotoxic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
25
20
15
10
5
0
Study I Study III
D1: Nutraceutical CSE Drink D2: Nutraceutical SFE Drink
Figure 5. Percent reduction in ALP levels as compared to control drink
23.75
16.11
2.02 2.97
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flavonoids which inhibits the lipid peroxidation and restores hepatocellular architecture.
Moreover, glycyrrhizic acid blocks the bioactivity of carbon tetrachloride by inhibiting the
activity of P4502E1 enzyme which is responsible for CCl4 metabolism.
Earlier, Huo et al. (2011) also revealed the therapeutic effect of licorice against CCl4 induced
hepatic damage in wister rats. The results delineated that provision of 100, 150 and
300mg/kg body weight of licorice water extract significantly reduced the serum ALP
concentration by 25.37, 21.12 and 50.60%, respectively. Whilst, 51.68% decline was
observed for bifendate, a reference drug used for comparison purpose. Later, Abdelrahman et
al. (2012) assessed the hepatoprotective effect of combined extracts of licorice and dates,
when orally administered to hepatotoxic dogs. CCl4 was used to induce hepatotoxicity which
resulted in significantly elevated serum ALP level (880.80±37.53 U/L). However, licorice
and dates based combined intervention efficiently counter the adverse effects of
hepatotoxicity and significantly reduced the serum ALP levels by 58.15%.
Likewise, Saleem et al. (2011) have also advocated the defensive role of licorice extract
against elevation in ALP level of albino mice. Aqueous extract of licorice was administrated
to test animals for one months at different dosses i.e. 0.2, 0.7 and 1 mg/mL/day. They
revealed that licorice extract supplementation resulted in a significant decline in serum ALP
in a dose dependent way. Provision of licorice extracts at a dose of 0.2, 0.7 and 1 mg/mL/day
decreased the ALP level by 10.10, 30.77 and 51.68%. They further elaborated that glycyrrhizin
is the major hepatoprotective agent in licorice extract which prevents the alternation in the
membrane permeability and increase the survival of hepatocytes under stress conditions.
It is concluded from the aforementioned discussion that dietary inclusion of licorice bioactive
components is helpful in alleviating toxins-induced hepatic damage. In current study, a
significant decrement in serum ALT, AST and ALP levels was noted as a result of licorice
based drinks administration however, drink containing supercritical fluid extract was more
effective. It is suggested that licorice should be an integral ingredient in diet based
hepatoprotective formulations.
4.7.1.4. Superoxide dismutase (SOD)
The statistical analysis regarding effect of different drink treatments on SOD activity level
exhibited non-significant effect of treatments in study I nevertheless, the effect was significant
83
in study III (Table 36). The mean values of liver SOD in study I were 11.37±0.43, 11.78±0.36
and 11.94±0.51 IU/mg protein in D0, D1 and D2, respectively. In study III, maximum SOD
activity (9.25±0.25 IU/mg protein) was observed in D2 followed by D1 (8.36±0.32 IU/mg
protein) and D0 (7.01±0.29 IU/mg protein). The graphical illustration (Figure 6) showed that
treatments D1 and D2 caused a non-significant elevation in liver SOD activity for study I
whereas in study III, significant increase was observed in D1 (19.26%) and D2 (31.95%)
groups.
Recently, Zhao et al. (2015) studied the effect of licorice derived isoliquiritigenin on by CCl4
induced hepatotoxicity through rodent modeling and reported that isoliquiritigenin effectively
restored liver SOD activity. CCl4 administration significantly reduced SOD activity from
33.12±5.02 U/mg protein in control group to 12.38±2.43 U/mg protein. Isoliquiritigenin
administration at a dose of 20 mg/kg momentously increased liver SOD activity (25.72±3.82
U/mg protein) towards normal value. In a previous study, Huo et al. (2011) elucidated that
pre-treatment with licorice water extract momentously improved the activities of various liver
endogenous enzymes including SOD, depending upon the active dose. In CCl4 treated
negative control group, the SOD activity was approximately decreased by 50% as compared
to the normal control group however, provision of licorice extract (100, 150 and 300 mg/kg
body weight) increased the hepatic SOD activity by 24.88, 27.34 and 47.74%, respectively.
Likewise, Yehuda et al. (2011) have documented the hepatoprotective potential of glabridin
due to upregulation of liver antioxidant enzymes under glucose stress. In their experiment,
they elucidated that the activity of manganese superoxide dismutase (Mn-SOD) decreased
under glucose stress in isolated monocyte cells. However, glabridin supplementation
moderately improved the mRNA expression of Mn-SOD, resulted in higher SOD activity. It
is clear from the above discussion that licorice extract and licorice based diet therapies have
potential to restore normal activity of hepatic SOD enzyme.
4.7.1.5. Catalase
The F values pertaining to the catalase activity delineated that this trait was affected non-
significantly in study I for treatment whereas in study III, catalase activity varied
significantly among different groups (Table 37). In study I, mean values regarding catalase
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Table 36. Effect of licorice drinks on SOD (IU/mg protein) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 11.37±0.43 11.78±0.36 11.94±0.51 1.07NS
Study III 7.01±0.39c 8.36±0.42b 9.25±0.45a 31.7**
NSNon-significant
**Highly significant
Study I : Normal rats
Study III: Hepatotoxic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
35
30
25
20
15
10
5
0
Study I Study III
D1: Nutraceutical CSE Drink D2: Nutraceutical SFE Drink
Figure 6. Percent increase in SOD levels as compared to control drink
31.95
19.26
3.61 5.01
Per
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activity were 15.63±0.72, 16.25±0.85 and 16.57±0.78 IU/mg protein in D0, D1 and D2 groups,
respectively. Likewise in study III, group D2 exhibited highest catalase activity (12.93±0.63
IU/mg protein) trailed by D1 (12.06±0.57 IU/mg protein) and D0 (10.28±0.46 IU/mg protein).
It is evident from the graphical representation that 25.78% increase was observed in study III
rats fed on drink containing supercritical fluid extract (D2) in contrast to drink containing
solvent extract (D1) which brought about 17.32% rise in catalase activity (Figure 7).The
findings of Huo et al. (2011) are in harmony with the current results, showing a significant
increase in liver catalase activity as a result of licorice extract supplementation in dose
dependent manner. Early treatment with 100, 150 and 300 mg/kg body weight of licorice
aqueous extract enhanced the catalase activity by 18.78, 30.36 and 80.57%, accordingly. The
mechanism behind this effect was believed to be the free radical stabilizing capacity of licorice
extract that can competently mitigate CCl4 induced lipid peroxidation and hepatocellular
damage.
In a bioefficacy trial, Zhao et al. (2015) probed the effect of licorice nutraceutics on CCl4
induced oxidative stress in rats and observed a marked escalation in catalase activity as a
result of isoliquiritigenin, supplementation. The research group observed a significant decline
in catalase activity (7.33±1.41 U/mg protein) in CCl4 administrated group as compared to the
control group (15.53±1.68 U/mg protein). Isoliquiritigenin supplementation competently
restored the catalase activity towards normal level as evident from the catalase activity value
in treatment group i.e. 13.96±2.33 U/mg protein. Earlier, Yehuda et al. (2011) have reported
that licorice derived flavonoid, glabridin, has potential to upregulate the mRNA expression of
catalase enzyme under glucose stress. Macrophage cells were selected as model to elaborate
the effect of high glucose stress and subsequent treatment with glabridin on the antioxidant
defense system. The results exhibited that chronic glucose stress down-regulated the mRNA
expression for catalase enzyme by 20%. It was noticed that inflammatory conditions had
further aggravated this effect. However, glabridin supplementation up-regulated the mRNA
expression and improved the enzymatic activity of catalase.
In another study, Kanimozhi and Karthikeyan (2011) evaluated the protective effect of licorice
leaves extract against 1,4-dichlorobenzene-induced liver carcinogenesis in rats. They affirmed
that treatment with 1,4-dichlorobenzene significantly suppressed the catalase activity.
However, 100 mg/kg body weight supplementation of licorice increased the catalase activity
86
Table 37. Effect of licorice drinks on catalase activity (IU/mg protein) of rats in different
studies
Studies
Treatments
D0 D1 D2
F value
Study I 15.63±0.72 16.25±0.85 16.57±0.78 1.49NS
Study III 10.28±0.46b 12.06±0.57ab 12.93±0.63a 50.1*
NSNon-significant
**Highly significant
Study I : Normal rats
Study III: Hepatotoxic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
30
25
20
15
10
5
0
Study I Study III
D1: Nutraceutical CSE Drink D2: Nutraceutical SFE Drink
Figure 7. Percent increase in catalase levels as compared to control drink
25.78
17.32
4.41 2.39
Per
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by 45.14%. They reported that hepatoprotective effect of licorice leaves extract may be due
to the bioactive components i.e. glycyrrhizin and glycyrhitic acid. The aforementioned
discussion has supported the role of licorice and its bioactive components in improving the
exogenous antioxidant system of the body by up-regulating the activity of antioxidant
enzymes.
4.7.1.6. Malondialdehyde (MDA)
It is evident from F value that treatments illustrated non-significant effect for MDA level in
study I whereas significant variation in MDA level was observed in among groups fed on
different drinks (Table 38). In study I, mean values for MDA level were 3.97±0.15, 3.85±0.12
and 3.78±0.18 nM/mg for D0, D1 and D2 groups, respectively. Similarly in study III, highest
MDA level was noted for D0 (8.14±0.42 nM/mg) which then significantly reduced to
6.45±0.31 in D1 group and 5.02±0.22 nM/mg in D2. Figure 8. showed percent reduction in
MDA levels as a result of licorice extracts supplemented drinks. In study III, 20.76 and 38.33%
decrement in MDA levels was observed for D1 and D2 groups respectively as compared to
control.
Thiobarbituric Acid Reactive Substances (TBARS) are formed as by-products during lipid
peroxidation when fats are degraded in this process. TBARS assay is commonly used to
measure these substances by using thiobarbituric acid as a reagent. Malondialdehyde (MDA)
is the major compound which is measured in TBARS assay as a sign of lipid peroxidation and
oxidative stress. The content of MDA increase with the increase in oxidative stress and
subsequent lipid peroxidation. Earlier research work has extensively focused on this biomarker
as an important indicator of hepatotoxicity (Trevisan et al., 2001).
The results of current study concerning significant decrease in MDA level as a result of
licorice drinks supplementation are in close agreement with the study of El-Tawil et al.
(2013). They studied the hepatoprotective effect of licorice extract against CCl4 induced
hepatotoxicity in isolated hepatocytes of rats. They noticed a significant elevation in TBARS
level of hepatocytes after 30 minutes of CCl4 administration. Contrarily, provision of licorice
extract significantly decreased the TBARS level in a time dependent way with maximum
decline at 120 minutes after intervention. They suggested that licorice has good potential to
curtail toxins-induced oxidative stress.
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Table 38. Effect of licorice drinks on MDA (nM/mg) level of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 3.97±0.15 3.85±0.12 3.78±0.18 2.05NS
Study III 8.14±0.42a 6.45±0.31b 5.02±0.22c 40.3**
NSNon-significant
**Highly significant
Study I : Normal rats
Study III: Hepatotoxic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
45
40
35
30
25
20
15
10
5
0
Study I Study III
D1: Nutraceutical CSE Drink D2: Nutraceutical SFE Drink
Figure 8. Percent reduction in MDA levels as compared to control drink
38.33
20.76
4.97 3.02
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The results of current investigation are well supported by the findings of Zhao et al. (2015)
who reported that CCl4 induced liver damage resulted in elevated MDA level, 9.18±1.81
nM/mg in comparison with the control group having 4.21±0.60 nM/mg MDA level.
Provision of isoliquiritigenin at 20 mg/kg dose for 3 consecutive days considerably reduced
the MDA level to 5.72±0.83 nM/mg. Earlier, Huo et al. (2011) also reported that CCl4
treatment elevated the level of hepatic MDA due to lipid peroxidation caused by free radicals
generated through CCl4 metabolism. Nevertheless, pre- feeding on licorice extract reduced
MDA concentration in dose dependent manner by 15.56, 42.10 and 104.8% at 100, 150 and
300 mg/kg dose, respectively. They proposed that the bioactive components of licorice
aqueous extract possess strong antioxidant potential and are involved in free radical
scavenging. This effect is possibly responsible for the prevention of lipid peroxidation and
reduction in MDA concentration in liver as MDA is a byproduct of reactions involving lipid
peroxidation. It is concluded from this discussion that licorice based drink is effective to
ameliorate lipid peroxidation under hepatic stress conditions.
4.7.1.7. Bilirubin
Statistical analysis regarding serum bilirubin level explicated non-significant effect of
treatments on this trait for study I while significant effect was observed for study III
(hepatotoxic rats). Means pertaining serum bilirubin levels (Table 39) in study I were
0.73±0.03, 0.71±0.02 and 0.70±0.02 mg/dL for D0, D1 and D2, respectively. Likewise in study
III, highest serum bilirubin (1.03±0.05 mg/dL) was detected in group fed on control drink (D0)
which then significantly reduced to 0.92±0.03 and 0.86±0.02 mg/dL in D1 and D2 groups,
respectively. Graphical representation (Figure 9) for percent reduction in serum bilirubin levels
expounded that licorice extracts containing drinks D1 and D2 caused 15.53% and 26.21%
reduction in serum bilirubin levels, respectively in hepatotoxic rats (study III).
These results for reduction in bilirubin levels are well supported by the findings of Al-
Razzuqi et al. (2012). During their trial, acute liver damage was induced in rabbit models
through intravenous injection of CCl4 and licorice aqueous extract was supplemented at a
single dose of 2gm/kg body weight. A significant decline in serum bilirubin concentration
was observed as it was 15.7% less in treatment group than CCl4 administrated negative control
group.
90
Table 39. Effect of licorice drinks on bilirubin level (mg/dL) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 0.73±0.03 0.71±0.02 0.7±0.02 0.34NS
Study III 1.03±0.05a 0.92±0.03ab
0.86±0.02b 6.17*
NSNon-significant
**Highly significant
Study I : Normal rats
Study III: Hepatotoxic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
30
25
20
15
10
5
0
Study I Study III
D1: Nutraceutical CSE Drink D2: Nutraceutical SFE Drink
Figure 9. Percent reduction in bilirubin levels as compared to control drink
26.21
15.53
4.11 2.74
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Earlier, Tominaga et al. (2009) has also reported the decreasing trend of serum bilirubin at
different doses of licorice flavonoid oil. Purposely, ethanolic extract of licorice was mixed with
medium chain (C8:C10) fatty acids and concentration of glabridin was adjusted to 3%. The
resulted product was termed as “licorice flavonoid oil” which was then administered to human
volunteers in the form of capsules at different dose levels (300, 600 and 900 mg). Their results
expounded that 300 mg was optimum dose which reduced the serum bilirubin level by 19.05%
in 8 weeks trial. In contrast, Fuhrman et al. (2002) reported that consumption of licorice extract
for 30 days has no effect on serum bilirubin in hypercholesterolemic subjects. However, an
increase in serum bilirubin level was detected in placebo group over one month bioefficacy
study.
The results of this study are also in close harmony with the findings of Aoki et al. (2013)
who evaluated physiological activities and antioxidant potential of glabridin rich licorice
flavonoid oil (LFO). Their results exhibited a decrement in total bilirubin from 0.77±0.06
mg/dL at the beginning of study to 0.74±0.05 at the termination as a result of 1200 mg daily
dose of LFO for four consecutive weeks. It is inferred from the above discussion that licorice
based nutraceutical drink has potential to curtail adverse effects of hepatotoxicity.
4.7.2. Hypocholesterolemic perspective
4.7.2.1. Total cholesterol
It is revealed from the statistical analysis (Table 40) that treatments have significant effect on
serum cholesterol levels in normal rats (study I) as well as in hypercholesterolemic rats (study
II). Means regarding serum cholesterol were 78.62±3.12, 75.84±3.25 and 74.32±2.97 mg/dL
for D0, D1 and D2 groups, respectively for study I. Serum cholesterol level was significantly
elevated in hypercholesterolemic rats (study II) as a result of high fat and cholesterol
supplemented diet. In study II, highest cholesterol level was recorded in D0 (152.38±5.34
mg/dL) which was then significantly reduced to 135.25±5.63 and 124.16±3.81 mg/dL in D1
and D2 groups, respectively. Figure 10 illustrated 3.54 and 5.47% decrement in serum
cholesterol levels for study I as a result of D1 and D2 drinks, accordingly. Likewise for study
II, drinks containing solvent extract (D1) and supercritical fluid extract (D2) resulted in 11.24
and 18.52% decline in total cholesterol.
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Table 40. Effect of licorice drinks on cholesterol (mg/dL) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 78.62±3.12a 75.84±3.25ab 74.32±2.97b 5.57*
Study II 152.38±5.34a 135.25±5.63b 124.16±3.81c 72.4**
*Significant
**Highly significant
Study I : Normal rats
Study II: Hypercholesterolemic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
20
18
16
14
12
10
8
6
4
2
0
Study I Study II
D1: Nutraceutical CSE Drink D2: Nutraceutical SFE Drink
Figure 10. Percent reduction in cholesterol levels as compared to control drink
18.52
11.24
5.47
3.54
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The current results regarding the serum cholesterol reduction by licorice extracts are in close
harmony with the earlier work of Ahn et al. (2013). During the trial, high fat diet induced obese
rats were fed on 0.1 and 0.25% supercritical fluid extract of licorice. At the end of 8 weeks
study, 32.33 and 20.4% decrement in total cholesterol was recorded in groups supplemented
with 0.1 and 0.25% extract, respectively. Recently, Mirtaheri et al. (2015) also reported the
hypolipidemic effect of glabridin rich dried ethanolic extract in overweight and obese subjects.
Licorice extract supplement at a dose of 1.5g/day for 8 weeks resulted in significantly lower
total cholesterol in intervention group as compared to control.
The results of current study are also supported by earlier work of Fuhrman et al. (2002) who
narrated the cholesterol lowering potential of licorice ethanolic extract. Their results exhibited
that dietary inclusion of 0.1g/day licorice extract for a period of 30 days significantly reduced
total cholesterol level in hypercholesterolemic subjects. One of their peers, Asgary et al. (2007)
also noted the hypocholesterolemic effect of licorice ethanolic extract in hypercholesterolemic
rabbits. The results showed a significantly lower total cholesterol in treatment group fed on
50mg/kg licorice extract for 60 days. Later, Lee et al. (2012) investigated the synergistic effect
of licorice, bitter gourd, red yeast rice, soy protein and chlorella in improving serum lipid
profile and addressing metabolic syndrome. Subjects with metabolic syndrome received 1g
combined extract of all the aforementioned commodities for a period of 12 weeks. At the end
of trial period, 18.52% decline in total cholesterol was noticed in treatment group.
A number of health complications are reported to exert a deleterious effect on serum lipid
profile including diabetes, liver disorders and metabolic syndrome. Bioactive components of
licorice have good potential to ameliorate such abnormalities in lipid profile. In this context,
Sen et al. (2011) have supported the potential of glycyrrhizin to mitigate the elevated
cholesterol level in streptozotocin (STZ) induced diabetic rats. An abrupt increase in serum
cholesterol concentration was observed after STZ administration however, a single dose of
100mg/kg body weight of glycyrrhizin effectively reduced the elevated cholesterol by ~66.8%.
They stated that poor consumption of serum glucose in diabetic situation encourages the
metabolism of lipids which ultimately increase the cholesterol level in blood. Glycyrrhizin
improved the glucose metabolism which in turn suppressed abnormally elevated lipid
metabolism and normalized the serum cholesterol concentration.
94
The results of current study are also in close agreement with the research outcomes of Saleem
et al. (2011) who reported a significant decline in serum cholesterol of albino mice as a result
of licorice extract supplementation. They reported that provision of licorice extract at varying
doses of 0.2, 0.7 and 1 mg/mL/day resulted in 13.74, 21.32 and 28.73% decrement in total
cholesterol of test animals. They documented that licorice extract is rich in phytosterols and
saponins (mainly glycyrrhizin) and these components are directly related with cholesterol
lowering potential of the extract. Therefore, one potential mechanism for the decrease of serum
cholesterol is its suppressed absorption from intestine because phytosterols have potential to
interrupt intestinal cholesterol and to hinder its absorption. Whereas, saponins have capacity
to interfere with enterohepatic circulation of bile acids. Moreover, they also cause
precipitation of cholesterol from micelles therefore making it inaccessible for absorption.
It is evident from the aforesaid discussion that bioactive components of licorice have
considerable potential to reduced elevated cholesterol level following different mechanisms.
Moreover, licorice based dietary interventions can effectively be used to modulate
dyslipidemia due to poor dietary practices.
4.7.2.2. High density lipoproteins (HDL)
The F value concerning serum HDL levels of different studies showed non-significant effect
of treatments in study I however, significant effect was observed in study II. In study I, mean
values for HDL were 35.48±1.37, 36.02±1.15 and 36.21±1.45 mg/dL in D0, D1 and D2 groups,
respectively (Table 41). Likewise in study II, mean value for HDL in control (D0) group was
52.39±1.88 mg/dL that improved significantly to 54.41±2.07 and 55.08±1.92 mg/dL in D1 and
D2 groups, respectively. It is evident from the graphical representation (Figure 11) that the
provision of licorice extracts based drinks D1 and D2 improved the serum HDL by 3.29 and
5.14%, respectively in study II.
Cholesterol is an essential compound and its integral part of all animal cell membranes.
Chemically it is lipid based compound and its transport in the body is mediated by different
biomolecules with varying size and chemistry. These molecules are named as lipoproteins.
Different carriers that facilitate the circulation of cholesterol includes HDL, LDL, IDL,
VLDL and chylomicrons. Among these, LDL and HDL are important indicators of
dyslipidemia condition as research has indicated a strong link between disturbance of serum
HDL and LDL levels and CVDs (Lewington et al., 2007). It is reported that low level
95
Table 41. Effect of licorice drinks on HDL (mg/dL) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 35.48±1.37 36.02±1.15 36.21±1.45 0.29NS
Study II 52.39±1.88b 54.41±2.07ab 55.08±1.92a 8.98*
NSNon-significant
*Significant
Study I : Normal rats
Study II: Hypercholesterolemic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
6
5
4
3
2
1
0
Study I Study II
D1: Nutraceutical CSE Drink D2: Nutraceutical SFE Drink
Figure 11. Percent increase in HDL levels as compared to control drink
5.14
3.29
2.08
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of HDL is associated with higher risk for CVDs however, an increase in HDL level is
regarded as favorable. It is evident from epidemiological studies that high serum HDL can
inhibit LDL-oxidation thereby has potential to overcome the deleterious effects caused by
oxidized LDL (Assmann and Nofer, 2003).
The results of current investigation are in close agreement with the pervious study of Asgary
et al. (2007), showed a significant increase in HDL level by licorice extract based dietary
intervention. In their study, fifteen male rabbits were divided in three test groups based on
different diets (normal diet, high fat diet, high fat diet + 50mg/kg licorice extract). Results
exhibited that provision of high fat diet resulted in significant decrement in serum HDL level
however, inclusion of licorice extract in diet ameliorated this deleterious effect. They reported
that serum HDL concentration was about 2.5 folds higher in intervention group as compared
to high fat fed negative control group. Moreover, nearly 20% higher HDL level was observed
in intervention group when compared with normal diet group.
In a trial, Saleem et al. (2011) assessed the therapeutic effect of licorice extract against
hypercholesterolemia and reported significant increase in serum HDL levels of albino rats in a
dose dependent manner. According to their results, oral administration of 0.2, 0.7 and 1
mg/mL/day licorice extract significantly decreased serum HDL by 33.91, 36.01 and 45.52%,
respectively. Recently, Mirtaheri et al. (2015) reported that licorice extract supplementation
significantly improve the serum lipid profile of obese/overweight subjects. Their results
explicated 11.08% increase in serum HDL level of individuals fed on 1.5g/day licorice extract
during 8 weeks bioevaluation trial. Moreover, LDL/HDL cholesterol ratio was also reduced
significantly which also confirmed an increase in HDL with the subsequent decrement in LDL
level.
Earlier, Gaur et al. (2014) examined the effect of licorice derived bioactive components on
glycemic and lipidemic parameters of STZ-induced diabetic rats. Results exhibited that dietary
inclusion of licorice derived bioactive moieties isoliquiritigenin, 2,4-dimethoxy-4-
hydroxychalcone and liquiritigenin-7,4-dibenzoate improved the serum HDL concentrations
by 55.65, 48.01 and 61.74%, respectively. It is clear from the discussion that bioactive moieties
of licorice have potential to improve serum HDL level of hypercholesterolemic subjects.
97
4.7.2.3. Low density lipoproteins (LDL)
Statistical analysis (Table 42) relating to the effect of licorice extracts based drinks on serum
LDL levels expounded significant effect of treatments on LDL levels in both studies. In study
I, the observed values of LDL for D0, D1 and D2 groups were 30.76±1.13, 29.63±0.82 and
28.05±1.06 mg/dL, respectively. Likewise in study II, highest value (61.53±2.89 mg/dL) for
LDL was noted in D0 that was decreased to 50.73±1.65 and 46.54±1.31 mg/dL in D1 and D2
groups, respectively. It is evident from the graph (Figure 12) that the provision of licorice
extract based drinks significantly reduced the serum LDL levels in both studies and drink
containing supercritical fluid extract (D2) showed greater reduction as compared to drink
containing solvent extract (D1). In study I, D1 and D2 reduced serum LDL by 3.68 and 8.81%,
respectively. Similarly in hypercholesterolemic rats (study II), 17.56 and 24.37% decrement in
serum LDL was noted for D1 and D2 groups, accordingly.
Abnormal increase in plasma LDL concentration followed by its oxidation, subsequent
aggregation and retention in arteries is considered as the major cause of atherosclerosis and
allied health complications. At earlier stage, LDL occupy the arterial walls where it combines
with the extra cellular matrix and its deposition starts (LDL retention). This phenomena
increases LDL susceptibility towards oxidation. Excessive oxidation of LDL leads to its
accumulation within the artery wall and results in foam cell formation, macrophage
cholesterol accumulation and subsequently in the development of atherosclerotic lesions.
These lesions then cause the narrowing of arteries which may lead to other deleterious effects
such as heart attacks. Recent research in this regard has focused on the bioefficacy of plant
based phytochemicals to ameliorate such abnormalities. Nutraceuticals from licorice root
have exhibited promising role in the protection of LDL and HDL against oxidation owing to
their free radical stabilizing mechanism (Fuhrman et al., 2002).
The current results regarding effect of licorice based dietary interventions on serum lipid
profile with special reference to LDL are in accordance with the research work of Asgary et
al. (2007). The study was carried out to explore the protective effect of licorice extract
against abnormal serum lipid profile and atherosclerosis. Results exhibited that licorice
extract at a dose of 50mg/kg significantly lowered down serum LDL level. Furthermore,
licorice extract supplementation significantly diminished LDL aggregation, oxidation and the
98
Table 42. Effect of licorice drinks on LDL (mg/dL) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 30.76±1.13a 29.63±0.82ab 28.05±1.06b 6.65*
Study II 61.53±2.89a 50.73±1.65b 46.54±1.31c 54.71**
*Significant
**Highly significant
Study I : Normal rats
Study II: Hypercholesterolemic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
30
25
20
15
10
5
0
Study I Study II
D1: Nutraceutical CSE Drink D2: Nutraceutical SFE Drink
Figure 12. Percent reduction in LDL levels as compared to control drink
24.37
17.56
8.81
3.68
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development of atherosclerotic lesions. The mechanism for this inhibitory effect includes the
possible binding of licorice polyphenols, especially glabridin, to the LDL molecules. Glabridin
has strong free radical scavenging capacity and it protects LDL from oxidation and
aggregation.
Likewise, research outcomes of Fuhrman et al. (2002) also illuminated the protective effect of
licorice extract against LDL oxidation and aggregation. Reportedly, licorice extract
supplementation increased the LDL resistance towards oxidation along with normalizing
serum lipid profile in hypercholesterolemic subjects. In their experiment,
hypercholesterolemic subjects (with serum cholesterol level of 220-260 mg/dL) were given
with dried ethanolic extract of licorice at a dose of 0.1 g/day for 30 days. Serum lipid profile
and LDL oxidation levels were measured at the termination of trail. It was evident from the
results that licorice consumption reduced the oxidation of plasma by 19% and increased the
resistance of LDL towards oxidation by 55%. Additionally, blood chemistry analysis showed
a significant reduction (~9%) in LDL cholesterol. One month consumption of licorice extract
reversed the biomarkers of hypercholesterolemia.
Recently, Mirtaheri et al. (2015) investigated the effect of licorice based therapeutic
intervention on serum lipid profile and atherogenic indices of overweight subjects. Purposely,
64 overweight and obese volunteers received 1.5g/day dried extract of licorice for 30
consecutive days. Serum lipid profiles were measured at base line and termination of
experiment. Results exhibited 8.18% decrement in LDL cholesterol level as a result of licorice
supplementation. Moreover, a significant decrease in LDL/HDL ratio was also observed which
confirmed a decline in serum LDL and improvement in HDL cholesterol as a result of licorice
based intervention.
Licorice based nutraceutical drinks effectively ameliorated the abnormalities is lipid
metabolism of hypercholesterolemic rats by controlling the elevated total cholesterol, LDL
and triglycerides and improving HDL levels. Conclusively, licorice based dietary
interventions are helpful to address lipid-related metabolic disorders.
4.7.2.4. Serum triglycerides
The F value (Table 43) relating to serum triglyceride levels exhibited non-significant effect of
treatments in study I whereas, significant effect of treatments was detected in study II. Means
100
regarding serum triglycerides in study I were 65.97±2.34, 64.18±1.97 and 63.01±2.26 mg/dL
for D0, D1 and D2 groups, respectively. Besides in study II, provision of hypercholesterolemic
diet increased the triglycerides level to 96.72±4.28 mg/dL in control group (D0) which was
subsequently reduced to 87.46±2.53 and 81.50±2.38 mg/dL in D1 and D2 groups, respectively.
Graph illustrating percent decrease (Figure 13) in triglyceride levels exhibited highest
reduction (15.74%) in group fed on supercritical fluid extract of licorice (D2) whereas drink
with conventional solvent extract (D1) resulted in 9.57% reduction.
Current findings regarding reduction in serum triglycerides level by licorice extracts are in
collaboration with the work of Ahn et al. (2013); reported significant decline in serum
triglycerides concentration upon oral intake of licorice supercritical CO2 extract. They
elucidated that supercritical CO2 extract at 0.1% and 0.25% concentrations instigated 7.79%
and 19.48% reduction in triglycerides level of hypercholesterolemic rats. Previously, Asgary
et al. (2007) have also reported similar results based on their research investigation, showing
a significant reduction in triglycerides level in rabbits fed on high fat diet. Their results
exhibited that oral administration of 50mg/kg body weight licorice extract resulted in 25.5%
reduction in triglycerides concentration as compared to control group in a 60 days trial.
One of the research group, Mirtaheri et al. (2015) probed the effect of dried ethanolic extract
of licorice on serum lipid parameters of overweight individuals. They found that licorice
extract inclusion at 1.5g/day effectively reduced serum triglycerides by 15.89% during 8 weeks
bioefficacy trial. Earlier, Sen et al. (2011) reported the ameliorating effect of glycyrrhizin
supplementation on STZ induced diabetes and allied abnormalities including disturbance in
serum lipid profile. They reported a marked increase in serum triglycerides level of STZ-
induced diabetic rats. However, supplementation with 100mg/kg body weight of glycyrrhizin
effectively reduced the elevated triglycerides concentration by 42.5%.
In a bioevaluation trial, Saleem et al. (2011) elucidated the effect of licorice extract on serum
lipid patterns of albino mice upon oral administration. Their results revealed that serum
triglycerides of mice decreased by 13.13, 25.42 and 41.48% as result of 0.2, 0.7 and 1
mg/mL/kg dose of licorice extract. They documented that saponins and phytosterols are the
major components of licorice extracts which are responsible for the aforementioned decline in
serum triglycerides. They concluded that saponins are involved in the reduction of triglycerides
101
Table 43. Effect of licorice drinks on triglycerides (mg/dL) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 65.97±2.34 64.18±1.97 63.01±2.26 1.39NS
Study II 96.72±4.28a 87.46±2.53b 81.50±2.38c 35.80**
NSNon-significant
**Highly significant
Study I : Normal rats
Study II: Hypercholesterolemic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
18
16
14
12
10
8
6
4
2
0
Study I Study II
D1: Nutraceutical CSE Drink D2: Nutraceutical SFE Drink
Figure 13. Percent reduction in triglycerides levels as compared to control drink
15.74
9.57
4.49
2.71
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by inhibiting pancreatic lipase activity. Whereas, phytosterols are involved in the metabolism
of triglycerides through their effect on the absorption of dietary cholesterol which is
decreased as the phytosterols content is increased. Additionally, licorice treatment also lowered
down serum VLDL which is the chief transporter of triglycerides in plasma, resulted in a
significant decline in the triglycerides level.
Likewise, Gaur et al. (2014) estimated the anti-diabetic and hypolipidemic effect of different
bioactive components of licorice namely isoliquiritigenin, 2,4-dimethoxy-4-hydroxychalcone
and liquiritigenin-7,4-dibenzoate in STZ-induced diabetic rats. They reported a significant
increase in the serum triglycerides from 98.10± 13.88 to 177.33±14.06 mg% as a result of STZ
induced diabetes. However, administration of licorice derived Isoliquiritigenin (200mg/kg
body weight), 2,4-dimethoxy-4-hydroxychalcone (50mg/kg body weight) and liquiritigenin-
7,4-dibenzoate (50mg/kg body weight) significantly reduced serum triglycerides by 38.41,
37.34 and 40.37%, respectively.
It is clear from the above discussion that licorice has potential to alleviate diet induced
abnormalities in the serum lipid profile. In current research, licorice based drinks significantly
modulated major biomarkers of hypercholesterolemia; decreased total cholesterol, LDL and
triglycerides along with improvement in HDL in treated groups. Moreover, it was observed
that drink containing supercritical fluid extract (nutraceuticalSFE) proved more effective in
curtailing the menace of dyslipidemia owing to its greater phytochemical content and higher
antioxidant activity. Thereby, it is deduced that use of licorice nutraceutics in dietary therapies
is a sustainable strategy to alleviate cardiovascular complications.
4.7.2.5. Glucose
It is revealed from the F value (Table 44) that serum glucose level was non-significantly
affected as a function of treatments in study I however, significant differences were observed
in study II. Means regarding this trait for study I were 81.65±2.06 (D0), 79.47±2.51 (D1) and
78.92±1.98 mg/dL (D2). Moreover in study II, control group (D0) exhibited highest value
(98.34±4.29 mg/dL) for serum glucose followed by D1 (93.25±3.62 mg/dL) and D2
(91.18±3.75 mg/dL). It is evident from the graph (Figure 14) that drinks D1 and D2 caused 5.17
and 7.28% decrease in serum glucose level, respectively in study II.
103
Table 44. Effect of licorice drinks on glucose (mg/dL) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 81.65±2.06 79.47±2.51 78.92±1.98 1.16NS
Study II 98.34±4.29a 93.25±3.62ab 91.18±3.75b 11.4*
NSNon-significant
*Significant
Study I : Normal rats
Study II: Hypercholesterolemic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
8
7
6
5
4
3
2
1
0
Study I Study II
D1: Nutraceutical CSE Drink D2: Nutraceutical SFE Drink
Figure 14. Percent reduction in glucose levels as compared to control drink
7.28
5.17
3.34
2.66
Per
cen
t R
edu
ctio
n
104
The present results pertaining to significant decrease in serum glucose levels as a result of
licorice extracts supplementation are in close agreement with the results of Sen et al (2011),
observed significant decline in serum glucose levels of STZ-induced diabetic rats. Provision
of 100mg/kg body weight glycyrrhizin through intraperitoneal injection to STZ-induced
diabetic wistar rats effectually reduced the serum glucose level by 54.7% as compared to
non-treated diabetic group. It was narrated that glycyrrhizin inhibits the glucose transport
mediated through sodium-glucose co transporter-I in the intestine thus lowering the glucose
concentrating in the blood. Earlier, Kalaiarasi and Pugalendi (2009) also reported that 18 β-
glycyrrhetinic acid has potential to decrease the blood glucose level and it also enhance the
insulin secretion in STZ induced diabetic rats.
Current results are also in line with the findings of Kataya et al. (2011) who probed the effect
of licorice extract on diabetic nephropathy. Purposely, STZ (60mg/kg body weight) was used
to develop diabetes in male wistar rats. The results of their study explicated that provision of
licorice extract (1g/kg body weight) for 60 days effectively reduced the adverse effects of
diabetes in test animals after the onset of diabetes. Licorice extract competently abridged the
elevated glucose level and a significantly lower glucose level was noticed in the serum of
diabetic rats receiving oral dose of licorice ethanolic extract.
The results regarding hypoglycemic effect of licorice extracts are also consistent with the
earlier study of Mae et al. (2003), reported that licorice ethanolic extract significantly
decreased the serum glucose level in genetically modified rats at a dose of 0.1-0.3g/100g diet
(approximately 100-300mg/kg body/day) during a 4 weeks bioefficacy study. They carried out
two experiments separately to check the ameliorative and preventive effect of licorice extract
against genetically induced diabetes. In preventive experiment, administration of licorice
extract at a dose of 0.1 and 0.2g/100 g diet reduced the serum glucose levels by 38.57% and
39.64%, respectively. Whereas in ameliorative experiment, 34.07% and 30.09% decline in
serum glucose concentration was reported at 0.1 and 0.3% licorice extract provision for 4
weeks. It is evident from the discussion that licorice based dietary intervention is effective to
ameliorate elevated glucose level under hypercholesterolemic conditions.
105
4.7.2.6. Insulin
Statistical analysis (F value) concerning the impact of different treatments on serum insulin
level delineated non-significant effect in study I however, significant effect was detected in
study II. Mean values regarding this trait for study I were 9.12±0.17, 9.26±0.21 and 9.37±0.24
µU/mL in D0, D1 and D2 groups. Likewise for study II, highest value for insulin was noted in
D2 group (11.54±0.39 µU/mL) followed by D1 (11.29±0.27 µU/mL) and D0 (10.93±0.32
µU/mL). It is evident from the graph (Figure 15) that provision of D1 and D2 drinks resulted in
3.29 and 5.63% decrement in insulin levels of hypercholesterolemic rats, respectively in study
II.
The instant outcomes regarding the increase in serum insulin level as the result of licorice
extract supplementation are supported by the bioevaluation trial of Sen et al. (2011). They
found that the treatment of STZ-induced diabetic rats with a single intraperitoneal injection
of glycyrrhizin (100mg/kg body weight) significantly improved serum insulin level. It was
explained that glycyrrhizin treatment also increased the volume and number of islets cells as
compared to non-treated diabetic group. Thus a possible mechanism through which
glycyrrhizin treatment rectify hyperglycemic condition is the regeneration and sensitization of
pancreatic β-cells which results in more insulin production and better glucose utilization.
The current results are well supported by the findings of Aoki et al. (2007), assessed the effect
of licorice flavonoid oil (medium chain fatty acids + 1.2% glabridin) on different serum
parameters in high fat diet induced-obese mice. Purposely, high fat diet was supplemented with
licorice flavonoid oil at different concentrations (0%, 0.5%, 1.0% and 2.0%) during 8 week
experimental period. Results delineated an increase in serum insulin concentration at a dose of
0.5% licorice flavonoid oil.
In conclusion, licorice based nutraceutical drink is an effective diet-based approach to alleviate
adverse effects of hypercholesterolemia by normalizing glucose & insulin levels. Moreover,
the use of novel extraction techniques like SFE should be encouraged to obtain better purity
and yield of desired bioactive components to increase the bioactivity.
106
Table 45. Effect of licorice drinks on insulin (µU/mL) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 9.12±0.17 9.26±0.21 9.37±0.24 0.31NS
Study II 10.93±0.32b 11.29±0.27ab 11.54±0.39a 5.57*
NSNon-significant
*Significant
Study I : Normal rats
Study II: Hypercholesterolemic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
6
5
4
3
2
1
0
Study I Study II
D1: Nutraceutical CSE Drink D2: Nutraceutical SFE Drink
Figure 15. Percent increase in insulin levels as compared to control drink
5.63
3.29
2.74
1.54 Per
cen
t In
crea
se
107
4.7.3. Safety assessment studies
Renal functioning indicators and hematological aspects were determined in all three studies to
assess the impact of licorice drinks on respective parameters.
4.7.3.1. Renal functioning tests
4.7.3.1.1. Urea
The F values concerning the effect of treatments on serum urea levels of normal (study I),
hypercholesterolemic (study II) and hepatotoxic rats (study III) delineated non-significant
impact of treatments on urea concentration in study I and II while, significant effect was
noted in study III. Mean values for study I exhibited 24.48±0.88, 23.82±0.75 and 23.55±0.81
mg/dL urea level in D0, D1 and D2 groups, respectively. Likewise in study II, the mean values
for D0, D1 and D2 were 26.75±0.92, 26.04±0.89 and 25.76±0.73 mg/dL, accordingly.
Likewise in study III, highest urea level (34.26±1.16 mg/dL) was noted in D0 group which
was then decreased to 31.38±1.04 and 29.61±1.17 mg/dL in D1 and D2 groups, respectively
(Table 46).
The current results are in collaboration with the research outcomes of Fuhrman et al. (2002),
investigated the effect of licorice extract supplementation on different biochemical parameters
in hypercholesterolemic subjects. All parameters were measured during 30 days experimental
period with licorice extract administration and another 30 days without licorice extract
provision to check the effectiveness of treatment. They found that blood urea nitrogen level
decreased by 7.14% after 30 days licorice intervention study however, same level was restored
when licorice treatment was halted for next 30 days. Later, Saleem et al. (2011) have also
reported a similar decreasing trend in serum urea level as the result of licorice extract
supplementation. In their experiment, albino mice were administrated with 0.2, 0.7 and 1
mg/mL/day licorice extract for one month. Results exhibited that urea levels of licorice treated
mice groups were significantly reduced by 24.94, 45.03 and 49.01% as a result of 0.2, 0.7 and
1 mg/mL/day extract supplementation, respectively.
4.7.3.1.2. Creatinine
It is observable from the F value (Table 47) that treatments affected serum creatinine levels
non-significantly in study I and II whereas, it was significantly changed in study III. Means
regarding the effect of licorice based drinks on serum creatinine levels in study I were
108
Table 46. Effect of licorice drinks on urea level (mg/dL) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 24.48±0.88 23.82±0.75 23.55±0.81 0.98NS
Study II 26.75±0.92 26.04±0.89 25.76±0.73 1.24NS
Study III 34.26±1.16a 31.38±1.04ab 29.61±1.17b 11.93*
NSNon-significant
*Significant
Study I : Normal rats
Study II: Hypercholesterolemic rats
Study III: Hepatotoxic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
109
0.84±0.02 (D0), 0.82±0.03 (D1) and 0.81±0.01 mg/dL (D2). Likewise in study II, 0.92±0.04,
0.89±0.02 and 0.87±0.02 mg/dL values were detected in D0, D1 and D2 groups. Creatinine level
was significantly high in hepatotoxic rats (study III) with maximum value in D0 group
(1.14±0.05 mg/dL) which was reduced to 1.02±0.03 and 0.94±0.04 mg/dL in D1 and D2 groups,
respectively.
The results of current study explicating a significant decrement in serum creatinine level as a
result of licorice extracts supplementation are in agreement with the outcomes of Saleem et
al. (2011). In their experiment, forty albino mice were divided in to four experimental groups
receiving 0, 0.2, 0.7 and 1 mg/mL/day oral dose of licorice extract for one month. At the
termination of study, collected serum was subjected to different biochemical analysis. Their
results showed that provision of 0.2, 0.7 and 1 mg/mL/day licorice extracts significantly
decreased the serum creatinine levels of treated mice by 16.15, 42.24 and 57.14%, respectively.
The major bioactive components of licorice, glycyrrhizin and glabridin, were reported to be
responsible for this effect following different mechanisms. Glabridin possesses anti-nephritis
activity and modulate the excretion of urinary proteins, blood urea nitrogen and serum
creatinine levels by strengthening glomerulus filtration system. Whereas, glycyrrhizin exhibits
anti-inflammatory activity by inhibiting glucocoticod metabolism.
Later, Gaur et al. (2014) have also documented a significant decrease in serum creatinine as a
result of licorice extract supplementation. The research group explored the effect of licorice
derived bioactive components, isoliquiritigenin, 2,4-dimethoxy-4-hydroxychalcone and
liquiritigenin-7,4-dibenzoate, on different biochemical parameters. Their results delineated
that serum creatinine level was significantly affected with the provision of all of the three test
compounds. A significant decrease of 8.70, 21.74 and 28.26% was observed when
isoliquiritigenin, 2,4-dimethoxy-4-hydroxychalcone and liquiritigenin-7,4-dibenzoate were
administrated at a dose of 200, 50 and 50 mg/kg body weight, respectively for 14 consecutive
days.
4.7.3.2. Hematological analyses
The F value regarding the effect of treatments on RBC count exhibited non-significant effect
in study I whereas, significant effect was for study II and III (Table 48). Means values
concerning the effect of licorice drinks on RBCs level in study I were 7.59±0.26, 7.81±0.18
110
Table 47. Effect of licorice drinks on creatinine level (mg/dL) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 0.84±0.02 0.82±0.03 0.81±0.01 1.14NS
Study II 0.92±0.04 0.89±0.02 0.87±0.02 1.72NS
Study III 1.14±0.05a 1.02±0.03ab 0.94±0.04b 9.36*
NSNon-significant
*Significant
Study I : Normal rats
Study II: Hypercholesterolemic rats
Study III: Hepatotoxic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
111
and 7.94±0.22 cells/pL in D0, D1 and D2 groups. Likewise in study II, highest RBCs count
(7.41±0.31 cells/pL) was observed in D2 group trailed by D1 (7.12±0.34 cells/pL) and D0
(6.85±0.14 cells/pL). Similarly, D0, D1 and D2 drinks resulted in 6.43±0.23, 7.06±0.30 and
7.36±0.35 cells/pL RBC count, respectively in study III.
Statistical analysis concerning the effect of different licorice based drinks on WBCs count
delineated significant effect study II and III during the course of study (Table 49). The mean
values for study I were 13.72±0.49, 13.06±0.52 and 12.94±0.38 cells/nL in D0, D1 and D2
groups, respectively. Similarly in study II, highest WBC count was observed in D0
(15.45±0.63 cells/nL) followed by D1 (14.86±0.47 cells/nL) and D2 (14.29±0.51 cells/nL).
Moreover in study III, 17.35±0.44, 15.41±0.60 and 14.78±0.35 cells/nL values of WBCs
were noted in D0, D1 and D2 groups, respectively.
It is clear from the F value that platelet count affected non-significantly as a result of
treatments in study I and II however, a significant effect was evident in study III (Table 50).
In study I, the observed values of platelet count in D0, D1 and D2 groups were 914.86±29.24,
925.23±25.81 and 933.45±32.15 x103/µL, respectively. Moreover, the recorded values of
platelet count for study II were 864.39±32.47, 896.51±37.63 and 908.73±21.72 x103/µL,
respectively. Likewise in study III, platelet count significantly increased from 776.14±18.16
x103/µL (D0) to 844.76±23.59 (D1) and 879.48±27.34x103/µL (D2).
The results regarding the effect of licorice based dietary interventions on the hematological
parameters are in agreement with the findings of Aoki et al. (2013). They evaluated the clinical
safety of licorice flavonoid oil (LFO) on different physiological parameters including
hematological attributes. Results depicted that RBCs count increased from 451±16 to 469±13
104/µL during four weeks at a daily dose of 300 mg LFO. Whereas, a decreasing trend in WBC
and platelet count was observed from 5526±331 to 5174±378 cells/µL and 22.6±1.4 to
23.5±1.6 104/µL, respectively. Moreover, all of the hematological and blood biochemistry
parameters remained within normal range.
In the nutshell, utilization of licorice nutraceutics in diet based therapies has proven safe as
there is no deleterious effect on kidney and hematological biomarkers. Moreover, licorice
based nutraceutical drinks effectively managed the adverse effects of hepatotoxicity and
abnormal serum lipid profile.
112
Table 48. Effect of licorice drinks on RBC (cells/pL) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 7.59±0.26 7.81±0.18 7.94±0.22 1.51NS
Study II 6.85±0.14b 7.12±0.34ab 7.41±0.31a 8.03*
Study III 6.43±0.23c 7.06±0.30b 7.36±0.35a 24.21**
NSNon-significant *Significant
**Highly significant
Study I : Normal rats
Study II: Hypercholesterolemic rats
Study III: Hepatotoxic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
113
Table 49. Effect of licorice drinks on WBC (cells/nL) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 13.72±0.49 13.06±0.52 12.94±0.38 2.33NS
Study II 15.45±0.63a 14.86±0.47ab 14.29±0.51b 5.21*
Study III 17.35±0.44a 15.41±0.60b 14.78±0.35c 65.8**
NSNon-significant *Significant
**Highly significant
Study I : Normal rats
Study II: Hypercholesterolemic rats
Study III: Hepatotoxic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
114
Table 50. Effect of licorice drinks on Platelets (103/µL) of rats in different studies
Studies
Treatments
D0 D1 D2
F value
Study I 914.86±29.24 925.23±25.81 933.45±32.15 0.22NS
Study II 864.39±32.47 896.51±37.63 908.73±21.72 1.45NS
Study III 776.14±18.16b 844.76±23.59ab 879.48±27.34a 8.70*
NSNon-significant *Significant
Study I : Normal rats
Study II: Hypercholesterolemic rats
Study III: Hepatotoxic rats
Do : Control drink
D1 : NutraceuticalCSE drink
D2 : NutraceuticalSFE drink
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CHAPTER 5
SUMMARY Recently, diet based therapies have gained special attention as an effective tool to address
various lifestyle related disorders. Plant derived bioactive components provides protection
against various health discrepancies and improve overall health status of the body. Herbal
plants have history of use for the treatment of various disorders and recent research has
revealed the presence of hundreds of phytochemicals in these plants. Licorice is one of the
commonly used herb in various herbal formulations and possess numerous health benefits. In
this context, current study was planned to explore the disease modulating potential of licorice
bioactive moieties with special reference to hepatic and lipidemic malfunctions. The project
was divided into three parts; firstly, licorice was subjected to extraction of bioactive moieties
through different extraction techniques followed by determination of antioxidant potential
and phytochemical content. Further, licorice based drink was developed using different levels
of two best selected extracts, one from each extraction mode. In last phase of the study,
hepatoprotective and hypocholesterolemic potential of developed drinks was evaluated using
model feed trial.
In extraction conditions optimization module, different conventional solvents including
ethanol, methanol and ethyl acetate were used at varying ratios with water 25:75, 50:50: and
75:25 to study their impact on extraction efficiency of nutraceutics. Amongst solvents,
aqueous ethanolic extract showed the highest values for TPC, TF, DPPH, FRAP and ABTS,
as 897.24±31.49 mg GAE/100g, 286.17±9.85 mg CE/100g, 72.65±2.45%, 451.52±15.73 μM
Fe2+/g and 11.02±0.46 μM Trolox/g respectively followed by aqueous methanolic extract
673.38±24.51 mg GAE/100g, 255.41±8.34 mg CE/100g, 66.22±2.84%, 369.91±10.64 mM
Trolox/g and 9.58±0.29 μM Trolox/g whilst, the lowest values were noted for aqueous ethyl
acetate extract as 555.07±17.35 mg GAE/100g, 229.86±9.81 mg CE/100g, 58.10±2.11%,
311.32±9.12 μM Fe2+/g and 8.66±0.22 μM Trolox/g, accordingly. Considering the effect of
solvent concentration, 75:25 solvent to water ratio showed the highest values for all
parameters, whereas solvent to water ratio of 25:75 exhibited least output. The detected values
at 75:25 for TPC, TF, DPPH, FRAP and ABTS were 859.47±21.26 mg GAE/100g,
289.02±7.24 mg CE/100g, 71.97±2.81%, 404.07±13.51 μM Fe2+/g and 10.98±0.29 μM
Trolox/g, respectively. At 50:50 solvent concentration, these assays exhibited values as
686.40±19.58 mg GAE/100g, 262.93±7.65 mg CE/100g, 67.33±2.76%, 374.25±12.04 μM
Fe2+/g and 9.85±0.38 μM Trolox/g, accordingly. Nevertheless, observed values for same traits
at 25:75 concentration were 579.82±16.23 mg GAE/100g, 219.48±6.32 mg CE/100g,
57.68±2.15%, 354.42±11.62 μM Fe2+/g and 8.42±0.026 μM Trolox/g respectively.
For comparison purpose, supercritical fluid extracts of licorice were obtained at 3500 (TSC1),
4500 (TSC2) and 5500 (TSC3) psi pressure against constant temperature of 40 oC. TSC3
exhibited highest values for TPC, TF, DPPH, FRAP and ABTS as 1532.75±36.84 mg
GAE/100g, 576.13±23.51 mg CE/100g, 88.26±3.255%, 743.45±19.38 μM Fe2+/g and
17.85±0.55 μM Trolox/g followed by TSC2 as 1475.28±47.62 mg GAE/100g, 531.64±21.46
mg CE/100g, 86.57±3.045%, 698.71±23.74 μM Fe2+/g and 16.09±0.47 μM Trolox/g. Whilst,
TSC1 showed minimum values for the same traits as 1286.51±41.15 mg GAE/100g,
462.87±17.59 mg CE/100g, 82.49±2.27%, 610.88±17.08 μM Fe2+/g and 14.62±0.62 μM
Trolox/g, respectively.
Afterwards, all the conventional solvent and supercritical fluid extracts were analyzed for their
glycyrrhizin and glabridin content via HPLC quantification. Results depicted that highest
concentrations of glycyrrhizin and glabridin were detected in supercritical fluid extract (SFE)
obtained at 5500 psi pressure (TSC3) as 5.02±0.031 and 2.97±0.012 mg/g, respectively.
However, minimum concentration of these components among SFE was observed in TSC1 as
3.87±0.034 and 1.64±0.014, accordingly. Amongst conventional solvent extracts, the highest
glycyrrhizin content was detected in 25% methanolic extract as 2.41±0.027 mg/g whereas,
highest glabridin content was observed in 75% ethanolic extract i.e. 1.13±0.010 mg/g. On the
basis of HPLC analysis, 75% ethanolic extract (nutraceuticalCSE) and TSC3 (nutraceuticalSFE)
were selected from conventional solvent and supercritical fluid extracts, respectively for
further investigation.
In product development module, licorice based nutraceutical drinks were prepared by adding
0.4% nutraceuticalCSE (T1), 0.2% nutraceuticalCSE (T2), 0.1% nutraceuticalSFE (T3), 0.2%
nutraceuticalSFE (T4) and control (T0) treatment without any extract. Mean squares regarding
color of licorice drinks showed significant effect of treatment and storage intervals on L*, a*,
b* and chroma values whereas, hue angle was affected non-significantly as a function of
116
117
storage intervals. Means pertaining L* values of licorice drinks explicated that control
treatment (T0) showed maximum L* value (79.65±2.84) whereas, minimum L* value
(52.42±1.49) was noted for licorice drink containing 0.4% solvent extract (T2). The mean
values of a* trait were 5.15±0.13, 7.24±0.18, 8.41±0.35, 6.86±0.18 and 7.42±0.37 for T0, T1,
T2, T3 and T4, correspondingly. During the course of time, a* values decreased from 7.53±0.34
to 6.53±0.14 during 60 days storage study. Likewise, The observed values for b* were
63.54±2.17, 58.24±2.36, 46.79±1.41, 61.37±2.25 and 61.16±2.01 for T0, T1, T2, T3 and T4,
respectively. A significant reduction from 59.92±1.74 to 56.51±1.88 was observed for this
parameter during storage study. Means concerning chroma values exhibited highest chroma
value (63.75±1.92) for control whereas, minimum value (47.54±1.76) for this character was
noted in T2. Likewise, means regarding hue angle showed 85.36±3.04, 82.91±2.85,
79.81±2.44, 83.62±3.28 and 83.09±3.37 values for T0, T1, T2, T3 and T4, respectively.
Mean squares indicated non-significant effect of treatments on pH, acidity and brix of
nutraceutical drinks whereas, significant differences were observed for pH and acidity as the
function of storage. Means related to the pH of licorice drinks depicted a decline in values from
4.48±0.02 at the initial day to 4.22±0.08 at 60th day. Moreover, means for acidity within the
treatments for T0, T1, T2, T3 and T4 were 0.14±0.01, 0.15±0.01, 0.15±0.02, 0.14±0.01 and
0.15±0.01, respectively. However, a significant rise in acidity was observed during storage
from 0.14±0.01 to 0.16±0.01. Likewise, the observed mean values (Table) for brix were
12.93±0.51, 13.23±0.57, 13.33±0.49, 13.09±0.39 and 13.15±0.32 for T0, T1, T2, T3 and T4,
respectively. Mean squares concerning the phytochemical screening assays and antioxidant
potential of licorice drinks explicated significant effect of treatments and storage. For treatment
effect the observed values for TPC in licorice drinks were 5.81±0.14 (T0), 15.93±0.54 (T1).
30.71±0.84 (T2), 18.17±0.66 (T3) and 35.28±1.22 mg GAE/100g (T4). Similarly, the values for
total flavonoids of licorice drink ranged from 2.37±0.10 mg CE/100g (T0) to 8.95±0.21 mg
CE/100g (T4). For DPPH free radical scavenging activity, maximum activity was noted in T4
(55.01±1.27%) followed by T2 (48.17±0.59%), T3 (43.89±1.12), T1 (34.97±085%) and T0
(7.42±0.15%). Likewise, same trend was observed for FRAP values with maximum value
observed for T4 (87.05±2.42 μM Fe2+/g) followed by T2 (77.04±2.38 μM Fe2+/g) whilst the
noted values for T0, T1 and T3 were 16.09±0.56, 53.31±2.12 and 64.17±2.68 μM Fe2+/g,
respectively. Moreover, a significant difference in ABTS values was detected as 1.23±0.04
118
µM TE/g (T0), 4.22±0.15 µM TE/g (T1), 5.34±0.17 µM TE/g (T2), 4.97±0.12 µM TE/g (T3)
and 5.84±0.19 µM TE/g (T4).
Means regarding effect of storage interval on TPC, total flavonoids, DPPH, FRAP and ABTS
assays are presented in Figure 2. A significant decline in TPC was observed during 60 day
storage study from 23.15±0.69 to 19.19±0.68 mg GAE/100g. The observed values for total
flavonoids at 0, 30th and 60th day of storage were 6.16±0.24, 5.53±0.21 and 5.09±0.18 mg
CE/100g. Similarly, the DPPH free radical scavenging activity of licorice extracts
supplemented drinks also exhibited a decreasing trend from 40.47±1.29% at initiation of study
to 35.18±1.09% at the termination of 60 days storage trial. Moreover, a significant decline in
FRAP and ABTS values was observed from 66.88±2.14 to 52.65±1.56 μM Fe2+/g and
4.65±0.16 to 3.93±0.18 µM TE/g, respectively.
All the prepared drinks were evaluated for their sensory attributes during storage interval of 60
days. Mean squares regarding sensorial attributes of licorice drinks exhibited significant effect
of treatment on color, taste, flavor, sweetness and overall acceptability whereas mouthfeel was
changed non-significantly as a function of treatment. Regarding storage interval, color and
overall acceptability explicated significant decline in sensory evaluation score while rest of the
parameters were non-significantly affected. Based on sensory evaluation scores, T1 (drink
containing 0.2% CSE) and T4 (drink containing 0.2% SFE) were selected for bioevaluation
trial.
Bioefficacy study was conducted to evaluate the therapeutic effect of selected drinks against
hepatotoxicity and dyslipidemia. Purposely, three studies were carried out i.e. normal rats
(study I), hypercholesterolemic rats (study II) and hepatotoxic rats (study III).
NutraceuticalCSE, nutraceuticalSFE and control drinks were given to all the respective groups
under each study. Regarding hepatoprotective effect, maximum ALT level was documented in
D0 (control drink) 78.62±3.12 IU/L that significantly reduced to 75.84±3.25 and 74.32±2.97
as a result of D1 (NutraceuticalCSE drink) and D2 (NutraceuticalSFE drink). In study III
(hepatotoxic rats), maximum reduction in serum ALT levels was observed in D2 (124.16±3.81
IU/L) trailed by D1 (135.25±5.63 IU/L) as compared to control treatment (152.38±5.34 IU/L).
In study I, D1 and D2 resulted in 2.09 and 3.66% decrease in serum ALT, respectively whereas
in study III, D2 resulted in 31.19% decline in serum ALT and 20.51% reduction was noted as
119
a result of D1. Likewise, serum AST level non-significantly affected by treatments in study I
however, effect of treatments for this trait was significant in study III. Means pertaining serum
AST in study I exhibited a minor decline from 93.87±4.01 IU/L to 90.92±3.82 and 89.75±3.74
IU/L as a result of D1 and D2 drinks, respectively. However, mean AST level for D0 in study
III was 182.45±8.68 IU/L which then reduced to 149.77±6.59 and 130.23±5.33 as a result of
D1 and D2, respectively. The highest percent increase in AST level was observed for Study III
i.e. 17.91 and 28.62% for D1 and D2, correspondingly.
Likewise, highest ALP level (165.48±7.59 IU/L) in study I was observed in D0 followed by D1
(162.14±6.98 IU/L) and D2 (160.57±7.05 IU/L). Likewise, means for study III reflected
maximum value for D0 (841.25±38.84 IU/L) that significantly reduced to 705.83±33.17 and
641.49±29.06 IU/L in D1 and D2, respectively. In study I, treatments D1 and D2 resulted in 2.02
and 2.97% decline in serum ALP, accordingly. Whereas, greater reduction in ALP level was
observed in hepatotoxic rats as 16.11 and 23.75% in D1 and D2, respectively. Moreover,
provision of licorice based drinks significantly increased the activity of liver antioxidant
enzymes. The mean values of liver SOD in study I were 11.37±0.43, 11.78±0.36 and
11.94±0.51 IU/mg protein in D0, D1 and D2, respectively. In study III, maximum SOD activity
(9.25±0.25 IU/mg protein) was observed in D2 followed by D1 (8.36±0.32 IU/mg protein) and
D0 (7.01±0.29 IU/mg protein). Treatments D1 and D2 caused a non-significant elevation (3.61
and 5.01% respectively) in liver SOD activity for study I whereas in study III, significant
increase was observed in D1 (19.26%) and D2 (31.95%) groups. Similarly, mean values
regarding catalase activity in study I were 15.63±0.72, 16.25±0.85 and 16.57±0.78 IU/mg
protein in D0, D1 and D2 groups, respectively. Likewise in study III, group D2 exhibited highest
catalase activity (12.93±0.63 IU/mg protein) trailed by D1 (12.06±0.57 IU/mg protein) and D0
(10.28±0.46 IU/mg protein). In study I, percent increase in catalase activity as a result of D1
and D2 drinks was 2.39 and 4.41% respectively. Whereas in study III, 25.78% and 17.32% rise
in catalase activity was recorded in D1 and D2 groups. Furthermore, mean values for MDA
level in study I were 3.97±0.15, 3.85±0.12 and 3.78±0.18 nM/mg for D0, D1 and D2 groups,
accordingly. Similarly in study III, highest MDA level was noted for D0 (8.14±0.42 nM/mg)
which then significantly reduced to 6.45±0.31 in D1 group and 5.02±0.22 nM/mg in D2.
Regarding percent decrease, 3.02 and 4.97% decrement in MDA levels was observed for D1
and D2 groups respectively in study I. Likewise in study III, D2 caused maximum reduction
120
(38.33%) followed by D1 (20.76%). Furthermore, means pertaining serum bilirubin levels in
study I were 0.73±0.03, 0.71±0.02 and 0.70±0.02 mg/dL for D0, D1 and D2 groups,
correspondingly. Likewise in study III, highest serum bilirubin (1.03±0.05 mg/dL) was
detected in group fed on control drink (D0) which then significantly reduced to 0.92±0.03 and
0.86±0.02 mg/dL in D1 and D2 groups, respectively.
Regarding hypolipidemic perspectives, means for serum cholesterol in study I indicated
78.62±3.12, 75.84±3.25 and 74.32±2.97 mg/dL values for D0, D1 and D2 groups, respectively.
In study II, highest cholesterol level was recorded in D0 (152.38±5.34 mg/dL) which was then
significantly reduced to 135.25±5.63 and 124.16±3.81 mg/dL in D1 and D2 groups,
respectively. Considering percent reduction, 3.54 and 5.47% decrement in serum cholesterol
levels was noted for study I as a result of D1 and D2 drinks, accordingly. Likewise for study II,
drinks containing solvent extract (D1) and supercritical fluid extract (D2) resulted in 11.24 and
18.52% decline in total cholesterol.
Serum HDL levels of different studies showed non-significant effect of treatments for this trait
in study I however, significant effect was observed in study II. In study I, mean values for HDL
were 35.48±1.37, 36.02±1.15 and 36.21±1.45 mg/dL in D0, D1 and D2 groups, respectively.
Similarly in study II, mean value for HDL in control (D0) group was 52.39±1.88 mg/dL that
improved significantly as 54.41±2.07 and 55.08±1.92 mg/dL in D1 and D2 groups, accordingly.
Regarding percent increase, HDL levels increased as 1.53 (D1) and 2.08% (D2) in study I. In
the same manner, 3.29 and 5.14% increase in HDL level was evident in D1 and D2 groups,
respectively in study II. Moreover, LDL level was significantly affected by licorice based
drinks in all studies. In study I, the observed values of LDL levels for D0, D1 and D2 groups
were 30.76±1.13, 29.63±0.82 and 28.05±1.06 mg/dL, respectively. Similarly in study II,
highest value (61.53±2.89 mg/dL) for LDL was noted in D0 that was decreased to 50.73±1.65
and 46.54±1.31 mg/dL in D1 and D2 groups, respectively. In study I, D1 and D2 reduced serum
LDL by 3.68 and 8.81%, respectively as compared to D0 (control). Similarly in
hypercholesterolemic rats (study II), 17.56 and 24.37% decrement in serum LDL was noted
for D1 and D2 groups, accordingly.
Means regarding serum triglycerides in study I indicated 65.97±2.34, 64.18±1.97 and
63.01±2.26 mg/dL values for D0, D1 and D2 groups, respectively. Besides in study II,
121
96.72±4.28, 87.46±2.53 and 81.50±2.38 mg/dL triglycerides level was observed in D0, D1 and
D2 groups, respectively. In study I, 2.71 and 4.49% decline in D1 and D2 groups was evident.
Likewise in study II, highest reduction (15.74%) was observed in D2 followed by D1 (9.57%).
Serum glucose level was non-significantly affected in study I however, significant differences
were observed in case of study II as a function of treatment. Means regarding this trait showed
81.65±2.06 (D0) to 79.47±2.51 (D1) and 78.92±1.98 (D3) mg/dL values in study I. Whereas in
study II, control group (D0) exhibited highest value (98.34±4.29 mg/dL) for serum glucose
followed by D1 (93.25±3.62 mg/dL) and D2 (91.18±3.75 mg/dL). For study I, 2.66 and 3.34%
decrease was noted in D1 and D2 groups, accordingly. Furthermore in study II, drinks D1 and
D2 caused 5.17 and 7.28% decrease in serum glucose level, respectively. Likewise for insulin,
D1 and D2 drinks caused 1.54 and 2.74% reduction in serum insulin level in study I,
respectively. A similar trend was observed in study II where the provision of D1 and D2 drinks
resulted in 3.29 and 5.63% decrement in insulin levels of hypercholesterolemic rats,
respectively. Additionally, values for safety assessment including kidney functioning and
hematology were within normal ranges without any adverse variation.
Conclusively, licorice has strong antioxidant potential owing to its rich phytochemistry.
Moreover, supercritical fluid extraction technique is a novel and safe alternate to conventional
solvent extraction for better recovery of biologically active components from licorice.
Furthermore, licorice supplemented drink is a convenient option to deliver valuable
nutraceuticals to masses with good antioxidant potential and sensory profile. Additionally,
licorice is easily available in Pakistan at very low price so its supplementation in drinks has
not significantly altered the cost of drink. This increase in cost is justified as licorice
supplemented drink has shown significant potential to curtail abnormalities in liver functions
due to hepatotoxicity and it can competently modulate serum lipid profile and hyperglycemic
conditions. Moreover, its regular consumption did not impart any hazardous effect on kidney
functions and hematological aspects. In the nutshell, nutraceutical compounds of licorice have
potential to combat various metabolic disorders and its regular use should be recommended to
alleviate lifestyle related disorders through diet based therapy.
122
Recommendations
Licorice based functional drink should be made a routine part of daily diet along with
other functional food products to get its full therapeutic potential
Licorice based functional/nutraceutical foods and beverages should be promoted to
alleviate hepatotoxicity and hypercholesterolemia
Novel extraction techniques should be adopted to improve the recovery of nutraceutics
at commercial scale
Food experts should work on the development of licorice based novel food items
Diet practitioners should encourage the use of licorice based therapeutic approaches to
address various metabolic disorders
Clinical trials using human subjects should be carried out to further explore the bioactivity
of licorice nutraceutics in vulnerable groups
Toxicological studies should be carried out to further explore the safety perspectives of
licorice based designer foods and beverages
Community based awareness programs should be launched to highlight the therapeutic
potential of functional foods and nutraceutics
Research should be carried out on the effectiveness of other extraction techniques for the
extraction of licorice bioactive components. Moreover, further research is needed to
explore the effect of harvesting stage, harvesting time and environmental conditions on
the bioactive components of licorice.
Government regulatory bodies, research institutes and food industry should work jointly
to implement the aforementioned recommendations through research based community
programs
123
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APPENDIX I
SENSORY EVALUATION PROFORMA
For Licorice Drink
Directions
Take these drinks one by one and evaluate them for the following parameters on Hedonic scale.
It is very important to rinse your mouth thoroughly with distilled water before taking each
sample.
Name of Judge Date
Character T0 T1 T2 T3 T4
Color
Flavor
Taste
Mouthfeel
Sweetness
Overall
acceptability
Scale for Evaluation Extremely poor 1 Very poor 2
Poor 3
Below fair above poor 4
Fair 5
Below good above fair 6
Good 7
Very good 8
Excellent 9