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

iv

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.

78

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

84

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

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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

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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.

88

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.

92

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

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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

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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

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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