Characterization of Mango Peel Mangiferin to Elucidate Its Nutraceutical Potential

Characterization of mango peel mangiferin to elucidate its nutraceutical potential

Muhammad Imran

M.Sc. (Hons.) Food Technology

A thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

FOOD TECHNOLOGY

NATIONAL INSTITUTE OF FOOD SCIENCE & TECHNOLOGY

UNIVERSITY OF AGRICULTURE, FAISALABAD

PAKISTAN

2013

To,

The Controller of Examinations,

University of Agriculture,

Faisalabad.

We, the Supervisory Committee, certify that the contents and form of this thesis submitted by Muhammad Imran, Reg. # 2002-ag-1661 have been found satisfactory, and recommend that it be processed for evaluation by the External Examiner(s) for the award of degree.

SUPERVISORY COMMITTEE:

Chairman:

(Prof. Dr. Masood Sadiq Butt)

Member:

(Prof. Dr. Faqir Muhammad Anjum)

Member:

(Prof. Dr. Javed Iqbal Sultan)

Dedicated

To Hazrat Muhammad

(Sallallahu Alaihi Wasallam) & MY FATHER (Late)

Khadim Hussain

DECLARATION I hereby declare that the contents of the thesis, studies on “Characterization of mango peel mangiferin to elucidate its nutraceutical potential” are the product of my own research and no part has been copied from any published source (expect the references, standard mathematical or genetic models/equations/formulas/protocols etc). I, further, declare that this work has not been submitted for the award of any other diploma/degree. The university may take action if the information provided found inaccurate at any stage. (In case of any default the scholar will be proceeded against as per HEC plagiarism policy).

Muhammad Imran

ACKNOWLEDGEMENTS

I feel myself inept to regard the Highness of Almighty ALLAH, my words have lost their expressions, knowledge is lacking and lexis scarce to express gratitude in the rightful manner to the blessings and support of Allah the Almighty who flourished my ambitions and helped me to attain goals. I present my humble gratitude from the deep sense of heart to the Holy Prophet MUHAMMAD that without him the life would have been worthless.

I have no words to express my gratitude to my honorable supervisor Prof. Dr. Masood Sadiq Butt, National Institute of Food Science and Technology, University of Agriculture, Faisalabad for his diligent cooperation and scrupulous support during the entire degree program. I expand my deepest appreciation and benedictions to Prof. Dr. Faqir Muhammad Anjum (T.I), Director General, National Institute of Food Science and Technology, University of Agriculture, Faisalabad for her great help. I also express my appreciation to Dr. Javed Iqbal Sultan, Professor, Institute of Animal Nutrition and Feed Technology for his compassionate attitude and valued suggestions. I am also obliged to Dr. Emilie Combet, Department of Human Nutrition, College of Medical, Veterinary and Life Sciences, Life Course Nutrition and Science, University of Glasgow, Scotland, United Kingdom and her team for their tremendous support.

I want pay my gratitude to Higher Education Commission (HEC), Pakistan for its support and encouragement to complete my task. I am grateful to HEC for providing me financial assistance in the form of indigenous scholarship and an esteemed opportunity of six month foreign training (IRSIP) to expand my erudite exposure.

I extend my obligations to my loving mother, sisters and brothers, without their moral and financial support, I wouldn’t have been at this position today. Lastly, I am also thankful to my friends and colleagues for their valuable critical discussions and endorsing support throughout research work and motivating me at times.

Muhammad Imran

CONTENTS

Sr. No. Title Page No.

I INTRODUCTION 1

II REVIEW OF LITERATURE 6

III MATERIALS AND METHODS 30

IV RESULTS AND DISCUSSION 43

V SUMMARY 130

RECOMMENDATIONS 136

LITERATURE CITED 137

LIST OF CONTENTS Acknowledgements i Contents ii List of Contents iii List of tables vi List of figures viii List of appendices ix Abstract x 1. INTRODUCTION 1 2. REVIEW OF LITERATURE 6 2.1. Nutraceutical and designer foods; an overview 7 2.2. Nutritional profiling of mango peel 9 2.3. Mangiferin: a unique nutraceutical compound 10 2.4. Isolation and quantification of mangiferin 13 2.5. In vitro antioxidants estimation 16 2.6. Pharmacokinetics aspects 18 2.7. Mangiferin against physiological malfunctionings 20 2.7.1. Oxidative stress related complications 20 2.7.2. Hypercholesterolemic perspectives 23 2.7.3. Hyperglycemia and insulin dysfunction 26 3. MATERIALS AND METHODS 30 3.1. Materials 30 3.2. Characterization of mango peel 30 3.2.1. Proximate analysis 30 3.2.1.1. Moisture content 31 3.2.1.2. Crude protein 31 3.2.1.3. Crude fat 31 3.2.1.4. Crude fiber 31 3.2.1.5. Total ash 31 3.2.1.6. Nitrogen free extract (NFE) 31 3.2.2. Mineral determination 32 3.2.3. Preparation of antioxidant extracts 32 3.2.3.1.Total phenolic content (TPC) 32 3.2.3.2. Antioxidant activity 34 3.2.3.3. DPPH 34

iii

3.2.3.4. Ferric reducing antioxidant power 34 3.3. Isolation of mangiferin 35 3.3.1. Characterization 35 3.3.2. Mangiferin analysis 35 3.4. Selection of best treatment 35 3.5. Preparation of nutraceutical/functional drink 37 3.5.1. Physicochemical analysis of functional drinks 37 3.5.1.1. Total soluble solids 37 3.5.1.2. pH 37 3.6.1.3. Total acidity 38 3.6. Sensory evaluation 38 3.7. In vivo studies 38 3.7.1. Study I: Normal rats 39 3.7.2. Physical parameters 40 3.7.2.1. Feed and drink intakes 40 3.7.2.2. Body weight gain 40 3.7.3. Serum separation 41 3.7.3.1. Serum lipid profile 41 3.7.3.2. Cholesterol 41 3.7.3.3. High density lipoproteins 41 3.7.3.4. Low density lipoproteins 41 3.7.3.5. Triglycerides 41 3.7.3.6. Hypoglycemic response 41 3.7.3.7. Antioxidant status 42 3.7.3.8. Liver and kidney functioning tests 42 3.7.3.9. Hematological aspects 42 3.7.3.10. Electrolytes balance 42 3.8. Statistical Analysis 42 4. RESULTS AND DISCUSSION 43 4.1. Proximate composition 43 4.2. Mineral analysis 45 4.3. Antioxidant extracts 47 4.4. HPLC characterization of mangiferin 53 4.5. Antioxidant indices of mangiferin 55 4.6. Functional/nutraceutical drink analysis 59 4.7. Sensory evaluation 62

4.8. Bioevaluation trials 67 4.8.1. Feed intake 68 4.8.2. Drink intake 71 4.8.3. Body weight 71 4.8.4. Cholesterol 77 4.8.5. Low density lipoprotein (LDL) 82 4.8.6. High density lipoprotein (HDL) 86 4.8.7. Triglycerides 90 4.8.8. Glucose 94 4.8.9. Insulin 99 4.8.10. Glutathione 103 4.8.11. TBARS 105 4.9. Liver functioning tests 109 4.9.1. Serum AST 109 4.9.2. Serum ALT 112 4.9.3. Serum ALP 112 4.10. Kidney functioning tests 117 4.10.1. Serum urea 117 4.10.2. Serum creatinine 117 4.11. Hematological aspects 118 4.11.1. Red blood cell (RBC) 118 4.11.2. Hemoglobin (Hb) 118 4.11.3. Hematocrit 121 4.11.4. Mean corpuscular volume (MCV) 121 4.11.5. White blood cell 121 4.11.6. Neutrophils: 124 4.11.7. Monocytes: 124 4.11.8. Lymphocytes: 125 4.12. Electrolytes balance: 125 4.12.1. Sodium 125 4.12.2. Potassium 127 4.12.3. Calcium 127 5. SUMMARY 130 RECOMMENDATIONS 136 LITERATURE CITED 137 APPENDICES 169

LIST OF TABLES

Sr. No. Title Page

1 Treatments used for estimation of extraction efficiency 33

2 Treatments used for the preparation of nutraceutical/functional drink 37

3 Different studies conducted in efficacy trial 39

4 Diets and functional drinks plan 40

5 Means squares for proximate composition of different mango peels 44

6 Proximate composition of different mango peels 44

7 Means squares for mineral contents different mango peels 46

8 Mineral profiling of different mango peels (mg/100g) 46

9 Means squares for antioxidant indices of mango peel extracts 48

10 Total phenolic contents (mg/100g GAE) of peel extracts 48

11 Free radical scavenging (DPPH %) activity of peel extracts 49

12 Antioxidant activity (β-carotene %) of peel extracts 51

13 Ferric reducing antioxidant power (FRAP mmol/100g) of peel extracts 51

14 Means squares for HPLC characterization of isolated mangiferin 54

15 HPLC characterization of isolated mangiferin (mg/g) 54

16 Means squares for different antioxidant indices of isolated mangiferin 56

17 Free radical scavenging (DPPH %) activity of isolated mangiferin 56

18 Antioxidant activity (β-carotene %) of isolated mangiferin 57

Ferric reducing antioxidant power (FRAP mmol/100g) of isolated 19 57 mangiferin

20 Mean squares for acidity, pH and TSS of functional drinks 60

21 Effect of treatments and storage on acidity (%) of functional drinks 60

22 Effect of treatments and storage on pH of functional drinks 61

23 Effect of treatments and storage on TSS of functional drinks 61

24 Mean squares for sensory evaluation of functional drinks 63

25 Effect of storage and treatments on the color 63

26 Effect of storage and treatments on the flavor 65

27 Effect of storage and treatments on the sweetness of the drinks 65

28 Effect of treatments and storage on sourness 66

29 Effect of storage and treatments on the overall acceptability 66

30 Effect of treatments and study weeks on feed intake (g/rat/day) 69

31 Effect of treatments and study weeks on drink intake (mL/rat/day) 72

32 Effect of treatments and study weeks on body weight (g/rat) 75

33 Effect of functional drinks on cholesterol (mg/dL) 78

34 Effect of functional drinks on LDL (mg/dL) 83

35 Effect of functional drinks on HDL (mg/dL) 88

36 Effect of functional drinks on triglycerides 92

37 Effect of functional drinks on glucose (mg/dL) 97

38 Effect of functional drinks on insulin (µU/mL) 101

39 Effect of functional drinks on serum glutathione (mg/L) 106

40 Effect of functional drinks on serum TBARS (µmol/L) 110

41 Effect of functional drinks on serum AST (IU/L) 114

42 Effect of functional drinks on serum ALT (IU/L) 115

43 Effect of functional drinks on serum ALP (IU/L) 116

44 Effect of functional drinks on serum urea (mg/dL) 119

45 Effect of functional drinks on serum creatinine (mg/dL) 120

46 Effect of functional drinks on red blood cell indices 122

47 Effect of functional drinks on red blood cell indices 123

48 Effect of functional drinks on white blood cell Indices 126

49 Effect of functional drinks on white blood cell Indices 128

50 Effect of functional drinks on electrolytes balance 129

vii

List of Figures

Sr. No. Title Page

1 Flow diagram for mangiferin extraction 36

2 Feed intake in study I, II and III (g/rat/day) 70

3 Drink intake in study I, II and III (mL/rat/day) 73

4 Body weight in study I, II and III (g/rat) 76

5 Percent reduction in cholesterol as compared to control 79

6 Percent reduction in LDL as compared to control 84

7 Percent increase in HDL as compared to control 89

8 Percent reduction in triglycerides as compared to control 93

9 Percent reduction in glucose as compared to control 98

10 Percent increase in insulin as compared to control 102

11 Percent increase in glutathione as compared to control 107

12 Percent reduction in TBARS as compared to control 111

viii

LIST of APPENDICES

Sr. No. Title Page

I Performa for sensory evaluation of the functional drink 169

II Composition of experimental diets 170

III Composition of salt mixture 171

IV Composition of vitamin mixture 172

ABSTRACT The present exploration was an attempt to investigate the therapeutic potential of mango peel extract and isolated mangiferin based functional/nutraceutical drinks against lifestyle related disorders. For the purpose, five different mango peels namely chaunsa, anwar ratol, langra, dusahri and desi were nutritionally characterized followed by mangiferin isolation & quantification, product development and lastly the bioevaluation trial to evaluate resultant drinks for the management of hypercholesterolemia and hyperglycemia. The nutritional analysis indicated that mango peel is a good source of moisture, protein and minerals. Amongst tested mango peels, ethanolic extract of chaunsa exhibited the highest TPC (75.35±3.96 mg/100g GAE), DPPH (59.28±3.69%) and β-carotene (57.33±4.14%) activities however, FRAP value (7.88±0.19 mmol/100g) was maximum in the acetone extract of chaunsa peel. The HPLC quantification of isolated mangiferin showed variations from chaunsa (5.53±0.31 mg/g) to desi peel (3.19±0.17 mg/g). In case of solvents, ethanolic extract yielded maximum mangiferin 5.25±0.01 mg/g followed by acetone 4.29±0.19 mg/g and water extract 3.44±0.21 mg/g. Besides, isolated mangiferin from chaunsa peel had the higher DPPH (77.22±3.96%), β-carotene (74.63±3.69%) and FRAP (12.30±0.24 mmol/100g) activities. During product development segment, three types of drinks containing whole mango peel (T1), mangiferin (T2) alongside control (T0) were prepared for comparison purpose. The formulated drinks were subjected to physicochemical analysis during two month storage that imparts substantial effect on pH and acidity whereas total soluble solids (TSS) affected non-momentously. Sensory scores of the prepared functional drinks decreased with the passage of time however, the scores were remained within acceptable range during storage. In the bioevaluation trial, male sprague dawley rats were involved. Accordingly, three types of studies were designed i.e. study I (normal rats), study II (hypercholesterolemic rats) and study III (diabetic rats). The bioefficacy trial was repeated for validity of the results. The body weights of rats were affected significantly due to functional drinks in all studies. The mangiferin supplemented drink (T2) imparted maximum decrease in cholesterol level during study II and III by 12.85 & 12.25% and 10.01 & 9.91% whilst for LDL by 14.52 & 15.21% and 10.21 & 11.25%, respectively (trial 1 & 2). Similarly, HDL and triglycerides levels were also affected momentously by the functional drinks in study II and III. The diminution in serum glucose and improvement in insulin level of the rats are the indicators showing the positive impact of mango peel polyphenols especially mangiferin based functional drink. In this context, mangiferin based functional drink (T2) showed better performance for glucose reduction 8.36 & 7.92% and 13.09 & 14.26% however, 4.01 & 4.65% and 7.15 & 7.98% enhancement for insulin was observed in study II & III (trial 1 & 2). Furthermore, glutathione level was improved and thiobarbituric acid reactive substances (TBARS) level was reduced by the therapeutic drinks. The normal ranges of liver and kidney function tests as well as hematological attributes proved the safety of resultant drinks. From the present investigation, it is concluded that drinks supplemented with mango peel polyphenols and mangiferin are effective to attenuate various metabolic syndromes.

CHAPTER 1

INTRODUCTION

Global nutritional scenario has motivated the researchers for the development of novel dietary approaches to combat various physiological threats in the vulnerable segments. Nutritional diversity is the vital component of food system focusing on balanced nutrition for holistic outcomes. The fruits and vegetables based nutraceutical/functional foods have enormous potential to cope with the dietary needs of target population owing to their innate therapeutic nature against degenerative disorders. Accordingly, the food based bioactive ingredients are one of the key priorities of the consumers from various socioeconomic communities due to their positive impact on health and longevity (Roller et al., 2007; Jenkins et al., 2008).

On worldwide scale, fruits and vegetables processing industry are generating million tons of agro-industrial waste/byproducts per annum that not only creating a disposal problem but also aggravates the environmental pollution. Thus, their efficient, inexpensive and proper disposal is one of the fundamental prerequisites for friendly ecosystem. Industrial residues especially fruits/vegetables peels are concentrated source of phytonutrients that have acquired core attention of the processors for their extraction and maximum recovery (Pinelo et al., 2006; Ajila et al., 2007).

Mango (Mangifera indica) is a popular fruit widely grown in tropical regions of the globe due to its sweet taste and high nutritive content (Kim et al., 2007; Palafox-Carlos et al., 2012). Currently, mango is considered as the 5th largest produced fruit throughout the world. Pakistan contributes 7.6% share in the world market with production of 177 thousand tons (Akhtar et al., 2009). The mango mainly constitutes pulp 33-70% followed by kernel 7-24% and peel 15-20% of the total fruit weight. Considering nutritional value, mango peel contains moisture, protein, ash, fibre & carbohydrates as 68.50, 2.05, 2.62, 5.40 & 26.5%, respectively and 453.92 kJ/100g energy (Bede, 2010; Ajila et al., 2007). Its peel is a promising source of phytonutrients such as polyphenols, carotenoids and vitamin E & C. Interestingly, higher polyphenol contents are present in mango peel than that of pulp (Ajila et al., 2007). Similarly, some other fruits like apple and pear peels have higher antioxidant activity and mineral contents compared to the pulp extracts (Manzoor et al., 2012; Leontowicz et al., 2003). The phytonutrients of mango byproduct are 1

affected by several factors including climatic conditions, agronomic practices and varietal differences (Tavarini et al., 2008; Manzoor et al., 2012).

Natural antioxidants are gaining popularity owing to their safe status and effectiveness in the physiological system. There is growing interest among the consumers against synthetic additives thereby diverting their trend towards natural counterparts (Siró et al., 2008; Sultan et al., 2009). These compounds act as free radical scavengers, metal chelators, free radical chain reaction & oxidative enzyme inhibitors and antioxidant enzyme cofactors (Karadag et al., 2009). During normal metabolic processes, free radicals are generated in the body that induce cellular damage in several ways. The most deleterious effect of free radicals i.e. singlet oxygen is the DNA damage (Van Langendonckt et al., 2002; Piconi et al., 2003). Besides, oxidized low density lipoprotein (LDL) is one of the causative agents for the development of coronary diseases (Pardo- Andreu et al., 2006). The diverse phenolic compounds of plant origin exhibit differential antioxidative activity against reactive oxygen species by scavenging hydroxyl & peroxy radicals and singlet oxygen quenching thereby inhibit lipid peroxidation (Severi et al., 2009; Wang and Jiao, 2000; Singh et al., 2009).

The physiological system of the body contains natural antioxidants that are divided into enzymatic and non-enzymatic groups to deal with the production of free radicals. In this connection, enzymatic antioxidants include superoxide dismutase, gluthathione peroxidise and catalase whilst β-carotene, vitamin C, vitamin E and selenium are the non- enzymatic antioxidants (Rojas and Brewer, 2008). The scavenging ability of mango peel polyphenols is owing to their molecular structure and degree of hydroxylation (Moure et al., 2001; Huang et al., 2005). Mango peel also exhibits significantly higher amount of phenolics as 19.06 to 23.90 mg/g and free radical scavenging activity i.e. 93.89 to 95.08% (Ribeiro et al., 2008).

Extraction is a crucial step for the recovery and purification of phytochemical from agro- industrial byproducts (Dorta et al., 2011). The isolation of phenolic compounds from mango peel powder is influenced by several factors like solvent, extraction time, solute/solvent ratio, temperature, efficiency of mass transfer and particle size. The sample preparation plays an exclusive role for the quantification of phytochemicals (Zhao et al., 2011; Haminiuk et al., 2011). Various methods are in practice to determine the antioxidants of mango peel powder. The frequently used assays are oxygen radical

absorbance capacity (ORAC), 1,1-diphenyls-2-picryl hydrazyl (DPPH) and ferric reducing antioxidant potential (FRAP). The measurement of total phenolics through spectrophotometer is a direct indicator of antioxidant capacity (Emoke et al., 2009; Ma et al., 2011).

The prophylactic effects of mango peel based phytochemicals like phenolic acid, gallic acid, flavonoids, tocopherols, mangiferin, quercetin and kaemperol are due to their antioxidative and immunomodulatory properties (Choi et al., 2007; Fan et al., 2007; Siddhuraju, 2007). These bioactive molecules play a key role by interferring the oxidation process, chelating catalytic metals and scavenging oxygen in food & biological systems (Kim, 2005). The polyphenolic extracts of various byproducts such as mango peel, kernel and bark showed the occurrence of xanthone C-glycosides, gallotannins and benzophenones (Barreto et al., 2008; Ajila et al., 2010b).

Mangiferin (2-β-D-glucopyranosyl-1,3,6,7-tetrahydroxy-9H-xanthen-9-one) is a xanthone that exhibits therapeutic potential with anti-tumor, antidiabetic, anti-inflammatory and antioxidant properties. Initially, it was isolated from the leaves of M. indica and a herb Anemarrhena asphodeloides (Garrido et al., 2004; Carvalho et al., 2005). The isomangiferin and homomangiferin (mangiferin monomethyl ether) are also present with mangiferin in the mango leaves and twigs (Muruganandan et al., 2005) that constitute 10% of the total phenolics (Sanchez et al., 2000). Mangiferin is an efficient iron chelator thereby prevents the generation of hydroxyl radical in fenton-type reactions (Matkowski et al., 2012).

Mango peel mangiferin alters the liver cholesterol metabolism resulting in low cholesterol accumulation and reduces the incidence of coronary complications (Zern et al., 2003; Vinson and Jang, 2001). It inhibits lipid peroxidase by scavenging lipid alkoxy and peroxy radicals thus prevents from cellular lipid hydrogen damage. Alongside, it significantly reduces the level of serum uric acid in experimental rats. Mangiferin decreases urate capacity and effectively scavenges mediated O2-production, the major reasons to lower the uric acid in mangiferin treated rats (Leiro et al., 2003; Wang et al., 2000).

Gallic acid and quercetin 3-O-galactoside are also the major polyphenols in mango peel that have anti-mutagenic, antioxidant and anti-inflammatory activities (Rastraelli et al., 2002; Kim et al., 2007). Earlier, Schieber et al. (2000) reported the quercetin and 3

associated glycosides in mango peel as quercetin 3-O-glucoside, quercetin 3-O- galactoside and quercetin 3-O-xyloside. In an investigation, quercetin decreases lipid oxidation and increases glutathione contents thereby protects the liver from oxidative damage (Molina et al., 2003; Hubbard et al., 2003).

Diabetes mellitus (DM) is a metabolic disorder growing rapidly as a consequence of changing dietary pattern and desk bound lifestyle (Mitra, 2007; Wang et al., 2011). For experimental purposes, several methods are in practice for the induction of diabetes however, injection of streptozotocin (STZ) is considered as one of the convenient options (Akbarzadeh et al., 2007). In the STZ treated rats, the concentration of insulin decreases rapidly after STZ-induced β-cell destruction with concomitant rise in blood glucose level (Cooper, 2011). The polyphenolic compounds from mango peel induce hypoglycemic effects in the diabetic rats. Among these antioxidants, mangiferin imparts antidiabetic activity by inhibiting intestinal glucose absorption and impairing glucosidase & amylase activity. Additionally, it has significant insulinotropic response by improving insulin sensitivity. Mangiferin also inhibits the nitric oxide synthase (NOS) overexpression and nuclear factor kappa B activation (Verrijn et al., 2012).

The blood cholesterol management is a cardinal issue in cardiovascular disease (CVD) prevention. Hypercholesterolemia and LDL oxidation play key role in the onset of atherosclerosis and related disorders. Owing to rich phytochemistry with special reference to polyphenols, natural products are one of the suitable preventive measures for coronary care and blood cholesterol regulation. The consumption of mango peel functional ingredients especially mangiferin and its other antioxidant moieties are important due to their cholesterol lowering potential (Singh et al., 2007; Matsuura et al., 2008). The mangiferin has ability to lessen serum cholesterol, low density lipoprotein and triglycerides. Nevertheless, it is associated with the augmentation of high density lipoprotein and decreasing glycemic index in diabetic subjects (Erlund et al., 2004).

Mangiferin inhibits enzymes that participate in the carbohydrates metabolism in the gut thereby slows down glucose absorption (Muruganandan et al., 2005). The triglycerides lowering ability of mangiferin contributes towards hypoglycemic activity via glucose fatty acid cycle. As per Randle glucose fatty acid cycle, consistent supply of plasma triglycerides enhances free fatty acid accessibility that impairs insulin sensitivity and glucose metabolism resulted in the development of hyperglycemia. Conclusively,

mangiferin provision facilitates glucose oxidation & utilization thereby alleviates hyperglycemia (Muruganandan et al., 2005).

Hepatic enzymes like alanine amino transferase (ALT) and aspartate amino transferase (AST) are higher in diabeties and oxidative stress state leading to lower hepatic efficiency. Mango peel antioxidants prevent the necrotic injury of the liver (Van Dam et al., 2002). The mangiferin has higher potential as antioxidant by inhibiting lipid peroxidation and neutralizing the reactive oxygen species (ROS) like superoxide anion, hydroxyl & peroxy radicals in biological system (Severi et al., 2009). Moreover, bioactive polyphenols have cytoprotective effect on rat hepatocytes injury induced by hydrogen peroxide or carbon tetrachloride (Yao et al., 2007; Zhao and Zhang, 2011).

The developing economies are facing the challenge of malnutrition among the masses due to lack of consistent supply of nutritious diet & allied healthy ingredients to combat various health related discrepancies. In Pakistan, several lifestyle related metabolic dysfunctions including diabetes, cardiovascular complications and oxidative stress have urged the researchers to develop effective dietary strategies to tackle the existing menace. In the instant exploration, mangiferin extraction from mango peel was carried out for its application as value added therapeutic ingredient in food system. The isolated mangiferin and mango peel extracts were characterized for their antioxidative potential followed by nutraceutical/functional drink development. Conclusively, the animal model study for the assessment of prophylactic impact of developed therapeutic drinks against various metabolic syndromes was the limelight of the investigation. The objectives set to be achieved are herein.

1. To optimize the extraction efficiency of various solvents for mango peel polyphenols

2. Isolation of mangiferin for the preparation of nutraceutical/functional drink

3. To elucidate the nutraceutical perspectives of mangiferin against hyperglycemia and hypercholesterolemia

CHAPTER 2

REVIEW OF LITERATURE

Plants are the assembled reservoirs of essential phytonutrients considered vital for the wellbeing of humans since archaic epoch. Nutrition and health are the interlinked factors influenced by human dietary lifestyle. In this relation, enormous phytochemicals and allied metabolites have emerged as disease preventive agents due to their therapeutic potential. The health augmenting abilities of such plant derived bioactive molecules have promoted their supplementation for the development of designer foods to address various lifestyle related disorders. In this milieu, mango peel is an example of phytonutrients dense source with special reference to mangiferin. Numerous studies have illuminated the potential of mangiferin to tackle various metabolic malfunctioning owing to its promising antioxidative perspectives. Different bioefficacy trials have elucidated the effectiveness of mangiferin against hypercholes terolemia, hyperglycemia, oxidative stress and several oncogenic events. Considering the facts, the current exploration was planned to investigate the nutraceutical potential of mangiferin, isolated from the peels of various mango varieties against metabolic syndromes. The literature pertaining to different aspects of the present study has been reviewed under the following headings.

2.1. Nutraceutical and designer foods; an overview

2.2. Nutritional profiling of mango peel

2.3. Mangiferin: a unique nutraceutical compound

2.4. Extraction and isolation of mangiferin

2.5. In vitro antioxidants estimation

2.6. Pharmacokinetics aspects

2.7. Mangiferin against physiological malfunctionings

2.7.1. Oxidative stress related complications

2.7.2. Hypercholesterolemic perspectives

2.7.3. Hyperglycemia and insulin dysfunction

2.1. Nutraceutical and designer foods; an overview

During the last few decades, the consumers interest has shifted towards the natural products owing to their health escalating perspectives. Amongst various bioactive molecules, the phenolic compounds attained the paramount position (Butt et al., 2009). These secondary plants metabolites are widely distributed in different fruits and vegetables (Butt and Sultan, 2009). Numerous techniques are available for the extraction of bioactive constituents like soxhlet, microwave assisted, heat reflux extraction and ultrasound assisted extraction (Xiao et al. 2008). These functional ingredients are essential for the vitality of life as they provide protection against plethora of diseases (Parr and Bolwell, 2000). The health promoting potential associated with the consumption of fruits and vegetables motivated the researchers for the identification, extraction and isolation of their allied functional ingredients (Wauthoz et al. 2007).

Firstly, Japanese scientists have promoted the concept of functional food in 1984 based on the relationships among nutrition, fortification, sensory satisfaction and modulation of physiological systems that further gained the legal status in 1991 as Foods for Specified Health Use (Sanders, 1998; Burdock et al., 2006). Designer foods are formed by the addition, alteration and modification of bioavailability of one or more food components and elimination of undesirable moieties through technological or biotechnological means (Prado et al., 2008). According to Diplock et al. (1999), food is considered as functional if it provides some health benefits beyond basic nutrition in the body. Additionally, these foods are characterized as medicinal, nutraceutical, prescriptive and therapeutic foods (Parr and Bolwell, 2000). Development and commerce of functional products is complex due to legislative and technological obstacles furthermore, consumer acceptance is also one of the factors affecting their production (Siro et al., 2008).

During processing of fruits and vegetables, enormous amount of agro-waste materials are generated annually. These byproducts/wastes cause severe environmental problems i.e. water pollution, vegetation damage and unpleasant odours (Zamorano et al., 2007). Nevertheless, these wastes hold ample amount of bioactive constituents that motivated the researchers for their efficient recovery, recycling and upgradation to transform them into value added products. Among different fruits parts peel, stone, hull and seed are rich in bioactive components thereby exhibit good antioxidant activities (Soong and Barlow, 2004; Peschel et al., 2007; Moure et al., 2006; Wolfe et al., 2003). However, peel has

gained special consideration due to the presence of high therapeutic polyphenols with easy recovery (Negro et al., 2003).

The mango (Mangifira Indica L.) is a popular fruit originated 4000 years ago from Asia mainly in the Indo-Burmese region. It is a rich source of phytochemicals and nutrients i.e. polyphenols, carotenoids, dietary fiber and vitamin C (Fowomola, 2010). Mango plays an important role in maintaining the health and normal physiological functioning due to the presence of essential vitamins and minerals (Singh et al., 2004; Chen et al., 2005).

Among different mango byproducts, peel is a considerable source of phytonutrients, i.e. polyphenols, omega-3 & -6 polyunsaturated fatty acids along with carbohydrates. Moreover, it provides dietary fiber and carotenoid pigments (Berardini, 2005; Ajila and Prasada, 2007). The byproducts of mango fruit are promising source of biologically active compounds that may be used for the preparation of a variety of value added commodities. Likewise, these byproducts enhanced water & oil retention, improved oxidative stability and emulsifying properties in the end product (Torres et al. 2002; Elleuch et al., 2011). Mango peel powder incorporation in macaroni uplifted the polyphenol concentration from 0.46 to 1.80 mg/g alongside carotenoid as 5 to 84 μg/g. Moreover, the nutritional, cooking, textural and sensory properties are also improved (Ajila et al., 2010a).

The bioactive molecules in peel performed multifarious functions such as hepatoprotective, anti-hemorrhagic, free radical scavenger and immunomodulatory activities (Abdalla et al., 2007; Nithitanakool et al., 2009). The phytochemistry of mango peel is dominated by gallic acid, p-coumaric acid, ellagic acid, protocatechuic acid and mangiferin that are present as 7.3, 20.9, 12.7, 5.4, and 4.8 mg/g, respectively (Hassan et al., 2007).

Human body produces reactive oxygen species (ROS) like peroxides by using metabolic oxygen that initiate the cascade of different metabolic disorders like cardiovascular complications and various oncogenic events. In this context, mango peel phenolic halts the pathogenesis of oxidative stress by quenching free radicals (Gorinstein et al., 2009).

During the storage of ready to drink (RTD) products; acidity, pH and total soluble solids (TSS) are the reliable tools to evaluate acceptability. Generally, pH of the RTD juice or drink diminishes during storage however, acidity increases whilst total soluble solids concentration depends upon the quantity of juice and nature of sugar. Recently, Ahmed et

al. (2008) evaluated the effect of storage on pH, acidity and TSS of green tea based functional drink. They observed a momentous decline in pH with significant enhancement in acidity. Nonetheless, storage imparted non-substantial differences on total soluble solids of functional drink. The pH decreased from 4.7 to 4.2 during two month storage. Likewise, 11% reduction in antioxidant concentration was noticed. Similarly, Hassan et al. (2007) documented a decline in pH and an increase in acidity of RTD juice during storage. They were of the view that decline in acidity and pH enhancement is due to citric and ascorbic acids breakdown.

Hedonic response is a key indicator used to determine the acceptability and preference of the product through trained taste panel. Sensory evaluation is a documented method to characterize the foods on the basis of natural senses including smell, taste, sight etc. carried out by trained taste panel (Kuti et al., 2004). The sensory assessment of a product is correlated with consumer approach, believe and awareness (Aaron et al., 1994). The products containing phytochemicals need careful assessment not only to evaluate consumer suitability but also find their effect on specific segment of population (Gylling et al., 1999; Quílez et al., 2003, 2006). Considering the importance, scientists working on the development of functional foods are not only emphasizing on their physiological functionality but also paid attention towards hedonic aspects.

2.2. Nutritional profiling of mango peel

Fruits byproducts are the potential candidates to be explored as nutraceuticals owing to the presence of an array of phytonutrients. In this reference, mango fruit & peel have enormous antioxidative potential thus improve overall health. During the processing of mango, peels are rich source of antioxidative compounds, dietary fiber and pectin (Rocha et al., 2007; Ajila and Prasada, 2011; Berardini, 2005). Whole mango fruit comprised of 15-20% peel portion that is enriched with different bioactive moieties like polyphenols with special reference to mangiferin, anthocyanins, gallic acid and querecetin thus imparting health promoting effect (Ajila et al., 2007; Ajila and Prasada, 2008; Berardini, 2005).

Regarding nutritional composition; moisture, protein, fat, crude fiber, ash and carbohydrate contents in different varieties of mango peel ranged from 66-75, 1.76-2.05, 2.16-2.66, 3.28-7.40, 1.16-3.0 and 20.8-28.2%, respectively (Balasubramanian, 1999). The earlier findings of Siddhuraju et al. (1996) and Ajila et al. (2007) documented that 9

mango peel is a good source of dietary fiber i.e. 45 to 78%. Likewise, Palafox-Carlos et al. (2011) probed the mango peels of different varieties for their fiber contents. They observed 16-28% soluble dietary fiber (SDF), 29-50% insoluble dietary fiber (IDF) and 45-78% of total dietary fiber (TDF).

The research investigations of Ojokoh (2008) affirmed that mango peel contains significant amount of crude fat, crude protein and dietary fiber by 5.1, 6.16 and 11.2%, respectively. The variations in the proximate and chemical composition of mango peel are dependent on climatic conditions, cultivars, agronomic practices and topographical locations (Granfeldt et al., 1992; Palafox-Carlos et al., 2011). Similarly, mango kernel powder has 91, 6, 11, 4 & 2% moisture, protein, fat, crude fiber and ash, respectively. Moreover, phenolic compounds ranged from 109-112 mg/100 g on dry weight basis (Zein et al., 2005; Fowomola, 2010).

The mango peel also contains substantial amount of minerals dominated by phosphorus (5.5-17.9 mg), calcium (6.1-12.8 mg) and iron (0.20-0.63 mg). Earlier, Ojokoh (2008) observed calcium, phosphorous and potassium contents in ripened mango peel from 145- 160, 26-35 and 190-211 mg/100g, respectively. Likewise, Burns et al. (2008) noticed 153-167, 31-41 and 194-217 mg/100g of calcium, phosphorous and potassium, respectively. One of the researchers groups, documented higher amount of potassium 192 mg/100g in mango peel followed by calcium 10-21mg/100g and phosphorous 17 mg/100g. Earlier, Akhtar et al. (2010) explored dusehri mango cultivar for its sodium, potassium and calcium contents. The results showed the concentrations of these minerals as 6.31, 38.40 and 7.15 mg/100g, respectively. Likewise, Chojnacka et al. (2006) evaluated different mango varieties for their mineral contents and revelead Fe, Zn, Ni, Pb and Cd by 2.81, 2.34, 5.31, 1.18 and 0.20 mg/100g, respectively.

2.3. Mangiferin: a unique nutraceutical compound

Mangiferin (MF) is a C-glucosylxanthone (1,3,6,7-tetrahydroxyxanthone-C2-β-D- glucoside) pharmacologically active flavonoid and a natural xanthone C-glycoside due to its oxygenated and heterocyclic nature. It was first isolated in 1908 as a coloring matter from the mango. The mango peel is a rich source of mangiferin however, also present in different parts like pulp, stem, bark and kernel (Andreu et al., 2005; Jagetia and Venkatesha, 2005). Several mangiferin isomers like isomangiferin and homomangiferin are regarded as potent antioxidants due to their diversity and mode of action. It is 10

pharmacologically active polyphenolic antioxidant that exhibits a wide range of therapeutic effects like anti-cancer, antioxidant, anti-atherosclerotic and anti- inflammatory alongside immunomodulatory perspectives (Muruganandan et al., 2005; Sanchez et al., 2000 and Prasad et al., 2007).

The mangiferin synthesis is carried out through friedel and craft reaction in which glycosyl donor and an electron rich aromatic compound joined through glycosidic linkage (Lee, 2009). In this context, aryl C-glycosylation without C-9 carbonyl function is required due to electron deficiency (Oyama et al., 2004; Furuta et al., 2009). Earlier, Faizi et al. (2006) synthesized mangiferin by the reaction of aglycone 1,3,6,7- tetrahydroxyxanthone with R-acetobromoglucose followed by hydrolysis of the formed O-glycosidic linkage.

Mangiferin is stable to enzymatic as well as acidic degradation thus acts as hydrolytically stable O-glucoside analogous for enzyme inhibitors (Yang et al., 2002). However, its metabolisim is carried out by intestinal microflora that interact with C-glucosyl bonds of various C-glucosides and transformed into corresponding aglycone (Li et al., 2008; Kim et al., 2007). Mangiferin has high bioavailability due to its chemical structure, low molecular weight below 500 dalton, less than 5 donor, 10 acceptor hydrogen bonds and log P potential. It is a potential antioxidant provides protection against cellular injury at low concentration alongside protecting the DNA damage. It imparts reduction in the localized oxygen concentration, producing phenoxy radicals, forming mangiferin metal complex, removing OH radicals and oxo-ferryl groups, modulating the polymer chain initiation reactions by halting the free radicals thus performed function as in vitro antioxidant.

The antioxidant activity of phenolic compounds is affected by their chemical configuration. Structure activity relationships have been used as a theoretical tool to determine antioxidant activity (Zheng, 2001). Previously, Oyama et al. (2004) investigated the antioxidant activity of mangiferin from different sources. They documented higher antioxidant potential of mangiferin due to its iron chelating ability. The higher polymerization degree, purity of active compounds, nature of molecules and applied methods are the cardinal factors to determine the antioxidant effectiveness of a biomolecules. Moreover, lipophilic nature of the antioxidant, interaction & isolation procedures and polarity of the extracting solvent are also important (Zheng, 2001).

Flavonoids with 2-3 double bond in conjugation with 4-carbonyl group exhibit lower IC50 values (indicative of stronger antioxidant activity) in microsomal system and optimize the phenoxyl radical-stabilizing effect of 3,4-catechol (Cholbi et al., 1991). Aglycones (mangiferin) are more potent antioxidants than their corresponding glycosides (Guo et al., 2011). One of their peers, Plumb et al. (1999) reported that the antioxidant properties of flavonol glycosides are directly associated with the number of glycosidic moieties. Aside from mere presence and total number, the position and structure of the sugar play an important role. Different other chemical modifications like methylation and O- glycosylation interfere with coplanarity of the B-ring with rest of the flavonoids thus change the overall structure of biomolecules (Bors et al., 1990). The glycosides bioavailability is enhanced by the addition of glucose moiety (Hollman et al., 2004).

The earlier study of Berardini et al. (2005) indicated higher phenolic, free radical scavenging and ferric reducing antioxidant power of mangiferin. They evaluated the antioxidant activity of bark, stem, peel and seed of different mango cultivars at varying extraction conditions. The research outcomes elucidated phenolic capacity of mangiferin as 1190-1690 mg/kg higher than that of other polyphenols. Likewise, DPPH, TEAC and FRAP values were also detected highest for magniferin by 370, 352 and 1056 Trolox/100g sample as compared to quercetin 3-O glucoside by 360, 319 and 756 Trolox/100g sample.

The previous investigation of Rajendran et al. (2008) expressed that mangiferin lowers the activities of electron transport chain and tricarboxylic acid (TCA) cycle key enzymes i.e. alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, isocitrate dehydrogenase and malate dehydrogenase in lung cancer treated animals. The findings elucidated the modulatory effect of mangiferin in preventing undesirable biochemical changes. One of the peers, Stoilova et al. (2005) illuminated the antioxidant activity of alcoholic extract of mangiferin in linoleic acid/water emulsion system through thiobarbituric acid reactive substances and by inhibiting conjugated dienes formation assay. The findings of this study elucidated mangiferin as a potent antioxidant. Likewise, one of researchers groups, Nishigaki et al. (2007) performed an in vitro study to determine the effectiveness of mangiferin on human umbilical vein endothelial cells against glycated protein iron chelate induced toxicity. They noticed enhancement in

antioxidant enzymes level and decline in lipid peroxidase due to glycated protein-iron chelate complex formation.

The mangiferin utilization proves beneficial against various metabolic disparities. In diabetic subjects, it modulates glucose metabolism moreover, ameliorates insulin resistance and enhances its production. During hypercholesterolemic phase, mangiferin causes diminishing effect on cholesterol synthesis by interfering the metabolic targets and prevents LDL oxidation thus alleviates lipid abnormalities. It also inhibits the expression of inducible nitric oxide synthase and tumor necrosis factor α (TNF- α) genes in experimental volunteers. In few in vitro and in vivo studies using different cancer cell lines have proven mangiferin effectiveness against a variety of malignancies including liver, colon, breast and lung (Liu, 2005). Overall, mangiferin may inhibit promotion and progression stages of various cancers by interfering with cell cycle regulation, signal transduction pathways, transcription and activating apoptosis (activation of pro-apoptotic genes and pro-apoptotic proteins) in neoplastic cells (Manach et al., 2005).

2.4. Isolation and quantification of mangiferin

Extraction is an important step in recovering and purifying phytochemicals from plant byproducts (González-Montelongo et al. 2010a; Daud et al. 2010). Different methods are employed for the extraction of biomolecules nonetheless, solvent extraction is a popular technique due to its ease, high recovery, low cost and better control. Among different solvents, ethanol, methanol, acetone and ethyl acetate are often used for the recovery of antioxidants from mango byproducts (Abdalla et al. 2007; Barreto et al. 2008; Nithitanakool et al. 2009).

The extraction of phenolic compounds is influenced by several variables like solvent, agitation, extraction time, solute⁄solvent ratio, temperature, efficiency of mass transfer and particle size. Although, sample preparation also plays a crucial role (Hurtado- Fernandez et al., 2010; Zhao et al., 2011; Haminiuk et al., 2011; Yang et al., 2011). In this context, freezing and drying processes are often used to protect fruit sample from degradation and microbial attack alongside they enhance the antioxidant concentration (Turkben et al., 2010).

The solvent partition method is commonly applied for the isolation of different nutraceutical components like theaflavin, catechins and mangiferin due to its low cost and

high efficiency. Accordingly, Jutiviboonsuk and Sardsaengjun, (2010) carried out solvent partition in order to isolate mangiferin from different mango varieties. For the purpose, they treated mango peel powder with different solvents like methanol, ethanol and 70% acetone. After filtration and rotary evaporation the resultant slurry was treated with petroleum ether to remove the fatty matter. Next, the aqueous layer was treated with cold acetone for the removal of impurities. Lastly, mangiferin was isolated by diluting water layer with 70% ethanol and resultant extract after concentration transformed into yellow color mangiferin through freeze drying.

The importance of lyophilisation (freeze dying) is well established to obtain pure compounds due to its ability to remove water from various food materials thus imparts long term stability to the end product (Kasper and Friess, 2011). For the extraction of bioactive components from mango peel, polar solvents enhance their liberation whilst alcoholic solvents cause rupture to the cell membrane thus cause adequate extraction of biomolecules (Lafka et al., 2007). For the extraction and isolation of mango polyphenols, polar solvents i.e. ethanol, methanol, water are more efficient than their non-polar counterpart. However, composition of the natural product, solvent property and diverse structure are the cardinal factors that ensure optimum recovery (Gonz´alez-Montelongo et al., 2010a, 2010b). Moreover, other factors including number of extraction cycles, solvent to plant material ratio, contact time & temperature and extraction technique significantly influence the isolation of mangiferin.

Liquid chromatography is an important separation technique carried out in the liquid phase where a mixture of compounds can easily be separated. Accordingly, two techniques such as liquid chromatography mass spectrometry (LC-MS) and HPLC are commonly used to extract and quantify mango peel polyphenols i.e. mangiferin (Wilkinson et al., 2008). The reverse phase high performance liquid chromatography (RP-HPLC) is a premium method used for the separation of phenolic compounds from mango byproducts in which the stationary phase is less polar than mobile phase. The stationary phase is generally made up of hydrophobic alkyl chain lengths i.e. C4, C8 and

C18 (Guzzi, 2008). The silica-bonded C18 column is commonly used to separate phenolic compounds. In RP-HPLC, retention time of phenolic compounds is higher for substances that are less polar (mangiferin, myricetin, quercetin, kaempferol) whilst, polar molecules are eluted easily (gallic acid, protocatechuic acid, epigallocatechin). Due to chemical

complexity, polyphenol compounds in mango peel are commonly identified and quantified through RP-HPLC using a gradient elution instead of isocratic mode (Merken and Beecher, 2001; Kim and Lee, 2001). Generally, gradient elution is carried out with polar solvent e.g. phosphoric, acetic, formic acids and less polar solvents i.e. acetonitrile and methanol (Kim and Lee, 2001).

Reverse phase high performance liquid chromatography (HPLC) methods coupled with various detectors including ultra voilet, UV-diode array, mass spectrometry and electrochemical have been developed for the quantification of mangiferin in mango peel, seed and kernel. Additionally, both gradient and isocratic mobile phases are in practice for the quantification of mangiferin & their derivatives alone and simultaneously with other compounds. The selection of mobile phase is mainly dependent upon the type of sample (Jong et al., 2006).

In this reference, Hassan et al. (2007) investigated mango peel for polyphenolic profile through HPLC. They utilized C18 column, UV-vis detector and maintained the temperature 40 οC with 1 mL/min flow rate. The outcomes of study showed 4.8 mg/g mangiferin alongside other 16 phenolics compounds. They were on the view that maturity stage, variety and climatic variations are the important factors that contribute to the peel phenolic composition. Earlier, Rivera et al. (2006) quantified mangiferin through HPLC in mango peel of four different Brazilian varieties namely Haden, Tommy Aktin, Palmer and Uba. They noticed variations in the mangiferin contents from 2.03 to 5.36 mg/g and inferred that varietal differences are responsible for these changes.

Likewise one of the scientific groups, Jutiviboonsuk and Sardsaengjun (2010) carried out the quantification of mangiferin in different thai mango varieties namely Nam Doc Mai, Keow Savoey and Gaew through chromatography. They extracted the bioactive moieties with different solvents like methanol, ethanol and 70% acetone. The evaluation of resultant extracts indicated highest mangiferin in methanolic extract of Nam Doc Mai variety (2.80 g/100g) followed by Keow Savoey (2.40 g/100g) and Gaew (1.30 g/100g). However, similar varieties exhibited relatively less mangiferin in acetone and ethanolic extracts. Likewise, Barretto et al. (2008) documented mangiferin as 4.94 g/kg dry weight in mango peel after quantification through HPLC at wavelength of 278 nm. Earlier, Andreu et al. (2005) investigated mangiferin contents in different mango byproducts like

peel, kernel and bark. The peel has the highest mangiferin 15.23 followed by kernel 8.98 g/kg whilst the least in bark 12.33 g/kg dry weight basis.

2.5. In vitro antioxidants estimation

The antioxidant capacity of a bioactive compound is assessed through its ability to decrease the initial amount of free radicals by 50% at appropriate concentration. Mango polyphenols have tendency to bind metals and free radicals. In this reference, structural variations, number & configuration of hydroxyl bonds and degree of methylation are the cardinal factors. Nonetheless, different physical variables like pH of the media, time & temperature, solvent and polyphenol structure are essential to determine the efficiency of process (Prior et al., 2005).

The antioxidant estimation is a multifaceted approach to draw irrefutable conclusions. A single system is not so successful to determine the activity because of complex interactions between bioactive moieties and food matrix (Frankel and Meyer 2000; Sanchez-Moreno, 2002). There are evident variations in the relative antioxidant potential when one model compound behaves as antioxidant and prooxidant under different situation. Lipophilic antioxidants are less effective than hydrophilic antioxidants in bulk oils, whilst showed higher activity in emulsions (Mishraa et al., 2006). The most frequently used methods for polyphenols estimation in mango peel are oxygen radical absorbance capacity (ORAC), 1,1-diphenyls-2-picryl hydrazyl (DPPH), 2,2'-azino-bis 3- ethylbenzothiazoline-6-sulphonic acid (ABTS) and ferric ion reducing antioxidant power (FRAP) methods (Dineshkumar et al., 2010). However, DPPH and FRAP attained paramount position due to their ease, economic, accuracy and reproducibility (Makan et al. 2003).

Earlier, Kim et al. (2007) evaluated the antioxidant capacity of raw and ripened mango peel of various varieties through total phenolic contents (TPC) estimation. The TPC was higher in ripened peel 90-110 mg GAE (gallic acid equivalent)/g as compared to raw 55- 85 mg GAE (gallic acid equivalent)/g dry peel. They were on the view that maturity stage, mango cultivar and applied methods are the key variables in this respect. Likewise, Larrauri et al. (1996) examined the ripened and raw peels of Hayden variety for total phenolic contents. Purposely, they extracted bioactive moieties through aqueous methanol and subjected to Folin-Ciocalteu assay. They recorded 70 mg GAE/g TPC in ripened as compared to 55 mg GAE/g in raw peel. Later, Nithitanakool et al. (2009) compared the 16

antioxidant potential of pomace and peels of different mango cultivars through phenolics estimation. The outcomes of the study indicated higher total polyphenol in peel (98.3 mg GAE/g) than that of pomace (68.8 mg GAE/g).

The solvent and extraction conditions like time and temperature are the major factors influencing polyphenol extraction. In this context, Ajila et al. (2007) compared different solvents including ethanol, acetone and water with respect to total phenolics in different mango cultivars. During experiment, organic solvents showed better performance as compared to water owing to their polarity differences. Moreover, acetone extract showed the highest polyphenols 110 mg/g GAE as compared to ethanol 92 mg/g GAE whilst, water extract revealed the lowest TPC by 55.06 mg/g GAE. Later, Ashoush et al. (2011) observed higher phenolic in methanol extract (19.06 trolox equivalent) as compared to water extract (3.06 trolox equivalent). They inferred that solvent imparts substantial effect on the content and composition of phenolics thus their activity may vary with solvent.

Earlier research investigations of Maisuthisakul and Gordon (2009) highlighted the importance of solvent for biomolecules extraction. They observed that ethanolic extract of mango seed kernel (MSK) exhibited four times higher DPPH scavenging activity

(IC50=0.56 μg/mL) as compared to MSK water extract (2.61 μg/mL). In another experimental, Vaghasiya and Chanda (2010) reported that acetone extract of mango seed kernel has higher antioxidant activity (IC50 = 11 μg mL-1) than the methanolic fraction

(IC50=12 μg mL-1).

One of the researchers groups, Kim et al. (2007) examined the effect of biomolecule concentration on free radical scavenging activity of ripened and unripened mango peels. They varied the concentration of unripened mango peel extract from 12.5 to 50 ug/mL and noticed enhancement in DPPH activity from 1.72 to 92.57%, respectively. However, ripened mango peel exhibited antioxidant activity at same concentrations as 3.99 to 81.86%, respectively.

There are several mechanistic approaches involved during free radical scavenging of antioxidant compounds. Accordingly, single electron transfer activity determines the potential of antioxidant to transfer one electron to reduce any compound including metal, carbonyl and radicals. Moreover, hydrogen atom transfer also delineated the capacity of an antioxidant to quench free radicals through hydrogen donation (Wildman et al., 2007).

Nonetheless, single electron transfer reactions are usually slow and require more time to reach completion as compared to hydrogen atom transfer (Prior, 2005).

In another research, Ayala-Zavala et al. (2009) observed 47.97% DPPH radical scavenging activity of mango peel powder at concentration of 322 mg/mL. Similarly, Rodrigo et al. (2007) noticed higher free radical scavenging activity of mango peel (53.3%) followed by seed (24.2%). Similarly, anthocyanin was detected highest in ripened mango peel 360-565 mg/100g as compared to raw form 203-326 mg/100g (Ajila et al., 2007).

Previously, Guo et al. (2011) explored the ferric reducing antioxidant potential of mango peel. They observed the value of 10.13 mmol/100g higher than pulp 0.38 mmol/100g. Mango peel indicated significant reducing power in term of FRAP. Among the different tropical and subtropical fruits, mango peel has higher antioxidant potential as compared to papaya and lemon. The antioxidant capacity of mango is also influenced by its genotype (Scalzo et al., 2005; Torunn et al., 2009). Furthermore, mango and its different by products like peel, bark, stem and seed hold rich antioxidative activity in term of β- carotene (Scalzo et al., 2005). Conclusively, in vitro antioxidant activity of mango peel is dependent on its iron-chelating and free radical scavenging properties.

2.6. Pharmacokinetics aspects

Globally, researchers have converged their attention towards the discovery of fruits/vegetables based phytonutrients with high antioxidative properties. Recently, healthcare professionals are also curious about the nutraceutical worth and pharmacokinetic facets of such bioactive moieties after consumption. The foods containing ample amount of polyphenols impart health benefits, usually correlated with their active metabolites available to the body (Manach et al., 2005; Palafox-Carlos et al., 2011). The absorption of mango peel polyphenols takes place by passive diffusion transversely in the gut epithelial cells membrane. During absorption, a large number of polyphenols penetrate in the gut wall due to their hydrophilic nature (Manach et al., 2005). One of the major reasons of mango peel health enhancing potential is its bioavailability and metabolic fate that primarily depend upon digestive immovability, discharge rate from the food matrix, dietary source and effectiveness of trans epithelial channel (Palafox-Carlos et al., 2011; Rodrigo et al., 2011; Tagliazucchi et al., 2010).

The mango fruit polyphenols are not evenly distributed in the peel, pulp & seeds and lacking in equal absorption efficiency throughout the body (Manach et al., 2005). There are multifarious factors including chemical structure and interaction with different macromolecules that interfere the release and absorption of mango peel polyphenols to various body organs (Palafox-Carlos et al., 2011; D’Archivio et al., 2010). Next, the interaction of polyphenols with fiber can lower bioavailability in the small intestine however, suppression of food matrix by reducing interaction between polyphenols & carbohydrate polymers may enhance their bioavailability (Parada and Aguilera, 2007).

Technically, the mango peel mangiferin is embedded within food matrix that interacts with enzymes and proteins. Likewise, several polyphenols from mango peel are available to the human body through the action of intestinal & hepatic enzymes and colonic microflora. Further, these polyphenols are absorbed and metabolized. Approximately, 48% of the dietary polyphenols are accessible in the small intestine whilst, 42% in the colon (Saura-Calixto et al., 2010). The mangiferin present in free form (aglycones) can conjugate in the small intestine and later in the liver by glucuronidation, methylation, sulphation or in combination. Conjugation reaction of bioactive molecules is generally carried out by different enzymes like cytosolic β-glucosidase (CBG) and lactase phloridizin hydrolase (LPH), positioned in the epithelial cells of the small intestine. Nevertheless, the catechol-O-methyltransferase (COMT), UDP glucuronosyl transferase (UDPGT) and phenol sulphotransferases (P-PST) are located in different body tissues (Scalbert and Williamson, 2000). The conjugation reaction significantly interferes with the metabolism of polyphenols by producing active metabolites or increasing their excretion rate (Scalbert and Williamson, 2000; Cano et al., 2002; D’Archivio et al., 2010). The polyphenols that are not absorbed in the small intestine reached the colon and metabolized by the colonic microflora (Scalbert and Williamson, 2000; D’Archivio et al., 2010). They further hydrolysed glycosides into aglycones followed by their degradation to phenolic acids (Aura et al., 2005; D’Archivio et al., 2010).

Generally, the free form of a drug is considered pharmacologically active that can easily diffuse through the cell membrane. In an investigation, high performance liquid chromatography mass spectrophotometry (HPLC-MS) method was used to determine the plasma profile following oral administration of mangiferin at single doses of 0.1, 0.3 and 0.9 g. The results elucidated maximum absorption of mangiferin at higher dose level

(Tagliazucchi et al., 2010). The findings of Yue et al. (2009) indicated the formation of complex between serum albumin & mangiferin however, the hydrophobic interaction dominated in the associated reaction. Later, Toutain et al. (2010) affirmed alteration in the metabolism of mangiferin due to its binding with serum albumin.

2.7. Mangiferin against physiological malfunctionings

The mangiferin exhibits various health augmenting properties required for vitality of life. Owing to free radical scavenging ability, it is effective to manage the health confronts with special reference to hypercholesterolemia and hyperglycemia. It also boosts the immune system through diverse pathways and controls various neurodegenerative disorders. The details of various aspects are described herein.

2.7.1. Oxidative stress related complications

During oxidative stress an imbalance between reactive oxygen species production and their quenching by endogenous antioxidants enzymes occurs that disturbs the normal removal of free radicals, generated in the living system. This situation triggers the abnormalities in redox potential and body capacity to tackle with obnoxious radicals resulting in cell damage (Butt and Sultan, 2009).

Reactive oxygen spices (ROS) are ubiquitous in nature and generated continuously inside the body nevertheless, some physical factors like poor dietary habits, lack of physical activity, smoking and environmental pollutants may enhance their generation. The prime objective of diet based therapy is to improve the defense system that in turn protects the body from free radicals thus ensures good health and longevity. In this scenario, consumption of polyphenols rich diet is a suitable strategy to augment the body antioxidative status (Koppula et al., 2012). Sedentary lifestyle coupled with poor dietary habits may initiate the plethora of disorders like cardiovascular diseases, diabetic complications and immune malfunctionings (Bárta et al., 2006). Mango polyphenols especially mangiferin has capacity to provide shield against the physiological threats by preventing lipid peroxidation (Wong et al., 2006; Seifried et al., 2007).

There are several routes that are involved in oxidative stress such as altered energy metabolism, non-specific chronic inflammation, reactive oxygen species (ROS) production and frequent utilization of anti-neoplastic drugs i.e. alkylating and cisplatin agents (Makan et al., 2003; Bakan et al., 2003). The cells have innate low molecular

weight enzymatic antioxidant compounds i.e. glutathione (GSH) with potential to defend against free radicals (Koppula et al., 2012). The glutathione peroxidase (GPx) is a selenoprotein, which reduces lipidic or non-lipidic hydroperoxides as well as H2O2 while oxidizing GSH (Koppula et al., 2012).

In recent years, researchers are emphasizing the identification of the naturally occurring bioactive compounds that increase the activity of endogenous antioxidant enzyme system (Finkel and Holbrook, 2000). Glutathione is synthesized by the action of glutathione synthetase and gamma glutamyl-cysteinyl-ligase (GCL). In mammalian cells, reduced glutathione (GSH) works as a prime cellular antioxidant and electron donor catalyzed by glutathione peroxidase (GPX) that reduces hydroperoxides into alcohols producing glutathione disulfide (GSSG), a substrate for glutathione reductase (GSSR). This nicotinamide adenine dinluetide phosphate (NADPH) dependent reaction regenerates glutathione, the only active form of the tripeptide. Inside cells, glutathione (GSH) is also consumed through direct non-enzymatic reactions involving radicals and by conjugating with electrophilic compounds catalyzed by glutathione transferase (Hayes and Pulford, 1995). After a decade, Prabhu et al. (2006) explored the role of mangiferin against myocardial infarction (ISPH) induced by isoproterenol. The oxidative stressed animals were treated with mangiferin @ 200 mg/kg body weight. The ISPH synthetic catecholamine and beta-adrenergic agonist caused reduction in the body antioxidant potential of the rats after 28 days. However, concurrent intake of mangiferin uplifted the concentration of different antioxidant enzymes like reduced glutathione, glutathione transferase and catalase by 34.17, 46.85 and 52.13%, respectively. They inferred that mangiferin enhances the activity of glutathione and other antioxidant enzymes by quenching free radicals and metal ions.

Likewise, De-Jian et al. (2003) carried out a 28 days rodent feeding trial to estimate the antioxidant and lipid peroxidation protecting ability of mangiferin. They induced oxidative stress through free radicals followed by mangiferin provision @ 100 mg/kg body weight. After the study period, examined biochemical parameters showed a marked increase in the activity of glutathione (GSH), glutathione S-transferase (GST) and catalase (CAT) as 52.38, 47.81 and 93.29%, respectively with 58.37% diminish for thiobarbituric acid reactive substances (TBARS).

Later, Pardo-Andreu et al. (2006) explored the protective role of Vimang mango derived mangiferin against mitochondrial oxidative stressed hypercholesterolemic mouse. They used TBARS assay for the detection of lipid peroxidation extent through malondialdehyde level. The results indicated a marked decline in TBARS level of mangiferin treated group from 6 to 4 nmole/mg protein with 33.33% reduction.

In an earlier exploration, Sanchez et al. (2000) probed the effectiveness of Mangifera indica L. stem bark extract, mangiferin and some selected antioxidants against 12-O- tetradecanoylphorbol-13-acetate (TPA) induced oxidative damage in serum, liver and brain in mice. The mice were administrated with a dose of 50–250, 50 and 100 mg/kg body weight of Vimang extract, mangiferin and vitamin E, respectively. The provision of respective treatments caused 70, 44 and 17% decline in ROS as compared to control alongside 55-73, 37 and 40% reduction in H2O2 contents.

In liver, carbon tetrachloride (CCl4) is accumulated in hepatic parenchyma cells and binds polyunsaturated fatty acids (PUFA), forming alkoxy (R•) and peroxy radicals (ROO•) associated with lipid peroxidation. These free radicals formed covalent bond with cellular protein, disrupt the cell membrane, alter the enzymatic activity and finally induce the hepatic necrosis (Pandit et al., 2004; Brautbar and Williams, 2002). Mangiferin exhibits assuaging role to CCl4 induced liver damage due to its antioxidative potential. The aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) are the sensitive indicators for the estimation of liver damage. As a result of hepatic injury, the disturbance in the function of hepatocytes causes leakage of enzymes from cells due to the altered permeability of membrane resulting in decreased AST, ALT and ALP concentrations in the hepatic cells whilst raised levels in the serum (Yadav and Dixit, 2003). However, pretreatment of mangiferin significantly prevents the malondialdehyde (MDA) elevation and antioxidant status depletion. It also maintains the cellular oxidant and antioxidant balance by decreasing the localized O2 concentration and generating mangiferin phenoxy radicals thus reduces free radical mediated lipid peroxidation (Ghosal et al., 1996). In a study, Sroka and Cisowski (2003) noticed a significant reduction in the concentration of liver enzymes like ALP, AST and ALT in rats after mango peel powder administration @ 30, 50 and 70 mg. Similarly, Rashad et al. (2008) and Hassan et al. (2007) observed significant decline in abnormal concentration of liver enzymes after mangiferin consumption. They were on the view that strong

antioxidative property of mango peel polyphenols halts the lipid peroxidation thereby ameliorates abnormal concentration of respective enzymes.

The mechanistic role of mangiferin as an antioxidant is primarily due to its radical captodative activity. Moreover, it acts as lipid peroxidation inhibitor by producing mangiferin phenoxy and oxoferryl radicals through polymer chain initiation by interacting with free radicals. The resultant complex initiates the polymerization of vinylic monomer methylmethacrylate (MMA). The mangiferin works as chain terminator by oxidizing other carbon radicals resulting in low molecular weight mangiferin-Fe-PMMA (polymethylmethacrylate)-polymer formation. Moreover, it neutralizes the alkoxy/lipid peroxy radicals and maintains cellular oxidant & antioxidant balance (Ghosal et al.,1996).

Mangiferin oxidizes Fe (II) and prevents Fe (III) reduction through ascorbates, reduces the provision of Fe (II) for the initiation of fenton reaction thus prevents ferrous iron induced lipid peroxidation in the mitochondria (Pardo et al., 2005). It stimulates Fe (III)- dependent ascorbate oxidation and significantly inhibits 2-deoxyribose degradation by Fe (III)-ethylenene diamine tetra acetic acid (EDTA) or Fe (III) citrate plus ascorbate with EDTA as iron co-chelator. Additionally, mangiferin showed dual mechanism of protection against iron related oxidative damage to 2-deoxyribose and ascorbate (Zhao et al., 1998).

2.7.2. Hypercholesterolemic role

Globally, dyslipidemia is one of the major risk factors for the development of cardiovascular disease (CVD) associated with elevated serum total cholesterol (TC), low density lipoprotein (LDL) and triglycerides (TG) levels along with suppressed high density lipoprotein (HDL). The hypercholesterolemia plays a major role in the progression of atherosclerosis due to the atheromas formation after cholesterol accumulation in the coronary arteries (Amrita et al., 2009; Anwer et al., 2012). Phytochemicals from fruits and vegetables byproducts are the potential candidates that provide protection against cardiovascular complications. Among different phytonutrients, mangiferin has hypocholesterolemic and hypoglycemic perspective thus imparts reduction in total cholesterol, LDL and triglycerides alongside an increment in HDL (Prasad et al., 2007).

Previously, Muruganandan et al. (2005) observed that mangiferin treatment resulted significant reduction in atherogenic index, total cholesterol, low density lipoprotein and total glycerides with immediate enhancement in high density lipoprotein (HDL) level of rats. Earlier, Miura et al. (2001b) carried out a two weeks rodent trial to explore the therapeutic worth of mango polyphenols especially mangiferin against lipid abnormalities. During the entire trial, experimental rats were fed on mangiferin (30 mg/kg) along with basal diet. The results regarding different lipids related parameters delineated that mangiferin caused 40 & 70% reduction in cholesterol and triglyceride levels, respectively.

Similarly, Dey et al. (1998) and Carmen et al. (1999) observed that mango peel water extract imparts significant reduction in abnormal lipid levels of hypercholesterolemic rats as compared to placebo. Similarly, Canal et al. (2000) noticed elevation in the HDL level of hypercholesterolemic rats by treating with 30, 50 & 70 mg aqueous mango peel and leaves extracts. Various researchers groups like Ho (2003) and Seth et al. (2004) reported beneficial impact of mango peel powder on hypertriglyceridemic rats. They documented a significant decline in plasma triacylglycerol, low density lipoprotein and cholesterol contents whilst an enhancement in high density lipoprotein. Later, Nair and Shyamala Devi (2006) expressed that mangiferin significantly reduces cholesterol, triglycerides and free fatty acid levels in experimental volunteers.

One of the scientific groups, Dineshkumar et al. (2010) probed mangiferin against lipid based abnormalities. They carried out a one month bio-efficacy trial using diabetic rats. The diabetes was induced by streptozotocin injection @ 65 mg/kg body weight (BW). They observed marked increase in LDL, cholesterol and triglyceride level of rats with 8.51% decline in HDL. However, mangiferin consumption @ 10 & 20 mg/kg BW, respectively resulted significant increase 23.64 and 26.31% in HDL, respectively. Similarly, total cholesterol and triglycerides were reduced by 34.53 & 60.41 and 20.79 & 19.06%, correspondingly. In a clinical trial, Aderibigbe et al. (1999) examined the potential of mangiferin in hypercholesterolemic rats during one month study. They observed 24.66 to 27.88% decline in the level of low density lipoprotein of diabetic rats with 10 & 20 mg/kg dose of mangiferin. Likewise, Morsy et al. (2010) also observed significant reduction for cholesterol, LDL and triglycerides in the Sprauge-Dawley rats treated with 30, 50 and 70 mg of mangiferin.

Guo et al. (2011) analyzed the effect of low and high doses of mangiferin administration i.e. 50 and 150 mg/kg, respectively on lipid profile of hamsters fed on high fat diet during 8 weeks trial. Accordingly, hamsters were divided into four groups on the basis of diet i.e. control, high fat (HF), HF+mangiferin 50 mg/kg and HF+mangiferin 150 mg/kg BW. The group receiving maximum dose of mangiferin showed higher reduction in LDL, cholesterol and triglycerides concentrations compared to remaining groups. They concluded that mangiferin treatment upregulates liver mRNA expression of peroxisome PPAR-α, fatty acid translocase (CD36) and carnitine palmitoyltransferase 1 (CPT-1) proteins that are involved in fatty acid oxidation and metabolism. Further, it downregulates the expression of proteins considered essential for fatty acid synthesis as acetyl Co-A carboxylase (ACC) and acyl Co-A diacylglycerol acyltransferase 2 (DGAT- 2). Earlier investigations showed that mangiferin exhibits suppressive effect on serum lipids in diabetes as well as cardioprotective role due to its antioxidant activity (Miura et al., 2001; Prabhu et al., 2006 a,b). Increased level of cholesterol and TG (triglycerides) is associated with cardiovascular disturbances where isoproterenol promotes lipolysis in myocardium (Sushamakumari et al., 1990). Accelerated lipolysis and plasma free fatty acids (FFA) may lead to an increase in hepatic TG synthesis and secretion (Pastwa et al., 2001).

Polyphenols like mangiferin from different plant sources alter the hepatic cholesterol metabolism resulting less accumulation of cholesterol in the aorta and significantly inhibit atherosclerosis thus decrease the incidence of coronary diseases (Zern et al., 2003; Vinson and Jang, 2001). Mangiferin inhibits lipid peroxidase (LPO) due to scavenging of lipid peroxy and alkoxy radicals thereby prevent abstraction of hydrogen from cellular lipids (Ghosal et al., 1996). Elevation of low density lipoprotein (LDL) is a key event in the development of atherosclerosis (Chisolm et al., 2000). This disorder is triggered by oxidative modifications of LDL after exposure to reactive oxygen species (ROS) from vascular wall cells (Wilkinson et al., 2008; Lamb et al., 1992). During hypercholesterolemic phase, elevated expression of LDL receptor (LDLr) indicates the accumulation of reactive oxygen spices in mitochondrian that make the subject susceptible to Ca2+ induced membrane permeability transition (MPT), a state associated with cell death (Paim et al., 2008).

Mitochondria can produce large amount of ROS and plays an important role for the vitality of cells. Thus, mitochondrial dysfunction and oxidative damage contribute to a number of diseases, including atherosclerosis (Vercesi et al., 2007). Previously, it was reported that high cholesterol de novo synthesis caused decline in mitochondrial NADPH activity thus increases the probability of oxidative stress and MPT (Kowaltowski et al., 2001). The mangiferin and its various isomers have potential to ameliorate the mitochondrial oxidative damage by lowering the activity of LDLr thus provide protection against lipid oxidation (Rodrigo et al., 2007).

2.7.3 Diabetes mellitus and mangiferin

The incidence of diabetes has been increased tremendously due to sedentary lifestyle, poor dietary habits, lack of exercise and high medication cost. According to International Diabetic Federation, the prevalence of diabetes among population will raise from 285 to 438 million from the year 2010 to 2030 (IDF, 2011). Diabetes is a metabolic syndrome characterized by hyperglycemia and insulin resistance due to disturbance in carbohydrate & fat metabolism (Wild et al., 2004; Fowler et al., 2007). Conventional therapies are frequently in practice to curtail this menace however, addition of diet based module amplified their therapeutic role against the existing peril. Various herbal by products are in use since ancient times as a diabetic cure. In this context, mango polyphenols especially mangiferin is in limelight to combat hyperglycemia, hyperinsulinemia and immune dysfunctions owing to its strong antioxidative potential. Growing evidences have elucidated that mangiferin modulates different metabolic expressions alongside imparts positive influence on functional and structural integrity of β-cells thus manages hyperglycemia (van Dam et al., 2002; Steyn et al., 2008; Trojan-Rodrigues et al., 2011).

One of the researchers groups, Sayed et al. (2011) conducted a bio-efficacy trial to investigate the hypoglycemic potential of mango polyphenols especially mangiferin. During experiment, they induce hyperglycemia in male albino rats by streptozotocin injection. The rats were treated with mangiferin water extract for two months and observed 42.02 to 51.08% reduction in glucose during two months study. Moreover, mangiferin administration caused marked enhancement in the insulin secretion i.e. 32.66%. They were on the view that mangiferin imparts positive impact on fat and carbohydrate metabolism, regulates the insulin transporters and ultimately manages the reduction in glucose. Likewise, Prasad et al. (2008) observed the blood glucose lowering

effect of mango peel extract in streptozotocin-diabetic rats. Purposely, rats were administrated with mangiferin dose of 250 mg/kg BW for four weeks and observed a significant decline in glucose concentration i.e. 17.55%. Similarly, Miura et al. (2001b) recorded a significant fall in blood glucose of experimental mice after ingestion of mangiferin @ 30 mg/kg BW for 3 weeks.

In a scientific exploration, (Arrowood, 1997) conducted a model feeding trial to evaluate the glucose lowering potential of mangiferin. The different streptozotocin doses were utilized to induce diabetes in Sprague Dawley rats. The diabetic rats were treated simultaneously with 30, 50 and 70 mg aqueous mango extract. The outcomes of study indicated a momentous dose dependent reduction in blood glucose level of rats. One of their peers, Dinesh-Kumar et al. (2010) noticed similar dose dependent glucose reduction in diabetic volunteers after consuming mangiferin @ 10 and 30 mg/kg BW.

Mangiferin has ability to inhibit the activity of intracellular enzyme tyrosine phosphatase 1B (PTP1B) and regulates the transactivation of peroxisome proliferator activated receptor isoforms (PPARs), consequently enhances the insulin sensitivity (Rau et al., 2006; Hu et al., 2007). The PTP1B acts as negative regulator of insulin signaling pathways (Aoki and Matsuda, 2000). In this context, previous investigations of Hu et al. (2007) elucidated that mangiferin and its derivatives have inhibitory effect on PTP1B protein expression.

The PPARα is a recognized regulator of fatty acid metabolism and often utilized in the treatment of hypertriglyceridemia (Berger et al., 2005a). Moreover, PPAR nuclear steroid hormone receptor also modulates glucose and lipid metabolism (Michalik et al., 2006). The reduction in the activity of PPAR is a key step to alleviate abnormalities in fatty acid and glucose metabolism. The research findings of Berger et al. (2005) and Wilkinson et al. (2008) affirmed that mangiferin and other mango polyphenolic compounds impede the activation of all three PPAR isoforms thus manage the abnormalities of fat and glucose metabolism, characteristic feature in type II diabetes. Inhibiting the activity of glucosidase is one of the mechanistic routes by which mango polyphenols tackle the peril of type II diabetes. In this regard, Prashanth et al. (2001) conducted a trial to validate the α-glucosidase inhibitory activity of mango polyphenols. They feed the experimental rats with ethanolic extract of mango peel rich in mangiferin and noticed a strong reduction in the concentration of enzyme. Likewise, Yoshikawa et al. (2001) also evaluated the

inhibitory action of mangiferin against glucosidase enzyme. The suppression in the activity of the enzyme causes reduction in carbohydrate breakdown and subsequently slower glucose absorption from the intestine.

Numerous evidences suggest that both pancreatic and extra pancreatic mechanisms might be involved in the management of anti-diabetic or hypoglycemic action. According to the Randle’s glucose-fatty acid cycle, increased supply of plasma triglycerides could constitute a source of increased free fatty acid availability and oxidation that can impair insulin action, glucose metabolism and utilization leading to the development of hyperglycemia. Therefore reduction of triglycerides with mangiferin facilitates glucose oxidation & utilization and subsequently ameliorates hyperglycemia (Muruganandan et al., 2005). The mangiferin exerts anti-diabetic properties by decreasing insulin resistance in non-insulin dependent KK/Ay mice. The chronic administration of mangiferin significantly improves the oral glucose tolerance in glucose fed rats (Miura et al., 2001; Murruganandan et al., 2005).

Streptozotocin induction decreases the nicotinamide-adenine dinucleotide (NAD) level in β-cells, causing histopathological defragmentation that initiates diabetes. In this phase, food & water consumption, urine excretion and glucose concentration are increased alongside decline in the level of insulin and C-peptide protein. The destruction of β-cells is the chief event responsible for these malfunctionings (Akbarzadeh et al., 2007). However, mangiferin provision regulates the structural and functional irregularities owing to its strong antioxidative, immunomodulatory and anti-inflammatory perspectives.

Free radicals initiate the cascade of adverse reactions in diabetic phase that modifies the glucose functionality resulting in cellular injury, nonetheless antioxidant treatment like mangiferin modulates the glucose metabolism, preserves insulin utilization and improves insulin secretion. Moreover, its ability to reduce atherogenic index is also helpful to alleviate glucose malfunctionings (Muruganandan et al., 2005; Ichiki et al., 1998).

Recently, Sarkar et al. (2004) carried out a bio-efficacy trial involving streptozotocin induced diabetic rats to evaluate the hypoglycemic response of mangiferin in four weeks trial. The mangiferin feeding at a dose of 40 mg/kg BW resulting 64.53% decline in rats glucose level. Moreover, insulin level was enhanced by 3 fold from 4.95±0.33 to 12.78±0.88. Previously, Sarkar et al. (2004) observed an increase in insulin level from 5.04±0.29 to 14.58±0.65 in diabetic rats treated with mangiferin. Later, Han et al. (2010) 28

noticed 23.37% reduction in glucose level of streptozotocin treated rats using mango polyphenols @ 300 mg/kg BW for a period of 14 days. Outcomes of different scientific explorations like Gouado et al. (2007) and Brakan et al. (2003) elucidated the effectiveness of mango peel polyphenols against insulin resistance. They observed improvement in insulin level by 33.18 and 35.43% in diabetic rats after taking 300 mg/kg BW dose of mangiferin at 14 and 21 days, respectively. Similarly, Scalbert and Williamson, (2007) also noticed 47.29% decline in glucose level of diabetic rats due to mango peel polyphenols treatment.

CHAPTER 3

MATERIALS AND METHODS

The present research project was carried out in the Functional and Nutraceutical Food Research Section, National Institute of Food Science and Technology (NIFSAT), University of Agriculture, Faisalabad. The in vitro antioxidant assay was conducted in NIFSAT and Department of Human Nutrition, University of Glasgow, Scotland, UK. In the current investigation, mango peel was subjected to characterization, antioxidant potential estimation and mangiferin isolation. Moreover, mango peel extract and mangiferin based functional/nutraceutical drinks were developed and evaluated against selected lifestyle related disorders. Materials used and protocols followed are described here in;

3.1. Materials

Different mango varieties i.e. Chaunsa, Anwar ratol, Langra, Dusehri and Desi were purchased from the local fruit market. The selected varieties were subjected to washing followed by peeling in the Canning Hall at NIFSAT. Afterwards, the separated peels of each variety were oven dried at 60 °C for 1 hr and ground to form respective powder. The analytical and HPLC grade standards were purchased from Merck (Merck KGaA, Darmstadt, Germany) and Sigma-Aldrich (Sigma-Aldrich Tokyo, Japan). For efficacy trial, male Sprague Dawley rats were housed in the Animal Room of NIFSAT. For biological assay, diagnostic kits were obtained from Sigma-Aldrich, Bioassay (Bioassays Chemical Co. Germany) and Cayman Chemicals (Cayman Europe, Estonia).

3.2. Characterization of mango peel

Initially, the mango peel samples were examined for various quality traits including proximate & mineral analysis, polyphenols estimation and mangiferin quantification. The procedures followed are given below;

3.2.1. Proximate analysis

The respective peel samples were evaluated for moisture, crude protein, crude fat, crude fiber, ash and nitrogen free extract (NFE) and results are expressed on fresh weight basis.

3.2.1.1. Moisture content

The moisture content in the mango peel was determined by drying sample in an air forced draft oven (Model: DO-1-30/02, PCSIR, Pakistan) by keeping temperature at 105±5 °C till constant weight according to the guidelines of AACC (2000).

3.2.1.2. Crude protein

The percentage of crude protein was estimated through Kjeltech Apparatus (Model: D- 40599, Behr Labor Technik, Gmbh-Germany) by adopting the protocol of AACC (2000).

Initially, sample was digested with conc. H2SO4 and digestion mixture for 6 hr till light greenish color. Afterwards, 250 mL dilution of digested sample was made. The diluted sample was distilled by taking 10 mL of sample and 10 mL of 40% NaOH solution in the distillation assembly. The liberated ammonia was trapped in 2% boric acid solution. Lastly, distillate was titrated against 0.1 N H2SO4 till golden brown end point.

3.2.1.3. Crude fat

Crude fat contents in peel samples were estimated using hexane as solvent in Soxtec System (Model: H-2 1045 Extraction Unit, Hoganas, Sweden) as described in AACC (2000).

3.2.1.4. Crude fiber

The mango peel were subjected to crude fiber content determination by digesting the fat free samples in 1.25% H2SO4 followed by 1.25% NaOH using Labconco Fibertech (Labconco Corporation Kansas, USA) following the protocol of AACC (2000).

3.2.1.5. Total ash

Total ash was estimated by direct incineration of dried sample in a Muffle Furnace (MF- 1/02, PCSIR, Pakistan) at 550°C after charring till grayish white residue by adopting the mentioned protocol of AACC (2000).

3.2.1.6. Nitrogen free extract (NFE)

The nitrogen free extract was calculated according to the expression given below

NFE % = 100 – (crude protein % + crude fat % + crude fiber % + ash %)

3.2.2. Mineral determination

The mango peel samples were subjected to mineral composition following the method of AOAC (2006). Minerals like calcium, magnesium, zinc, iron and phosphorous were estimated by Atomic Absorption Spectrophotometer (Varian AA240, Australia), while sodium and potassium were determined through Flame Photometer-410 (Sherwood Scientific Ltd., Cambridge).

3.2.3. Preparation of antioxidant extracts

During the extraction of antioxidants from peel powder samples various solvents such as acetone, ethanol and water were used to assess their extraction efficiency (Table 1). Purposely, prepared samples were subjected to orbital shaker for 7 hr followed by centrifugation (15 min) at 7000 rpm. The resultant extracts were filtered using vacuum filtration assembly and solvents were recovered by Rotary Evaporator (EYELA, N-N series, Japan) at 40°C (Rusak et al., 2008). The extracts were evaluated for various antioxidant assays including total phenolic contents (TPC), β-carotene antioxidant activity, free radical scavenging activity by DPPH (1,1-diphenyl-2-picrylhydrazyl) and ferric reducing antioxidant power (FRAP) as discussed below.

3.2.3.1. Total phenolic content (TPC)

Total phenolic contents in the resultant extracts were estimated by Folin-Ciocalteu method (Singleton et al., 1999). Accordingly, 125 µL sample was taken in a test tube followed by the addition of 500 µL distilled water and 125 µL of Folin-Ciocalteu reagent. Afterwards, 1.25 mL of 7% sodium carbonate was further added. Final volume upto 3 mL solution was made by adding distilled water and allowed to stand for 90 min. The absorbance of the antioxidant extracts was measured at 765 nm using UV-vis spectrophotometer (CECIL CE7200). Calibration/standard curve for gallic acid was drawn with concentrations of 0.05, 0.10, 0.15, 0.20, 0.25 and 0.30 mg/mL.

C=c×V/m C = total content of phenolic compounds in mg/g plant extract, in GAE c = the concentration of gallic acid calculated from the calibration curve in mg/mL V = the volume of extract in mL m = the weight of plant methanolic extract in g

Table 1. Treatments used for estimation of extraction efficiency Treatments Mango peel varieties Solvents

T1 Chaunsa Water

T2 Dusehri Water

T3 Desi Water

T4 Langhra Water

T5 Anwar Ratool Water

T6 Chaunsa Ethanol

T7 Dusehri Ethanol

T8 Desi Ethanol

T9 Langhra Ethanol

T10 Anwar Ratool Ethanol

T11 Chaunsa Acetone

T12 Dusehri Acetone

T13 Desi Acetone

T14 Langhra Acetone

T15 Anwar Ratool Acetone

3.2.3.2. Antioxidant activity

Antioxidant activity of the resultant extracts were evaluated using assay based on coupled oxidation of β -carotene and linoleic acid following the protocol of Taga et al. (1984). For the purpose, ß-carotene (2 mg) was dissolved in 20 mL of chloroform. Later, 3 mL aliquot of the solution was placed in 50 mL beaker and added 40 mg linoleic acid and 400 mg Tween 20. Afterwards, chloroform was removed by purging with nitrogen. Oxidation of ß-carotene emulsion was monitored spectrophotometrically by measuring absorbance at 470 nm. The results was presented in percent inhibition using the following expression

In (a/b) × 1/t = sample degradation rate

In = natural log a = initial absorbance (470 nm) at time zero b = absorbance (470 nm) after 40 min t = time (min)

3.2.3.3. DPPH scavenging activity

The free radical scavenging activity of mango peel extracts was determined according to the method of Brand-williams et al. (1995). For experimental, fresh methanolic solution of DPPH (1,1-diphenyl-2-picrylhdrazyl) was prepared before assay. Various concentrations of each sample (40, 80, 120, 160, 200 and 240 μg/mL) were added to 1 mL DPPH solution. The reaction mixtures were shaken gently and allowed to stand for 30 min at ambient temperature. The absorbance of the samples was measured at 520 nm by spectrophotometer.

Reduction of absorbance (%) = [(AB - AA) / AB] × 100

AB = absorbance of blank sample (t = 0 min) AA = absorbance of tested extract solution (t = 15 min)

3.2.3.4. Ferric reducing antioxidant power

Ferric reducing antioxidant power of extracts was estimated by adapting the protocol of Sun et al. (2010). The peel extract (0.5 mL) was mixed with phosphate buffer (1.25 mL, 0.2 M, pH 6.6) and potassium ferricyanide (1.25 mL, 1%). After incubation, 10% TCA (1.25 mL) along with 0.1% ferric chloride were added in the mixture and then left at room temperature for 10 min. Sample absorbance was measured at 700 nm.

3.3. Isolation of mangiferin

Mangiferin from mango peel extracts (Table 1) was isolated through solvent partition method following the guidelines of Mitra (2007). Purposely, the resultant extracts were first treated with petroleum ether for 6 hr to remove fatty matter followed by cold acetone extraction (24 hr) that separated the tannins. Lastly, the samples were subjected to 70% ethanol extraction (6 hr) for mangiferin isolation. The collected ethanolic fractions that contained mangiferin were concentrated under reduced pressure using freeze drier (CHIRST, Alpha 1-4 LD plus, Germany) and mangiferin was separated as a yellow color powder (Figure 1).

3.3.1. Characterization

Isolated mangiferin was quantified through HPLC (PerkinElmer, Series 200, USA) using UV-visible detector (Model: HP 481) and C18 column (250 mm x 4.6 mm, 5.0 μm particle size). A 10 µL aliquot of sample was taken through autosampler (WISP Model 710) and maintained the column temperature 40 oC throughout the analysis. During mangiferin quantification, mobile phase was composed of acetonitrile and 0.05% acetic acid in ratio of 90:10. The flow rate was maintained at 1 mL/min followed by quantification with UV/vis detector at wavelength ranged from 278 to 370 nm (Jacqueline et al., 2008).

3.3.2. Mangiferin analysis

Antioxidant profiling of mangiferin including free radical scavenging activity, β- carotene & linoleic acid and ferric reducing antioxidant potential were carried out by respective methods (Muller et al., 2011; Taga et al., 1984; Sun et al., 2010).

3.4. Selection of best treatment

On the basis of extraction efficiency, HPLC characterization and in vitro tests, one best treatment of peel extracts along with mangiferin were selected for the development of nutraceutical/functional drinks.

Mango peel powder (50g) Mango peel powder (50g)

Distilled water (150mL) Solvents (150mL)

Extraction through orbital shaker Extraction through orbital shaker

Filtration Concentration through rotary evaporator

Partition with boiling petroleum ether

Petroleum ether layer Water layer

Cold acetone treatment

Cold acetone layer Water layer

Partition with 70% ethanol

Water layer Ethanol layer

Concentrated through rotary evaporator

Freeze drying

Mangiferin

Figure 1. Flow diagram for mangiferin extraction

3.5. Preparation of nutraceutical/functional drink

During this segment, three types of nutraceutical/functional drinks were prepared, first sample (T1) contains mango peel extract while other supplemented with mangiferin (T2) alongside control (T0) for comparison purpose. All the treatments had same recipe except the presence of specific functional ingredient (Table 2). The main ingredients used for the preparation of functional drinks were aspartame, citric acid, sodium benzoate, carboxy methyl cellulose (CMC), food grade color and flavor. After processing of drinks, active ingredients including whole mango peel extract and isolated mangiferin were added individually @ 300 mg/500 mL in respective drinks considering stability of the ingredients.

Table 2. Treatments used for the preparation of nutraceutical/functional drink Treatments Description

T0 Control

T1 Drink containing mango peel extract

T2 Drink containing mangiferin

3.5.1. Physicochemical analysis of functional drinks

Functional drinks were analyzed for their total soluble solids, pH and acidity during two month storage at specific intervals i.e. 0, 30 and 60 days.

3.5.1.1. Total soluble solids

Total soluble solids of functional/nutraceutical drinks were determined by hand refractometer (TAMCO, Model No. 90021, Japan) and results were expressed as °Brix as mentioned in AOAC (2006).

3.5.1.2. pH

Functional drinks were taken in 50 mL beakers and pH was evaluated directly though calibrated pH meter (InoLab 720, Germany) adopting the guidelines of AOAC (2006).

3.6.1.3. Total acidity

Total acidity of functional drinks was estimated by titrating drink samples against 0.1N sodium hydroxide solution to persistent pink color following the protocols of AOAC (2006).

3.6. Sensory evaluation

The prepared functional/nutraceutical drinks (T0, T1, T2) were evaluated for sensory response using 9-points hedonic scale ranged from extremely liking to disliking (9 = like extremely; 1 = dislike extremely) as mentioned in Appendix-I, following the guidelines of Meilgaard et al. (2007). The sensory behavior of functional drinks for various characteristics like color, flavor, sweetness, sourness and overall acceptability was assessed on monthly basis during storage. The sensory profiling was performed in the Sensory Evaluation Laboratory at NIFSAT, University of Agriculture Faisalabad. Each panelist was provided with written instructions to award scores for the developed drinks. During hedonic evaluation, panelists were seated in separate booths equipped with white fluorescent light and drinks were presented in polystyrene cups (40 mL in 120 mL cup, topped with transparent vented lid) labeled with random codes at room temperature. For enhancing the accuracy, evaluators were provided water and unsalted crackers to neutralize their mouth feel between samples testing. Samples were presented to the judges randomly to avoid any biasness and asked to rate their acceptance by assigning score for selected parameters. Moreover, panelists names were concealed to maintain secrecy.

3.7. In vivo studies

To investigate the therapeutic potential of developed functional drinks against lifestyle related disorders with special reference to hypercholesterolemia and hyperglycemia, a bioefficacy trial was conducted. For the purpose, 100 experimental rats were housed in the Animal Room of NIFSAT, University of Agriculture, Faisalabad. The rats were acclimatized by feeding basal diet for a period of one week. The environmental conditions as temperature (23±2 ºC) and humidity (55±5%) were maintained throughout the study duration with 12 hr light-dark period. At the initiation of study, some rats were sacrificed to establish a baseline trend. During efficacy trial, three independent studies were carried out involving normal, hyperglycemic and hypercholesterolemic rats (Table 3). Each study was comprised of 30 rats, divided in three equal groups, ten in each. In

study I, rats were fed on normal diet whilst, in study III diabetic rats [streptozotocin (STZ) @ 65 mg/kg body weight] were involved relied on normal diet. However in study II, high cholesterol diet was given to the rats. Accordingly, control, mango peel extract supplemented and mangiferin based drinks were given to the respective groups (Table 3). During 8 weeks of efficacy trial, instantaneous administration of functional drinks was assured to evaluate their therapeutic effects. Physical parameters like feed & drink intakes and body weight were also recorded. At the end of study, the overnight fasted rats were decapitated and blood was collected in EDTA coated tubes. Initially, blood samples were tested for different hematological characteristics like red & white blood cells indices. Later, the collected samples were subjected to centrifugation for serum separation. The respective sera from each group were analyzed for different biomarkers by Microlab 300 (Merck, Germany). The respective commercial kits were used for the estimation of various biochemical parameters like total cholesterol, LDL, HDL, triglycerides, glucose and insulin levels. Additionally, liver and kidney soundness tests were also performed to estimate the safety & renal modulating perspectives of functional drinks. The entire biological trial was repeated to validate the data.

The details of these studies are herein;

Table 3. Different studies conducted in efficacy trial

Study I Normal rats

Study II Hypercholesterolemic rats

Study III Diabetic rats

3.7.1. Study I: Normal rats

In this study, 30 rats were divided into three homogeneous groups fed on normal diet with simultaneous intake of nutraceutical/functional drinks. The experimental diet (Appendix II) comprised of corn oil (10%), protein (10%), corn starch (66%), cellulose (10%), mineral (3%) and vitamin mixture (1%).

Following similar approach, two other studies were conducted to determine the impact of functional drinks against respective disorders i.e. hypercholesterolemia and hyperglycemia in separate rodent modeling (Table 4).

Study II: Hypercholestrolemic rats

In study III, hypercholesterolemia in rats was induced by providing diet containing 1.5% cholesterol and cholic acid (0.5%) in order to raise their lipid profile marker. The periodic examination was conducted to assess the induction of hypercholesterolemia. The functional drinks were administrated to the rats simultaneously for evaluating their effect on the respective group.

Study III: Diabetic rats

Diabetes was induced in rats by a single intraperitoneal injection of streptozotocin (STZ) @ 65 mg/kg, dissolved in citrate buffer pH 4.5. Afterwards, respective functional drinks alongside normal diet were provided to the diabetic rats to evaluate their therapeutic role.

Table 4. Diets and functional drinks plan Study I Study II Study III Studies Normal rats Hypercholesterolemic rat Diabetic rats

Groups 1 2 3 1 2 3 1 2 3

Drinks T0 T1 T2 T0 T1 T2 T0 T1 T2

T0: Control T1: Drink containing mango peel extract T2: Drink containing mangiferin

3.7.2. Physical parameters

3.7.2.1. Feed and drink intakes

The net feed intake was recorded on daily basis by excluding spilled diet from the total diet. Likewise, functional drink intake of each rat was also measured daily by monitoring the differences in the graduated bottles (Wolf and Weidbrode, 2003).

3.7.2.2. Body weight gain

Weight gain of experimental rats was determined on weekly basis throughout the study period to evaluate any suppressing effect of functional drinks on the body weight.

3.7.3. Serum separation

For serum separation, blood samples were collected in commercially available red topped tubes. The respective samples were allowed to clot at room temperature for 30 min. Further, clotted part was removed after centrifugation through Centrifugal Machine (Model: 800, China) @ 4000 rpm for 6 min (Uchida et al., 2001; Adkins et al., 2002).

3.7.3.1. Serum lipid profile

Lipid related biomarkers including cholesterol, low density lipoproteins (LDL), high density lipoproteins (HDL) and triglycerides (TG) were estimated by their respective protocols using commercial kits.

3.7.3.2. Cholesterol

Serum cholesterol level was determined by using CHOD–PAP method following the guidelines of Kim et al. (2011).

3.7.3.3. High density lipoproteins

High density lipoprotein (HDL) was estimated by Cholesterol Precipitant method as elaborated by Alshatwi et al. (2010)

3.7.3.4. Low density lipoproteins

Low density lipoproteins (LDL) in sera samples were recorded following the protocols of Kim et al. (2011).

3.7.3.5. Triglycerides

Triglycerides level was measured by liquid triglycerides (GPO–PAP) method (Kim et al. 2011).

3.7.3.6. Hypoglycemic response

Hypoglycemic response of functional drinks was examined by measuring the serum glucose and insulin levels of rats. In each study, rats sera samples were monitored for glucose concentration by GOD-PAP method adopting the guidelines of Kim et al. (2011). Likewise, insulin level was estimated by following the protocol of Ahn et al. (2011).

3.7.3.7. Antioxidant status

Glutathione contents were determined following the instructions of Feng et al. (2011). The colored product of GSH + DTNB in the protein free supernatant was recorded at 412 nm and calculated as nmol/mg protein. Similarly, indicator of lipid peroxidation i.e. thiobarburic acid reactive species (TBARS) was also measured (Huang et al., 2011)

3.7.3.8. Liver and kidney functioning tests

Liver function tests including aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were performed. The levels of AST and ALT were assessed by the dinitrophenylhydrazene (DNPH) method using Sigma Kits 59-50 and 58-50, respectively whilst ALP by Alkaline Phosphates–DGKC method (Basuny, 2009). Moreover, the urea and creatinine concentrations were also recorded by GLDH and Jaffe-method, respectively using commercial kits (Jacobs et al. 1996; Thomas, 1998) to examine the renal functionality of tested rats groups.

3.7.3.9. Hematological aspects

Initially, the collected blood samples were analyzed for red and white blood cells indices. Accordingly, red blood cells indices comprised of total red blood cells (TRBCs), hemoglobin (Hb), hematocrit (Ht) and mean corpuscular volume (MCV) were estimated. Likewise, white blood cell profiling i.e. white blood cells, lymphocytes, monocytes and neutrophils was carried out through Automatic Blood Analyzer (Nihon Kohden, Japan) following the guidelines of Caduff et al. (2011).

3.7.3.10. Electrolytes balance

The electrolytes Like Na, K and Ca in the tested samples were estimated by following the directions of Al Haj et al. (2011).

3.8. Statistical Analysis

The collected data were subjected to statistical analysis using completely randomized design (CRD) through statistical software Cohort version 6.1 (Co Stat, 2003). Furthermore, analysis of variance (ANOVA) technique was applied to determine the level of significance (Steel et al., 1997).

CHAPTER 4

RESULTS AND DISCUSSION

Dietary phytonutrients provide protection against various metabolic disparities and improve the overall health status. In this milieu, mango peel is a potential source of bioactive moieties that has ability to ameliorate various lifestyle related disorders. In current study, different mango peels were analyzed for compositional & nutritional assay, antioxidant and mangiferin isolation & quantification. During product development, three types of functional/nutraceutical drinks were prepared by supplementation of whole mango peel extract and mangiferin alongside control. Lastly, the prepared functional drinks were evaluated against hypercholesterolemia and diabetes through a model feeding trial. The results and discussion of examined parameters are elaborated herein:

4.1. Proximate composition

Proximate composition is important to estimate the quality of raw material. Mean squares in Table 5 showed that protein, fiber and ash contents varied significantly in different peel samples however, non-significant variations were noticed for moisture, fat and NFE.

The means elucidated highest moisture in the peel of desi mango 71.38±2.05 followed by anwar ratol, chaunsa, langra, and dusahri as 71.01±3.91, 70.74±4.01, 69.86±5.20 and 68.33±4.14%, respectively. Moreover, protein contents were recorded as 2.36±0.01, 2.25±0.05, 2.20±0.04, 2.06±0.05 and 1.94±0.04% in anwar ratol, chaunsa, langra, dusahri and desi, correspondingly. Similarly, fat and fiber contents in respective varieties were 2.31±0.14 & 5.01±0.25, 2.26±0.10 & 5.47±0.31, 2.25±0.17 & 4.88±0.12, 2.18±0.18 & 4.69±0.17 and 2.11±0.12 & 4.53±0.18%. Besides, the ash contents ranged from 2.59±0.03 (chaunsa) to 1.84±0.02% (desi), respectively. Likewise, the recorded NFE values for respective samples were 87.87±6.87, 87.60±3.41, 88.86±5.20, 89.09±3.85 & 89.58±2.89, respectively (Table 6).

The results of present investigation are in accordance with the previous findings of Ajila et al. (2007b). They carried out proximate profiling of different mango peels and observed moisture, protein, fat, fiber and ash contents in the range of 66-75, 1.76-2.05, 2.16-2.66, 3.28-7.40, and 1.16-3.0%, respectively. Similarly, Ojokoh (2008) recorded the values for crude fat, crude protein and dietary fiber by 5.1, 6.16 and 11.2%, respectively.

Table 5. Means squares for proximate composition of different mango peels

SOV df Moisture Protein Fat Fiber Ash NFE

Varieties 4 3.4841NS 0.07977* 0.03024 NS 0.38481* 0.25745* 2.21124 NS

Error 10 3.46316 0.01373 0.01526 0.05116 0.06568 0.25634

NS=Non-significant *=Significant

Table 6. Proximate composition of different mango peels

Parameters Chaunsa Anwar ratol Langra Dusahri Desi

Moisture 70.74±4.01 71.01±3.91 69.86±5.20 68.33±4.14 71.38±2.05

Protein 2.25±0.05a 2.36±0.01a 2.20±0.04ab 2.06±0.05b 1.94±0.04c

Fat 2.31±0.14 2.26±0.10 2.25±0.17 2.18±0.18 2.11±0.12

Fiber 5.01±0.25a 5.47±0.31a 4.88±0.12ab 4.69±0.17b 4.53±0.18c

Ash 2.59±0.03a 2.31±0.02ab 2.21±0.19b 1.98±0.12c 1.84±0.02d

NFE 87.84±6.87 87.60±3.41 88.86±5.20 89.09±3.85 89.58±2.89

The variations in the proximate composition of different peel samples are due to varietal differences, climatic conditions, topographic locations and agronomic practices (Granfeldt et al., 1992; Palafox-Carlos et al., 2010). Earlier, Zein et al. (2005) reported 77, 2, 6, 11 and 2% moisture, crude fiber, protein, fat and ash, respectively in mango peels. Likewise, Prasad et al. (2007) characterized mango peel and noticed protein 1.76%, fiber 7.4%, moisture 75.25%, fat 2.66% and ash 1.30%.

Previously, Ashoush et al. (2011) estimated the ash, fat, protein and crude fiber contents of mango peel powder by 3.88, 1.23, 3.6 and 9.33%, respectively. Likewise, Chau and Huang (2003) observed 2.24, 2.82, 10.35 & 4.23% fat, protein, fiber & ash respectively, in mango peel powder.

4.2. Mineral analysis

Mean squares explicated significant variations in the mineral contents of different mango peel samples (Table 7).

In the present case, the highest K content was observed in chaunsa (18.78±1.26 mg/100g) followed by desi (18.76±0.96 mg/100g), anwar ratol (17.73±1.21 mg/100g), dusahri (17.16±1.02 mg/100g) and langra (16.21±1.12 mg/100g). Similarly, Mg and Ca were recorded as 56.11±4.21 & 87.46±6.32, 54.73±3.69 & 82.72±4.18, 52.54±1.16 & 79.81±3.85, 50.25±1.52 & 75.08±4.10 and 56.83±2.32 & 78.39±5.02 mg/100g in chaunsa, anwar ratol, langra, dusahri and desi mango peels, respectively. Likewise, Na and Cr contents were 18.07±0.85 & 0.273±0.005, 17.77±1.41 & 0.263±0.006, 17.23±1.11 & 0.261±0.001, 16.50±1.01 & 0.256±0.003 and 17.02±0.96 & 0.258±0.004 mg/100g in respective peel samples, correspondingly. Moreover, maximum Cu content (0.076±0.002 mg/100g) was noticed in anwar ratol followed by desi (0.069±0.001 mg/100g), langra (0.066±0.006 mg/100g) and dusahri (0.063±0.002 mg/100g), whilst minimum (0.056±0.003 mg/100g) in chaunsa. Additionally, Fe and Mn contents were 8.826±0.25 & 0.043±0.005, 9.596±0.16 & 0.030±0.004, 7.896±0.21 & 0.026±0.006, 5.346±0.31 & 0.046±0.001 and 6.510±0.21 & 0.033±0.003 mg/100g in the respective peel samples (Table 8). The values regarding mineral composition in the instant research are in line with the earlier findings of Peter et al. (2007) and Gopalan et al. (1999), they explored the ripend mango for calcium, phosphorous and potassium contents and observed variations from 145 to 160, 26 to 35, and 180 to 211 mg/100 g, respectively.

Table 7. Means squares for mineral contents of different mango peels

SOV df K Mg Ca Na

Varieties 4 0.07977* 0.03024* 3.48413** 0.38481*

Error 10 0.01373 0.01526 3.46316 0.05116

SOV df Cr Cu Fe Mn

Varieties 4 0.00749* 8.900* 8.87908** 2.233*

Error 10 0.00571 4.733 0.26019 4.867

*=Significant **= Highly significant

Table 8. Mineral profiling of different mango peels (mg/100g)

Minerals Chaunsa Anwar ratol Langra Dusahri Desi

K 18.78±1.26a 17.73±1.21ab 16.21±1.12c 17.16±1.02b 18.76±0.96a

Mg 56.11±4.21a 54.73±3.69b 52.54±1.16bc 50.25±1.52c 56.83±2.32a

Ca 87.46±6.32a 82.72±4.18ab 79.81±3.85bc 75.08±4.10c 78.39±5.02b

Na 18.07±0.85a 17.77±1.41ab 17.23±1.11b 16.50±1.01d 17.02±0.96c

Cr 0.273±0.005a 0.263±0.006ab 0.261±0.001b 0.256±0.003c 0.258±0.004c

Cu 0.056±0.003d 0.076±0.002a 0.066±0.006ab 0.063±0.002b 0.069±0.001c

Fe 8.826±0.25ab 9.596±0.16a 7.896±0.21b 5.346±0.31d 6.510±0.21c

Mn 0.043±0.005a 0.030±0.004b 0.026±0.006c 0.046±0.001a 0.033±0.003b

Likewise, Burns et al. (2003) also recorded calcium, phosphorous and potassium in mango peel that ranged from 153 to 167, 31 to 41 and 194 to 217 mg/100 g, respectively. In another study, Mahdavian and Somashekar (2008) reported Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn by 2.14, 85.71, 14.22, 189.31, 39.31, 14.06, 9.52 and 32.67 ug/g dry weight of mango, respectively. The results of present exploration concerning sodium, potassium and calcium are in harmony with the work of Akhtar et al. (2010), examined Na, K and Ca contents of dusahri mango peel and compiled the values as 6.33, 38.48 and 7.18 mg/100g, for respective minerals. The compositional variations among different peel samples regarding proximate and mineral assays are possibly due to varietal differences, soil type, environmental & climatic conditions and fruit maturity stage.

4.3. Antioxidant extracts

Mean squares elucidated that antioxidant indices of mango peel extracts significantly affected by treatments and solvents however, their interaction was non-momentous (Table 9).

The means for mango peel varieties (Table 10) showed that the highest TPC 75.35±3.96 mg/100g GAE was observed in chaunsa peel followed by 68.34±2.85 mg/100g GAE anwar ratol, 64.95±1.89 mg/100g GAE langra, 61.89±3.85 mg/100g GAE dusahri and the lowest output 58.85±2.45 mg/100 g GAE in desi mango peel. Means regarding solvents exposed the maximum TPC in ethanol 80.17±5.16 mg/100g GAE followed by acetone 62.28±3.12 mg/100g GAE and water extract 55.17±2.41 mg/100g GAE.

Likewise, chaunsa peel exhibited the highest DPPH activity 59.28±3.69% than that of anwar ratol 57.49±1.48%, langra 53.34±2.98%, dusahri 53.20±1.45% and desi 49.64±2.74%. The mean values for solvents showed maximum DPPH activity in ethanolic extract 60.42±2.41% followed by acetone 56.36±3.69% and water 46.98±2.78% (Table 11). The antioxidant activity β-carotene% and FRAP values for different mango peels i.e. chaunsa, anwar ratol, langra, dusahri & desi were 57.33±4.14% & 8.88±0.62 mmol/100g, 54.45±3.96% & 7.80±0.45 mmol/100g, 49.12±3.10% & 7.54±0.25 mmol/100g, 48.14±1.18% & 7.12±0.32 mmol/100g and 45.38±2.50% & 6.24±0.29 mmol/100g. Likewise, ethanolic extract had the maximum β-carotene value 58.59±3.69% followed by acetone 51.98±2.11% and water extract 42.08±1.85%. In contrary, highest

Table 9. Means squares for antioxidant indices of mango peel extracts

SOV df TPC DPPH ß-carotene FRAP

Treatments (A) 4 624.21** 337.971** 588.339** 18.2696*

Solvent (B) 2 2486.01** 743.764** 409.828** 0.7085**

A x B 8 13.15 NS 7.087NS 8.956 NS 0.4888 NS

Error 30 1.00 1.452 0.0380 2.35

NS=Non- significant **=Highly significant

Table 10. Total phenolic contents (mg/100g GAE) of peel extracts

Parameters Ethanol Acetone Water Mean

Chaunsa 87.67±4.12 71.25±3.69 67.12±2.98 75.35±3.96a

Anwar ratol 81.09±4.85 64.29±3.01 59.63±3.41 68.34±2.85b

Langra 79.52±4.45 60.96±3.14 54.36±2.65 64.95±1.89c

Dusahri 77.28±4.10 58.86±3.29 49.52±2.91 61.89±3.85cd

Desi 75.28±4.29 56.03±3.21 45.25±2.25 58.85±2.45d

Mean 80.17±5.16a 62.28±3.12b 55.17±2.41c

Table 11. Free radical scavenging (DPPH %) activity of peel extracts Parameters Ethanol Acetone Water Mean

Chaunsa 65.23±4.11 60.25±4.23 52.36±2.12 59.28±3.69a

Anwar ratol 62.25±3.32 60.63±3.18 49.58±3.30 57.49±1.48ab

Langra 60.96±3.20 54.36±3.01 44.69±2.14 53.34±2.98b

Dusahri 58.32±2.45 54.69±2.15 46.58±2.89 53.20±1.45b

Desi 55.35±2.25 51.89±3.29 41.69±1.35 49.64±2.74c

Mean 60.42±2.41a 56.36±3.69b 46.98±2.78c

FRAP activity was noticed in acetone 7.88±0.19 mmol/100g followed by ethanol 7.52±0.12 mmol/100g and water extract 7.13±0.21 mmol/100g (Table 12 & 13).

The results regarding TPC contents in the current exploration are comparable with the findings of Kim et al. (2007). They examined the antioxidant capacity of ripend and unripend peels of different mango cultivars through total phenolics estimation. They observed higher TPC 90-110 mg/g GAE in ripend peels as compared to raw 55-85 mg/g GAE on dry basis. They further expressed that TPC contents were affected by maturity stage, cultivar type and agronomic practices. Likewise, Barretto et al. (2008) investigated the polyphenolic concentration in the peels of different mango cultivars including Embrapa-141-Roxa, Fafa, Van Dyke, Tommy Atkins, Amrapali & Kent and noticed 24.24, 52.28, 59.09, 25.13, 18.12 and 91.21 g/kg TPC values, respectively. One of the researchers groups, Nithitanakool et al. (2009) examined the antioxidant potential of mango peel and pomace. Purposely, they conducted polyphenolic estimation and detected higher value in peel 98.3 mg GAE/g as compared to pomace 68.8 mg GAE/g.

Previously, Ajila et al. (2007b) compared different solvents like acetone, ethanol and water for total phenolic contents of mango peel. They were of the view that ethanol and acetone are more efficient than water due to their polarity differences. The recorded polyphenols in ethanol, acetone and water were 92.62, 90.02 and 55.05 mg GAE/g, respectively. Likewise in another experiment, Ajila et al. (2007b) explicated that acetone exhibits better affinity for mango polyphenol extraction than water and recorded 54.67, 90.18, 100.00 and 109.70 mg/g TPC for badami ripe, badami raw, raspuri ripe and raw, correspondingly.

Earlier, Larrauri et al. (1996) probed raw and ripend peels of hayden variety for their total phenolic contents. They used aqueous methanol for antioxidant extraction and then subjected to Folin-Ciocalteu assay. They observed higher TPC contents 70 mg GAE/g in ripend peel as compared to 55 mg GAE/g for raw peel. Likewise, Ueda et al. (2000) concluded that peel is a better source of antioxidant than pulp. Similarly, Jung et al. (2008) and Liu et al. (2008) reported a correlation between antioxidant activity and type of solvent for mango polyphenols extraction. The results concerning DPPH activity in instant study are in agreement with the outcomes of Ayala-Zavala et al. (2010). They evaluated the effect of different concentrations on DPPH activity of mango peel. They were of the view that polyphenol concentration has linear association with DPPH activity

Table 12. Antioxidant activity (β-carotene %) of peel extracts Parameters Ethanol Acetone Water Mean

Chaunsa 63.59±5.01 59.68±4.23 48.71±2.12 57.33±4.14a

Anwar ratol 61.68±4.30 57.12±3.01 44.56±2.45 54.45±3.96b

Langra 57.25±2.25 49.16±3.01 40.96±2.24 49.12±3.10c

Dusahri 55.48±2.48 48.26±2.05 40.69±1.87 48.14±1.18c

Desi 54.96±1.78 45.68±1.15 35.51±0.96 45.38±2.50d

Mean 58.59±3.69a 51.98±2.11b 42.08±1.85c

Table 13. Ferric reducing antioxidant power (FRAP mmol/100g) of peel extracts

Parameters Ethanol Acetone Water Mean

Chaunsa 8.96±0.42 9.63±0.21 8.05±0.12 8.88±0.62a

Anwar ratol 7.89±0.52 8.25±0.14 7.25±0.19 7.80±0.45b

Langra 7.51±0.17 7.69±0.31 7.42±0.18 7.54±0.25c

Dusahri 7.02±0.21 7.39±0.05 6.96±0.21 7.12±0.32d

Desi 6.25±0.11 6.48±0.15 5.98±0.25 6.24±0.29e

Mean 7.52±0.12b 7.88±0.19a 7.13±0.21c

and observed 67.97% free radical inhibition at highest polyphenolic concentration (322 mg/mL). Previously, Ribeiro et al. (2008) observed that mango peel has higher free radical scavenging activity 53.3% than that of seed 24.2%.

Later, Kim et al. (2010) investigated the effect of polyphenolic concentration on free radical scavenging activity. They varied the concentration of mango peel extracts containing bioactive moieties from 12.5 to 50 ug/ml and observed a linear association between DPPH activity and polyphenolic concentration as 1.72 to 92.57 and 3.99 to 81.86% in unripend and ripend mango peel, respectively. Among the various mechanistic routes regarding antioxidant action of mango polyphenols, single electron transfer activity is promising due to its ability to donate one electron thus reduces metals, carbonyls and free radicals moreover, hydrogen atom transfer also helps to quench free radicals through hydrogen donation (Wright et al., 2001).

The instant results regarding FRAP activity of mango peel are in accordance with the conclusions of Guo et al. (2003), evaluated ferric reducing antioxidant power of various mango byproducts i.e. peel & pulp. They observed higher FRAP value for peel 10.13 mmol/100g as compared to pulp 0.38 mmol/100g on wet weight basis. Different scientific groups like Scalzo et al. (2005) and Torunn et al. (2009) documented the higher FRAP activity of mango peel than papaya and lemon peels. The higher ferric reducing activity of mango polyphenols is due to their ability to chelate metal ions and free radicals. Likewise, Berardini et al. (2004) evaluated the FRAP activity of mango peel water and ethanolic extracts. They noticed higher activity in ethanolic extract 436 µmolTrolox/100g in comparison to water 361 µmolTrolox/100g. In another study, Kawpoomhae et al. (2010) observed higher ferric reducing antioxidant power in ethanolic extract. Recently, Joona et al. (2013) noticed the FRAP activity in methanol extract of mango leaves as 0.85 ug/mL. Moreover, mango byproducts i.e. peel, seed, stem and bark possess high antioxidant activity in term of β-carotene that ranged from 42 to 71% (Scalzo et al. 2005).

From the above discussion, it is inferred that antioxidant indices of mango peel are influenced by the type of solvent and variety. Conclusively, all tested extracts exhibited good antioxidant ability however, ethanolic extract showed better performance as compared to acetone and water extracts. Amongst various mango peels, chaunsa showed better performance regarding polyphenol profile.

4.4. HPLC characterization of mangiferin

Mean squares in Table 14 showed significant effect of treatments and solvents on the mangiferin concentration. However, their interactive effect exhibited non-momentous variations.

The means for treatments indicated highest mangiferin in chaunsa 5.53±0.31 mg/g trailed by anwar ratol 4.88±0.30 mg/g, langra 4.09±0.18 mg/g and dusahri 3.97±0.23 mg/g whilst lowest value 3.19±0.17 mg/g was observed in desi mango peel. Considering the solvents, the mean mangiferin values for ethanol, acetone and water extracts were 5.25±0.01, 4.29±0.19 and 3.44±0.21 mg/g, respectively (Table 15).

The current results are synchronized with the explorations of Luo et al. (2012), they probed mango peels of different varieties i.e. LPM, ZHM and XH-2 for their mangiferin contents through high performance liquid chromatography. For the purpose, they utilized reverse phase HPLC with C18 column and noticed maximum mangiferin concentration in LPM (7.49 mg/g) than that of ZHM (7.34 mg/g) and XH-2 (1.04 mg/g) peels. In another research, Reibero et al. (2008) carried out HPLC quantification of mangiferin in four Brazilian mango varieties and detected the range 1.05 to 12.4 mg/kg. One of the researchers groups, Manthey et al. (2009) observed mangiferin as 227.4–996.1 μg/g in mango puree of Mexico Ataulfo cultivar. They inferred that mangiferin concentration depends upon the cultivar, harvesting season, maturity stage, agro climatic variations etc.

Afterwards, Jutiviboonsuk and Sardsaengjun, (2010) quantified mangiferin from the peels of different Thai mango varieties including Nam Doc Mai, KeowSavoey and Gaew. They carried out HPLC quantification of methanol, ethanol and acetone extracts for mangiferin concentration and noticed the highest mangiferin (2.80 g/100g) in Nam Doc Mai followed by KeowSavoey (2.40 g/100g) and Gaew variety (1.30 g/100g). Regarding solvents, acetone showed the highest mangiferin concentration 0.66 g/100g as compared to ethanol and methanol by 0.15 and 0.13 g/100g, respectively. Earlier, Barretto et al. (2008) performed HPLC analysis of mango peel for mangiferin contents and reported its concentration 4.94 g/kg at 278 nm. Similarly, Barretto et al. (2008) and Pott et al. (2003) examined different mango byproducts including peel, kernel and bark for their mangiferin

Table 14. Means squares for HPLC characterization of isolated mangiferin SOV Df Mangiferin

Treatments (A) 4 18.9538*

Solvent (B) 2 14.7520**

A x B 8 1.5451NS

Error 30 0.9745

NS=Non- significant *= Significant **=Highly significant

Table 15. HPLC characterization of isolated mangiferin (mg/g)

Parameters Ethanol Acetone Water Mean

Chaunsa 7.21±0.15 5.23±0.15 4.15±0.19 5.53±0.31a

Anwar ratol 6.12±0.41 4.56±0.32 3.96±0.14 4.88±0.30b

Langra 4.63±0.15 4.12±0.39 3.51±0.18 4.09±0.18c

Dusahri 4.33±0.11 4.02±0.24 3.56±0.11 3.97±0.23d

Desi 3.96±0.18 3.55±0.31 2.05±0.05 3.19±0.17e

Mean 5.25±0.01a 4.29±0.19b 3.44±0.21c

potential. They established that mango peel has the highest mangiferin contents 15.23 g/kg as compared to kernel 12.33 g/kg and bark 8.98 g/kg. Likewise, they also determined the other isomers of mangiferin like homomangiferin in peel (0.26 g/kg), isomangiferin (0.79 g/kg) and mangiferin gallate in bark (1.36 g/kg dry weight). Likewise, De Silva et al. (2006) also revealed mangiferin in the range of 6.40 to 18.33 g/kg on dry weight basis in different mango byproducts i.e. peel, kernel and stem.

4.5. Antioxidant indices of mangiferin

Means squares (Table16) pertaining to DPPH, β-carotene and FRAP activity of isolated mangiferin showed that treatments and solvents imparted significant differences on these traits. Means for DPPH activity of mangiferin are presented in Table 17. The ethanolic extract showed the highest value 75.05±4.12% followed by acetone 68.67±3.25% and water extract 59.51±2.45%. Similarly, means regarding varieties indicated the maximum DPPH activity in chaunsa 77.22±3.96% trailed by anwar ratol 70.26±4.89%, langra 64.58±2.69% and dusahri 64.90±4.12% whilst the minimum value was noticed in the peel of desi cultivar 61.75±3.85%. The means in Table 18 depicted that β-carotene activity was higher in the peel of chaunsa, anwar ratool, langra and dusahri by 74.63±3.69, 67.27±4.12, 63.77±2.33 and 62.52±2.85%, respectively than that of desi peel 61.64±3.69%. Among the solvents, maximum β-carotene activity was reported in acetone (71.49±3.65%) followed by ethanol (65.25±2.85%) and water (61.14±3.87%), correspondingly.

The FRAP values of the peels of chaunsa, anwar ratol, langra, dusahri and desi were 12.30±0.24, 10.56±0.32, 10.46±0.18, 10.26±0.32 and 8.33±0.24 mmol/100g, respectively. Likewise, the recorded FRAP values in ethanol acetone and water extracts were 12.02±0.28, 9.41±0.48 & 8.39±0.57 mmol/100g in respective manner (Table 19).

The higher DPPH and FRAP values of isolated mangiferin are in agreement with the earlier findings of Crozier et al. (2009), they probed the antioxidant potential of mangiferin in terms of free radical scavenging activity and ferric reducing antioxidant power. The DPPH and FRAP values of the tested compounds have linear association with polyphenols and they observed 55 to 68.03% DPPH activity and 7.03 to 13.02 mmol/100g FRAP at the concentration range of 10 to 50μmol/L. They expressed that

Table 16. Means squares for different antioxidant indices of isolated mangiferin

SOV df DPPH ß- carotene FRAP

Treatments (A) 4 724.342** 429.711** 322.984*

Solvent (B) 2 889.870** 978.020** 257.787**

A x B 8 9.459NS 6.214 NS 211.173NS Error 30 1.090 1.583 225.489

NS=Non- significant **=Highly significant

Table 17. Free radical scavenging (DPPH %) activity of isolated mangiferin Parameters Ethanol Acetone Water Mean

Chaunsa 83.20±6.01 78.21±4.23 70.26±4.12 77.22±3.96a

Anwar ratol 80.41±3.32 70.52±3.96 59.85±3.80 70.26±4.89b

Langra 72.15±4.20 64.25±4.01 57.33±2.14 64.58±2.69c

Dusahri 71.23±5.15 67.23±2.25 56.25±4.25 64.90±4.12c

Desi 68.18±3.56 63.18±2.25 53.89±3.28 61.75±3.85d

Mean 75.05±4.12a 68.67±3.25b 59.51±2.45c

Table 18. Antioxidant activity (β-carotene %) of isolated mangiferin Parameters Ethanol Acetone Water Mean

Chaunsa 75.1±4.23 80.19±5.65 68.52±3.12 74.63±3.69a

Anwar ratol 66.45±2.01 73.25±2.60 62.12±4.30 67.27±4.12ab

Langra 62.23±4.01 70.96±2.25 58.12±2.14 63.77±2.33b

Dusahri 62.25±4.20 66.12±4.15 59.18±2.90 62.52±2.85bc

Desi 60.18±3.25 66.96±1.89 57.78±1.25 61.64±3.69c

Mean 65.25±2.85b 71.49±3.65a 61.14±3.87c

Table 19. Ferric reducing antioxidant power (FRAP mmol/100g) of isolated mangiferin Parameters Ethanol Acetone Water Mean

Chaunsa 14.25±0.96 12.41±0.41 10.25±0.12 12.30±0.24a

Anwar ratol 12.42±0.45 11.05±0.52 8.21±0.45 10.56±0.32ab

Langra 12.02±0.59 11.25±0.32 8.12±0.36 10.46±0.18b

Dusahri 11.96±0.48 10.47±0.49 8.35±0.38 10.26±0.32c

Desi 9.45±0.69 8.52±0.41 7.02±0.25 8.33±0.24d

Mean 12.02±0.28a 9.41±0.48b 8.39±0.57c

isolation procedures and solvent polarity are the factors of prime importance. Similarly, Rao and Gianfreda (2000) noticed higher antioxidant activity in poly-aromatic polyphenolic compounds including mangiferin that are affected by several factors i.e. resonance energy, delocalisation of the phenoxy radical, O-H bond dissociation and steric hindrance. In another research, Amin and Mulhizah (2006) observed significant differences in the antioxidant activity (β-carotene) of methanolic and water extract of mango polyphenol by 58 and 20%, respectively. Previously, Ajila et al. (2007) polyphenols purity, concentration and freedom from interacting compounds are the cardinal factors that enhance antioxidant capacity of bioactive moieties.

Later, Vaghasiya and Chanda (2010) observed low IC50 value (11 μg/mL) for acetone extract of mangiferin than water extract (12 μg/mL) thus proved the higher antioxidative potential of mangiferin due to its purity and concentration. Previously, Abdalla et al. (2007) highlighted the factors that affect the antioxidant activity of bioactive molecules. In this context, extraction procedure, nature of the molecules, interaction behavior investigated the effect of variety on the DPPH activity of the polyphenols. They extracted mangiferin from raspuri & badami ripe and unripe mango peels and noticed lower IC50 values for raspuri peel from 1.83 to 1.98 than that of badami peel by 3.67 to 4.54 ug/g GAE.

Previously, Guo et al. (2003) documented the value 10.13 mmol/100g for ferric reducing antioxidant power of mangiferin. One of the researchers groups, Kim et al. (2010) evaluated the effect of mangiferin concentration on free radical scavenging activity. They observed that DPPH value varied from 1.72 to 92.57% at mangiferin concentrations 12.5 to 50 ug/mL.

The reaction of catechol moiety with 6,7-dihydroxylated structure alongside number of hydroxyl groups in side chain are responsible for the free radical scavenging activity of mangiferin (Sato et al., 1992). Later, Kano et al. (2005) estimated the concentration- response curves for the DPPH scavenging activity of mangiferin. They reported IC50 values for mangiferin and ascorbic acid as 55.90 and 13.74%, respectively. Previously, Dar et al. (2005) observed the DPPH scavenging activity of mangiferin water extract as 50.21±3.11%.

Masibo and He (2008) & Ghosal and Rao (1996) reported that mangiferin performs antioxidant action by decreasing the localized O2 concentration, generating mangiferin phenoxy radicals and binding metal ions (Fe 2+/3+) in the form of mangiferin-iron complex. Moreover, mangiferin regulates polymer chain reaction, production of feebly reactive oxo-ferryl radical, neutralizing alkoxy radicals and maintains cellular oxidation- antioxidant balance. Decisively, the isolated mangiferin showed higher antioxidant due to the presence of more hydroxyl groups and catechol moiety. Additionally, the ability to form a complex with metal ion further advocates its antioxidant capacity.

4.6. Functional/nutraceutical drink analysis

Mean squares (Table 20) indicated non-significant effect of treatments on the acidity, pH and TSS of the resultant drinks whereas, storage significantly affected these parameters except for TSS. The acidity in functional/nutraceutical drinks including T0, T1 and T2 was observed as 0.176±0.001, 0.173±0.002 & 0.170±0.004%, respectively. However, storage caused a significant enhancement in acidity from 0.161±0.001 to 0.187±0.005% at 0 and th 60 day, correspondingly (Table 21). Likewise, the recorded pH of drinks i.e.T0, T1 and

T2 was 4.58±0.04, 4.56±0.06 & 4.57±0.05, respectively. During storage, pH momentously decreased from 4.67±0.10 to 4.45±0.09 (Table 22). The total soluble solids (TSS) of the functional drinks T0, T1 and T2 were 1.77±0.07, 1.78±0.06 and 1.80±0.04, respectively. Likewise, storage also revealed non-significant decline in TSS and observed values at 0 and 60th days were 1.80±0.05 and 1.77±0.08, respectively (Table 23). The possible mechanism behind decline in pH with increased acidity is due to the citric acid breakdown in drinks while, the acidic nature of added artificial sweetner may alter the acidity during storage. The current results regarding acidity, pH and TSS of functional drinks are in harmony with the findings of Omodamiro et al. (2012), they observed a declining trend in pH and elevation in acidity of the functional drinks during storage. Previously, Ayub et al. (2010) documented that storage intervals impart momentous effect on the acidity of juice stored under ambient conditions. They observed variations in acidity of strawberry juice from 1.59 to 2.14% after 3 months storage. One of their peers, Hussain et al. (2003) delineated a non-substantial increase in total soluble solids of apple and apricot blend.

Similarly, Majumdar et al. (2010) prepared a polyphenolic enriched functional beverage with gourd-basil leaves extract and noticed an inverse association between acidity and pH 59

Table 20. Mean squares for acidity, pH and TSS of functional drinks SOV df Acidity pH TSS

Treatments (A) 2 0.00154 NS 0.11937NS 0.00267 NS

Days (B) 2 0.00008** 0.00090 ** 0.00170NS

A x B 4 0.00002 NS 0.00605 NS 0.00003 NS

Error 18 0.00001 0.00852 0.00146

NS=Non- significant *= Significant **=Highly significant

Table 21. Effect of treatments and storage on acidity (%) of functional drinks

Storage Treatments Means intervals

(days) T0 T1 T2

0 0.163±0.001 0.160±0.005 0.162±0.005 0.161±0.001c

30 0.175±0.003 0.168±0.001 0.166±0.003 0.169±0.003b

60 0.190±0.002 0.188±0.004 0.183±0.006 0.187±0.005a

Means 0.176±0.001 0.173±0.002 0.170±0.004

T0= Control T1=Drink containing mango peel extract T2=Drink containing mangiferin

Table 22. Effect of treatments and storage on pH of functional drinks

Treatments Storage Means intervals (days) T0 T1 T2

0 4.71±0.21 4.65±0.32 4.66±0.30 4.67±0.10a

30 4.64±0.30 4.59±0.21 4.58±0.25 4.60±0.07a

60 4.40±0.15 4.46±0.14 4.49±0.17 4.45±0.09b

Means 4.58±0.04 4.56±0.06 4.57±0.05

Table 23. Effect of treatments and storage on TSS of functional drinks

Treatments Storage Means intervals (days) T0 T1 T2

0 1.79±0.02 1.80±0.03 1.82±0.05 1.80±0.05

30 1.78±0.01 1.79±0.01 1.81±0.04 1.79±0.06

60 1.75±0.08 1.77±0.04 1.79±0.05 1.77±0.08

Means 1.77±0.07 1.78±0.06 1.80±0.04

T0= Control T1=Drink containing mango peel extract T2=Drink containing mangiferin

during storage with increase in TSS from 11.32 to 11.50% after 6 months storage. One of the researchers groups, Dhaliwal and Hira (2004) also reported a decline in pH and elevation in the acidity of polyphenolic beverage as a function of storage. Earlier, Majumdar et al. (2008) observed significant incline in acidity from 0.25 to 0.36 g/100ml in cucumber-basil juice during storage. Previously, Krishnaveni et al. (2001) observed slight changes in acidity from 0.25-0.27% of jackfruit based beverage. They inferred that enhancement in acidity and reductions in pH are due to breakdown of carbohydrates by the action of microorganisms that produce acids in the drink. One of the researchers groups, Saron et al. (2007) evaluated the effect of storage on acidity, pH and total soluble solids of polyphenol supplemented drinks. They recorded increasing trend for TSS probably due to loss in moisture content whilst, acidity and pH have inverse correlation with storage. In this reference, Nunes et al. (1995) observed an elevation in acidity of strawberry juice after two month storage due to the breakdown of pectin into pectenic acid. Likewise, Riaz et al. (1988) documented reduction in pH during storage from 4.7 to 4.2 in fruit juices. Supraditareporn and Pinthong (2012) also noticed a decline in pH and increment in the acidity of orange juice subjected to storage. Recently, Rossana et al. (2012) also observed an inverse relation between acidity and pH of yogurt-based drink supplemented with cereals and grapes during 30 days. One of their peers, Ahmad et al. (2012), explicated an inverse correlation between acidity and pH of the polyphenol enriched functional drink during storage. They noticed significant increase in acidity from 0.14±0.003 to 0.21±0.006% from 0 to 60th day however treatments exerted non- significant differences. Similarly, pH decreased from 4.7 to 4.2 during storage.

4.7. Sensory evaluation

Means squares in Table 24 showed that all of the sensory attributes affected non- significantly with treatments however, storage intervals imparted substantial differences except for flavor.

Means for color scores (Table 25) indicated non-substantial variations from 7.50±0.03 to

7.59±0.06 in T0 and T2, respectively. Likewise, storage showed momentous decline in color scores from 7.70±0.01 at 0 day to 7.38±0.03 at 60th day. Statistical interpretation elucidated that flavor was affected non-significantly by treatments and storage. The maximum flavor scores were recorded in T1 7.62±0.06 followed by T2 7.61±0.04 and T0 7.58±0.02 (Table 26). The assigned scores for flavor at 0 and 60th days were 7.66±0.02 &

Table 24. Mean squares for sensory evaluation of functional drinks Overall SOV df Color Flavor Sweetness Sourness Acceptability

Treatments (A) 2 0.01749 NS 0.00127NS 0.00930 NS 0.02187 NS 0.02312NS

Days (B) 2 0.23250* 0.02960NS 0.09513* 0.05951* 0.21803*

A x B 4 0.00166 NS 0.00080NS 0.00456 NS 0.00391 NS 0.00341NS

Error 18 0.02232 0.01992NS 0.02010 0.02064 0.01762NS

Table 25. Effect of storage and treatments on the color

Storage Treatments

intervals Means (days) T0 T1 T2

0 7.68±0.01 7.70±0.04 7.72±0.03 7.70±0.01a

30 7.52±0.03 7.58±0.01 7.62±0.02 7.57±0.02ab

60 7.32±0.02 7.38±0.05 7.45±0.07 7.38±0.03b

Means 7.50±0.03 7.55±0.05 7.59±0.06

T0= Control T1=Drink containing mango peel extract T2=Drink containing mangiferin

7.55±0.05, respectively. The sweetness differed non-significantly in treatment T0, T1 and

T2 as 7.34±0.05, 7.39±0.02 & 7.43±0.03, respectively. A significant declining trend was noticed for sweetness (Table 27), at the initiation, recorded scores were 7.52±0.02 that decreased to 7.26±0.05 at the termination of study.

Likewise, sourness varied non-substantially among the functional drinks T0, T1 and T2 as 7.54±0.04, 7.56±0.01 and 7.58±0.03, in respective drinks. However during storage, scores for sourness decreased significantly from 7.70±0.01 to 7.45±0.04 at the initiation till the termination of trial (Table 28).

The observed scores for overall acceptability in functional/nutraceutical drinks T0, T1 and

T2 were 7.50±0.03, 7.55±0.04 and 7.59±0.02, respectively. Nevertheless, 60 days storage resulted a significant reduction in overall acceptability scores from 7.67±0.05 to 7.37±0.08 (Table 29).

The current findings regarding sensory response are in harmony with the conclusions of Ahmad et al. (2012), investigated the hedonic response of polyphenol enriched functional beverage (green cool) during storage. They prepared three types of drinks with varying concentration of green tea functional ingredients alongside control and stored for two months. There was observed non-significant differences in sensory traits including color, flavor, sourness and overall acceptability with treatments however, storage caused significant decline in the sweetness level of the drinks. One of their peers, Mishra et al. (2012) reported decline in the flavor and overall acceptability scores of ascorbic acid supplemented functional drinks as a function of storage. Previously, Aziah and Komathi (2009) carried out a trial to assess the effect of mango peel supplementation on the sensory profile of biscuits. They supplemented flour with different concentrations of mango peel powder and observed darkening of biscuits due to their strong coloring ability. They also noticed a significant decline in color, flavor, texture and overall acceptability scores during storage. Earlier, Ajila et al. (2007) and Ribeiro et al. (2008) deduced that mango peel powder (MPP) addition improves the taste and flavor profile of biscuits.

Some other scientists like Ahmad et al. (2012), Ahmed et al. (2008) & Jan and Masih (2012) are of the view that sweetness of the functional beverage is decreased whilst, sourness increased during storage due to the breakdown of acidic compounds, their notion

Table 26. Effect of storage and treatments on the flavor

Treatments Storage Means intervals (days) T0 T1 T2

0 7.65±0.03 7.68±0.06 7.66±0.07 7.66±0.02

30 7.58±0.02 7.62±0.03 7.60±0.01 7.60±0.04

60 7.52±0.05 7.55±0.02 7.58±0.03 7.55±0.05

Means 7.58±0.02 7.62±0.06 7.61±0.04

Table 27. Effect of storage and treatments on the sweetness of the drinks

Treatments Storage Means intervals (days) T0 T1 T2

0 7.52±0.01 7.55±0.06 7.50±0.01 7.52±0.02a

30 7.31±0.03 7.38±0.05 7.40±0.02 7.36±0.06b

60 7.20±0.06 7.26±0.02 7.32±0.04 7.26±0.05b

Means 7.34±0.05 7.39±0.02 7.43±0.03

T0= control T1=Drink containing mango peel extract T2=Drink containing mangiferin

Table 28. Effect of treatments and storage on sourness

Treatments Storage Means intervals (days) T0 T1 T2

0 7.72±0.03 7.68±0.02 7.70±0.04 7.70±0.01a

30 7.50±0.05 7.54±0.04 7.56±0.02 7.63±0.06a

60 7.40±0.01 7.46±0.03 7.49±0.06 7.45±0.04b

Means 7.54±0.04 7.56±0.02 7.58±0.03

Table 29. Effect of storage and treatments on the overall acceptability

Treatments Storage Means intervals (days) T0 T1 T2

0 7.66±0.09 7.65±0.07 7.70±0.03 7.67±0.05a

30 7.52±0.02 7.60±0.03 7.66±0.01 7.59±0.06a

60 7.30±0.06 7.40±0.05 7.42±0.08 7.37±0.08b

Means 7.50±0.03 7.55±0.04 7.59±0.02

T0= control T1=Drink containing mango peel extract T2=Drink containing mangiferin

is also supporting the instant results. Moreover, nature of functional ingredients is also a key factor in this regard. Previously, Manzano et al. (1997) compared the sensory characteristics of dusheri and chaunsa mango varieties after heat treatment. They observed that acidity was increased with the passage of time that influenced the flavor of the product. They found that chaunsa mango exhibited better hedonic response. Another factor that accelerates the hedonic changes is the conversion of complex compounds into esters, aldehydes and acids during storage (Weichman et al., 1987). Additionally, Sistrunk and Morris (1985) observed a non-substantial decline in flavor of apple and grape juices during storage up to 12 months. Earlier, Din et al. (2011) recorded a significant decline in the overall acceptability of bitter gourd based functional beverage during storage. In another research, Rathore et al. (2007) observed significant increase in the sourness of mango juice and attributed this as a function of citric acid degradation. Earlier, Murtaza et al. (2004) prepared strawberry juice and observed variations in the sensory profile in terms of color, flavor and taste during three month storage. They ascribed these changes to the non-enzymatic browning reaction of reducing sugars and amino acids. In the current study, mango peel extract and mangiferin based functional drinks have performed better regarding hedonic response. The storage caused significant decline in the color, sweetness, sourness and overall acceptability scores of the tested drinks nonetheless, all values were within acceptable ranges thus prove their suitability for further utilization in the bioevaluation trial. 4.8. Bioevaluation trials Bioefficacy study was conducted to investigate the functional/nutraceutical worth of mango polyphenols especially mangiferin against lifestyle related disorders involving experimental Sprague Dawley rats. The trials were carried out on rodents rather than humans due to convenient management, closed supervision, control diet and environmental conditions. In the current explorations, efficacy trial was divided into three segments. In study I, normal rats were used whereas in study II and III hypercholestrolemic and diabetic rats were involved, correspondingly. Additionally, rats were provided respective diets along with concurrent intake of functional drinks (T0, T1 and T2) to assess their therapeutic potential. At the commencement of study, some rats were sacrificed to establish the baseline trend whilst rests were dissected at the end. Feed and drink intakes were measured on daily basis however, body weights were recorded 67

weekly. The mango peel extract and mangiferin containing drinks were examined against hypercholesterolemia and diabetes. For better understanding, the results of all tested attributes in different studies are elaborated collectively. 4.8.1. Feed intake Mean squares (Table 30) elucidated non-significant effect of treatments on feed intake however, study intervals imparted momentous differences on this parameter in all studies (trial 1 & 2). The feed intake increased as a function of time and at 1st week it was recorded as 15.13±0.82, 14.65±0.39 and 14.69±0.66 g/rat/day in T0, T1 and T2 groups that successively elevated to 20.71±1.01, 19.96±1.07 and 20.10±0.96 g/rat/day, respectively at the 8th week (study I; trial 1). Likewise increasing tendency for feed intake was noticed in trial 2. The initial values for T0, T1 and T2 groups were 15.20±0.74, 14.63±0.43, 14.71±0.72 g/rat/day that increased to 20.78±1.02, 20.05±1.76 and 20.35±1.19 g/rat/day, respectively at the end of study I (Figure 2).

In study II, the highest feed intake was observed in T0 group by 16.96±1.03 & 16.98±0.92 st g/rat/day at 1 week however, values in T1 and T2 groups were 16.55±1.01 & 16.64±0.81 and16.85±0.76 & 16.91±0.89 g/rat/day (trial 1 & 2). Time span improved the feed intake, at termination the observed values were 24.01±0.66 & 23.96±0.83, 23.78±1.05 &

23.75±1.19 and 23.81±0.87 & 23.89±0.14 g/rat/day in groups T0, T1 and T2 in respective trials. Likewise in study III, feed consumption in T0 group was increased from 16.55±1.55

& 16.50±0.85 to 22.12±1.65 & 22.09±1.14 g/rat/day at start tills the end, respectively.

Similarly, in other groups i.e. T1 and T2 enhancement in feed intakes were recorded from 16.35±0.36 & 16.39±0.78 to 21.98±1.45 & 21.90±0.95 and 16.41±0.62 &16.48±0.45 to 22.01±0.96 & 22.05±0.85 g/rat/day at 1st and 8th week, respectively during trial 1 & 2 (Figure 2). The results of present exploration are in agreement with the findings of Guo et al. (2011), observed non-significant effect of mangiferin administration @ 50 and 150 mg on feed consumption of hyperlipidemic rats. Recently, Freitas et al. (2012) documented non- momentous variations in feed intake of rats relied on mango peel polyphenols. Different researchers including Vasant et al. (2011) and Swamy et al. (2011) expounded non- significant effect of polyphenols intake on feed consumption. They were of the view that the polyphenols enhance the feed efficiency rather suppressing the appetite.

Table 30. Effect of treatments and study weeks on feed intake (g/rat/day) Study II Study I Study III (Hypercholesterolemic (Normal rats) (Diabetic rats) SOV df rats) Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2

Treatments 3 4.231 NS 7.932 NS 2.021 NS 3.957 NS 4.056 NS 6.052 NS

Weeks 7 29.321** 31.361** 06.12** 8.56** 13.012** 18.254**

Error 21 1.003 0.959 0.218 0.389 0.196 0.201

NS=Non-significant **=Highly significant

Study I 25

20 To(Tr1) To(Tr2) 15 T1(Tr1) 10 T1(Tr2) g/rat/day 5 T2(Tr1) T2(Tr2) 0 week 1 week2 week3 week4 week5 week6 week7 week8

Study II 30

25 To(Tr1) 20 To(Tr2)

15 T1(Tr1)

g/rat/day T1(Tr2) 10 T2(Tr1) 5 T2(Tr2) 0 weeks1 week2 week3 week4 week5 week6 week7 week8

Study III

20 To(Tr1) To(Tr2) 15 T1(Tr1) 10

g/rat/day T1(Tr2)

5 T2(Tr1) T2(Tr2) 0 week 1 week2 week3 week4 week5 week6 week7 week8

Figure 2. Feed intake in study I, II and III (g/rat/day)

4.8.2. Drink intake

Mean squares for drink intake exhibited non-substantial effect of treatments whereas, study span (weeks) showed momentous variations (Table 31).

Drink intake in Figure 3 depicted that in the beginning (study I; trial 1), the drink intake in different rats groups T0, T1 and T2 were 20.05±0.43, 20.04±0.74 and 20.06±0.98 mL/rat/day, respectively that progressively increased to 26.71±0.39 mL/rat/day (T0),

26.48±1.04 mL/rat/day (T1) and 26.59±0.58 mL/rat/day (T2) at the termination. Likewise, in study I (trial 2) the values were 20.07±0.74 & 26.69±0.63, 20.03±1.19 & 26.58±0.34 st th and 20.05±1.01 & 26.63±0.83 mL/rat/day for T0, T1 and T2 at 1 and 8 week, respectively. Means for drink intake (study II; trial 1) were 22.91±0.56, 22.82±0.43 and st th 22.78±0.24 mL/rat/day in T0, T1 and T2 groups at 1 week whilst at 8 week 29.31±0.72, 28.78±1.16 and 28.88±0.23 mL/rat/day for respective groups. Similarly, the maximum drink intake was observed in T0 followed by T1 and T2 i.e. 29.35±0.41, 28.88±0.97 and 28.99±0.28 mL/rat/day, respectively during the entire trial.

Moreover, in study III recorded values for T0, T1 and T2 were 20.92±0.63, 20.75±0.056 and 20.81±0.48 mL/rat/day that uplifted to 27.42±1.04, 27.22±0.62 and 27.29±0.87 mL/rat/day during entire period (trial 1). Similarly, in groups relied on T0, T1 and T2 elevation in drink intake was noticed from 20.89±0.26 to 27.37±0.65, 20.78±0.21 to 27.24±0.76 and 20.92±0.92 to 27.48±0.63g/rat/day at 1st and 8th week, respectively (trial 2).

The results of this study are consistent with the earlier work of Alshatwi et al. (2011), who noticed a non-significant effect of polyphenols consumption on drink intake of experimental animals. Numerous researchers have also reported a non-momentous effect of polyphenols for this trait (Kuo et al., 2005; Uchiyama et al., 2011). Similarly, Bock et al. (2008) illuminated that mangiferin consumption did not impart any suppressing effect on drink intake.

4.8.3. Body weight

Mean squares (Table 32) indicated that the treatments and study weeks imparted significant differences on body weights of rats in all studies.

The Figure 4 showed that at the initiation of study I (trial 1 & 2), the body weights in different rats groups T0, T1 and T2 were 132±5.32 & 130±1.12, 128±3.85 & 129±6.42 and 71

Table 31. Effect of treatments and study weeks on drink intake (mL/rat/day) Study II Study I Study III (Hypercholesterolemic (Normal rats) (Diabetic rats) SOV df rats) Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2

Treatments 3 0.451 NS 0648 NS 0.654 NS 0.541 NS 0.468 NS 0.564 NS

Weeks 7 6.253** 7.546** 4.526** 4.563** 6.458** 8.254**

Error 21 0.212 0.301 0.213 0.185 0.196 0.222

NS=Non-significant **=Highly significant

Study I 30

25 To(Tr1) 20 To(Tr2)

15 T1(Tr1) T1(Tr2)

mL/rat/day 10 T2(Tr1) 5 T2(Tr2) 0 week1 week2 week3 week4 week5 week6 week7 week8

Study II 35 30 To(Tr1) 25 To(Tr2) 20 T1(Tr1) 15 T1(Tr2) 10

mL/rat/day T2(Tr1) 5 T2(Tr2) 0 week 1 week2 week3 week4 week5 week6 week7 week8

Study III 30

25 To(Tr1) 20 To(Tr2) 15 T1(Tr1) T1(Tr2)

mL/rat/day 10 T2(Tr1) 5 T2(Tr2) 0 week 1 week2 week3 week4 week5 week6 week7 week8

Figure 3. Drink intake in study I, II and III (mL/rat/day)

131±6.24 & 133±3.32 g/rat, that significantly uplifted to 233±17.41 & 236±9.41,

223±13.02 & 226±9.41 and 228±7.21 & 227±8.41 g/rat in T0, T1 and T2, respectively at the termination. Similar enhancement for rats body weights was recorded with the passage of time in study 2 (trial 1), for T0 at first and last weeks by 132±4.02 &

262±15.03 g/rat, respectively whereas, the groups relied on T1 and T2 had lower body weights than that of control as 131±6.32 & 235±13.21 and 133±8.21 & 240±10.05 g/rat.

Similarly in trial 2 (study II), the noticed weights in control group (T0) were higher

129±4.03 & 259±12.63 than that of T1 and T2 134±6.58 & 229±11.58 and 136±9.58 & st th 238±14.58 g/rat, respectively at 1 and 8 week.

Likewise in study III (trial 1 & 2), the recorded weights for T0, T1 and T2 were 136±4.21 & 134±4.22, 130±6.19 & 132±3.01 and 134±5.36 & 136±5.04 g/rat at first week that enhanced to 248±7.14 & 241±10.41, 220±7.31 & 221±11.02 and 236±9.21 & 232±8.78 g/rat, respectively at the last week (Figure 4). The present results regarding body weight are comparable with the earlier findings of Dineshkumar et al (2010), they noticed significant reduction in the body weight of streptozotocin-induced diabetic rats due to intake of mangiferin @ 10 and 20 mg/kg. They inferred that mangiferin diminishes the activity of amylase and glucosidase enzymes, enhances thromogenesis alongside accelerates the faecal bile excretion thus manages the body weight. Earlier, Yoshikawa et al. (2001) suggested that mangiferin lowers the body weight by inhibiting the activity of glucosidase enzymes including sucrase, isomaltase and maltase. These enzymes actively participate in carbohydrates metabolism and reduce the digestion of polymers into monomers in intestine by interrupting or retarding the carbohydrate breakdown (Singh et al., 2009). Earlier, Muruganandan et al. (2005) observed significant decline in the body weight from 206.67±13.18 to 191.67±15.35 g in strptozotocin induced diabetic Wistar rats after the provision of mangiferin (10 and 20 mg/day) for 28 days. Further they concluded that mangiferin significantly reduces the incidence of hyperglycemic and hyperlipidemic related variables. Similarly, Morsy et al. (2010) evaluated the effect of oral administration of mango leaves extract @ 30, 50 and 70 mg containing mangiferin on body weight of diabetic Sprague Dawley rats. They deduced that tested extracts momentously reduce the body weight as 20.42, 23.70 and 27.13%, respectively by managing the carbohydrate metabolism owing to its antioxidant potential.

Table 32. Effect of treatments and study weeks on body weight (g/rat) Study II Study I Study III (Hypercholesterolemic (Normal rats) (Diabetic rats) rats) SOV df Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2

Treatments 3 169.32* 225.61* 1002.63** 12025.71** 651.01* 996.12*

Weeks 7 4201.01* 4725.5* 5523.2** 5963.1** 2452.2** 4745.9**

Error 21 43.251 81.02 65.21 69.03 36.03 41.06

*=Significant **=Highly significant

Study I 250

200 T0(Tr1) TO(Tr2) 150 T1(Tr1) 100 T1(Tr2) g/rat/week 50 T2(Tr1) T2(Tr2) 0 weeks1 week2 week3 week4 week5 week6 week7 week8

Study II 300

250 T0(Tr1) 200 TO(Tr2)

150 T1(Tr1) T1(Tr2) g/rat/week 100 T2(Tr1) 50 T2(Tr2) 0 weeks1 week2 week3 week4 week5 week6 week7 week8

Study III 300

250 T0(Tr1) 200 TO(Tr2)

150 T1(Tr1)

g/rat/day T1(Tr2) 100 T2(Tr1) 50 T2(Tr2) 0 weeks1 week2 week3 week4 week5 week6 week7 week8

Figure 4. Body weight in study I, II and III (g/rat)

Afterwards, Guo et al. (2011) conducted a rodent experiment to elucidate the effect of mangiferin on hyperlipidemia in high fat fed animals. They expressed that mangiferin @ 50 and 150 mg/kg for 8 weeks reduces the body weight by interferring the lipid metabolism. Moreover, they observed that mangiferin enhances the expression of peroxisome proliferator-activated receptor-a (PPAR-a), carnitine palmitoyltransferase 1 (CPT-1) and fatty acid translocase (CD36) however, suppresses the activity of sterol regulatory element-binding protein 1c (SREBP-1c), acetyl CoA carboxylase (ACC), acyl- CoA: diacylglycerol acyltransferase 2 (DGAT-2) and microsomal triglyceride transfer protein (Freitas et al., 2012). 4.8.4. Cholesterol

In the present research, effect of mango peel extract and mangiferin supplementation was evaluated on lipid profile including total cholesterol, LDL, HDL and triglycerides in normal, hypercholesterolemic and diabetic rats.

The F value in Table 33 showed that treatments imparted significant effect on cholesterol in all studies except for study I. In this case (trial 1), the observed cholesterol level in T0 groups was 78.26±3.98 mg/dL higher than that of T1 and T2 76.08±5.24 & 75.72±6.18 mg/dL, respectively. Likewise in trial 2, the reported value for T0 was (81.23±4.88 mg/dL) that non-momentously reduced in T1 (79.13±5.89 mg/dL) and T2 groups (78.78±5.43 mg/dL). However means for cholesterol in study II (trial 1 & 2), indicated maximum values for T0 (141.25±8.01 & 145.85±6.25 mg/dL) that significantly decreased in groups relied on T1 and T2 as 128.28±6.08 & 132.78±6.84 mg/dL and 123.10±6.48 & 127.98±4.32, respectively. Likewise in study III (trial 1), the highest cholesterol level

98.42±4.64 mg/dL was recorded in T0 followed by T1 91.81±6.32 whilst the lowest value in T2 88.30±5.34 mg/dL. Likewise in subsequent trial, T0 showed the maximum value

(101.25±4.92 mg/dL) that substantially suppressed in T1 (93.92±6.21 mg/dL) and T2 (90.89±4.52 mg/dL), respectively (Table 33).

It is evident from the Figure 5 that in study II (trial 1), drinks T2 (mangiferin supplemented drink) and T1 (mango peel extract based drink) resulted 12.85 & 9.18% reduction in cholesterol whereas in trial 2, same treatments exhibited 12.25 & 8.96% decline, respectively. Moreover, in study III (trial 1 & 2), T2 caused highest reduction

10.01 & 7.01% trailed by T1 9.9 & 6.32%, respectively as compared to control.

Table 33. Effect of functional drinks on cholesterol (mg/dL) Treatments F value

T0 T1 T2 Study I (Trial 1) 78.26±3.98 76.08±5.24 75.72±6.18 0.89NS (Trial 2) 81.23±4.88 79.13±5.89 78.78±5.43 0.95NS

Study II (Trial 1) 141.25±8.01a 128.28±6.08b 123.10±6.48c 80.2** (Trial 2) 145.85±6.25a 132.78±6.84b 127.98±4.32c 65.3**

Study III (Trial 1) 98.42±4.64a 91.81±6.32b 88.30±5.34c 28.1** (Trial 2) 101.25±4.92a 93.92±6.21b 90.89±4.52c 29.7**

NS= Non-significant **= Highly significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

T1 T2

14 12.85 12.25 12 9.9 10.01 10 9.18 8.96

8 7.01 6.32 6

Percent decrease 3.25 3.01 4 2.79 2.58 2

0 Study1(Tr 1) Study 1(Tr 2) Study 2( Tr 1) Study 2(Tr 2) Study 3(Tr 1) Study 3(Tr 2)

Figure 5. Percent reduction in cholesterol as compared to control

The outcomes of instant investigations are in agreement with the findings of Guo et al. (2011), they evaluated the effect of low and high concentration of mangiferin i.e. 50 and 150 mg/kg, respectively on the lipid profile of hamsters fed on high fat diet for 8 weeks. Accordingly, hamsters were divided into four groups on the basis of diet i.e. control, high fat (HF), HF+mangiferin 50 mg/kg and HF+mangiferin150 mg/kg body weight (BW). The high dose treatment of mangiferin caused maximum reduction in cholesterol 38.25% as compared to control group. They were of the view that mangiferin administration upregulates liver mRNA expression of peroxisome PPAR-α, fatty acid translocase (CD36) and carnitine palmitoyltransferase 1 (CPT-1) proteins, involved in fatty acid oxidation and metabolism. Additionally, it down regulates the expression of proteins like acetyl Co-A carboxylase (ACC) and acyl Co-A diacylglycerol acyltransferase 2 (DGAT- 2) considered essential for fatty acid synthesis.

Earlier, Morsy et al. (2010) also noticed a momentous decline in cholesterol, low density lipoprotein and triglycerides level in the experimental rats treated with mangiferin @ 30, 50 and 70 mg. Previously, Miura et al. (2001) and Prabhu et al. (2006 a,b) explicated the lipid modulating and cardioprotective role of mangiferin in diabetic subjects owing to its antioxidant activity.

Afterwards, Muruganandan et al. (2005) observed that mangiferin treatment resulted significant reduction in atherogenic index, total cholesterol, low density lipoprotein and total glycerides with simultaneous enhancement in high density lipoprotein (HDL) level in rats. Earlier, Miura et al. (2001b) conducted a two weeks model feeding trial to investigate the therapeutic potential of mango polyphenols against lipid related abnormalities. During the entire trial, experimental rats were fed on mangiferin (30 mg/kg) along with basal diet. The results regarding different lipids related parameters delineated that mangiferin caused 40 & 70% reduction in cholesterol and triglyceride levels, respectively.

Various researchers groups like Ho (2003) and Seth et al. (2004) reported soothing impact of mango peel powder in hypertriglyceridemic rats. They documented a significant decline in plasma triacylglycerol, low density lipoprotein and cholesterol contents whilst an enhancement in high density lipoprotein. Later, Nair and Shyamala Devi (2006) expressed that mangiferin and other promising mango peel polyphenols like

qurecitin significantly reduce cholesterol, triglycerides and free fatty acid levels in the experimental volunteers. One of the researchers groups, Dineshkumar et al. (2010) probed the lipid lowering role of mangiferin in diabetic rats. To induce diabetes, streptozotocin injection @ 65 mg/kg BW was used. They noticed an increase in LDL, cholesterol and triglyceride level of rats with 8.51% reduction in HDL. Nonetheless, mangiferin provision @ 10 & 20 mg/kg BW resulted significant decline in cholesterol and triglycerides levels by 34.53 & 60.41 and 20.79 & 19.06%, respectively. Earlier, Aderibigbe et al. (2001) conducted a one month rodent trial to investigate the cholesterol lowering effect of mangiferin in hypercholesterolemic rats. The mangiferin treatment at a dose of 10 & 20 mg/kg resulted 12.02 & 16.18% reduction for cholesterol in dose dependent manner.

Mangiferin ability to alter the hepatic cholesterol metabolism is one of the promising mechanisms by which it tackles the elevated cholesterol level. Due to hindrance in cholesterol synthesis by mangiferin resulting less accumulation of cholesterol in the aorta thereby inhibits atherosclerosis and other coronary complications (Zern et al., 2003; Vinson and Jang, 2001). Moreover, mangiferin inhibits the activity of lipid peroxidase (LPO) after scavenging the peroxy and alkoxy radicals thus halts the liberation of hydrogen from cellular lipids (Ghosal et al., 1996). Low density lipoprotein (LDL) enhancement is a key episode in the development of atherosclerosis (Chisolm et al., 2000). This syndrome is accelerated by oxidative modification of LDL due to reactive oxygen species (ROS) from vascular wall cells (Witztum and Steinberg, 1991; Lamb et al., 1992). During hypercholesterolemia, enhanced expression of LDL receptor (LDLr) reflects the accumulation of reactive oxygen species in mitochondria thus increases the probability of membrane oxidation. In this context, mangiferin protects the cells from oxidation and manages the normal ratio of lipid profile (Paim et al., 2008). High cholesterol de novo synthesis causes reduction in mitochondrial NADPH activity resultantly increases the incidence of oxidative stress and MPT (Kowaltowski et al., 2001). The mangiferin and its various isomers have ability to protect the mitochondria from oxidative damage by lowering the activity of LDLr thus proved suitable to alleviate lipid peroxidation (Rodrigo et al., 2007).

From the above mentioned discussion, it is inferred that mango peel extract and mangiferin based therapeutic drinks have potential to alleviate abnormal cholesterol 81

levels however, mangiferin is more promising in this regard.

4.8.5. Low density lipoprotein (LDL)

The statistical analysis (F values) revealed significant effect of treatments on LDL level in all studies except for study I (Table 34).

The recorded means for LDL in study I were 29.32±1.48 & 27.85±1.08, 28.36±1.64 &

26.97±2.13 mg/dL and 28.16±2.19 & 26.88±1.32 for T0, T1 and T2 groups, respectively

(trial 1 & 2). However in study II, T0 had highest LDL level as 58.96±3.49 mg/dL that significantly reduced in T2 & T1 groups by 50.40±4.23 & 52.94±3.92 mg/dL, respectively (trial 1). Likewise in the follow up trial, the observed LDL concentrations were

60.21±4.64, 53.43±2.13 & 51.05±4.90 mg/dL for T0, T1 and T2, respectively. Similarly in study III, (trial 1 & 2), mean LDL values for T0, T1 and T2 differed substantially i.e. 47.21±4.21 & 45.25±3.32, 43.68±4.28 & 41.63±3.94 and 41.90 ±3.74 & 39.69±3.32 mg/dL, correspondingly (Table 34).

The Figure 6 elucidated that in study I (trial 1 & 2), the developed functional drinks T1 &

T2 caused reduction in LDL by 3.28 & 3.15% and 3.96 & 3.48%, respectively. In study

II, mangiferin based drink (T2) imparted maximum diminish in LDL value 14.52 &

15.21% as compared to mango peel extract (T1) 10.21 & 11.25%, correspondingly in both trials. Accordingly, the recorded LDL decrease for T1 & T2 groups was 7.48 & 8.01% and 11.25 & 12.29 %, respectively in continous trials of study III.

Low density lipoprotein (LDL) known as bad cholesterol is a principal carrier of cholesterol in the blood. It mainly comprised of apo-B100 protein, cholesterol esters and triglycerides alongside linoleate that combines with cholesterol esters and makes it prone to oxidation. Further oxidation of LDL by free radicals initiates the cascade of abnormal macrophage modification resulting foam cell deposition thereby accelerates the coronary complications. Numerous scientific explorations are in favour that mangiferin has ability to ameliorate the cardiovascular disorders owing to its strong free radical scavenging activity (Bor et al., 1999).

The current investigation is accordance with the earlier conclusions of Muruganandan et al. (2005), they observed a substantial decline in LDL in hypercholestrolemic and diabetic male Wistar rats after mangiferin treatment @ 10 & 20 mg/kg BW for 28 days.

Table 34. Effect of functional drinks on LDL (mg/dL) Studies Treatments F value T0 T1 T2 Study I (Trial 1) 29.32±1.48 28.36±1.64 28.16±2.19 2.36NS (Trial 2) 27.85±1.08 26.97±2.13 26.88±1.32 1.66NS

Study II (Trial 1) 58.96±3.49a 52.94±3.92b 50.40±4.23c 75.4** (Trial 2) 60.21±4.64a 53.43±2.13b 51.05±4.90c 86.9**

Study III (Trial 1) 47.21±4.21a 43.68±4.28b 41.90 ±3.74c 39.2** (Trial 2) 45.25±3.32a 41.63±3.94b 39.69±3.32c 48.5**

NS= Non Significant **= Highly significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

15.21 16 14.52 14 12.29 12 11.25 11.25 10.21 10 8.01 7.48 T1 8 T2 6 percentdecrease 3.96 3.48 4 3.28 3.15

0 Study1(Tr 1) Study 1(Tr 2) Study 2( Tr 1) Study 2(Tr 2) Study 3(Tr 1) Study 3(Tr 2)

Figure 6. Percent reduction in LDL as compared to control

The recorded LDL decline in these phases was 59.53 & 54.98% and 37.07 & 51.98%, respectively. Previously, Aderibigbe et al. (2001) reported 24.66 and 27.88% reduction in low density lipoprotein in diabetic rats after mangiferin treatment @ 10 & 20 mg/kg BW.

Likewise, Dineshkumar et al. (2010) noticed the LDL lowering potential of mangiferin in diabetic rats. For the intention, experimental rats were provided mangiferin @ 20 mg/kg BW for 28 days. The serum analysis for LDL revealed 31.34% reduction in the treated group as compared to control. They deduced that antioxidative action and fatty acid synthesis enzymes suppressive behaviour of mangiferin are the leading routes by which it tackles the lipid related abnormalities. Elevation of low density lipoprotein (LDL) is a key event in the development of atherosclerosis (Chisolm et al., 2000). This disorder is triggered by oxidative modification of LDL after exposure to reactive oxygen species (ROS) from vascular wall cells (Witztum and Steinberg, 1991; Lamb et al., 1992). During hypercholesterolemic phase, elevated expression of LDL receptor (LDLr) indicates the accumulation of reactive oxygen spices in mitochondria that make the subjects susceptible to Ca2+ induced membrane permeability transition (MPT) (Paim et al., 2008). In this context, mangiferin has proven beneficial to suppress the LDLr expression and scavenge the free radicals thus protects the cell death by shielding the membranes from oxidation.

One of the researchers groups, Pardo-Andreu et al. (2008) investigated the effect of oral administration of mangiferin on LDL receptor activity (LDLr-/-) in mice. The mice were treated with 40 mg/kg dose of mangiferin for 7 days and observed reduction in ROS production, efflux of calcium ions and MPT incidence when compared to negative control. Additionally, LDLr-/- mice also possessed high cholesterol level, leading to excessive utilization of NADPH reducing equivalents, which are mitochondrial antioxidant defence compounds. However, animals receiving mangiferin treatment have less NADPH requirement resulting increased antioxidant defence ability consequently protects mitonchondria from oxidative stress.

Later, Guo et al. (2011) examined the effect of different concentrations of mangiferin i.e. 50 and 150 mg/kg on the lipid profile of hamsters fed on high fat diet. They concluded that mangiferin caused dose dependent reduction in low density lipoprotein (LDL) due to its oxidation protective role. In a clinical trial, Aderibigbe et al. (2001) examined the LDL

reducing capacity of mangiferin in hypercholesterolemic rats during one month study. They observed 24.66 to 27.88% decline in the level of low density lipoprotein of hypercholestrolemic rats after mangiferin treatment @ 10 & 20 mg/kg.

It is concluded from the above discussion that mango peel polyphenols supplemented functional/nutraceutical drinks with special reference to mangiferin have capacity to curtail the menace of LDL and other lipid related variables.

4.8.6. High density lipoprotein (HDL)

The F values (Table 35) showed that HDL level was affected significantly due to treatments in study II & III however, non-momentous differences were observed in study I. In study I (trial 1), HDL level was non-significantly increased from 33.23±3.15 mg/dL in T0 to 33.78±1.16 and 34.07±2.64 mg/dL in T1 and T2 groups, respectively. Likewise in trial 2, this trait was non-substantially increased from 35.21±2.78 (T0) to 36.24±2.87 (T2) mg/dL. However in study II (trial 1), T0 group exhibited the lowest HDL level

(27.25±1.11 mg/dL) that elevated momentously in T1 & T2 as 28.24±1.46 and 28.78±1.47 mg/dL, respectively. Similarly in trial 2, a substantial enhancement in HDL was recorded from 26.21±1.62 (T0) to 27.60±1.98 (T2) mg/dL. In study III (trial 1 & 2), T0 had the lowest HDL level as 30.12±2.18 & 31.25±2.89 mg/dL than that of T1 & T2 31.03±1.09 & 32.18±2.82 and 31.43±2.12 & 32.50±2.43 mg/dL, respectively (Table 35).

It is depicted from the Figure 7 that in both trials of study I, treatments T1 and T2 caused a non-significant increase in HDL level (1.65 & 1.96 and 2.52 & 2.92%), respectively as compared to control. In contrary, for study II & III (trial 1 & 2) the significant elevation was noticed in T2 (5.61 & 5.30 and 4.34 & 4.01%) followed by T1 (3.65 & 4.01 and 3.01 & 2.96%), respectively.

Being lipophilic, cholesterol is water insoluble and required different lipoproteins for its circulation in the blood. The cholesterol transportation system consists of chylomicron (CM), very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) however, HDL and LDL are the most promising carriers that determine the efficiency of lipid profile (Yang et al., 2012). Various research evidences have elucidated that an imbalance between LDL and HDL enhances the probability of cardiovascular complications (Michos et al., 2012). The HDL is known as “good

cholesterol” owing to its ability to facilitate reverse cholesterol transportation (RCT), collects the extra cholesterol from arteries and tissues and sends back to liver where it is absorbed and excreted in the form of bile acid. It prevents cholesterol deposition in the medium calibre artery by collecting it from sub-endothelial space, the place for atheroma formation. Recently, the role of HDL has been highlighted to manage the cardiovascular health due to its inverse association with LDL (McEneny et al., 2012; Gadi et al., 2012).

In the module of diet based therapy, polyphenols have gained immense attention as coronary curing agent due to their cholesterol and LDL lowering abilities. Numerous polyphenols are considered important for curtailing this menace however, mangiferin has attained special position owing to its unique structure and mode of action. Numerous investigations have shown the positive impact of mangiferin against abnormal lipid profile by protecting the LDL from oxidation and enhancing the HDL level in obese diabetic model (Hou et al., 2009; Deka and Vita, 2011; Al-Attar and Zari, 2010; Bahorun et al., 2012). The significant effect of mango peel extract and mangiferin based functional drinks on the HDL level of rats fed on high cholesterol diet are in harmony with the earlier findings of Muruganandan et al. (2005), observed significant elevation in HDL concentration (10.90%) of hypercholestrolemic rats treated with mangiferin. Moreover decline in cholesterol, atherogenic index, low density lipoprotein and triglycerides are also noticed.

Different scientific groups including Dey et al. (1998) and Carmen et al. (1999) investigated the HDL boosting potential of mango peel extract rich in mangiferin through a bioefficacy trial and observed a significant reduction in cholesterol and LDL with an increase in HDL. Similarly, Canal et al. (2000) delineated a beneficial role of mangiferin supplementation @ 30, 50 & 70 mg/Kg BW for HDL enhancement in hypercholesterolemic rats.

The observations of Guo et al. (2011) further strengthened the results of current study regarding HDL elevation as they noticed a significant effect of mangiferin on HDL level. They treated high fat fed hamsters with mangiferin @ 50 and 150 mg/kg, respectively for two months and inferred that mangiferin causes HDL enhancement in dose dependent manner due to its ability to alter the fatty acid metabolism, provides protection to LDL from oxidation and modulates the HDL/LDL ratio.

Table 35. Effect of functional drinks on HDL (mg/dL) Studies Treatments F value T0 T1 T2 Study I (Trial 1) 33.23±3.15 33.78±1.16 34.07±2.64 34.3 NS (Trial 2) 35.21±2.78 35.90±2.98 36.24±2.87 28.0 NS

Study II (Trial 1) 27.25±1.11b 28.24±1.46b 28.78±1.47a 24.3* (Trial 2) 26.21±1.62b 27.26±0.63b 27.60±1.98a 25.6*

Study III (Trial 1) 30.12±2.18b 31.03±1.09ab 31.43±2.12a 11.9* (Trial 2) 31.25±2.89b 32.18±2.82ab 32.50±2.43a 10.0*

NS= Non Significant * = Significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

6 5.61 5.3

5 4.34 4.01 4.01 4 3.65

2.92 3.01 2.96 3 2.52 T1 T2 1.96

Percent increase 2 1.65

0 Study1(Tr 1) Study 1(Tr 2) Study 2( Tr 1) Study 2(Tr 2) Study 3(Tr 1) Study 3(Tr 2)

Figure 7. Percent increase in HDL as compared to control

The significant effect of mangiferin on HDL elevation in diabetic rats is supported by the work of Dineshkumar et al. (2010). They conducted a model feeding trial to investigate the lipids assuaging ability of mangiferin in streptozotocin induced diabetic rats. The rats were provided mangiferin @ 10 & 20 mg/kg BW for one month and observed increase in HDL level by 23.64 and 26.31%, respectively. They deduced that due to strong antioxidant potential, mangiferin halts the process of lipid peroxidation thus improves HDL concentration. Previously, Miura et al. (2001) and Prabhu et al. (2006 a,b) documented that mango polyphenols uplift the HDL level in diabetic rats due to their antioxidative potential. One of their peers, Nair and Shyamala Devi (2006) have observed an increase in HDL and decrease in abnormal cholesterol & triglyceride levels in the volunteers after mangiferin treatment. The present results are advocating the importance of mangiferin to curtail the complications of dyslipidemia.

4.8.7. Triglycerides

The F values in Table 36 showed that treatments imparted significant effect on the triglycerides level of rats during all studies except for study I.

In normal rats (study I), mean triglycerides values were 67.12±2.38 & 64.23±3.42,

65.21±2.38 & 62.34±2.86 and 65.10±3.32 & 62.14±2.96 mg/dL in T0, T2 and T1 groups, respectively (trial 1 & 2). Likewise in study II, highest triglycerides level was observed in

T0 (95.23±3.87 & 97.15±4.09 mg/dL) that momentously reduced to 90.23±4.87 &

91.31±3.67 and 85.73±5.36 & 87.43±4.90 mg/dL in groups relied on T1 and T2. Likewise in study III, the highest value was noticed in T0 (75.25±4.62 mg/dL) followed by T1

(72.02±4.32 mg/dL) and T2 groups (69.79±3.76 mg/dL) (trial 1). In the follow up trial, the triglycerides concentration significantly reduced from 72.56±3.23 T0 to 69.55±3.56 T1 and 67.59±3.98 mg/dL in T2 (Table 36).

It is evident from the Figure 8 that drinks T2 and T1 caused highest decline in triglycerides level by 9.98 & 10.01% and 5.25 & 6.01%, respectively (study II; trial 1 & 2). Likewise, the recorded reduction in triglycerides for T2 and T1 groups in study III (trial 1 & 2) was 7.25 & 6.85 and 4.29 & 4.15 %, respectively.

In the instant study, significant diminish in triglycerides level of hypercholestrolemic rats (study II) is corroborated with the findings of Guo et al. (2011), tested mangiferin against abnormal lipid profile of hypercholestrolemic hamsters. The animals were treated with 90

different concentrations of mangiferin i.e. 50 and 150 mg/kg BW, respectively for a period of 56 days and observed a dose dependent decline in triglycerides and LDL concentrations by 23 & 30%, respectively. They inferred that mangiferin reduces the triglycerides by inhibiting the expression of fatty acid synthesis proteins like acetyl Co-A carboxylase (ACC) and acyl Co-A diacylglycerol acyltransferase 2 (DGAT-2). Similarly, Dey et al. (1998) and Carmen et al. (1999) observed reduction in the triglycerides level of hypercholesterolemic rats due to mangiferin administration.

The results of current exploration are in line with the investigations of Miura et al. (2001b), they launched a two weeks efficacy study to evaluate the triglycerides lowering ability of mangiferin. For the intention, the rats were fed on mangiferin (30 mg/kg) along with basal diet during the entire trial and noticed 70% reduction in triglyceride levels of tested animals. In another study, Dineshkumar et al. (2010) probed mangiferin against lipids related malfunctionings in diabetic rats. The diabetes was induced through a streptozotocin injection @ 65 mg/kg body weight BW) and treated with mangiferin @ 10 & 20 mg/kg BW for the period of 30 days. The mangiferin provision resulted a significant reduction in triglyceride level of rats by 20.79 & 19.06%, correspondingly. Earlier, Garcia et al. (2003) evaluated the effect of water extract of mango bark containing mangiferin against lipid profile indicators and noticed 54% decline in triglycerides level of diabetic rats. They were of the view that due to strong antioxidant potential mangiferin protects the LDL from oxidation moreover, it influences different molecular targets involved in triglyceride synthesis. Likewise, Moharib (2006) documented the triglycerides suppressing ability of mangiferin in diabetic rats model. Similarly, Seth and Sharma (2004) also found significant decrease in triglycerides of hypertriglyceridemic rats after intraperitoneal injection of aqueous extract of mango leaves rich in mangiferin. One of the researchers groups, Morsy et al. (2010) observed a momentous reduction in triglycerides level of streprozotocin diabetic rats after treatment with mango polyphenol extract. They recorded dose dependent diminish in triglycerides level by 23.87, 25.54 and 61.02% @ 30, 50 and 70 mg of mango polyphenols, respectively. They were of the view that mango peel polyphenols especially mangiferin has potential to tackle cardiovascular complications by lowering arthrogenic index and effecting the activity of triglycerides synthesis and metabolism.

Table 36. Effect of functional drinks on triglycerides Studies Treatments F value T0 T1 T2 Study I (Trial 1) 67.12±2.38 65.21±2.38 65.10±3.32 1.00NS (Trial 2) 64.23±3.42 62.34±2.86 62.14±2.96 1.28NS

Study II (Trial 1) 95.23±3.87a 90.23±4.87b 85.73±5.36c 28.2** (Trial 2) 97.15±4.09a 91.31±3.67b 87.43±4.90c 28.6**

Study III (Trial 1) 75.25±4.62a 72.02±4.32b 69.79±3.76c 12.6** (Trial 2) 72.56±3.23a 69.55±3.56b 67.59±3.98c 10.8**

NS= Non Significant **= Highly significant

Study I : Normal diet Study II : High cholesterol diet Study III:Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

12 9.98 10.01 10

7.25 8 6.85 6.01 6 5.25 T1 4.29 4.15 T2 4 3.01 2.95 3.25 Percent decrease 2.85

0 Study1(Tr 1) Study 1(Tr 2) Study 2( Tr 1) Study 2(Tr 2) Study 3(Tr 1) Study 3(Tr 2)

Figure 8. Percent reduction in triglycerides as compared to control

Triglycerides are converted into fatty acids and monoglycerols in the lumen of small intestine by the action of pancreatic lipase. Further they reassembled in the chylomicrons that enter the lymphatic system and hydrolyzed to free fatty acids by lipases enzymes activities (Volek et al., 2001). These free fatty acids move towards hepatic cells & oxidized in the mitochondria to form ATP and produce triglycerides for storage in very low density lipoprotein particles (Browning and Horton, 2004). In liver, lipid metabolism is dependent on the activity of different expressions like mRNA genes and lipogenic genes. In this context, mangiferin halts the activity of hepatic lipogenic genes thus reduces triglycerides and free fatty acids levels in serum and liver (Choi et al., 2007). The Diacylglycerol acyltransferase (DGAT) microsomal enzyme join acyl CoA to 1,2- diacylglycerol and completes the TG biosynthesis. In mammals, microsomal enzyme comprises of two isoforms including DGAT-1 and DGAT-2, encoded by distinctive genes, present at high concentration in white adipose tissues (Wang et al., 2010). Previously, Brown and Goldstein (1997) have explicated that mangiferin supplementation reduces liver triglycerides level by inhibiting DGAT-2 gene expression. Another promising route by which mangiferin manages the triglycerides elevation is its effect on microsomal triglyceride transfer protein (MTP) that plays a key role in very low density lipoprotein synthesis by facilitating the transfer of hepatic lipids to nascent apolipoprotein B (Qin et al., 2008). The mangiferin significantly inhibits MTP mRNA expression and reduces serum TG level in hamsters fed on high fat diet (Avramoglu and Adeli, 2004).

It is concluded that whole mango peel extract and mangiferin based functional/ nutraceutical drinks are effectual against lipid related variables. Nevertheless, the resultants drinks showed better performance in hypercholestrolemic and diabetic phases. Furthermore, mangiferin based drink was more efficient to ameliorate the threat of hypercholesterolemia as compared to sample containing whole mango peel extract thus can be utilized as dietary intervention against lifestyle related disorders with special reference to lipid related abnormalities.

4.8.8. Glucose

The statistical analysis (F values) revealed that treatments imparted significant differences on the glucose level in different groups of rats during all studies excluding study I (Table 37).

The recorded mean glucose levels in study I (trial 1 & 2) for T0, T1 and T2 groups were 82.58±5.23 & 85.96±4.83, 80.23±5.62 & 84.03±7.32 and 79.68±7.12 & 83.22±6.34 mg/dL, respectively (Table 37). However in study II (trial 1 & 2), maximum glucose level was noticed in T0 group 101.25±7.64 & 99.63±6.86 mg/dL that momentously declined to

93.58±8.35 & 92.70±7.26 and 92.79±6.68 & 91.74±7.04 mg/dL in T1 and T2 groups.

Likewise in study III, T0 group showed the highest glucose level 214.96±8.34 &

216.41±7.56 mg/dL that significantly decreased in T2 and T1 groups as 186.82±8.98 & 185.55±8.46 and 197.51±8.53 & 196.26±7.15 mg/dL, respectively (trial 1 & 2).

It is evident from the Figure 9 (study II; trial 1 & 2) that drinks T1 and T2 resulted 7.58 & 6.96 and 8.36 & 7.92% reduction in glucose level, respectively. Similarly in study III, maximum decline in glucose 13.09 & 14.26% was observed in T2 followed by 8.12 &

9.31% in T1 (trial 1 & 2).

The instant results are in accordance with the findings of Prasad et al. (2009), observed blood glucose lowering effect of mangiferin in streptozotocin-diabetic rats. They delineated that administration of mangiferin @ 250 mg/kg for four weeks resulted significant diminish in glucose concentration (17.55%). Likewise, Miura et al. (2001b) reported significant reduction in serum glucose of experimental mice after ingestion of mangiferin @ 30 mg/kg for 3 weeks. In other study, Gupta et al. (2011) reported 23.37% decrease in glucose level of diabetic rats by the supplementation of mango polyphenols @ 300 mg/kg BW for a period of 14 days. Earlier, Scalia et al. (2007) also recorded 47.29% decline in glucose level of streptozotocin induced diabetic rats with mangiferin treatment. Later, Sayed et al. (2011) conducted a bioefficacy trial involving male diabetic Albino rats to explore the role of mango peel polyphenols especially mangiferin against glucose related abnormalities and indicated 51.08 & 42.02% glucose reduction after first & second month. In this connection, Prashanth et al. (2001) carried out a rodent trial to validate the α-glucosidase inhibitory activity of mangiferin. They used ethanolic extract of mango peel rich in mangiferin and observed a significant reduction in the concentration of this enzyme. One of their peers, Yoshikawa et al. (2001) also documented the inhibitory action of mangiferin against glucosidase enzyme. They further reported that suppression in the enzyme activity causes reduction in carbohydrate breakdown thereby slows down glucose absorption from the intestine. Later, Muruganandan et al. (2005) described the ability of mangiferin to modulate both

pancreatic and extra pancreatic activities involved in the management of diabetes. According to the Randle’s glucose-fatty acid cycle, higher supply of plasma triglycerides leads to increase free fatty acids availability & oxidation that impairs insulin action, glucose metabolism and utilization leading to diabetes. However, mangiferin reduces triglycerides level and facilitates glucose oxidation & utilization subsequently alleviates hyperglycemia. Additionally, mangiferin exhibits anti-diabetic response by decreasing the insulin resistance in non-insulin dependent KK/Ay mice. The chronic supplementation of mangiferin momentously enhances the oral glucose tolerance in glucose fed rats.

One of the researchers groups, Akbarzadeh et al. (2007) have established that induction of streptozotocin lowers the nicotinamide adenine dinucleotide (NAD) level in β-cells, causing histopathological defragmentation that develops hyperglycemia in experimental volunteers. Moreover, utilization of food & water, urine excretion and glucose concentration are enhanced along with decline in the level of insulin and C-peptide protein. The β-cells damage plays a key role in the onset of this event. Nevertheless, mangiferin provision regulates the structural and functional abnormalities of β-cells through its strong antioxidant, immunomodulatory and anti-inflammatory properties.

Previously, Ichiki et al. (1998) determined that reactive oxygen species initiate the cascade of adverse reactions that modify the glucose functionality resulting in diabetes however, mangiferin treatment modulates the glucose metabolism, preserves insulin utilization & improves insulin secretion thus alleviates glucose malfunctionings. In another scientific exploration, Morsy et al. (2010) noticed significant effect of different mangiferin aqueous extract i.e. 30, 50 and 70 mg/kg BW with 31.85, 37.14 and 43.11% reduction in serum glucose level of Sprauge-Dawley diabetic rats.

The findings of Dinesh-Kumar et al. (2010) elucidated that mangiferin doses @ 10 and 20 mg/kg BW suppressed glucose level by 8.86 and 26.82% in adult male Wistar rats. Afterwards, Sellamuthu et al. (2012) conducted a rodent trial involving streptozotocin induced diabetic rats to evaluate the hypoglycemic response of mangiferin during four weeks. The mangiferin feeding at a dose of 40 mg/kg BW was resulted 64.53% decline in glucose level. It is inferred from the above debate that mango peel extract and mangiferin based functional drinks are effective to address the glucose related abnormalities nonetheless, mangiferin is more capable to curtail this threat.

Table 37. Effect of functional drinks on glucose (mg/dL) Studies Treatments F value T0 T1 T2 Study I (Trial 1) 82.58±5.23 80.23±5.62 79.68±7.12 1.53NS (Trial 2) 85.96±4.83 84.03±7.32 83.22±6.34 1.41NS

Study II (Trial 1) 101.25±7.64a 93.58±8.35b 92.79±6.68b 22.2** (Trial 2) 99.63±6.86a 92.70±7.26b 91.74±7.04b 12.2**

Study III (Trial 1) 214.96±8.34a 197.51±8.53b 186.82±8.98c 56.02** (Trial 2) 216.41±7.56a 196.26±7.15b 185.55±8.46c 70.01**

NS= Non Significant **= Highly significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

T1 T2 16 14.26 13.09 14

12 9.31 10 8.36 8.12 7.58 7.92 8 6.96

Perent Perent decrease 3.51 2.85 3.19 4 2.24 2

0 Study1(Tr 1) Study 1(Tr 2) Study 2( Tr 1) Study 2(Tr 2) Study 3(Tr 1) Study 3(Tr 2)

Figure 9. Percent reduction in glucose as compared to control

4.8.9. Insulin

The F values in Table 38 indicated momentous variations for insulin level due to treatments in study II and III whereas, non-significant effect was observed in study I.

In study I (trial 1), mean insulin values for T0, T1 and T2 groups were 7.58±0.42, 7.66±0.38 and 7.76±0.23 µU/mL whilst in 2nd trial 8.01±0.35, 8.15±0.46 and 8.25±0.48

µU/mL for respective groups. Similarly, in study II (trial 1 & 2), T0 group elucidated the lowest insulin level 7.05±0.16 & 7.35±0.49 µU/mL that was significantly increased to

7.22±0.42 & 7.53±0.52 and 7.33±0.19 & 7.69±0.13 µU/mL in T1 and T2 groups, respectively. Moreover, diabetic rats (study III; trial 1 & 2) had the lowest insulin level

5.21±0.29 and 5.03±0.14 µU/mL in T0 group that was significantly enhanced in T1 and T2 groups as 5.49±0.38 & 5.33±0.34 and 5.58±0.23 & 5.43±0.37 µU/mL, respectively (Table 38 ).

The Figure 10 depicted the percent increase in insulin; in study II (trial 1 & 2) mangiferin supplemented drink (T2) led to 4.01 & 4.65% incline whereas T1 (whole mango peel) drink resulted 2.35 & 2.45% increase for this trait. Similarly in study III (trial 1 & 2), the recorded uplift for insulin in T2 was 7.15 & 7.98% higher than that of T1 5.36 & 6.01%, respectively.

The current results regarding insulin enhancement are in harmony with the conclusions of Sellamuthu et al. (2012), explored the hypoglycemic response of mangiferin in streptozotocin induced diabetic rats model. The mangiferin administration @ 40 mg/kg BW for 28 days caused improvement in insulin level by 3 fold from 4.95±0.33 to 12.78±0.88. Earlier, Sellamuthu et al. (2009) observed an increase for insulin level from 5.04±0.29 to 14.58±0.65 in diabetic rats after mangiferin supplementation. Likewise, Birnbaum (2001) and Evans (2002) conferred that mangiferin lessens the risk of insulin resistance by quenching free radicals and restoring mitochondrial redox homeostasis. One of the scientific groups, Gupta et al. (2011) probed mango polyphenols against impaired insulin level in diabetic rats. They reported 32.89 and 35.43% boost in insulin levels after treatment with 300 mg/kg BW of polyphenolic extract enriched with mangiferin at 14th and 21th day, respectively.

Protein tyrosine phosphatase 1B (PTP1B) has a critical role in type-2 diabetes as a negative regulator of insulin signaling pathway. It is primarily responsible for the

dephosphorylation of activated insulin receptor thereby downregulates the insulin signaling (Klaman et al., 2000; Pei et al. 2004). In this connection, Rau et al. (2006) and Hu et al. (2007) noted the beneficial role of mangiferin for the inhibition of PTP1B expression moreover, it regulates the transactivation of peroxisome proliferator activated receptor isoforms (PPARs) by increasing insulin sensitivity. Earlier, Saxena and Vikram (2004) determined that insulin acts as antilipolytic agent by inhibiting the activities of adenyl cyclase, activation of the cAMP phosphodiesterase that leads to cAMP degradation and the promotion of glucose uptake & lipid storage. The findings of Sayed et al. (2011) elucidated the hypoglycemic potential of mango peel polyphenols especially mangiferin as it induced marked enhancement in the insulin secretion (32.66%) of diabetic male Albino rats. They inferred that mangiferin exhibits positive impact on fat & carbohydrate metabolism thus regulates the insulin transporters. Likewise, Dieni and Storey (2011) documented that hexokinase plays a prime role in the glucose homeostasis maintenance through catalyzes the phosphorylation of glucose to glucose-6-phosphate in the streptozotocin induced diabetic volunteers. Moreover, higher hexokinase activity causes decline in the insulin concentration. They observed that mangiferin administration to the diabetic rats suppresses the hexokinase activity by activation of glycolysis and enhances the glucose utilization thus improves insulin secretion. In another research, Periyar et al. (2009) inferred that pyruvate kinase activity is significantly enhanced in streptozotocin diabetic rats due to decrease in glucose utilization. The mangiferin lowers the activity of pyruvate kinase during diabetic phase thus accelerates the glucose consumption and insulin production.

Later, Fridly and Philipson (2010) have unveiled that lactate dehydrogenase (LDH) activity is increased during hyperglycemic phase however, mangiferin imparts diminishing effect on LDH expression. Besides, Pessôa et al. (2012) observed the gluconeogenic enzymes lowring potential of mangiferin in STZ-induced diabetic rats. Similarly, Zhaoyun et al. (2010) reported that activity of glucose-6-phosphate dehydrogenase is enhanced during diabetes that suppresses the functioning of HMP shunt and the production NADH and NADPH nevertheless, mangiferin brings glucose- 6- phosphate dehydrogenase activity to normal level by improving the formation of NADPH, increases lipogenesis and provides substitute channel to dispose off the excess glucose via HMP pathway.

100

Table 38. Effect of functional drinks on insulin (µU/mL) Studies Treatments F value T0 T1 T2 Study I (Trial 1) 7.58±0.42 7.66±0.38 7.76 ±0.23 14.9 NS (Trial 2) 8.01±0.35 8.15±0.46 8.25±0.48 6.06 NS

Study II (Trial 1) 7.05±0.16b 7.22 ±0.42a 7.33±0.19a 12.06* (Trial 2) 7.35±0.49b 7.53 ±0.52a 7.69±0.13a 8.66*

Study III (Trial 1) 5.21±0.29b 5.49±0.38a 5.58±0.23a 25.65** (Trial 2) 5.03±0.14b 5.33±0.34a 5.43±0.37a 30.66**

* = Significant **= Highly significant NS= Non Significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

101

9 7.98 8 7.15 7 6.01 6 5.36 4.65 5 4.01 T1 4 T2 2.96

Percent increase 3 2.36 2.35 2.45 1.69 2 1.02 1

0 Study1(Tr 1) Study 1(Tr 2) Study 2( Tr 1) Study 2(Tr 2) Study 3(Tr 1) Study 3(Tr 2)

Figure 10. Percent increase in insulin as compared to control

102

Different scientists including Petersen & Shulman (2006) and Evan (2002) expounded that mitochondrial dysfunctions caused a decline in the expressions of nuclear encoded genes which modulate the mitochondrial homeostasis like PPAR-ϒ. The elevated levels of intracellular fatty acids metabolites increased the magnitude of oxidative stress due to higher production of free radicals. During oxidative stress, reactive oxygen species activate the protein kinase signaling cascades and serine phosphorylation of IRS-1 thus impair insulin sensitivity. In this reference, Bhowmik et al. (2009) described the assuaging role of mangiferin in the oxidative stress induced insulin resistance by its interference with intestinal glucose absorption in gut after stimulating peripheral glucose utilization and glycemia reduction through inhibition of glucose intake.

Decisively, mango peel polyphenols especially mangiferin based functional/nutraceutical drinks have potential to manage glucose and insulin related malfunctions thus introduce in the diet based therapy against these perils.

4.8.10. Glutathione

It is evident from the statistical analysis (F values) that glutathione level was affected substantially due to treatments in the entire studies (Table 39).

Means regarding glutathione level in study I (trial 1 & 2) showed the lowest value

47.56±3.22 & 49.65±3.68 mg/L in T0 that significantly uplifted to 50.00±3.21 &

52.11±3.02 and 50.92±3.93 & 52.96±2.25 mg/L in T1 and T2 groups, respectively.

Likewise in study II, the lowest glutathione level was observed in T0 (39.63±2.32 mg/L) that significantly enhanced in T1 (43.64±3.29 mg/L) and T2 (45.66±2.45 mg/L). Similar increasing trend was reported during the 2nd trial, the glutathione level was uplifted from

37.89±1.65 T0 to 43.53±1.25 mg/L in T2. Moreover in study III (trial 1 & 2), the recorded values were 42.85±2.09 & 44.17±3.96, 45.64±2.57 & 47.27±3.95 and 47.25±3.24 &

49.31±2.62 mg/L for T0, T1 and T2, respectively (Table 39).

It is obvious from the Figure 11 that mangiferin based functional drink (T2) resulted significant rise in glutathione content during entire efficacy trial. In study II (trial 1 & 2),

T2 showed 15.21 & 14.89% incline whereas, T1 drink caused 10.12 & 9.63% enhancement for this attribute. Likewise in study III (trial 1 & 2), the observed uplift for glutathione contents in T2 was 10.26 & 11.63% higher than that of T1 6.52 & 7.01%, respectively.

103

During oxidative stress, reactive oxygen spices (ROS) are abundantly generated and produce hydrogen peroxide that promotes several undesirable reactions. The antioxidant glutathione halts this process by converting the hydrogen peroxide into water and helps the body to maintain its normal oxidation potential. Numerous physical factors like poor dietary habits, lack of physical activity, smoking and environmental pollutants may accelerate the production of several noxious radicals however, polyphenols like mangiferin quench these deleterious moieties, improve the body defense system and ensures good health & longevity (Wong et al., 2006; Seifried et al., 2007).

The cells contain low molecular weight endogenous antioxidant glutathione (GSH) that has capacity to protect from free radicals. It is a major endogenous antioxidant that actively participates in protecting cellular components from oxidative damage (Power and Blumbergs, 2009). Chemically, glutathione is composed of cysteine, glutamic acid and glycine and exists in reduced (GSH) and oxidized states (GSSG). Numerous scientific explorations advocate the beneficial impact of glutathione during oxidative stress phase. It quenches free radicals by conjugating the electrophiles controlled by glutathione transferase and oxido-reduction cyclic glutathione transformation by glutathione peroxidase and glutathione reductase thus regulates the imbalance between body redox potential and reactive oxygen species (Wong et al., 2006; Seifried et al., 2007).

The instant results are in harmony with the previous findings of Prabhu et al. (2006), they carried out a model feeding trial to evaluate the effective role of mangiferin against myocardial infarction (ISPH) oxidative stress phase. The mangiferin treatment @ 200 mg/kg body weight for 28 days enhanced the concentration of glutathione, glutathione transferase and catalase by 34.17, 46.85 and 52.13%, respectively. They were of the view that free radical scavenging activity of mangiferin is responsible for this uplift. Being a strong antioxidant, mangiferin has ability to enhance the activities of glutathione and other antioxidant enzymes. Additionally, it quenches the free radicals (hydroxyl and superoxide anions) due to strong radicals catching ability. Mangiferin has proven antioxidant that neutralizes the noxious radicals due to its unsaturated 2–3 bond and 4- oxo function 3’,4’-catechol groups (Gao et al., 1999).

In another study, De-Jian et al. (2003) conducted a bioefficacy trial to examine the glutathione enhancing perspectives of mangiferin in experimental rats. The oxidative

104

stress was induced through free radicals with concomitant provision of mangiferin @ 100 mg/kg body weight for 28 days. They observed a significant increase in the activity of glutathione (GSH), glutathione S-transferase (GST) and catalase (CAT) by 52.38, 47.81 and 93.29%, respectively.

One of the researcher groups, Pardo-andreu et al. (2008) observed that mango polyphenols especially mangiferin has potential to protect the body from oxidative stress. They treated experimental rats with 40 mg/kg BW of mangiferin for 7 days and noticed significant increase in the activities of different endogenous antioxidant enzymes like GSH, superoxide dismutase (SOD) with marked decline in lipid peroxidation. Earlier, Combs and Gray (1998) documented that mangiferin has potential to refurnish the activity of selenium dependent antioxidant enzymes like glutathione peroxidase. Later, Akila and Devaraj (2008) described that mangiferin increases the GSH activity thereby upregulates the GPx concentration.

Mangiferin tackles the oxidative stress related disparities by protecting lipid peroxidation and formation of mangiferin phenoxy and oxo-ferryl radicals through modification of polymer chain initiation by interacting with free radicals. This complex induces vinylic monomer methylmethacrylate (MMA) polymerization. Likewise, chain terminators initiate the mangiferin-Fe-PMMA-polymer formation and neutralize the alkoxy/lipid peroxy radicals, considered as the promising route by which mangiferin ameliorates oxidative stress (Ghosal and Rao, 1996).

Decisively, mango peel polyphenols and mangiferin based functional/nutraceutical drinks have potential to enhance the serum glutathione level thus useful for the management of oxidative stress related variables.

4.8.11. TBARS

The F values in Table 40 explicated significant effect of treatments on serum thiobarbituric acid reactive substances (TBARS) level of rats during the entire bioevaluation trial.

In study I (trial 1 & 2), the highest TBARS value was reported in T0 (6.55±0.43µmol/L) that momentously lowered in T1 (6.22±0.52 µmol/L) and T2 (6.10±0.28 µmol/L) groups.

105

Table 39. Effect of functional drinks on serum glutathione (mg/L) Treatments Studies F value T0 T1 T2 Study I (Trial 1) 47.56±3.22b 50.00±3.21ab 50.92±3.93a 24.5* (Trial 2) 49.65±3.68b 52.11±3.02ab 52.96±2.25a 22.2*

Study II (Trial 1) 39.63±2.32b 43.64±3.29ab 45.66±2.45a 180.02** (Trial 2) 37.89±1.65b 41.54±3.41ab 43.53±1.25a 170.6**

Study III (Trial 1) 42.85±2.09c 45.64±2.57b 47.25±3.24a 42.3** (Trial 2) 44.17±3.96c 47.27±3.95b 49.31±2.62a 51.2**

* = Significant **= Highly significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

106

T1 T2

15.21 16 14.89

14 11.63 12 10.12 10.26 9.63 10 7.06 7.01 8 6.66 6.52 6 5.12 4.96 Percent increase 4

0 Study1(Tr 1) Study 1(Tr 2) Study 2( Tr 1) Study 2(Tr 2) Study 3(Tr 1) Study 3(Tr 2)

Figure 11. Percent increase in glutathione as compared to control

107

Whilst, in the 2nd trial, observed TBARS values were 7.01±0.35, 6.60±0.32 and

6.52±0.19µmol/L in T0, T1 and T2 groups, respectively. Similarly in study II (trial 1 & 2), the reported TBARS values were 10.29±0.10 & 11.01±0.62, 9.24±0.42 & 9.61±0.63 and

8.62±0.21 & 9.12±0.32 µmol/L in T0, T1 and T2 groups. Moreover, TBARS values in study III (trial 1) varied from 9.36±0.38 µmol/L (T0) to 8.46±0.12 µmol/L (T2), respectively. Similar trend was observed in the trial 2 for T0, T1 and T2 group as 8.42±0.23, 7.76±0.32 and 7.58±0.15 µmol/L (Table 40).

The Figure 12 elucidated that functional drink containing mangiferin showed significant reduction in TBARS value during respective studies. In study I, the reported values were

5.06 & 5.89% (T1) and 6.89 & 7.01% (T2) during trial 1 & 2. Similarly, the highest decline regarding this parameter was observed in study II (trial 1) as 16.23 and 10.25% in

T2 and T1, respectively whereas 17.21 & 12.69% in the subsequent trial. In study III, maximum TBARS reduction 10.02 & 9.63% was noticed in T2 followed by 7.85 & 6.23% in T1, correspondingly (trial 1 & 2). During lipid peroxidation, cellular membrane integrity is damaged due to reaction between polyunsaturated fatty acids of the membrane and free radicals resulting in the production of lipid hydroperoxides and malondialdehyde (MDA). In this connection, mangiferin performed assuaging action on the abnormal TBARS and other lipid peroxidation byproducts by hampering the formation of superoxide, chelateing metal ions and decreasing the mitochondrial oxidation (Prabhu et al., 2006; Garrido et al., 2004).

The findings of current exploration regarding TBARS level are in accordance with the previous conclusions of De-Jian et al. (2003), they have conducted 28 days bioefficacy trial to examine the lipid peroxidation protecting ability of mangiferin. The oxidative stressed rats were treated with mangiferin @ 100 mg/kg BW. They noticed a significant decline 58.37% in thiobarbituric acid reactive substances (TBARS) as compared to control and inferred that mangiferin provides shield against lipid peroxidation owing to its chemical structure, metal chelating ability and radical quenching perspectives.

One of the researchers groups, Pardo-Andreu et al. (2008) have reported the therapeutic role of mangiferin against mitochondrial oxidative stress in hypercholestrolemic rats. For the intention, they carried out TBARS assay and lipid peroxidation extent measurement through malondialdehyde level. The experimental rats were provided mangiferin (Vimang) and observed 33.33% reduction in TBARS level. They were of the view that

108

mangiferin mitigates oxidative stress by inhibiting Fe (II)-citrate-mediated mitochondrial swelling, maintains transmembrane potential, suppresses the TBARS formation and enhances the O2 consumption (Castilho et al., 1994).

In another research, Zasada et al. (2011) documented that mangiferin (300 mg/kg) caused a decline in TBARS concentration by 38% in experimental rats. Likewise, different scientific groups including Schulze and Lee (2005) and Lee & Prasad (2003) ascribed the free radical scavenging ability and lipid peroxidation lowering potential of mangiferin in atherosclerosis and hypercholestrolemic phases. During these conditions, atherogenic diet leads to enhancement in lipid peroxidation because of higher production of free radicals and lipid peroxidative products. However, mangiferin ameliorates these abnormal events by lowering the TBARS level and enhancing the activity of glutathione and other antioxidant enzymes. Lipid peroxidation is initiated after the reaction of free radicals and polyunsaturated fatty acids (PUFA), forming alkoxy (R•) and peroxy radicals (ROO•). These obnoxious radicals develop covalent bonds with cellular protein, disrupt the cell membrane integrity, enzyme activity and subsequently cause lipid peroxidation (Pandit et al., 2004; Brautbar and Williams, 2002). However, mangiferin exhibits soothing action on free radicals mediated lipid peroxidation by preventing the malondiladehyde (MDA) elevation & antioxidant status depletion, maintains the cellular oxidant-antioxidant balance by decreasing the localized O2 concentration and generates mangiferin phenoxy radicals. In the nutshell, mango peel extract and mangiferin based functional drinks are valuable against oxidative stress and allied complication.

4.9. Liver functioning tests

4.9.1. Serum AST

The F values showed substantial effect of treatments on serum AST level in study II & III whereas non-momentous variations were observed in study I (Table 41).

In study I (trial 1& 2), the recorded AST values for T0, T1 and T2 were 109.05±6.32 &

104.42±7.43, 103.25±7.82 & 100.62±7.02 and 105.15±7.72 & 101.80±6.92 IU/L, respectively. Likewise in study II (trial 1), value for this attribute was highest in T0

(141.77±7.62 IU/L) that significantly lowered in T1 (125.19±9.62 IU/L) and T2 (128.01±8.43 IU/L) groups. Similar response was observed in trial 2; the maximum value

109

Table 40. Effect of functional drinks on serum TBARS (µmol/L) Studies Treatments F value

T0 T1 T2 Study I (Trial 1) 6.55±0.43a 6.22±0.52ab 6.10±0.28b 11.3* (Trial 2) 7.01±0.35a 6.60±0.32ab 6.52±0.19b 13.5*

Study II (Trial 1) 10.29±0.10a 9.24±0.42b 8.62±0.21c 95.51** (Trial 2) 11.01±0.62a 9.61±0.63b 9.12±0.32c 76.02**

Study III (Trial 1) 9.36±0.38a 8.78±0.40b 8.46±0.12c 26.6** (Trial 2) 8.42±0.23a 7.76±0.32b 7.58±0.15c 31.3**

* = Significant **= Highly significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

110

T1 T2

17.21 18 16.23 16 14 12.69

12 10.25 9.63 10.02 10 7.85 8 6.89 7.01 5.89 6.23 6 5.06 Percent decrease 4 2 0 Study1(Tr 1) Study 1(Tr 2) Study 2( Tr 1) Study 2(Tr 2) Study 3(Tr 1) Study 3(Tr 2)

Figure 12. Percent reduction in TBARS as compared to control

111

was noticed in T0 as 135.81±8.23 IU/L followed by T2 122.38±9.75 IU/L and T1 120.20±9.34 IU/L, respectively. Moreover in study III (trial 1 & 2), serum AST concentrations were declined to 110.92±8.63 & 101.00±9.55 and 113.69±8.12 &

103.50±7.23 IU/L in T1 and T2 groups, respectively as compared to 119.27±8.51 &

113.11±6.68 (IU/L) in T0 (Table 41).

4.9.2. Serum ALT

The F values in Table 42 explicated that ALT level was affected non-significantly with treatments in study I whereas momentous variations were observed in study II and III.

Mean ALT values in study I (trial 1) for T0, T1 and T2 groups were 48.65±2.12, 46.78±3.12 and 47.56±3.81 IU/L, correspondingly. Likewise in trial 2, the recorded values were 46.24±3.11, 44.92±3.02 and 45.18±3.16 IU/L in T0, T1 and T2 groups, respectively. However in study II (trial 1 & 2), the maximum ALT value was observed in

T0 (52.76±2.82 & 55.99±3.43 IU/L) than that of T1 (49.20±3.96 & 51.90±3.58 IU/L) and

T2 (50.25±4.03 & 52.68±3.20 IU/L) groups, respectively. In study III (trial 1), recorded

ALT values in T0, T1 and T2 groups were 46.55±2.12, 42.21±3.10 and 45.15±2.89 IU/L whereas in subsequent trial 44.02±2.53, 41.18±2.41 and 42.19±3.16 IU/L, respectively (Table 42).

4.9.3. Serum ALP

It is obvious from the F values that treatments momentously affected serum ALP level in all studies except for study I (Table 43).

In study I (trial 1 & 2), T0 had the highest ALP level 150.68±8.95 & 145.79±9.12 IU/L than that of T1 and T2 groups as 147.58±12.43 & 142.13±11.73 and 148.17±13.22 & 143.56±14.15 IU/L, respectively (Table 43). Similarly, in study II (trial 1), ALP level in

T0 was 202.89±14.20 IU/L that varied significantly in T1 and T2 groups as 190.03±15.81 and 195.25±12.63 IU/L, respectively. The subsequent trial also showed a momentous decline from 212.41±15.23 IU/L in T0 to 201.63±13.63 & 205.29±13.38 IU/L in T1 & T2, respectively. Similarly in study III (trial 1), maximum ALP level 216.39±16.15 IU/L was reported in T0 that momentously reduced to 206.50±10.72 IU/L in T1 and 210.22±13.33

IU/L in T2 groups. In the next trial, recorded values for T0, T1 and T2 groups were 220.21±14.12, 208.23±15.87 and 211.06±14.12 IU/L, correspondingly.

112

Various scientific studies have highlighted a linear association between high fat & sugar rich diets and hepatotoxicity. These diets are the gateways for reactive oxygen species (ROS) that react with membrane polyunsaturated fatty acids and impart structural and functional damages thus enhancing the leakage of AST and ALT from liver to the serum. In this context, polyphenols like mangiferin diminish this elevation of ALT and AST level due to its antioxidant and anti-inflammatory capacity (Pardo-andreu et al., 2008; Ojewole, 2005; Izunya et al., 2011).

Earlier, Iman et al. (2011) elucidated the suppressing role of mangiferin against abnormal AST, ALT and ALP levels of hypercholestrolemic and hyperglycemic rats. The mangiferin and Psidium guajava extract treatment in the form of a gavage for one month caused a significant reduction in hepatic enzymes concentration. One of their peers, Rai et al. (2010) also noticed 25.18% reduction in ALP level of rats after treatment with mango peel extract @ 250 mg/kg. Different researcher groups, Rawi et al. (1995) and Sheela and Augusti (1992) observed enhancement in ALT, AST and ALP concentrations of diabetic rats by 53.36, 84.38 and 87.41% however, mango polyphenols supplementation resulted a marked decline in the abnormal values. They deduced that strong antioxidative potential of mango polyphenols has tendency to maintain the structural integrity of liver. Later, Mahmood et al. (2002) noticed a reduction in AST activity by 28.48% after mango peel polyphenols treatment.

Previously, Rashad et al. (2008) and Hassan et al. (2007) documented that supplementation of mangiferin significantly reduces the abnormal concentrations of liver enzymes. They concluded that strong antioxidative properties of mango peel polyphenols are responsible to inhibit lipid peroxidation thereby alleviate abnormal enzyme concentrations.

113

Table 41.Effect of functional drinks on serum AST (IU/L) Studies Treatments F value T0 T1 T2 Study I (Trial 1) 109.05±6.32 103.25±7.82 105.15±7.72 5.42NS (Trial 2) 104.42±7.43 100.62±7.02 101.80±6.92 4.67NS

Study II (Trial 1) 141.77±7.62a 125.19±9.62b 128.01±8.43b 48.5 * (Trial 2) 135.81±8.23a 120.20±9.34b 122.38±9.75b 47.9*

Study III (Trial 1) 119.27±8.51a 110.92±8.63b 113.69±8.12b 27.36* (Trial 2) 113.11±6.68a 101.00±9.55b 103.50±7.23b 37.60*

* = Significant NS= Non Significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

114

Table 42.Effect of functional drinks on serum ALT (IU/L) Studies Treatments F value T0 T1 T2 Study I (Trial 1) 48.65±2.12 46.78±3.12 47.56±3.81 0.87NS (Trial 2) 46.24±3.11 44.92±3.02 45.18±3.16 0.67NS

Study II (Trial 1) 52.76±2.82a 49.20±3.96b 50.25±4.03ab 10.6* (Trial 2) 55.99±3.43a 51.90±3.58b 52.68±3.20ab 14.1*

Study III (Trial 1) 46.55±2.12a 42.21±3.10c 45.15±2.89b 28.1* (Trial 2) 44.02±2.53a 41.18±2.41c 42.19±3.16b 23.24*

NS= Non Significant * = Significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

115

Table 43.Effect of functional drinks on serum ALP (IU/L) Studies Treatments F value T0 T1 T2 Study I (Trial 1) 150.68±8.95 147.58±12.43 148.17±13.22 0.12NS (Trial 2) 145.79±9.12 142.13±11.73 143.56±14.15 0.22NS

Study II (Trial 1) 202.89±14.20a 190.03±15.81b 195.25±12.63ab 8.77* (Trial 2) 212.41±15.23a 201.63±13.63b 205.29±13.38ab 4.72*

Study III (Trial 1) 216.39±16.15a 206.50±10.72b 210.22±13.33ab 3.47* (Trial 2) 220.21±14.12a 208.23±15.87b 211.06±14.12ab 6.03*

* = Significant NS= Non Significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

116

4.10. Kidney functioning tests

4.10.1. Serum urea

The F values elucidated (Table 44) that treatments imparted significant effect on serum urea concentrations of rats in all studies excluding study I. In study I (trial 1 & 2), the observed values for serum urea level were 19.98±1.43 & 22.25±1.78, 18.99±0.86 &

21.35±1.12 and 19.25±1.39 & 21.96±0.94 mg/dL in T0, T1 and T2 groups, respectively.

However in study II (trial 1), urea level in T0 group was 27.95±1.64 mg/dL that decreased significantly in T1 and T2 as 25.78±1.49 and 26.85±0.34 mg/dL, respectively. Similar response was noticed in the following trial; the highest value was recorded in T0 as

29.47±1.98 mg/dL followed by T1 27.35±1.96 mg/dL and T2 28.64±1.62 mg/dL, respectively. Likewise in study III (trial 1 & 2), a substantial decline was observed from

30.25±2.21 & 32.55±1.46 mg/dL (T0) to 28.96±1.21 & 30.15±1.91 (T1) during both trials (Table 44).

4.10.2. Serum creatinine

The F values indicated substantial effect of functional drinks on serum creatinine in study II & III however, non-significant variations were noticed in study I (Table 45). In study I

(trial 1 & 2), mean creatinine values in T0, T1 and T2 groups were 0.79±0.04, 0.77±0.01 and 0.78±0.04 mg/dL whereas 0.81±0.03, 0.79±0.04 and 0.80±0.03 mg/dL for respective trial, correspondingly. Likewise in study II, the values for this trait significantly reduced from 0.85±0.03 to 0.80±0.04 mg/dL and 0.91±0.06 to 0.84±0.01 mg/dL in T0 to TI groups (trial 1 & 2), respectively. In study III, reported creatinine levels were 0.98±0.02,

0.91±0.02 & 0.94±0.06 mg/dL in T0, T1 & T2 groups. Similar in trial 2, maximum value was noticed in T0 0.96±0.03 mg/dL followed by T2 0.92±0.05 mg/dL and T1 0.90±0.06 mg/dL (Table 45).

During normal state, kidney executes various vital functions like body homeostatic, regulation of electrolyte balance, blood pressure and removal of toxins (Garcia et al., 2012). However in diseased condition, the kidneys ability to remove metabolic wastes like urea and creatinine is hampered. The creatinine is a break down product of creatinine phosphate and its normal range indicates the normal functioning of kidney however, during diseased phase its level is abnormally high. The elevated blood creatinine level is an indicator of impaired glomerulus filtration. The mango peel polyphenols especially 117

mangiferin has tendency to ameliorate kidney related malfunctionings by improving the overall antioxidant status and reducing toxins produced by reactive oxygen species (Muruganandan et al., 2002).

Earlier, Bibu et al. (2010) also illuminated the urea and creatinine lowering potential of mango peel extract. They observed that ethanolic extract of mango peel (100 mg/kg) inhibited gentamicin induced proximal tubular necrosis and decreased the serum urea and creatinine concentrations in kidney. Likewise, Al-Majed et al. (2002) and Yaman et al. (2010) observed that mango polyphenols caused decline in urea and creatinine levels in GM-induced renal injury rats owing to their antioxidative and anti-inflammatory potential. Later, Kemasari et al. (2011) elucidated the effective role of mango peel extract against abnormal urea and creatinine levels of alloxan induced diabetic rats. One of the researchers groups, Hu et al. (2010) expounded that mangiferin significantly reduced serum uric acid, creatinine and urea levels of hyperuricemic mice.

4.11. Hematological aspects

4.11.1. Red blood cell (RBC)

The F values in Table 46 showed that treatments imparted non-significant effect on red blood cells in all studies (I, II & III). In study I (trial 1 & 2), the mean RBC values for T0,

T1 and T2 groups were 6.20±0.29 & 7.01±0.24 cells/pL, 6.45±0.31 & 7.08±0.52 and 6.29±0.12 & 7.06±0.41 cells/pL, respectively (Table 46). Moreover in study II (trial 1), the minimum RBC concentration was reported in T0 (5.25±0.34 cells/pL) that non- substantially increased in T1 (5.44±0.24 cells/pL) and T2 (5.35±0.33 cells/pL). Likewise pattern was observed in the next trial. Similarly in study III, the RBC values varied non- significantly in T0, T1 and T2 groups as 4.10±0.26 & 5.01±0.32, 4.19±0.19 & 5.25±0.28 and 4.15±0.16 & 5.20±0.13 cells/pL, respectively (trial 1 & 2).

4.11.2. Hemoglobin (Hb)

It is revealed from the F values (Table 46) that treatments imparted substantial effect on hemoglobin level in study II and III whereas non-significant variations were observed in study I. Means regarding Hb in study I (trial 1 & 2) showed the values 12.11±0.12 &

10.89±0.65 g/L for T0 that non-momentously enhanced to 12.25±0.91 & 11.10±0.72 and

12.20±0.41 & 10.94±0.73 g/L in T1 and T2 groups, respectively. Nevertheless in study II, the lowest hemoglobin value was noticed in T0 (9.81±0.64 g/L) that momentously

118

Table 44. Effect of functional drinks on serum urea (mg/dL) Studies Treatments F value T0 T1 T2 Study I (Trial 1) 19.98±1.43 18.99±0.86 19.25±1.39 4.51NS (Trial 2) 22.25±1.78 21.35±1.12 21.96±0.94 2.94NS

Study II (Trial 1) 27.95±1.64a 25.78±1.49b 26.85±0.34ab 15.1* (Trial 2) 29.47±1.98a 27.35±1.96b 28.64±1.62ab 13.4*

Study III (Trial 1) 30.25±2.21a 28.96±1.21b 29.12±1.93ab 3.18* (Trial 2) 32.55±1.46a 30.15±1.91b 31.01±1.62ab 3.42*

NS= Non Significant * = Significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

119

Table 45. Effect of functional drinks on serum creatinine (mg/dL) Studies Treatments F value T0 T1 T2 Study I (Trial 1) 0.79±0.04 0.77±0.01 0.78±0.04 0.64NS (Trial 2) 0.81±0.03 0.79±0.04 0.80±0.03 0.87NS

Study II (Trial 1) 0.85±0.03a 0.80±0.04b 0.82±0.05ab 7.16* (Trial 2) 0.91±0.06a 0.84±0.01b 0.87±0.04ab 14.60*

Study III (Trial 1) 0.98±0.02a 0.91±0.02b 0.94±0.06ab 12.1* (Trial 2) 0.96±0.03a 0.90±0.06b 0.92±0.05ab 8.51*

* = Significant NS= Non Significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

120

increased in T2 (9.99±0.68 g/L) and T1 (10.85±0.67 g/L) groups (trial 1). Likewise increasing trend was observed during the 2nd trial, the hemoglobin level enhanced from

10.88±0.52 to 11.96±0.83 g/L in T0 and T1 groups, respectively. In study III, the reported values for this trait in T0 group was 9.55±0.69 & 8.64±0.48 g/L that differed momentously in T1 and T2 groups by 10.44±0.43 & 10.60±0.32 and 9.99±0.48 & 9.95±0.38 g/L, in respective trials (Table 46).

4.11.3. Hematocrit

The F values (Table 47) explicated that hematocrit concentration affected momentously by the treatments in all studies except for study I. The means in Table 47 (study I; trial 1) showed non-momentous differences in hematocrit level from 45.10±1.12% (T0) to

46.75±3.12% (T1) and 45.91±2.81% (T2). During the next trial, observed values were 46.72±2.51, 47.16±2.43 and 46.96±3.42% for respective groups. However, in study II

(trial 1) hematocrit value in T0 was 37.33±1.12% that significantly enhanced to

40.02±2.92 and 38.05±2.51% in T1 and T2 groups, respectively. In the subsequent trial, similar trend was observed that validates the data. In study III, the hematocrit level noticed as 33.63±2.31, 35.74±2.71 & 34.75±1.92% (trial 1) and 35.85±1.72, 38.99±1.13

& 36.39±1.81% in T1, T2 and T3 groups, respectively (trial 2).

4.11.4. Mean corpuscular volume (MCV)

The F values (Table 47) showed that mean corpuscular volume was affected non- momentously by functional drinks in all studies. In study I (trial 1), the mean MCV values were reported as 52.90±3.21, 53.32±4.11 and 52.94±3.10 fL in T0, T1 and T2 groups, respectively. Likewise pattern was observed during trial 2. Moreover in study II

(trial 1 & 2), the observed MCV values for T0, T1 and T2 groups were 45.63±2.94 & 43.78±2.76, 46.12±1.62 & 44.26±3.30 and 45.96±3.91 & 43.92±2.76 fL, correspondingly. In study III, the lowest MCV value was 42.96±3.13 in T0 that uplifted non-momentously by 43.68±3.91 and 43.10±3.32 fL in T1 and T2 groups, respectively (trial 1). Likewise pattern was observed in the subsequent trial (Table 47).

4.11.5. White blood cell

The F values in Table 48 explicated that treatments imparted non-substantial variations on white blood cells in all studies excluding study III. In study I, mean WBC values were recorded as 13.71±0.18, 12.96±0.64 and 13.29±0.63 cells/nL in T0, T1 and T2 groups,

121

Table 46.Effect of functional drinks on red blood cell indices RBC (cells/pL) Treatments F value T0 T1 T2 Study I (Trial 1) 6.20±0.29 6.45±0.31 6.29±0.12 9.85 NS (Trial 2) 7.01±0.24 7.08±0.52 7.06±0.41 11.13 NS Study II (Trial 1) 5.25±0.34 5.44±0.24 5.35±0.33 5.81 NS (Trial 2) 6.15±0.26 6.43±0.12 6.30±0.22 8.16 NS Study III (Trial 1) 4.10±0.26 4.19±0.19 4.15±0.16 2.82 NS (Trial 2) 5.01±0.32 5.25±0.28 5.20±0.13 3.63 NS

Hemoglobin Treatments F value (g/L) T0 T1 T2 Study I (Trial 1) 12.11±0.12 12.25±0.91 12.20±0.41 1.08NS (Trial 2) 10.89±0.65 11.10±0.72 10.94±0.73 1.86NS

Study II (Trial 1) 9.81±0.64b 10.85±0.67a 9.99±0.68ab 20.4* (Trial 2) 10.88±0.52b 11.96±0.83a 11.01±0.80ab 24.3*

Study III (Trial 1) 9.55±0.69b 10.44±0.43a 9.99±0.48ab 1.08* (Trial 2) 8.64±0.48b 10.60±0.32a 9.95±0.38ab 1.83*

NS=Non-significant *= Significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

122

Table 47.Effect of functional drinks on red blood cell indices Hematocrit (%) Treatments F value T0 T1 T2 Study I (Trial 1) 45.10±1.12 46.75±3.12 45.91±2.81 3.81NS (Trial 2) 46.72±2.51 47.16±2.43 46.96±3.42 2.69NS

Study II (Trial 1) 37.33±1.12b 40.02±2.92a 38.05±2.51b 10.8* (Trial 2) 38.98±2.43b 41.12±3.01a 39.50±1.83b 14.6*

Study III (Trial 1) 33.63±2.31b 35.74±2.71a 34.75±1.92ab 6.56* (Trial 2) 35.85±1.72b 38.99±1.13a 36.39±1.81ab 7.36*

MCV (fl) Treatments F value T0 T1 T2 Study I (Trial 1) 52.90±3.21 53.32±4.11 52.94±3.10 1.83 NS (Trial 2) 51.99±3.52 53.01±4.53 52.29±3.64 3.99 NS

Study II (Trial 1) 45.63±2.94 46.12±1.62 45.96 ±3.91 2.23 NS (Trial 2) 43.78±2.76 44.26±3.30 43.92±2.76 2.04 NS

Study III (Trial 1) 42.96±3.13 43.68±3.91 43.10±3.32 2.85 NS (Trial 2) 43.90±3.52 44.42±3.44 43.99±2.86 2.15 NS

NS=Non-significant *= Significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

123

respectively. Likewise trend was observed in the trial 2. Similarly, the highest value

(study II, trial 1) was noticed in T0 (15.47±0.66 cells/nL) that non-momentously reduced in T2 (15.02±0.32 cells/nL) and T1 (14.96±0.32 cells/nL), respectively. The T0 group in trial 2 showed maximum WBC value (14.74±0.87 cells/nL) followed by T2 (14.12±0.13 cells/nL) and T1 (13.25±0.82 cells/nL). During study III (trial 1 & 2), the WBC values varied non-significantly in T0, T1 and T2 groups as 17.51±0.43 & 19.00±0.19, 14.23±0.48 & 17.12±0.65 and 15.25±0.97 & 18.46±0.85 cells/nL, respectively (Table 48).

4.11.6. Neutrophils

It is evident from the F values that treatments imparted significant differences on the neutrophils in all studies except for study I (Table 48). In this case (trial 1), mean neutrophils values for T0, T1 and T2 groups were 72.23±3.86, 74.20±4.82 and

73.56±5.63%, correspondingly. Likewise, the lowest value was reported in T0

(61.32±3.47%) that non-substantially increased to 62.25±4.67 and 63.08±3.04% in T2 and

T1, respectively (trial 2). During study II, the T0 group had the minimum value for this trait as 62.25±5.32% that significantly increased in T2 67.56±3.61% and T1 68.23±2.81%.

Similarly, mean values (trial 2) for T0, T1 and T2 groups were 67.23±4.46, 72.12±3.46 and

70.32±1.47%, respectively. Moreover, neutrophils level for T0 group in study III was

55.36±4.63% that enhanced non-momentously in T2 and T1 groups by 57.25±3.47 and

59.15±2.09%, respectively (trial 1). Likewise, the observed values for T0, T1 and T2 groups were 57.22±2.46, 60.30±1.49 and 59.81±2.96% in respective trial, correspondingly (Table 48). 4.11.7. Monocytes The statistical interpretation (F values) regarding monocytes in Table 49 explicated non- momentous effect of treatments in all studies. During study I (trial 1), the monocytes values in T0, T1 and T2 were 7.70±0.12, 8.10±0.22 and 7.80±0.43%, respectively. Similar pattern was observed during the next trial. Similarly in study II (trial 1), T0 had the lowest value as 5.70±0.21% than that of T1 6.14±0.10% and T2 5.91±0.32%, whereas during the 2nd trial, levels for this trait were 4.10±0.37, 5.01±0.31 and 4.78±0.23%. In study III

(trial 1 & 2), the recorded monocytes level in T0 group was 4.15±0.26 & 5.45±0.36% that non-significantly uplifted in T2 4.96±0.24 & 5.96±0.41% and T1 5.45±0.42 & 6.32±0.18%, respectively (Table 49).

124

4.11.8. Lymphocytes The F values indicated non-significant effect of treatments on lymphocytes during the entire experiment (Table 49). In the initial study, the reported lymphocytes for T0 group were 38.26±1.96% that non-substantially inclined in T2 38.75±2.12% and T1

39.20±0.63%. Likewise, non-momentous increase was noted in trial 2 for T0, T1 and T2 groups as 40.99±1.74, 42.06±1.25 and 41.03±1.98%, correspondingly. Moreover in study

II, a non-significant uplift in lymphocytes level was reported; T1 (35.05±1.47 &

32.42±1.69%) and T2 (34.73±1.30 & 31.01±1.59%) as compared to group T0 (34.55±1.13 & 30.90±1.34%), respectively. In study III (trial 1 & 2), the noticed differences in lymphocytes were 32.96±2.59 & 30.61±1.54, 33.36±1.90 & 31.13±2.08 and 33.03±1.56

& 30.99±1.03% in T0, T1 and T2 groups, respectively (Table 49).

Erythrocytes and allied membranes have a high ratio of polyunsaturated fatty acids to the total lipids, indicating the susceptibility of lipid peroxidation. Moreover, RBCs are highly prone to lipid peroxidation due to constant exposure to oxygen and pro-oxidants (Pawlak et al., 1998). During hypercholesterolemic and hyperglycemic phases, irregularities in both white and red blood cells indices are observed by different scientists. They inferred that elevation in microvescicles production, formation of excessive toxins and membrane oxidations are the key factors in this regard (Kumar, 2000; Hoffman et al., 2004; Madjid et al., 2004).

The findings of different scientists including Muruganandan et al. (2005); Muruganandan et al. (2002) and Kemasari et al. (2011) delineated that mango peel polyphenols impart positive impact on red blood cells, hemoglobin, hematocrit and MCV levels of experimental rats due to their membrane protective and antioxidant perspectives.

4.12. Electrolytes balance

4.12.1. Sodium

The results in Table 50 presented that treatments imparted non-significant effect on the serum sodium level of rats in all studies. During study I (trial 1 & 2), mean values for sodium in T0, T1 and T2 were 110.23±8.69 & 112.63±8.87, 112.25±10.03 & 114.21±9.64 and 111.12±7.71 & 113.26±8.64 mEq/L, respectively (Table 50). Moreover in study II (trial 1 & 2), the values for sodium were 103.26±9.42 & 105.25±8.63, 107.23±9.25 &

111.63±10.36 and 104.65±8.87 & 109.31±7.42 mEq/L in T0, T1 and T2, respectively.

125

Table 48.Effect of functional drinks on white blood cell Indices WBC(cells/nL) Treatments F value T0 T1 T2 Study I (Trial 1) 13.71±0.18 12.96±0.64 13.29±0.63 5.91 NS (Trial 2) 12.96±0.72 12.30±0.12 12.50±0.27 4.81 NS

Study II (Trial 1) 15.47±0.66 14.96±0.32 15.02±0.32 32.35 NS (Trial 2) 14.74±0.87 13.25±0.82 14.12±0.13 30.4 NS

Study III (Trial 1) 17.51±0.43a 14.23±0.48b 15.25±0.97ab 30.01* (Trial 2) 19.00±0.19a 17.12±0.65b 18.46±0.85ab 33.35*

Neutrophils Treatments F value (%) T0 T1 T2 Study I (Trial 1) 72.23±3.86 74.20±4.82 73.56±5.63 9.7 NS (Trial 2) 61.32±3.47 63.08±3.04 62.25±4.67 8.13 NS

Study II (Trial 1) 62.25±5.32b 68.23±2.81a 67.56±3.61ab 26.27* (Trial 2) 67.23±4.46b 72.12±3.46a 70.32±1.47ab 24.4*

Study III (Trial 1) 55.36±4.63b 59.15±2.09a 57.25±3.47ab 19.6* (Trial 2) 57.22±2.46b 60.30±1.49a 59.81±2.96ab 15.2*

NS=Non-significant *= Significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

126

Similarly in study III, noticed values in T0, T1 and T2 groups were 107.12±9.45 & 109.23±8.56, 112.25±8.60 & 113.36±9.36 and 109.36±7.64 & 110.12±6.32mEq/L, respectively (trial 1 & 2).

4.12.2. Potassium

The statistical analysis (F values) revealed that serum potassium concentrations were non- momentously affected by treatments in the entire efficacy trial (Table 50).

In study I, mean potassium values (Table 50) were 5.91±0.12 & 6.01±0.21, 6.06±0.32 &

6.26±0.41 and 6.01±0.43 & 6.09±0.30 mEq/L in T0, T1 and T2 groups, respectively (trial 1 & 2). Similarly, lowest potassium level (4.50±0.16 & 5.71±0.24 mEq/L) was recorded in T0 that uplifted non-momentously in T1 (5.15±0.32 & 6.02±0.35 mEq/L) and T2 (4.80±0.22 & 5.99±0.34 mEq/L), respectively (study II; trial 1 & 2). Likewise during study III (trial 1 & 2), highest values were noticed in T1 (5.85±0.39 & 4.35±0.26 mEq/L) followed by T2 (5.45±0.19 & 4.25±0.23 mEq/L) and T0 (5.10±0.10 & 4.13±0.02 mEq/L).

4.12.3. Calcium

The results in Table 50 elucidated that treatments imparted non-momentous effect on serum calcium concentrations in respective studies.

Mean calcium levels in study I were 12.41±0.56 & 14.99±0.54 mEq/L (T0), 13.01±0.62 &

15.21±0.35 mEq/L (T1) and 12.65±0.75 & 15.04±0.66 mEq/L (T2), respectively (trial 1 &

2). Likewise in study II (trial 1), mean values in T0, T1, and T2 groups were 11.88±0.96, 12.25±0.31 and 12.01±0.36 mEq/L whilst in 2nd trial 12.46±0.74, 13.01±0.43 and 12.59±0.62 mEq/L, respectively. Similarly, calcium concentrations in study III were

10.77±0.62, 11.01±0.60 and 10.85±0.42 mEq/L in T0, T1 and T2 groups, respectively (trial 1). Likewise pattern was observed in trial 2 (Table 50).

Electrolytes perform numerous life sustaining processes like homeostasis maintenance, ensures proper acid base ratio and oxygen balance. In contrast, their imbalance resulting oxidative stress and kidney malfunctionings (Paudel and Karma, 2003). There are strong evidences supporting the mango peel polyphenols ability to modulate electrolytes balance by managing the activity of glands involved in sodium and potassium secretions (Toda et al., 1991; Mulder et al., 2001).

127

Table 49.Effect of functional drinks on white blood cell Indices Monocytes (%) Treatments F value T0 T1 T2 Study I (Trial 1) 7.70±0.12 8.10±0.22 7.80±0.43 84.9 NS (Trial 2) 6.48±0.05 7.01±0.37 8.78±0.52 86.12 NS

Study II (Trial 1) 5.70±0.21 6.14±0.10 5.91±0.32 26.3 NS (Trial 2) 4.10±0.37 5.01±0.31 4.78±0.23 28.02 NS Study III (Trial 1) 4.15±0.26 5.45±0.42 4.96±0.24 14.8 NS (Trial 2) 5.45±0.36 6.32±0.18 5.96±0.41 16.25 NS Lymphocytes (%) Treatments F value T0 T1 T2 Study I (Trial 1) 38.26±1.96 39.20±0.63 38.75±2.12 7.14 NS (Trial 2) 40.99±1.74 42.06±1.25 41.03±01.98 7.61 NS Study II (Trial 1) 34.55±1.13 35.05±1.47 34.73±1.30 2.92 NS (Trial 2) 30.90±1.34 32.42±1.69 31.01±1.59 4.23 NS Study III (Trial 1) 32.96±2.59 33.36±1.90 33.03±1.56 2.47 NS (Trial 2) 30.61±1.54 31.13±2.08 30.99±1.03 3.15 NS

NS=Non-significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin

128

Table 50.Effect of functional drinks on electrolytes balance Sodium (Na) Treatments F values mEq/L T0 T1 T2 Study I (Trial 1) 110.23±8.69 112.25±10.03 111.12±7.71 3.83 NS (Trial 2) 112.63±8.87 114.21±9.64 113.26±8.64 2.94 NS Study II (Trial 1) 103.26±9.42 107.23±9.25 104.65±8.87 9.22 NS (Trial 2) 105.25±8.63 111.63±10.36 109.31±7.42 12.7 NS Study III (Trial 1) 107.12±9.45 112.25±8.60 109.36±7.64 12.7 NS (Trial 2) 109.23±8.56 113.36±9.36 110.12±6.32 9.08 NS

Potassium (K) Treatments F values mEq/L T0 T1 T2 Study I (Trial 1) 5.91±0.12 6.06±0.32 6.01±0.43 28.6 NS (Trial 2) 6.01±0.21 6.26±0.41 6.09±0.30 29.31 NS

Study II (Trial 1) 4.50±0.16 5.15±0.32 4.80±0.22 18.26 NS (Trial 2) 5.71±0.24 6.02±0.35 5.99±0.34 20.01 NS

Study III (Trial 1) 5.10±0.10 5.85±0.39 5.45±0.19 16.02 NS (Trial 2) 4.13±0.02 4.35±0.26 4.25±0.23 15.01 NS

Calcium (Ca) Treatments F values mEq/L T0 T1 T2 Study I (Trial 1) 12.41±0.56 13.01±0.62 12.65±0.75 12.8 NS (Trial 2) 14.99±0.54 15.21±0.35 15.04±0.66 13.33 NS

Study II (Trial 1) 11.88±0.96 12.25±0.31 12.01±0.36 6.95 NS (Trial 2) 12.46±0.74 13.01±0.43 12.59±0.62 7.02 NS

Study III (Trial 1) 10.77±0.62 11.01±0.60 10.85±0.42 4.62 NS (Trial 2) 12.79±0.76 13.12±0.45 13.01±0.53 6.00 NS

NS=Non-significant

Study I : Normal diet Study II : High cholesterol diet Study III: Diabetic rats

T0 : Control drink (without active ingredients) T1 : Drink containing mango peel extract T2 : Drink containing mangiferin 129

CHAPTER 5

SUMMARY

Dietary phytonutrients provide protection against various physiological ailments owing to their health enhancing potential. In this context, mango peel polyphenols especially mangiferin has tendency to ameliorate various lifestyle related disorders. In the present research, five different mango peels were analyzed for compositional & nutritional assay, antioxidant and mangiferin isolation & quantification. Afterwards, three types of functional/nutraceutical drinks were prepared by the supplementation of whole mango peel extract (T1) and mangiferin (T2) alongside control (T0). Lastly, the formulated functional drinks were tested against the threats of hypercholesterolemia and hyperglycemia through a model feeding trial. Mean squares regarding proximate composition indicated that protein, fiber and ash contents varied significantly among different peel samples however, non-substantial variations were observed for moisture, fat and NFE. The means elucidated highest moisture in the peel of desi mango 71.38±2.05 followed by anwar ratol, chaunsa, langra and dusahri as 71.01±3.91, 70.74±4.01, 69.86±5.20 and 68.33±4.14%, respectively. Moreover, protein contents were observed as 2.36±0.01, 2.25±0.05, 2.20±0.04, 2.06±0.05 and 1.94±0.04% in anwar ratol, chaunsa, langra, dusahri and desi, correspondingly. Similarly, fat and fiber contents in respective varieties were 2.31±0.14 & 5.01±0.25, 2.26±0.10 & 5.47±0.31, 2.25±0.17 & 4.88±0.12, 2.18±0.18 & 4.69±0.17 and 2.11±0.12 & 4.53±0.18%. Besides, the ash contents ranged from 2.59±0.03 (chaunsa) to 1.84±0.02% (desi), respectively. Likewise, the recorded NFE values for respective samples were 87.87±6.87, 87.60±3.41, 88.86±5.20, 89.09±3.85 & 89.58±2.89%, respectively. Moreover, tested mango peels contain appreciable amount of minerals especially calcium (Ca), magnesium (Mg) and potassium (K). The antioxidant indices of mango peel extracts showed significant variations due to treatments and solvents however, their interactive effect was non-momentous. The maximum TPC was observed in chaunsa peel (75.35±3.96 mg/100g GAE) trailed by anwar ratol (68.34±2.85 mg/100g GAE), langra 64.95±1.89 mg/100g GAE) and dusahri (61.89±3.85 mg/100g GAE) whilst the minimum output was noticed in the peel of desi mango (58.85±2.45 mg/100 g GAE). Means regarding solvents exposed the maximum

130

TPC in ethanol 80.17±5.16 mg/100g GAE followed by acetone 62.28±3.12 mg/100g GAE and water extract 55.17±2.41 mg/100g GAE. Likewise, chaunsa peel exhibited the highest DPPH activity 59.28±3.69% than that of anwar ratol 57.49±1.48%, langra 53.34±2.98%, dusahri 53.20±1.45% and desi 49.64±2.74%. The mean values for solvents showed maximum DPPH activity in ethanolic extract 60.42±2.41% followed by acetone 56.36±3.69% and water 46.98±2.78%. The antioxidant activity (β-carotene) and FRAP values for different mango peels i.e. chaunsa, anwar ratol, langra, dusahri & desi were 57.33±4.14% & 8.88±0.62 mmol/100g, 54.45±3.96% & 7.80±0.45 mmol/100g, 49.12±3.10% & 7.54±0.25 mmol/100g, 48.14±1.18% & 7.12±0.32 mmol/100g and 45.38±2.50% & 6.24±0.29 mmol/100g, respectively. Similarly, ethanolic extract had the maximum β-carotene value 58.59±3.69% followed by acetone 51.98±2.11% and water extract 42.08±1.85%. However, the highest FRAP activity was observed in acetone 7.88±0.19 mmol/100g followed by ethanol 7.52±0.12 mmol/100g and water extract 7.13±0.21 mmol/100g. The DPPH, β-carotene and FRAP activity of isolated mangiferin were affected significantly by treatments and solvents. The ethanolic extract indicated the highest DPPH value 75.05±4.12% followed by acetone 68.67±3.25% and water extract 59.51±2.45%. Similarly, the maximum DPPH activity was recorded in mangiferin isolated from chaunsa peel 77.22±3.96% trailed by anwar ratol 70.26±4.89%, langra 64.58±2.69% and dusahri 64.90±4.12% while the minimum value was noticed for the mangiferin of desi peel 61.75±3.85%. The β-carotene activity in the peel of chaunsa, anwar ratool, langra, dusahri and desi peel were 74.63±3.69, 67.27±4.12, 63.77±2.33, 62.52±2.85 and 61.64±3.69%. Among the solvents, maximum β-carotene activity was reported in acetone (71.49±3.65%) followed by ethanol (65.25±2.85%) and water extract (61.14±3.87%). The FRAP values of mangiferin isolated from the peels of chaunsa, anwar ratol, langra, dusahri and desi were 12.30±0.24, 10.56±0.32, 10.46±0.18, 10.26±0.32 and 8.33±0.24 mmol/100g, respectively. Likewise, the recorded FRAP values for ethanol, acetone & water extracts were 12.02±0.28, 9.41±0.48 & 8.39±0.57 mmol/100g in respective manner. Mean squares showed significant effect of treatments and solvents on the mangiferin concentrations nevertheless, their interactive effect exhibited non-momentous variations. The means for treatments indicated the highest mangiferin in chaunsa peel 5.53±0.31 mg/g trailed by anwar ratol 4.88±0.30 mg/g, langra 4.09±0.18 mg/g and dusahri 131

3.97±0.23 mg/g whilst the lowest value 3.19±0.17 mg/g was observed in desi mango peel. Considering the solvents, the mean mangiferin values for ethanol, acetone and water extracts were 5.25±0.01, 4.29±0.19 and 3.44±0.21 mg/g, respectively. The statistical analysis showed non-significant effect of treatments on acidity, pH and TSS of the prepared drinks whereas, storage imparted momentous effect on these parameters except for TSS. The acidity in functional/nutraceutical drinks i.e. T0, T1 and T2 was observed as 0.176±0.001, 0.173±0.002 and 0.170±0.004%, respectively. However, storage caused a significant enhancement in acidity from 0.161±0.001 to 0.187±0.005% th at 0 and 60 day, respectively. Likewise, the recorded pH values of drinks i.e. T0, T1 and

T2 were 4.58±0.04, 4.56±0.06 & 4.57±0.05, correspondingly. During storage, pH significantly decreased from 4.67±0.10 to 4.45±0.09. The total soluble solids (TSS) of the functional drinks T0, T1 and T2 were 1.77±0.07, 1.78±0.06 and 1.80±0.04, respectively. Likewise, storage also revealed a non-significant decline in TSS and the values at 0 and 60th days were 1.80±0.05 and 1.77±0.08, respectively. Means squares pertaining to sensory response showed non-substantial effect due to treatments however, storage intervals imparted momentous differences except for flavor.

Means for color scores varied from 7.50±0.03 to 7.59±0.06 in drinks T0 and T2, respectively. Likewise, storage exhibited a momentous decline in color scores from 7.70±0.01 at 0 day to 7.38±0.03 at 60th day. Statistical interpretation elucidated that flavor was affected non-momentously due to treatments and storage. The sweetness differed non-significantly in treatment T0, T1 and T2 with scores 7.34±0.05, 7.39±0.02 & 7.43±0.03, respectively. However during storage, a significant declining trend was observed for sweetness and recorded scores were 7.52±0.02 at the initiation of study that decreased to 7.26±0.05 at the termination. Bioefficacy trial was carried out to explore the functional/nutraceutical worth of mango peel polyphenols especially mangiferin against lifestyle related disorders involving Sprague Dawley rats. In the current exploration, efficacy trial was divided into three phases. In study I, normal rats were used whereas in study II and III hypercholestrolemic and diabetic rats were involved, respectively. Additionally, rats were provided respective diets along with concurrent intake of functional drinks (T0, T1 and T2) to assess their therapeutic potential. Mean squares indicated that the treatments and study weeks imparted significant differences on body weights of rats in the entire studies. At the initiation of study I (trial 132

1) the mean body weights in different rats groups T0, T1 and T2 were 132±5.32, 128±3.85 and 131±6.24 g/rat, that significantly uplifted to 233±17.41, 223±13.02 and 228±7.21 g/rat in the respective groups at the termination. Similar enhancement for rats body weights was recorded with the passage of time in study 2 (trial 1), for T0 at first and last weeks by 132±4.02 & 262±15.03 g/rat, respectively whereas, the groups relied on T1 and

T2 had lower body weights than that of control as 131±6.32 & 235±13.21 and 133±8.21

& 240±10.05 g/rat. Likewise in study III (trial 1 & 2), the recorded weights for T0, T1 and

T2 were 136±4.21 & 134±4.22, 130±6.19 & 132±3.01 and 134±5.36 & 136±5.04 g/rat at the first week that enhanced to 248±7.14 & 241±10.41, 220±7.31 & 221±11.02 and 236±9.21 & 232±8.78 g/rat, respectively at the last week. In the present research, effect of mango peel extract and mangiferin supplementation was evaluated on lipid profile including total cholesterol, LDL, HDL and triglycerides in normal, hypercholesterolemic and diabetic rats. The F values showed that treatments imparted significant effect on cholesterol, LDL, HDL and triglycerides in all studies except for study I. The means for cholesterol in study II (trial 1 & 2), showed the highest values for T0 (141.25±8.01 & 145.85±6.25 mg/dL) that significantly decreased in groups relied on T1 and T2 as 128.28±6.08 & 132.78±6.84 mg/dL and 123.10±6.48 & 127.98±4.32, respectively. During study III (trial 1), the highest cholesterol level

98.42±4.64 mg/dL was observed in T0 followed by T1 91.81±6.32 whilst the lowest value was recorded in T2 88.30±5.34 mg/dL. Likewise in the subsequent trial, T0 showed the maximum value (101.25±4.92 mg/dL) that substantially suppressed in T1 (93.92±6.21 mg/dL) and T2 (90.89±4.52 mg/dL), respectively.

The present reduction for cholesterol in groups T2 and T1 was 12.85 & 9.18% whereas in trial 2, same groups exhibited 12.25 & 8.96% decline, respectively. Likewise in study III

(trial 1 & 2), T2 group showed the highest reduction 10.01 & 7.01% trailed by T1 9.9 & 6.32%, respectively as compared to control.

In study II, T0 had the highest LDL level as 58.96±3.49 mg/dL that significantly reduced in T2 & T1 groups by 50.40±4.23 & 52.94±3.92 mg/dL, respectively (trial 1). Likewise in the next trial, the observed LDL concentrations were 60.21±4.64, 53.43±2.13 and

51.05±4.90 mg/dL for T0, T1 and T2 groups, respectively. Similarly in study III, mean LDL values for respective groups differed substantially i.e. 47.21±4.21 & 45.25±3.32, 43.68±4.28 & 41.63±3.94 and 41.90 ±3.74 & 39.69±3.32 mg/dL, correspondingly. The graphical depiction elucidated that T1 & T2 groups had reduction in LDL by 3.28 & 133

3.15% and 3.96 & 3.48%, respectively (study I; trial 1 & 2). In study II, maximum diminish in LDL value was 14.52 & 15.21% in group T2 as compared to 10.21 & 11.25% in T1 group, correspondingly in both trials. Accordingly, the recorded LDL decline for T1

& T2 groups was 7.48 & 8.01% and 11.25 & 12.29%, respectively (study III; trial 1 & 2).

In study II (trial 1), T0 group exhibited the minimum HDL level (27.25±1.11 mg/dL) that elevated momentously in T1 & T2 as 28.24±1.46 and 28.78±1.47 mg/dL, respectively.

Similarly in trial 2, a substantial enhancement in HDL was recorded from 26.21±1.62 (T0) to 27.60±1.98 (T2) mg/dL. In study III (trial 1 & 2), T0 had the lowest HDL level as

30.12±2.18 & 31.25±2.89 mg/dL than that of T1 & T2 31.03±1.09 & 32.18±2.82 and 31.43±2.12 & 32.50±2.43 mg/dL, respectively. Considering the percent increase, a significant improvement was observed in T2 (5.61 & 5.30 and 4.34 & 4.01%) followed by

T1 (3.65 & 4.01 and 3.01 & 2.96%) in study II & III, respectively.

In hypercholesterolemic rats (study II), the highest triglycerides level was recorded in T0 (95.23±3.87 & 97.15±4.09 mg/dL) that momentously reduced to 85.73±5.36 &

87.43±4.90 and 90.23±4.87 & 91.31±3.67 mg/dL in T2 and T1 groups, correspondingly.

Likewise in study III (trial 1), the maximum value was observed in T0 (75.25±4.62 mg/dL) followed by T1 (72.02±4.32 mg/dL) and T2 group (69.79±3.76 mg/dL). In the successive trial, similar declining trend was observed in respective groups. The statistical analysis revealed that treatments imparted significant variations on glucose level in different groups of rats in all studies excluding study I. In study II (trial 1 & 2), maximum glucose level was recorded in T0 group 101.25±7.64 & 99.63±6.86 mg/dL that momentously declined to 93.58±8.35 & 92.70±7.26 and 92.79±6.68 & 91.74±7.04 mg/dL in T1 and T2 groups. Likewise in study III, T0 group showed the highest glucose level

214.96±8.34 & 216.41±7.56 mg/dL that significantly decreased in T2 and T1 groups as 186.82±8.98 & 185.55±8.46 and 197.51±8.53 & 196.26±197.51 mg/dL, respectively

(trial 1 & 2). Considering the percent reduction (study II; trial 1 & 2), T1 and T2 groups showed 7.58 & 6.96 and 8.36 & 7.92% decline in glucose level, respectively. Similarly in study III, maximum decrease in glucose 13.09 & 14.26% was observed in T2 followed by

8.12 & 9.31% in T1 group (trial 1 & 2). The F values indicated momentous variations in insulin level due to treatments in study II and III whereas, non-significant effect was observed in study I. In study II (trial 1 & 2),

T0 group elucidated the lowest insulin level 7.05±0.16 & 7.35±0.49 µU/mL that significantly increased to 7.22±0.42 & 7.53±0.52 and 7.33±0.19 & 7.69±0.13 µU/mL in 134

T1 and T2 groups, respectively. Moreover, the diabetic rats (study III; trial 1 & 2) showed the lowest insulin level 5.21±0.29 and 5.03±0.14 µU/mL in T0 group that momentously increased in T1 and T2 groups by 5.49±0.38 & 5.33±0.34 and 5.58±0.23 & 5.43±0.37 µU/mL, respectively.

Regarding percent increase in insulin (study II: trial 1 & 2), group T2 led to 4.01 & 4.65% enhancement whereas T1 showed 2.35 & 2.45% increase for this trait. However in study

III (trial 1 & 2), the recorded uplift for insulin in T2 group was 7.15 & 7.98% higher than that of T1 5.36 & 6.01%, respectively. The functional drinks improved the glutathione activity and reduced the serum TBARS level in rats during all studies. In study II (trial 1 & 2), maximum glutathione enhancement was observed in T2 group as 15.21 & 14.89% followed by T1 10.12 & 9.63%, respectively. Likewise in study III (trial 1 & 2), the observed uplift for glutathione content in T2 group was 10.26 & 11.63% higher than that of T1 6.52 & 7.01%, respectively. The maximum TBARS reduction was observed in T2 group 16.23 & 17.21% than that of T1 10.25 & 12.69%, correspondingly (study II; trial 1 & 2). In study III, the highest decline 10.02 & 9.63% was observed in T2 followed by 7.85 & 6.23% in T1, respectively (trial 1 & 2). It has been observed that mango peel extract and mangiferin based functional drinks did not impart any adverse effect on the red and white blood cells indices alongside electrolytes balance. In the nutshell, mango peel polyphenolic extract and mangiferin based therapeutic drinks are effective to ameliorate various physiological disorders. However, mangiferin based drink performed better against hypercholesterolemia and hyperglycemia as compared to mango peel extract based drink with special reference to manage the elevated serum cholesterol and glucose concentrations. The functional drinks enhanced the overall antioxidant status after diminishing the body lipid peroxidation. Conclusively, mango peel polyphenols especially isolated mangiferin is effectual to curtail various metabolic dysfunctions therefore should be encouraged in the diet based regimen for the vulnerable segment.

135

RECOMMENDATIONS

 Locally available nutrient dense byproducts must be explored for the preparation of functional foods to enhance the health status of the target population

 Mango polyphenols based therapeutic drinks especially rich in mangiferin should be encouraged in the daily diet as safeguard against various metabolic disorders

 Novel isolation techniques like super critical fluid extraction should be adopted to seprate the nutraceuticals for further use in designers food

 Dietitians should advice mango polyohenols based therapeutic foods for the vulnerable segment to curtail dyslipidemia and hyperglycemia

 Different promising nutraceuticals like mangiferin must be proposed against oxidative stress and related complications

 Community based research trials should be planned to enhance the meticulousness regarding the lifestyle related syndromes

 Awareness regarding diet based therapy ought to be launched among the public through mass media campaigns

136

LITERATURE CITED

AACC. 2000. Approved Methods of American Association of Cereal Chemists. The American Association of Cereal Chemists, Inc. St. Paul. Minnesota. Aaron, J.I., D.J. Mela and R.E. Evans. 1994. The influences of attitudes, beliefs and label information on perceptions of reduced-fat spread. Appetite. 22:25-37. Abdalla, A.E.M., S.M. Darwish, E.H.E. Ayad and R.M. El-Hamahmy. 2007. Egyptian mango by-product 1. Compositional quality of mango seed kernel. Food Chem. 103:1134-1140. Aderibigbe, A.O., T.S. Emudianughe and B.A.S. Lawal. 2001. Evaluation of anti-diabetic action of Mangifera indica in mice. Phytother. Res. 15:456-458. Adkins, J.L. 2002. Toward a human serum blood proteome: Analysis by multidimensional separation coupled with mass spectrometry. Mol. cell Proteomics. 1:947-955. Ahmad, R.S., M.S. Butt, N. Huma and M.T. Sultan. 2012. Green tea catechins based functional drink (Green cool) improves the antioxidant status of SD rats fed on high cholesterol and sucrose diets. Pak. J. Pharm. Sci. Still in press. Ahmed, M., A. Ahmad, Z.A. Chatha and S.M.R. Dilshad. 2008. Studies on preparation of ready to serve mandarin (Citrus reticulata) diet drink. Pak J. Agri. Sci. 45(4):470- 476. Ahn, J., W. Choi, S. Kim and T. Ha. 2011. Anti-diabetic Effect of Watermelon (Citrullus vulgaris Schrad) on Streptozotocin-induced Diabetic Mice. Food Sci. Biotechnol. 20(1):251-254. Ajila, C. M., M., Aalami, K. Leelavathi and U.J.S.P. Rao. 2010a. Mango peel powder: A potential source of antioxidant and dietary fiber in macaroni preparations. Inn. Food Sci. Emer. Technol. 11(1): 219-224. Ajila, C.M., K.A. Naidu, S.G. Bhat and U.J.S.P. Rao. 2007b. Bioactive compounds and antioxidant potential of mango peel extract. Food Chem. 105:982-988. Ajila, C.M., L. Jaganmohan Rao, U.J.S. Prasada Rao. 2011. Characterization of bioactive compounds from raw and ripe Mangifera indica L. peel extracts. Food Chem. Toxicol. 48(12):3406-3411. Ajila, C.M., S.G. Bhat and U.J.S.P. Rao. 2007a. Valuable components of raw and ripe peels from two indian mango varieties. Food Chem. 102:1006-1011.

137

Akbarzadeh, A., D. Norouzian, M.R. Mehrabi, S. Jamshidi, A. Farhangi, A. Allah, S. Mofidian and R.B. Lame. 2007. Induction of diabetes by streptozotocin in rats. IJCB. 22:60-64. Akhtar, S., S. Mahmood, S. Naz, M. Nasir and M.T. Sultan. 2009. Sensory evaluation of mangoes (Mangifera indica.L) grown in different regions of Pakistan. Pak J. Botany. 41(6):2821-2829. Akhtar, S., S. Naz, M.T. Suoltan, S. Mahmood, M. Nasir and A. Ahmad. 2010. Physico- chemical attributes and heavy metals content of mangoes (Magnifera Indica) cultivated in different regions of Pakistan. Pak. J. Bot. 42(4): 2691-2702. Akila, V. and H. Devaraj. 2008. Synergistic effect of tincture of Crataegus and Mangifera indica L. extract on hyperlipidemic and antioxidant status in atherogenic rats. Vasc. Pharmacol. 49:173-177. Al Haj, M., E. Kazzam, N.J. Nagelkerke, F. Nyberg, M.G. Nicholls and A. Adem. 2011. Effect of dehydration in the presence and absence of the angiotensin receptor blocker losartan on blood constituents in the camel. J. Med. Sci. 4:73-78. Al-Attar, A.M. and T.A. Zari. 2010. Influences of crude extract of tea leaves, Camellia sinensis, on streptozotocin diabetic male albino mice. Saudi J. Biol. Sci. 17:295- 301. Al-Majed, A.A., A.M. Mostafa, A.C. Al-Rikabi and O.A. Al-Shabanah. 2002. Protective effects of oral arabic gum administration on gentamicin induced nephrotoxicity in rats. Pharmacol. Res. 46(5):445-451. Alshatwi, A.A., M.A. Al Obaaid, S.A. Al Sedairy, E. Ramesh and K.Y. Lei. 2011. Black and green tea improves lipid profile and lipid peroxidation parameters in Wistar rats fed a high-cholesterol diet. J. Physiol. Biochem. 67:95-104. Alshatwi, A.A., M.A. Al-Obaaid, S.A. Al-Sedairy, A.H. Al-Assaf, J.J. Zhang and K.Y. Lei. 2010. Tomato powder is more protective than lycopene supplement against lipid peroxidation in rats. J. Nutr. Res. 30:66-73. Amin, I. and O. Mukhrizah. 2006. Antioxidant capacity of methanolic and water extracts prepared from food-processing by-products. J. Sci. 86:5. Amrita B, A. Liakot, A. Masfida and R. Begum. 2009. Studies on the antidiabetic effects of Mangifera indica stem-barks and leaves on nondiabetic, type 1 and type 2 diabetic model rats. Bangladesh J. Pharmacol. 4:110-4.

138

Andreu, G.P., R. Delgado, J.A. Velho, C. Curti and E.V. Anibal. 2005. Iron complexing activity of mangiferin, a naturally occurring glucosylxanthone, inhibits mitochondrial lipid peroxidation induced by Fe2+-citrate. Euro. J. Pharmacol. 513(1-2):47-55. Anis, A. and M.S. Anne. 2009. Quantitative Prediction of Cell Wall Polysaccharide Composition in Grape (Vitis vinifera L.) and Apple (Malus domestica) Skins from Acid Hydrolysis Monosaccharide Profiles. J. Agric. Food Chem. 57(9):3611- 3619. Anwer, T., M. Sharma, K.K. Pillai, G. Khan and M.M. Safhi. 2012. Antihyperglycemic, antidyslipidemic and antioxidant activity of Rhus coriaria in STZ-induced type 2 diabetic rats. Afr. J. Pharma. Pharacol. 6(41):2851-2855. AOAC. 2006. Official Methods of Analysis of Association of Official Analytical Chemists International. In: Horwitz, W. (Ed.), 18th ed. AOAC Press, Arlington, VA, USA. Aoki, N. and T. Matsuda. 2000. A cytosolic protein-tyrosine phosphatase PTP1B specifically dephosphorylates and deactivates prolactin activated STAT5a and STAT5b. J. Biol. Chem. 275: 39718-39726. Arrowood, M. J. 1997. Diagnosis. In: Fayer R (ed) Cryptosporidium and ryptosporidiosis. CRC. Press, New York, USA. Ashoush, I.S. and M.G.E. Gadallah. 2011. Utilization of mango peels and seed kernels powders as sources of phytochemicals in biscuit. World J. Dairy Food Sci. 6(1):35-42. Aura, A.M., P. Martin-Lopez and K.A. O’Leary. 2005. In vitro metabolism of anthocyanins by human gut microflora. Eu. J. Nutr. 44:133-142. Avramoglu, R.K. and K. Adeli. 2004. Hepatic regulation of apolipoprotein. B. Rev. Endocr. Metab. Disord. 5:293-301. Ayala-Zavala, J., C. Rosas-Domínguez, V. Vega-Vega and G.A. González-Aguilar. 2010. Antioxidant enrichment and antimicrobial protection of fresh-cut fruits using their own byproducts: Looking for integral exploitation. J. Food Sci. 75(8):175-181. Ayala-Zavala, J.F., G.A. Gonzalez-Aguilar and L. Del-Toro-Sanchex. 2009. Enhancing safety and aroma appealing of fresh cut fruits and vegetables using the antimicrobial and aromatic power of essential oils. J. Food Sci. 74:84-91.

139

Ayub, M., J. Ullah, A. Muhammad and A. Zeb. 2010. Evaluation of strawberry juice preserved with chemical preservatives at refrigeration temperature. Int. J. Nutr. Metab. 2(2):027-032. Aziah, N.A.A. and C.A. Komathi, 2009. Acceptability attributes of crackers made from different types of composite flour. Int. Food Res. J. 16:479-482. Bahorun, T., A. Luximon-Ramma, V.S. Neergheen-Bhujun, T.K. Gunness, K. Googoolye, C. Auger, A. Crozier and O.I. Aruoma. 2012. The effect of black tea on risk factors of cardiovascular disease in a normal population. Prev. Med. 54:98-102. Bakan, N., S. Taysi, O. Yilmaz, E. Bakan, S. Kuskay and N. Uzun. 2003. Glutathione peroxidase, glutathione reductase, Cu-Zn superoxide dismutase activities, glutathione, nitric oxide, and malondialdehyde concentrations in serum of patients with chronic lymphocytic leukaemia. Clin. Chem. Acta. 338:143-9. Balasubramanian, S., C. Frier, J.G. Harrison. 1999. Thermodynamic Melting Studies on Oligonucleotide-Peptide Conjugates. Nucleos. Nucleot. 18:1477. Barreto, J.C., M.T. Trevisan and W.E. Hull. 2008. Characterization and quantitation of polyphenolic compounds in bark, kernel, leaves, and peel of mango (Mangifera indica L.). J. Agric. Food Chem. 56(14):5599-5610. Barta J. 2006. Tarositas vizelvonassal in: Horvanthne, Barta J.: Feldolgozastechnologia es minoseg. BCE Elelmiszertudomanyi Kar es Mezogazda Kiado, Budapest, Hungary. 51-54. Basuny, A.M., A.M. Gaafar and S.M. Arafat. 2009. Tomato lycopene is a natural antioxidant and can alleviate hypercholesterolemia. Afr. J. Biotechnol. 8(23):6627-6633. Bede, N.O. 2010. Nutritional implications of projects giving high priority to the production of staples of low nutritive quality: The case for cassava (Manihot esculenta, Crantz) in the humid tropics of West Africa. Int. Inst. Tropical Agri. Ibadan, Nigeria. Berardini, N., M. Knoedler, A. Schieber and R. Carle. 2005a. Utilization of mango peels as a source of pectin and polyphenolics. Inn. Food Sci. Emerg. Tech. 6:442-52. Berger, J.P., T.E. Akiyama and P.T. Meinke. 2005. PPARs: therapeutic targets for metabolic disease. Trends. Pharmacol. Sci. 26: 244-251.

140

Bhowmik, A., A.K. Liakot, A. Masfida and R. Begum. 2009. Studies on the antidiabetic effects of M. indica stem-barks and leaves on non-diabetic type I and type II diabetic model rats. Bangl. J. Pharmacol. 4:110-114. Bibu, K.J., A.D. and K.A. Mercey. 2010. Therapeutic effect of ethanolic extract of Hygrophila spinosa T. Anders on gentamicin-induced nephrotoxicity in rats. Indian J. Exp. Biol. 48(9):911-917. Birnbaum, M.J. 2001. Turning down insulin signaling. J. Clin. Invest. 108:655 -659. Bock, C., K.H. Waldmann and W. Ternes. 2008. Mangiferin and hesperidin metabolites are absorbed from the gastrointestinal tract of pigs after oral ingestion of a Cyclopia genistoides (honeybush tea) extract. Nutr. Res. 28(12):879-891. Bor, M.V., C. Cevik and I. Uslu. 1999. Selenium levels and glutathione peroxidase activities in patients with acute myocardial infarction. Acta. Cardiol. 54(5):271- 276. Bors, W. W. Heller, C. Michel and M. Saran. 1990. Flavonoids as antioxidants: determination of radical-scavenging efficiencies. Methods Enzymol. 186:343-355. Brand-Williams, W., M.E. Cuvelier and C. Berset. 1995. Use of a free radical method to evaluate antioxidant activity. Lebensm. Wiss. Technol. 28:25-30. Brautbar, N. and J. Williams. 2002. Industrial solvents and liver toxicity: risk assessment, risk factors and mechanisms. Int. J. Hyg. Environ. Health. 205:479-491. Brown, M.S. and J.L. Goldstein. 1997. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell. 89:331-340. Browning, J.D. and J.D. Horton. 2004. Molecular mediators of hepatic steatosis and liver injury. J. Clin. Invest. 114:147-152. Burdock, G.A., I.G. Carabin and J.C. Griffiths. 2006. The importance of GRAS to the functional food and nutraceutical industries. Toxicol. 221:17–27. Burns, J., David, A.S.G. Mary, A.F. Mackintosh, S.G. Ball and J.P. Greenwood. 2008. The Effect of Chronic Anti-Hypertensive Therapywith Bendrofl umethiazide on Sympathetic Drive. Clin. Med. Cardiol. 2:263-269. Burns, J., P.D. Fraser and P.M. Bramley. 2003. Identification and quantification of carotenoids, tocoferols and chlorophyll in commonly consumed fruits and vegetables. Phytochem. 62:939-947.

141

Butt, M.S and Sultan M.T. 2009. Green tea: nature's defense against malignancies. Crit. Rev. Food Sci. Nutr. 49(5):463-73.

Butt, M.S., M.T. Sultan, M.S. Butt and J. Iqbal. 2009. Garlic; nature’s protection against physiological threats. Cri. Rev. Food Sci. Nutr. 49:538-551. Caduff, A., H.U. Lutz, L. Heinemann, G.D. Benedetto, M.S.Talary and S. Theander. 2011. Dynamics of blood electrolytes in repeated hyperand/or hypoglycaemic events in patients with type 1 diabetes. Diabetologia. 10:2210-2219. Canal, J.R., M.D. Torres, J.M. Romiro and C. Perez. 2000. A chloroform extract obtained from a decoction of Ficus carica leaves improves the cholesterolaemic status of rats with Streptozotocin induced diabetes. Pharm. Biol. 38:181-186. Cano, A., M.B. Arnao, G. Williamson and M.T. Garcia-Conesa. 2002. Superoxide scavenging by polyphenols: effect of conjugation and dimerization. Redox Report. 7:379-383. Carmen, P., R.C. Jose, J.E. Campillo, R. Adolaida and M.D. Torres. 1999. Hypotriglyceridaemic activity of Ficus carica leaves in experimental hypertriglyceridemic rats. Phytother. Res. 13(3):188-191. Carvalho, A.C.S., M.M. Guedes, A.L. Souza, M.T.S. Trevisan, A.F. Lima, F.A. Santos and V.S.N. Rao. 2005. Gastroprotective effect of mangiferin, a xanthonoid from Mangifera indica, against gastric injury induced by ethanol and indomethacin in rodents. Planta Med. 73:1372-1376. Castilho, R.F., A.R. Meinicke, A.M. Almeida, M. Hermes-Lima and A.E. Vercesi. 1994. Oxidative damage of mitochondria induced by Fe(II)citrate is potentiated by Ca2+ and includes lipid peroxidation and alterations in membrane proteins. Arch. Biochem. Biophys. 308:158-163. Caughron, K.F. and E.L. Smith. 2002. Definition and description of diabetes mellitus. South Med. J. 95(1):35-49. Chau, C.F. and Y.L. Huang. 2003. Comparison of the chemical composition and physicochemical properties of different fibers prepared from the peel of Citrus sinensis L. Cv. Liucheng. J. Agric. Food Chem. 51(9):2615-2618. Chen, C. and T. Kong. 2005. Dietary cancer-chemoprevention compounds: from signaling and gene expression to pharmacological effects. Trends Pharm. Sci. 26: 318-326. 142

Chisolm, G.M. and D. Steinberg. 2000. The oxidative modification hypothesis of atherogenesis: an overview. Free Radic. Biol. Med. 28:1815-1826.

143

Choi, C.S., D.B. Savage, A. Kulkarni and X.X. Yu. 2007. Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance. J. Biol. Chem. 282:22678-22688. Chojnacka, K., H.G´orecka and H. G´orecki. 2006. The effect of age, sex, smoking habit and hair color on the composition of hair. Environ. Toxicol. Pharmacol. 22:52-57 Cholbi, M.R., M. Paya and M.J. Alcaraz. 1991. Inhibitory effects of phenolic compounds

on CCl4-induced microssomal lipid peroxidation. Experientia. 47:195-199. Combs, G.F. and W.P. Gray. 1998. Chemoprotective agents: selenium. Pharmacol. 79:179-192. Cooper, G.J. 2011. Therapeutic potential of copper chelation with triethylenetetramine in managing diabetes mellitus and Alzheimer’s disease. Drugs. 71:1281-1320. Crozier, A., I.B. Jaganath and M.N. Clifford. 2009. Dietary phenolics: Chemistry, bioavailability and effects on health. Nat. Prod. Rep. 26:1001-1043. D’Archivio, M., C. Filesi, R. Vari, B. Scazzocchio and R. Masella. 2010. Bioavailability of the polyphenols: status and controversies. Int. J. Mol. Sci. 11:1321-1342. Dar, A., S. Faizi, S. Naqvi, T. Roome, S. Zikr-ur-Rehman, M. Ali, S. Firdous and S.T. Moin. 2005. Analgesic and antioxidant activity of mangiferin and its derivatives: The structure activity relationship. Biol. Pharm. Bull. 28:596-600. Daud, N.H., C.S. Aung, A.K. Hewavitharana, A.S. Wilkinson, J.T. Pierson, S.J. Roberts- Thomson, P.N. Shaw, G.R. Monteith, M.J. Gidley and M.O. Parat. 2010. Mango extracts and the mango component mangiferin promote endothelial cell migration. J. Agric. Food Chem. 58:5181-5186. de Freitas, V. and N. Mateus. 2012. Protein/polyphenol interactions: past and present contributions. Mechanisms of astringency perception. Curr. Org. Chem. 16:724- 746. De Silva, J.P., F.M. Areias, F.M. Proenca and O.P. Coutinho. 2006. Oxidative stresss protection by newly synthesized nitrogen compounds with pharmacological potential. Life Sci. 78:1256-1267. De-jian, J., T. Gui-shan, Y. Feng, D. Yan-hua, X. Kang-ping and L. Yuan-jian. 2003. Protective effect of xanthones against myocardial ischemia-reperfusion injury in rats. Acta. Pharmacol. 24:175.

144

Deka, A. and J.A. Vita. 2011. Tea and cardiovascular disease. Pharmacol. Res. 64:136- 145. Dey, N.D., K. Sharma and A. Pattanaik. 1998. Effect of dietary inclusion of Ficus infectoria leaves as a protectant of proteins on the performance of lambs. Small Rumin. Res. 75(2-3):105-114. Dhaliwal, M. and C.K. Hira. 2004. Effect of storage on physico-chemical and nutritional characteristics of carrot-spinach and carrot-pineapple juices. J. Food Sci. Technol. 41:613-617. Dieni, C.A. and K.B. Storey. 2011. Regulation of hexokinase by reversible phosphorylation in skeletal muscle of a freeze-tolerant frog. Comp. Biochem. Physiol. Biochem. Mol. Biol. 159:236-243. Din, A., S.A.H. Bukhari, A. Salam and B. Ishfaq. 2011. Development of functional and dietetic beverage from bitter gourd. Internet. J. Food Safety. 13:355-360. Dineshkumar, B. M. Analava and M. Manjunatha. 2010. Studies on the anti-diabetic and hypolipidemic potentials of mangiferin (Xanthone Glucoside) in streptozotocin- induced Type 1 and Type 2 diabetic model rats. Int. J. Adv. Pharm. Sci. 1:75-85. Diplock, A.T., P.J. Agget, M. Ashwell, F. Bornet, E.B. Fern and M.B. Roberfroid. 1999. Scientific concepts of functional foods in Europe: Consensus Document. British J. Nutr. 81:1-27. Dorta, E., M.G. Lobo and M. Gonzalez. 2011. Reutilization of Mango Byproducts: Study of the Effect of Extraction Solvent and Temperature on Their Antioxidant Properties. J. Food Sci. 77(1):C80-C88. Elleuch, M., D. Bedigian, O. Roiseux, S. Besbes, C. Blecker and H. Attia. 2011. Dietary fibre and fibre-rich by-products of food processing: Characterisation, technological functionality and commercial applications: A review. Food Chem.124(2):411-421. Emoke, P., I. Groza, Olga Soriþãu, C. Tomuleasa, M. Cenariu, D. Groza and T. Vlasiu. 2009. Rat bone marrow mesenchymal stem cells isolation, cultivation and differentiation. Vet. J. 15(1):29-33. Erlund I., R. Koli, G. Alfthan, J. Marniemi, P. Puukka and P. Mustonen. 2008. Favorable effects of berry consumption on platelet function, blood pressure and HDL cholesterol. Am. J. Clin. Nutr. 87:323-331.

145

Evans, J.L., I.D. Goldfine, B.A. Maddux and G.M. Grodsky. 2002. Oxidative stress and stress-activated signalling pathways: A unifying hypothesis of type 2 diabetes. Endocr. Rev. 23:599-622. Faizi, S., A. Zikr-ur-Rehman, M. Ali and A. Naz. 2006. Magn. Reson. Chem. 44: 838. Fan, J., X. Ding and W. Gu. 2007. Radical-scavenging proanthocyanidins from sea buckthorn seed. Food Chem. 102:168-177. Feng, C.H., H.Y. Huang and C.Y. Lu. 2011. Quantitation of the glutathione in human peripheral blood by matrix assisted laser desorption ionization time-of-ﬂight mass spectrometry coupled with micro-scale derivatization. Anal. Chem. 690(2):209- 214. Finkel, T. and N.J. Holbrook. 2000. Oxidants, oxidative stress and the biology of ageing. Nature. 408(6809):239-47. Fowler, M.J. 2007. Diabetes Treatment, Part 2: Oral agents for glycemic management. Clin. Diabetes. 25:131-134. Fowomola, M.A. 2010. Some nutrients and antinutrients contents of mango (Magnifera indica) seed. Afr. J. Food Sci. 4(8):472-476. Frankel E.N. and A.S. Meyer. 2000. The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. J. Sci. Food Agric. 80:1925-1941. Fridlyand L.E. and L.H. Philipson. 2010. Glucose sensing in the pancreatic beta cell: a computational systems analysis. Theor. Biol. Med. Model. 7:15. Furuta, T., M. Nakayama, H. Suzuki, H. Tajimi, M. Inai, H. Nukaya, T. Wakimoto and T. Kan. 2009. Org. Lett. 11:22-33. Gadi, R., A. Amanullah and V.M. Figueredo. 2012. HDL-C: Does it matter? An update on novel HDL-directed pharmaco-therapeutic strategies. Int. J. Cardiol. Still in press. Gao, Z., K. Huang, X. Yang and H. Xu. 1999. Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi. Biochem. Biophys. Acta. 1472(3):643-650. Garcia, D., J. Leiro and R. Delgado. 2003. Mangifera indica L. extract (Vimang) and mangiferin modulate mouse humoral immune responses. Phytother. Res. 17:1182- 1187.

146

Garcia, G.G., P. Harden and J. Chapman. 2012. Global role of kidney transplantation, for the World Kidney Day Steering Committee. Hong Kong J. Nephrol. 14:1-6. Garrido, G., D. González, Y. Lemus, D. Garcia, L. Lodeiro and G. Quintero. 2004. In vivo and in vitro anti-inflammatory activity of Mangifera indica L. extract (VIMANG). Pharmacol. Res. 50:143-149. Ghosal, S. and G. Rao. 1996. A plausible chemical mechanism of the bioactivities of mangiferin. Indian J. Chem. 35:561-566. González-Montelongo R., M.G. Lobo and M. González. 2010a. The effect of extraction temperature, time and number of steps on the antioxidant capacity of methanolic banana peel extracts. Sep. Purif. Technol. 71:347-55. Gopalan, C., B.V. Ramasastri and S.C. Balasubramaniam. 1999. Nutritive value of Indian foods. National Institute of Nutrition, ICMR, India. Gorinstein, S., R. Haruenkit, S. Poovarodom, Y. Park, S. Vearasilp, M. Suhaj, K.S. Ham, B.G. Heo, J.Y. Cho and H.G. Jang. 2009. The comparative characteristics of snake and kiwi fruits. Food Chem. Toxicol. 47:1884-1891. Gorinstein, S., Z. Zachwieja, M. Folta, H. Barton, J. Piotrowicz, M. Zemser, M. Weisz, S. Trakhtenberg and O. Martın-Belloso. 2001. Comparative contents of dietary fiber, total phenolics, and minerals in persimmons and apples. J. Agric. Food Chem. 49:952-957. Gouado, I., F.J. Schweigert, R.A. Ejoh, M.F. Tchouanguep and J.V. Camp. 2007. Systemic levels of carotenoids from mangoes and papaya consumed in three forms (juice, fresh and dry slice). Eur. J. Clin. Nutr. 61(10):1180–1188. Granfeldt, Y., I. Bjorck, A. Drews and J. Tovar. 1992. An in vitro procedure based on chewing to predict metabolic responses to starch in cereal and legumes products. Eur. J. Clin. Nutr. 46:649-660. Guo, C., J. Yang, J. Wei, Y. Li, J. Xu and Y. Jiang. 2003. Antioxidant activities of peel, pulp and seed fractions of common fruits as determined by FRAP assay. Nutr. Res. 23:1719-1726. Guo, C.J. and J.J. Yang. 2001. Progress in the study of antioxidant capacity of fruits and vegetables. China Public Health.17:87-88. Guo, F. 2011. Beneficial effects of mangiferin on hyperlipidemia in high-fat-fed hamsters. Mol. Nutr. Food Res. 55(12):1809-1818.

147

Gupta, R. and R.S. Gupta. 2011. Antidiabetic efficacy of Mangifera indica seed kernels in rats: a comparative study with glibenclamide. Diabetologia Croatica 40-44. Guzzi, G., and C.A. La Porta. 2008. Molecular mechanisms triggered by mercury. Toxicology. 244:1-12. Gylling, H., P. Puska, E. Vartiainen and T.A. Miettinen. 1999. Retinol, vitamin D, carotenes and a-tocopherol in serum of moderately hypercholesterolemic population consuming sitostanol ester margarine. Atherosclerosis. 145:279-285. Haminiuk, C.W.I. 2006. Influence of temperature on the rheological behavior of whole araça pulp (Psidium cattleianum sabine). LWT-Food Sci. Technol. 39(4):426-430. Haminiuk, C.W.I., M.S.V. Plata-Oviedo, A.R. Guedes, A.P. Stafussa, E.Bona and S.T. Carpes. 2011.Chemical, antioxidant and antibacterial study of Brazilian fruits. Inter. J. Food Sci. Technol. 46:1529-1537. Hassan, S.W., M. Lawal, B.V. Muhammed, R.A. Umar and V. Saidu, 2007. Effects of anthraquinone glycosides and aqueous ethanol extracts of Ficus sycomorus L. (Moraceae) on rat liver and kidney functions. Asian J. biochem., 2(2):136-141. Hayashi, K., R. Kojima and M. Ito. 2006. Strain differences in the diabetogenic activity ofstreptozotocin in mice. Biol. Pharmaceut. Bull. 29:1110-1119. Hayes, J.D. and D.J. Pulford. 1995. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol. 30:445-600. Ho, L. 2003. Studies on mangiferin production from mango leaves. Dissertation; Plovdiv, Bulgaria. Adv. Food Sci. 28:46-5. Hoffman, M., A. Blum, R. Baruch, E. Kaplan and M. Benjamin. 2004. Leukocytes and coronary heart disease. Atherosclerosis. 172:1-6. Hollman, P.C.H. 2004. Absorption, Bioavailability, and Metabolism of Flavonoids. Pharm Biol. 42:74-83 Hou, Y., W. Shao, R. Xiao, K. Xu, Z. Mad, B.H. Johnstone and Y. Du. 2009. Pu-erh tea aqueous extracts lower atherosclerotic risk factors in a rat hyperlipidemia model. Exp. Gerontol. 44:434-439. Hu, H.G., M.J. Wang, Q.J. Zhao, H.L. Liao, L.Z. Cai, Y. Song, J. Zhang, S.C. Yu, W.S. Chen, C.M. Liu and Q.Y. Wu. 2007. Synthesis of mangiferin derivatives as protein tyrosine phosphatase 1B inhibitors.Chem. Nat. Compd. 43:663-666.

148

Hu, Q.H., X. Zhang, Y. Wang and L.D. Kong. 2010. Mangiferin promotes uric acid excretion and kidney function improvement and modulates related renal transporters in hyperuricemic mice. Yao Xue Xue Bao. 45(10):1239-1246. Huang, C.S., M.C. Yin and L.C. Chiu. 2011. Antihyperglycemic and antioxidative potential of Psidium guajava fruit instreptozotocin-induced diabetic rats. Food Chem. 49(9):2189-2195. Huang, D., B. Ou, and R.L. Prior. 2005. The chemistry behindantioxidant capacity assays. J. Agric. Food Chem. 53:1841-1856. Hubbard, E.M., V.S. Ramachandran and G.M. Boynton. 2003. ‘Cortical cross-activation as the locus of grapheme-color synesthesia’, 3rd Annual Meeting of the Vision Sciences Society, Sarasota, FL. Hurtado-Fernandez, E., M. Gomez-Romero, A. Carrasco-Pancorbo and A. Fernandez- Gutierrez. 2010. Application and potential of capillary electroseparation methods to determine antioxidant phenolic compounds from plant food material. J. Pharma. Biomed. Anal. 53:1130-1160. Hussain, I., A. Zaib and M. Ayub. 2010. Quality attributes of apple and apricot blend juice preserved with potassium sorbate during storage at low temperature. Internet. J. Food Sci. 12:80-86. Hussain, S., S. Rehman, M.A. Randhawa and M. Iqbal. 2003. Studies on Physico- chemical, microbiological and sensory evaluation of mango pulp storage with chemical preservatives. J. Res. Sci. 14:01-09. Ichiki, H., T. Miura and K. Kubo. 1998. New antidiabetic compunds, mangiferin and its glucoside. Biol. Pharm. Bill. 21:1389-1390. Iman, M., M.R. Sayed, Mourad and D.A. Sayed. 2011. Biochemical changes in experimental diabetes before and after treatment with Mangifera indica and psidium guava extracts. Int. J. Pharm. Biomed. Sci. 2(2):29-41. International Diabetes Federation (IDF). 2011. http://www.idf.org/global-diabetes-plan- 2011-2021. Accessed online on Iqbal, S. and Bhanger M.I. 2007. Stabilization of sunflower oil by garlic extract during accelerated storage. Food Chem. 100:246-254.

149

Izunya, A.M., A.O. Nwaopara, A. Aigbiremolen, M.A.C. Odike, G.A. Oaikhena, J.K. Bankole and P.A. Ogarah. 2010. Morphological and biochemical effects of crude aqueous extract of Mangifera indica L. (Mango) stem bark on the liver in Wistar rats. Res. J. Appl. Sci. Engr. Technol. 2(5):460-465. Jacobs, D.S., W.R. DeMott, H.J. Grady, R.T. Horvat, D.W. Huestis and B.L. Kasten. 1996. Laboratory test handbook, 4th ed. Lexi-comp Inc., Hudson (Cleveland). Jacqueline, C.B., M.T.S. Trevisan, W.E. Hull, G. Erben, S. Edy, D.E. Brito and B. Pfudstein. 2008. Characterization and quantification of polyphenolic compounds in bark, kernel, leaves and peel of Mango (Mangifera indica L.). J. Agric. Food Chem. 56:5599-5610. Jagetia, G.C. and V.A. Venkatesha. 2005. A glucosylxanthone, protects against the radiation-induced micronuclei formation in the cultured human peripheral blood lymphocytes. Int. Congr. Ser. 1276:195-6. Jagetia, G.C. and V.A. Venkatesha. 2005. Effect of mangiferin on radiation-induced micronucleus formation in cultured human peripheral blood lymphocytes. Environ. Mol. Mutagen. 46:12-21. Jan, A. and E.D. Masih. 2012. Development and quality evaluation of pineapple juice blend with carrot and orange juice. Int. J. Sci. Res. 2(8):2250-3153 Jenkins, D.J., C.W. Kendall, T.H. Nguyen, A. Marchie, D.A. Faulkner, C. Ireland, A.R. Josse, E. Vidgen, E.A. Trautwein, K.G. Lapsley, C. Holmes, R.G. Josse, L.A. Leiter, P.W. Connelly and W. Singer. 2008. Effect of plant sterols in combination with other cholesterol-lowering foods. Metab. 57(1):130-139. Jong, D.J.T.A., L.M. Widrow and P. Cseresnjes. 2006. A&A. 446:855. Joona, K., C. Sowmia, K.P. Dhanya and M.J. Divya. 2013. Preliminary phytochemical investigation of Mangifera indica leaves and screening of antioxidant and anticancer activity. Res. J. Pharm. Biol. Chem. Sci. 4(1):1112. Jung, M.J., S. Heo and M. Wang. 2008. Free radical scavenging and total phenolic contents from methanolic extracts of Ulmus davidiana. Food Chem. 108:482-487. Jutiviboonsuk, A. and C. Sardsaengjun. 2010. Mangiferin in leaves of three Thai mango (Mangifera indica L.) varieties. IJPS. 6(3):122-129. Kano, M., T. Takayanagi, K. Harada, K. Makino and F. Ishikawa. Antoxidant activity of anthocyanins from purple sweet potato, Ipomoera batatas cultivar ayamurasaki. Biosci. Biotechnol. Biochem. 69(5):979-988. 150

Karadag, A., B. Ozcelik and S. Saner. 2009. Review of methods to determine antioxidant capacities. Food Anal. Methods. 2:41-60. Kasper, J.C. and W. Friess. 2011. The freezing step in lyophilization: physico-chemical fundamentals, freezing methods and consequences on process performance and quality attributes of biopharmaceuticals. Eur. J. Pharma. Biopharma. 78:248-263. Kawpoomhae, K., M. Sukma, T. Ngawhirunpat, P. Opanasopit and A. Sripattanaporn. 2010. Antioxidant and neuroprotective effects of standardized extracts of Mangifera indica leaf. Thai J. Pharm. Sci. 34: 32-43. Kemasari, P., S. Sangeetha and P. Venkatalakshmi. 2011. Antihyperglycemic activity of Mangifera indica L. in alloxan induced diabetic rats. J. Chem. Pharm. Res. 3(5):653-659. Kim, Y., J.K. Brecht and S.T. Talcott. 2007. Antioxidant phytochemical and fruit quality changes in mango (Mangifera indica L.) following hot water immersion and controlled atmosphere storage. Food Chem. 105(4): 1327–1334 Kim, H., J.Y. Moon, H. Kim, D. Lee, H. Cho, M. Choi, Y.S. Kim, A. Mosaddik and S.K. Cho. 2010. Antioxidant and antiproliferative activities of mango (Mangifera indica L.) flesh and peel. Food Chem. 121:429-436. Kim, J.I., J.K. Paik, O.Y. Kim, H.W. Park, J.H. Lee, Y. Jang and J.H. Lee. 2011. Effects of lycopene supplementation on oxidative stress and markers of endothelial function in healthy men. Atherosclerosis. 215:189-195. Kim, J.S. 2005. Radical scavenging capacity and antioxidant activity of E vitamin fraction in rice bran. J. Food Sci. 70:208-213. Kim, Y. and J. Lee. 2001. On posterior consistency of survival models. Ann. Statist. 29:666-686. Kim, Y., K.J. Brecht and S.T. Talcott. 2007. Antioxidant phytochemical and fruit quality changes in Mango (Mangifera Indica L.) following hot water immersion and controlled atmosphere storage. Food Chem. 10:1016. Klaman, L.D., O. Boss, O.D. Peroni, J.K.J. Kim, L. Martino, J.M. Zabolotny, N. Moghal, M. Lubkin, Y.B. Kim, A.H. Sharpe, A. Stricker-Krongrad, G.I. Shulman, B.G. Neel and B.B. Kahn. 2000. Mol. Cell. Biol. 20:5479. Kolawole, O.M., Kayode, R.M.O. and Akinduyo, B.K. 2007. Proximate and microbial analyses of burukutu and pito produced in Ilorin, Nigeria. Africa J. Biotechnology, 6 (5): 587-590. 151

Koppula, S.H., Kumar, S., V., More, B.W., Kim, I.S., Kim, and D.K. Choi. 2012. Recent Advances on the Neuroprotective Potential of Antioxidants in Experimental Models of Parkinson’s Disease. Int. J. Mol. Sci. 13(8):10608–10629. Kowaltowski, A.J., R.F. Castilho and A.E. Vercesi. 2001. Mitochondrial permeability transition and oxidative stress. FEBS Lett. 495:12-15. Krishnaveni, A., G. Manimegalai and R. Saravanakumar. 2001. Storage stability of jack fruit RTS beverage. J. Food Sci. Technol. 38:601-602. Kumar, P. 2000. Impact of anaemia in patients with head and neck cancer. Oncol. 5:13- 18. Kuo, K.L., M.S. Weng, C.T. Chiang, Y.J. Tsai, S.Y. Lin-shiau and J.K. Lin. 2005. Comparative studies on the hypolipidemic and growth suppressive effects of oolong, black, pu-erh, and green tea leaves in rats. J. Agric. Food Chem. 53:480- 489. Kuti, T., A. Hegyi and B. Kemeny. 2004. Analysis of sensory data of different food products by ANOVA. Chemometrics Intelligent Lab. Syst. 72:253-257. Lafka, T.I., V. Sinanoglu and E.S. Lazos. 2007. On the extraction and antioxidant activity of phenolic compounds from winery wastes. Food Chem. 104:1206-1214. Lamb, D.J., G.M. Wilkins and D.S. Leake. 1992. The oxidative modification of low density lipoprotein by human lymphocytes. Atherosclerosis. 92:187-192. Larrauri, J.A., P. Ruperez, B. Borroto and F. Saura-calixto. 1996. Mango peel as a new tropical fibre: preparation and characterization. Lebensmittel Wissenschaft Und Technology. 29: 729-733. Lee, J.W., J.K. Kim, P. Srinivasan, J. Choi, J.H. Kim, S.B. Han, D.J. Kim and M.W. Byun. 2009. Effect of gamma irradiation on microbial analysis, antioxidant activity, sugar content and color of ready-to-use tamarind juice during storage. Food Sci. Technol. 42:101-105. Lee, P. and K. Prasad. 2003. Effects of flaxseed oil on serum lipids and atherosclerosis in hypercholesterolemic rabbits. J. Cardiovasc. Pharmacol. Therapeut. 8:61-69. Leiro, J.M., E. Álvarez, J.A. Arranz, I.G. Siso and F. Orallo. 2003.In vitro effects of mangiferin on superoxide concentrations and expression of the inducible nitric oxide synthase, tumor necrosis factor and transforming growth factor- genes. Biochem. Pharmacol. 65:1361-1371.

152

Leontowicz, M., S. Gorinstein, H. Leontowicz, R. Krzeminski, A. Lojek, E. Katrich, M. Ciz, O. Martin-Belloso, R. Soliva-Fortuny and R. Haruenkit. 2003. Apple and pear peel and pulp and their influence on plasma lipids and antioxidant potentials in rats fed cholesterol-containing diets. J. Agric. Food Chem. 51:5780-5785. Li, J. H., C.L. Ren, Y.H. Zhang, X.Y. Liu, X.J. Yao and Z. D. Hu. 2008. J. Mol. Struct. 885:64-69. Liu, H.X., J.J. Zhang, P. Zheng and Y. Zhang. 2005. Altered expression of MAP-2, GAP- 43, and synaptophysin in the hippocampus of rats with chronic cerebral hypoperfusion correlates with cognitive impairment. Brain Res. Mol. Brain Res. 139:169-177. Liu, J.Q., J.N. Tian, Z.D. Hu and X.G. Chen. 2008. Bioorg. Med. Chem. 73:443-450. Luo, F., Q. Lv, Y. Zhao, G. Hu, G. Huang, J. Zhang, C. Sun, X. Li and K. Chen. 2012. Quantification and purification of mangiferin from Chinese mango (Mangifera indica L.) cultivars and its protective effect on human umbilical vein endothelial cells under H2O2-induced stress. Int. J. Mol. Sci. 13:11260-11274. Ma, Q., R.H. Tipping and N.N. Lavrentieva. 2011. Pair identity and smooth variation rules applicable for the spectroscopic parameters of H2O transitions involving high-J states. Mol. Phys. 109:1925-1941. Madjid, M., I. Awan, J.T. Willerson and S.W. Casscells. 2004. Leukocyte count and coronary heart disease: implications for risk assessment. J. Am. Coll. Cardiol. 44:1945-1956. Mahdavian, E.S. and R.K. Somashekar. 2008. Heavy metals and safety of fresh fruits in Bangalore city, India - A case study. Kathmandu Univ. J. Sci. Eng. Technol. I:17- 27. Mahmoud, M.R., H.S. EL-Abhar and S. Saleh. 2002. The effect of Nigella Sativa oil against the liver damage induced by Schistosoma mansoni infection in mice. J. Ethnopharmacol. 55:141-145. Maisuthisakul, P. and M.H. Gordon. 2009. Antioxidant and tyrosinase inhibitory activity of mango seed kernel by product. Food Chem. 117:332-341. Majumdar, T.K., C.R. Vasudish, K.S. Premavalli and A.S. Bawa. 2008. Studies on processing and storage stability of ashgourd-mint leave juice. J. Food Proc. and Preserv. 34:549-556.

153

Majumdar, T.K., D.D. Wadikar and A.S. Bawa. 2010. Development, stability and sensory acceptability of cucumber-basil juice blend. Afr. J. Food. Agric. Nutr. Dev. 10:4093-4104. Majumdar, T.K., D.D. Wadikar, C.R. Vasudish, K.S. Premavalli and A.S. Bawa. 2011. Effect of storage on physico-chemical, microbiological and sensory quality of bottlegourd-basil leaves juice. Am. J. Food Technol. 6(3):226-234. Makan, B., Y. Pillay, P. Barron and B. Asia. 2003. Service Level Agreement Memorandum & Pro Forma Guidelines. Chief Directorate, District and Development Cluster. National Department of Health. Pretoria. Manach, C., G. Williamson, C. Morand, A. Scalbert and C. Remesy. 2005. Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am. J. Clin. Nutr. 81:230-242. Manthey, J.A. and P. Perkins-Veazie. 2009. Influences of harvest date and location on the levels of beta-carotene, ascorbic acid, total phenols, the in vitro antioxidant capacity, and phenolic profiles of five commercial varieties of mango (Mangifera indica L.). J. Agric. Food. Chem. 57:10825-10830. Manzano, J.E, Y. Perez and E. Rojas. 1997. Coating waxes on haden mango fruits (Mangifera indica L.) cultivar for export. Acta Hort. 455:738-746. Manzoor, M., F. Anwar, N. Saari and M. Ashraf. 2012. Variations of antioxidant characteristics and mineral contents in pulp and peel of different Apple (Malus domestica Borkh.) cultivars from Pakistan. Molecules. 17:390-407. Marthac, R. and B. Msusan. 2008. Effect of natural antioxidants on oxidative stability of frozen, vacuum-packaged beef and pork. J. Food Quality. 31(2):173-188. Masibo, M and Q. He. 2009. Mango bioactive compounds and related nutraceutical properties- A review. Food Rev. Int. 25:346-370. Masibo, M. and H. Qian. 2008. Major mango polyphenols and their potential significance to human health. Comp. Rev. Food Sci. Food Safety. 7:309-319. Matkowski, A., P. Kuśś, E. Góralska, and D. Woźniak. 2012. Mangiferin-a bioactive xanthonoid, not only from mango and not just antioxidant. Mini. Rev. Med. Chem. 13(3):439-55. Matsuura, E., G.R. Hughes and M.A. Khamashta. 2008. Oxidation of LDL and its clinical implication. Autoimmun. Rev. 7:558-566.

154

McEneny, J., P. McPherson, M. Spence, U. Bradley, S. Blair, M. McKinley, I. Young and S. Hunter. 2012. Does a diet high or low in fat influence the oxidation potential of VLDL, LDL and HDL subfractions? Nutr. Metabol. Cardiovasc. Dis. 1-7. Meilgaard, M.C., G.V. Civille and B.T. Carr. 2007. Sensory evaluation techniques, 4th ed. C.R.C. Press L.L.C., New York. Merken, H.M., C.D. Merken and G.R. Beecher. 2001. Kinetic method for the quantitation of flavonoids as aglycones in foods. J. Agric. Food Chem. 49(6):2727-2732. Michalik, L., J. Auwerx, J.P. Berger, V.K. Chatterjee, C.K. Glass, F.J. Gonzalez, P.A. Grimaldi, T. Kadowaki, M.A. Lazar, S. O’Rahilly, C.N.A. Palmer, J. Plutzky, J.K. Reddy, B.M. Spiegelman, B. Staels and W. Wahli. 2006. Peroxisome proliferator- activated receptors, international union of pharmacology. LXI. Pharmacol. Res. 58:726-741. Michos, E.D., C.T. Sibley, J.T. Baer, M.J. Blaha and R.S. Blumenthal. 2012. Niacin and statin combination therapy for atherosclerosis regression and prevention of cardiovascular disease events. J. Am Coll. Cardiol. 59(23). Mishra, V., V. Puranik, V. Singh, M. Verma, N. Yadav and G.K. Rai. 2012. Development of Vitamin C rich value added beverage. Am. J. Food Technol. 7(4):222-229. Mitra, A. 2007. Effects of Composite of tulsi leaves, amla, bitter gourd, gurmur leaves, jamun fruit and seed in type 2 diabetic patients. J. Clin. Diagn. Res. 1:511-520. Mitrakou, A. 2011. Kidney: its impact on glucose homeostasis and hormonal regulation. Diabetes Res. Clin. Prac. 93:66-72. Miura, T., H. Ichiki, N. Iwamoto, M. Kato, M. Kubo, H. Sasaki, M. Okada, T. Ishida, Y. Seino and K. Tanigawa. 2001. The suppressive effect of mangiferin with exercise on blood lipids in type 2 diabetes. Biol. Pharm. Bull. 24(9):1091-1092. Moharib, S.A. 2006. Hypolipidemic effect of dietary fibre in rats. Adv. Food Sci. 28:46- 55. Molina, L., F. Constantinescu, L. Michel, C. Reimmann, B. Duffy and G. Defago. 2003. Degradation of pathogen quorum-sensing molecules by soil bacteria: a preventive and curative biological control mechanism. FEMS Microbiol. Ecol. 45:71-81. Morsy, M.D., O.A. Mostafa and W.N. Hassan. 2010. RAes eparochtential protective effect of α-tocopherol on vascular complication in spinal cord reperfusion injury in rats. J. Biomed. Sci. 17(55):2-9

155

Morsy, R.M.Y., N.R. EL-Tahan and A.M.A. El-Hadad. 2010. Effect of aqueous extract Mangifera Indica leaves, as functional foods. J. Appl. Sci. Res. 6(6):712-721. Moure, A., H. Dominguez and J.C. Parajo. 2006. Antioxidant properties of ultrafiltration recovered soy protein fractions from industrial effluents and their hydrolysates. Process Biochem. 41(2):447-456. Moure, A., J.M. Cruz, D. Franco, J.M. Dominguez, J. Sineiro, H. Dominguez, M.J. Nunez and J.C. Parjo. 2001. Natural antioxidants from residual sources. Food Chem. 72:145-171. Mulder, T.P., C.J. van Platerink, P.J.W. Schuyl and J.M. van Amelsvoort. 2001. Analysis of theaflavins in biological fluids using liquid chromatography-electrospray mass spectrometry. J. Chromatogr B Biomed Sci. Appl. 760:271-279. Muller, L., K. Frohlich and V. Bohm. 2011. Comparitive antioxidant activities of carotenoids measured by ferric reducing antioxidant power (FRAP), ABTS bleaching assay (alpha-TEAC), DPPH assay and peroxyl radical scavenging assay. Food Chem. 129:139-148. Murtaza, M.A., N. Huma, J. Javaid, M.A Shabbir, G.M. Din and S. Mahmood. 2004. Studies on stability of strawberry drink stored at different temperatures. Int. J. Agric. Biol. 6(1):58-60. Muruganandan, S., K. Scrinivasan, S. Gupta, P.K. Gupta and J. Lal. 2005. Effect of mangiferin on hyperglycemia and atherogenicity in streptozotocin diabetic rats. J. Ethnopharmacol. 97:497-501. Muruganandan, S., K. Scrinivasan, S. Gupta, P.K. Gupta and J. Lal. 2005. Effect of mangiferin on hyperglycemia and atherogenicity in streptozotocin diabetic rats. J. Ethnopharmacol. 97:497-501. Muruganandan, S., K. Srinivasan, S. Gupta, J. Gupta and J.J. Lal. 2005. Effect of mangiferin on hyperglycemia and atherogenicity in streptozotocin diabetic rats. J. Ethnopharmacol. 97:497-501 Muruganandan, S., S. Gupta, M. Kataria, J. Lal and P.K. Gupta. 2002. Mangiferin protects the streptozotocin-induced oxidative damage to cardiac and renal tissues in rats. Toxicol. 176:165-173. Nair, P.S. and D.C.S. Shyamala. 2006. Efficacy of mangiferin on serum and heart tissue lipids in rats subjected to isoproterenol induced cardiotoxicity. Toxicol. 228(2- 3):135-139. 156

Negro, D.R., A. Rossi and I. Cerveri. 2003. The burden of COPD in Italy:results from the Confronting COPD survey. Respir. Med. 97:s43-50. Nishigaki, I., R. Venugopal Sakthisekaran D, Rajkapoor B. 2007. In vitro protective effect of mangiferin against glycated protein-iron chelate induced toxicity in human umbilical vein endothelial cells. J.Biol. Sci. 7(7):1227-32. Nithitanakool, S., P. Pithayanukul and R. Bavovada. 2009. Antioxidant and hepatoprotective activities of Thai mango seed kernel extract. Planta Med. 75:1118-1123. Nunes, M.C.N and Morais, A.M.M.B. 1995. Quality of strawberries after storage in controlled atmosphere at above optimum storage tempratures. Proc. Fla. State. Hort. Soc.108:273-278. Ojewole, J. 2005. Anti-inflammatory, analgesic and hypoglycemic effects of Mangifera indica Linn. (Anacardiaceae) stem-bark aqueous extract. methods find exp. Clin. Pharmacol. 27:547-554. Ojokoh, B.A., O.S. Adewale and S.O. Falaki. 2008. Improving on the smoothing technique for obtaining emission probabilities in hidden Markov models. Oriental J. Comp. Sci. Technol. 1(1):15-24. Omodamiro, R.M., C. Aniedu, U. Chijoke and E. Oti. 2012. Storage and consumer acceptability of fruit: Ginger based drinks for combating micronutrient deficiency. J. Stored Prod. Postharvest Res. 3(6):80-82. Oyama, K. and T. Kondo. 2004. Total synthesis of flavocommelin, a component of the blue supramolecular pigment from Commelina communis, on the basis of direct 6- C-glycosylation of flavan. J. Org. Chem. 69:5240-5246. Paim, B.A., J.A. Velho, R.F. Castilho, H.F.C. Oliveira and A.E. Vercesi. 2008. Oxidative stress in hypercholesterolemic LDL receptor knockout mice is associated with low content of mitochondrial NADP-linked substrates and is partially reversed by citrate replacement. Free Radic. Biol. Med. 44:444-451. Palafox-Carlos, H., J.F. Ayala-Zavala and G.A. Gonza´ lez-Aguilar. 2011. The role of dietary fiber in the bioaccessibility and bioavailability of fruit and vegetable antioxidants. J. Food Sci. 76:6-15. Pan, Z., R.W. Arritt, M. Segal, T.C. Chen and S.P. Weng. 2000. Effects of quasi- stationary large-scale anomalies on some mesoscale features associated with the 1993 flood: A regional model simulation. J. Geophys. Res. 105(24):551-29. 157

Pandit, S., T.K. Sur, U. Jana, P.K. Debnath, S. Sen and D. Bhattacharyya. 2004. Prevention of carbon tetrachloride-induced hepatotoxicity in rats by Adhatoda vasica leaves. Indian J. Pharmacol. 36(5):312-320. Parada, J. and J.M. Aguilera. 2007. Food microstructure affects the bioavailability of several nutrients. J. Food Sci. 72:R21-R32. Pardo, A.G., R. Delgado, J. Velho, M.I. Natalia, C. Curti and A.E. Vercesi. 2005.

Mangifera indica L. extract (Vimang) inhibits Fe2+-citrate-induced lipoperoxidation in isolated rat liver mitochondria. Pharmacol. Res. 51:427-35. Pardo-Andreu, G.L., R.A. Cavalheiro, D.J. Dorta, Z. Naal, R. Delgado, A.E. Vercesi and C. Curti. 2006. Fe (III) Shifts the Mitochondria Permeability Transition-Eliciting Capacity of Mangiferin to Protection of Organelle. J. Pharmacol. Exp. Ther. 320(2):646-53. Pardo-Andreua, G.L., B.A. Paim, R.F. Castilho, J.A. Velho, R. Delgado, A.E. Vercesi and H.C.F. Oliveirac. 2008. Mangifera indica L. extract (Vimang®) and its main polyphenol mangiferin prevent mitochondrial oxidative stress in atherosclerosis- prone hypercholesterolemic mouse. Pharmacol. Res. 57:332-338. Parr, S. and B. Bolwell. 2000. Phenols in the plant and in man. The potential for possible nutritional enhancement of the diet by modifying the phenols content or profile. J. Sci. Food Agric. 80:985-1012. Pastwa, E., R.D. Neumann and T.A. Winters. 2001. In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts. Nucleic Acids Res. 29:2-10. Paudel, B.H. and S. Kamar. 2003. Effect of water and saline load on urinary output in healthy undergraduate medical students. J. Nepal Med. Ass. 42:23-26. Pawlak, W., J. Kedziora, K. Zolynski, K. Kedziora-Kornatowska, J. Blaszczyk, P. Witkowski and J. Zieleniewski. 1998. Effect of long term bed rest in men on enzymatic antioxidative defense and lipid peroxidation in erythrocytes. J. Gravit. Physiol. 5(1):163-164. Pei, Z., G. Liu and T.H. Lubben. 2004. Szczepankiewicz, B. G. Curr. Pharm. Des. 10:3481. Periyar S.S., P.M. Balu, M.P. Sathiya and K. Murugesan. 2009. Antihyperglycemic effect of mangiferin in streptozotocin induced diabetic rats. J. Health Sci. 55:206-214.

158

Peschel, W., W. Dieckmann, M. Sonnenschein and A. Plescher. 2007. High antioxidant potential of pressing residues from evening primrose in comparison to other oilseed cakes and plant antioxidants. Ind. Crop Prod. 25:44-54. Pessôa B.S., E.B. Peixoto, A. Papadimitriou, J.M. Lopes de Faria and J.B. Lopes de Faria. 2012. Spironolactone improves nephropathy by enhancing glucose-6- phosphate dehydrogenase activity and reducing oxidative stress in diabetic hypertensive rat. J. Renin Angiotensin Aldosterone Syst. 13:56-66. Peter, M., F. Leonard, C. Bernard, K. Joyce, G. Victor and M. Kaswija. 2007. Physical and chemical characteristics of off vine ripened mango (Mangifera indica L.) fruit (Dodo). Afr. J. Biotechnol. 6(21):2477-2483. Petersen, K.F. and G.I. Shulman. 2006. Etiology of insulin resistance. Am. J. Med. 119(5):10-16. Piconi, L., L. Quagliaro and A. Ceriello. 2003. Oxidative stress in diabetes. Clin. Chem. Lab. Med. 41:1144-1149. Pinelo, M., A. Arnous and A.S. Meyer. 2006. Upgrading of grape skin: Significance of plant cell-wall structural components and extraction techniques for phenol release. Trends Food Sci. Technol. 17:579-590. Plumb, J.A., A.D. Noyes, S. Graziano, J. Wang, J. Mao and V.G Chinchar. 1999. Isolation and identification of viruses from adult largemouth bass during a 1997- 1998 survey in the southeastern United States. J. Aquat. Anim. Health. 11:391- 399. Pott, I., M. Marx, S. Neidhart, W. Mühlbauer and R. Carle. 2003. Quantitative determination of B-carotene stereoisomers in fresh, dried and solar-dried mangoes (Mangifera Indica L.). J. Agric. Food Chem. 51:4527-4531. Power, J.H.T. and P.C. Blumbergs. 2009. Cellular glutathione peroxidase in human brain: cellular distribution, and its potential role in the degradation of Lewy bodies in Parkinson’s disease and dementia with Lewy bodies. Acta. Neuropathologica. 117(1):63-73 Prabhu, S., J. Mallika, K.E. Sabitha and C.S.S. Devi. 2006a. Cardioprotective effect of mangiferin on isoproterenol induced myocardial infarction in rats. Ind. J. Exp. Biol. 44(3):209-215.

159

Prabhu, S., M. Jainu, K.E. Sabitha and C.S.S. Devi. 2006b. Role of mangiferin on biochemical alterations and antioxidant status in isoproterenol induced myocardial infarction in rats. J. Ethnopharmacol. 107(1):126-133. Prado, F.C., J.L. Parada, A. Pandey and C.R. Soccol. 2008. Trends in non-dairy probiotic beverages. Food Res. Int. 41:111-123. Prasad, K.N. H., Xie, J. Hao, B. Yang, S. Qiu, X. Wei, F. Chen and Y. Jiang. 2010. Antioxidant and anticancer activities of 8 - hydroxypsoralen isolated from wampee [Clausena lansium (Lour.) Skeels] peel. Food Chem.118:62-66. Prasad, S., N. Kalra and Y. Shukla. 2007. Hepatoprotective effects of lupeol and mango pulp extract of carcinogen induced alteration in Swiss albino mice. Mol. Nutr. Food Res. 51:352-9. Prasad, S., N. Kalra, M. Singh and Y. Shukla. 2008. Protective effects of lupeol and mango extract against androgen induced oxidative stress in Swiss albino mice. Asian J. Androl. 10(2):313–8. Prasad, S.K., A. Kulshreshtha and N.Q. Taj. 2009. Antidiabetic activity of some herbal plants in STZ- induced diabetic albino rats. Pak J. Nutr. 8:551-557. Prashanth, D., A. Amit, D.S. Samiulla, M.K. Asha and R. Padmaja. 2001. α-Glucosidase inhibitory activity of Mangifera indica bark. Fitoterapia. 72(6):686-688. Prior, R.L., X.L. Wu and K. Schaich. 2005. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food Chem. 53:4290-4302. Qin, B., R.A. Anderson and K. Adeli. 2008. Tumor necrosis factoralpha directly stimulates the overproduction of hepatic apolipoprotein B100-containing VLDL via impairment of hepatic insulin signaling. Am. J. Physiol. Gastrointest. Liver Physiol. 294:1120-1129. Quílez, J., J.A. Ruiz, G. Brufau and M. Rafecas. 2006. Bakery products enriched with phytosterols, α-tocopherol and β-carotene. Sensory evaluation and chemical comparison with market products. Food Chem. 94:399-405. Quílez, J., M. Rafecas, G. Brufau, P. García-Lorda, I. Megías, M. Bulló J.A. Ruiz and J. Salas-Salvadó. 2003. Bakery products enriched with phytosterol esters, alpha- tocopherol and beta-carotene decrease plasma LDL-cholesterol and maintain plasma beta-carotene concentrations in normocholesterolemic men and women. J. Nutr. 133(10):3103-3109. 160

Rai, P.K., D. Jaiswal, S. Mehta, D.K. Rai, B. Sharma and G. Watal. 2010. Effect of Curcuma longa freeze dried rhizome powder with milk in STZ- induced diabetic rats. Indian J. Clin. Biochem. 2:175-181. Rajendran, P., G. Ekambaram and D. Sakthisekaran. 2008. Effect of mangiferin on benzo(a)pyrene induced lung carcinogenesis in experimental Swiss albino mice. Nat. Prod. Res. 22(8):672-680. Rao, M.A. and L. Gianfreda. 2000. Properties of acid phosphatase–tannic acid complexes formed in the presence of Fe and Mn. Soil Biol. Biochem. 32:1921-1926. Rashad, M.M. and S.A. Moharib. 2008. Studies of the effect of some plant leaf fibres on the key liver enzymes intermediate carbohydrate and lipid metabolism in rats. Adv. Food Sci. 1:1-8. Rastraelli, L., A.J.N. Selles, H.T.V. Castro, J. Aguero-Aguero, J. Gonzalez-Gonzalez, F. Naddeo and F.D. Simone. 2002. Isolation and quantitative analysis of phenolic antioxidants, free sugars,and polyols from mango (Mangifera Indica L.) stem bark aqueous decoction used in Cuba as a nutritional Supplement. J. Agric. Food Chem. 50:762-766. Rathore, H.A., T. Masud, S. Sammi, A.H. Soomro. 2007. Effect of storage on physic- chemical composition sensory properties of mango (Mangifera indica) variety dusehri. Dep. of food Technol, Univ College of Agric, Rawlakot. 479-482. Rau, O., M. Wurglics, A. Paulke, J. Zitzkowski, N. Meindl, A. Bock, T. Dingermann, M. Abdel-Tawab and M. Schubert-Zsilavecz. 2006. Carnosic acid and carnosol, phenolic diterpene compounds of the labiate herbs rosemary and sage, are activators of the human peroxisome proliferator-activated receptor gamma. Planta. Med. 72:881-887. Rawi, S.M. 1995. Studies of the ability of sulfur containing compounds to block diabetogenic effect of alloxan in albino rats. Proc. Zool. Soc. A. R. Egypt. 26:244- 259. Riaz, R.A., A. Ali and M. Saleem. 1988. Studies on the preparation and storage stability of comminuted kinow fruit beverage bees. Pak. J. Sci. Ind. Res. 32(8):574-578. Ribeiro, S.M.R., L.C.A. Barbosa, J.H. Queiroz, M. Knodler and A. Schieber. 2008. Phenolic compounds and antioxidant capacity of Brazilian mango (Mangifera indica L.) varieties. Food Chem. 110:620-626.

161

Rivera, D.G., I.H. Balmaseda, A.A. Leon, B.C. Hernandez, L.M. Montiel, G.G. Garrido, S. Cuzzocrea and R.D. Hernandez. 2006. Anti-allergic properties of Mangifera indica L. extract (Vimang) and contribution of its glucosylxanthone mangiferin. J. Pharm. Pharmacol. 58:385-392. Rocha, J. D.L., B. Doiron, E. Shea-Brown, K. Josic and A. Reyes. 2007. Correlation between neural spike trains increase with firing rate. Nature. 448:802. Rodrigo, R., C. Guichard and R. Charles. 2007. Clinical pharmacology and therapeutic use of antioxidant vitamins. Fundam. Clin. Pharmacol. 21:111–27. Rojas, M.C. and M.S. Brewer. 2007. Effect of natural antioxidants on oxidative stability of cooked, refrigerated beef and pork. J. Food Sci. 72(4), S282–S288. Roller, M., Y. Clune, K. Collins, G. Rechkemmer and B. Watzl. 2007. Consumption of prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis has minor effects on selected immune parameters in polypectomised and colon cancer patients. Br. J. Nutr. 97: 676-684. Rossana, C., A. Lanera, A. Trani, M. Gobbetti and R.D. Cagno. 2012. Yogurt-like beverages made of a mixture of cereals, soy and grape must:Microbiology, texture, nutritional and sensory properties. Int. J. Food Microbiol. 155:120-127. Rusak, G., D. Komes, S. Likic, D. Horzic and M. Kovac. 2008. Phenolic content and antioxidative capacity of green and white tea extracts depending on extraction conditions and solvent used. Food Chem. 110:852-858. Sanchez, G.M., L. Re, A. Giuliani, A.J. Nunez-Selles, G.P. Davison and O. Leon- Fernandez. 2000. Protective effects of Mangifera Indica L. extract, mangiferin and selected antioxidants against TPA-induced biomolecules oxidation and peritoneal macrophage activation in mice. Pharmacol. Res. 42:565-573. Sanchez-Moreno, C. 2002. Review: methods used to evaluate the free radical scavenging activity in foods and biological systems. Food Sci. Technol. Int. 8(3):121-37. Sanders, M.E. 1998. Overview of functional foods: Emphasis on probiotic bacteria. Int. Dairy J. 8:341-347. Sarkar, A., Y., Sreenivasan, G.T., Ramesh, and Manna, S.K. 2004. Beta-D-glucoside suppresses tumor necrosis factor-induced activation of nuclear transcription factor kappaB but potentiates apoptosis. J. Biol. Chem. 279: 33768-33781.

162

Saron, E.S., S.T. Dantas, H.C.D. Menezes, B.M.C. Soares and M.F. Nunes. 2007. Sensorial stability of ready to drink passion fruit juice packaged in tinplate cans. Ciencia e Tecnologia de Alimentos. 27(4):772-778. Sato, T., A. Kawamoto, A. Tamura, Y. Tatsumi and T. Fujii. 1992. Mechanism of antioxidant action of pueraria glycoside (PG)-1 (an isoflavonoid) and mangiferin (a xanthonoid). Chem. Pharm. Bull. 40(3):721-724. Saura-Calixto, F., J. Perez-Jimenez and S. Tourino. 2010. Proanthocyanidin metabolites associated with dietary fibre from in vitro colonic fermentation and proanthocyanidin metabolites in human plasma. Mol. Nutr. Food Res. 54:939-946. Saxena, A. and N.K. Vikram. 2004. Role of selected Indian plants in management of type II diabetes. J. Alt. Complement Med. 10:369-378. Sayed, M.R., I.M. Mourad and D.A. Sayed.2011. Biochemical changes in experimental diabetes before and after treatment with Mangifera indica and Psidium guava extracts. Int. J. Pharm. Biomed. Sci. 2(2):29-41. Scalbert, A. and G. Williamson. 2000. Dietary intake and bioavailability of polyphenols. J. Nutr. 130:2073-2085. Scalia, R., Y. Gong, B. Berzins, L.J. Zhao and K. Sharma. 2007. Hyperglycemia is a major determinant of albumin permeability in diabetic microcirculation: the role of μ-calpain. Diabetes. 56:1842-1849. Scalzo, J., A. Politi, N. Pellegrini, B. Mezzetti and M. Battino. 2005. Plant genotype affects total antioxidant capacity and phenolic contents in fruit. Nutr. 21(2):207- 213. Schieber A., W. Ullrich and R. Carle. 2000. Characterization of polyphenols in mango puree concentrate by HPLC with diode array and mass spectrometric detection. Innov. Food Sci. Emerg. 1:161–166. Schieber, A., F.C. Stintzing and R. Carle. 2001. By-products of plant food processing as a source of functional compounds- recent developments. Trends Food Sci. Technol. 12:401-413. Schulze, P.C. and R.T. Lee. 2005. Oxidative stress and atherosclerosis. Curr. Atheroscler. Rep. 7:242-248. Seifried, H.E., D.E. Anderson, E.I. Fisher and J.A. Milner. 2007. A review of the interaction among dietary antioxidants and reactive oxygen species. J. Nutr. Biochem. 18:567-579. 163

Sellamuthu, P.S., B.P. Muniappan, S.M. Perumal and M. Kandasamy. 2009. Antihyperglycemic effect of mangiferin in streptozotocin diabetic rats. J. Health Sci. 55(2):206-214. Sellamuthu, P.S., P. Arulselvan, B.P. Muniappan and M. Kandasamy. 2012. Effect of mangiferin isolated from Salacia chinensis regulates the kidney carbohydrate metabolism in streptozotocin-induced diabetic rats. Asian Pac. J. Tropic. Biomed. 1583-1587. Seth, S.D. and B. Sharma. 2004. Medicinal plants of India. Indian J. Med. Res. 120:9-11. Severi, J.A., Z.P. Lima, H. Kushima, A.R.M.S. Brito, L.C. dos Santos, W. Vilegas and C.A. Hiruma-Lima. 2009. Polyphenols with antiulcerogenic action from aqueous decoction of mango leaves (Mangifera Indica L.). Mol. 14:1098-1110. Sheela, C.G. and K.T. Augusti. 1992. Antidiabetic effects of S. allylNv cysteine sulfoxide from garlic Allium sativum linn. Indian J. Exp. Biol. 30:523-526. Siddhuraju, P. and K. Becker. 2007. The antioxidant and free radical scavenging activities of processed cowpea (Vigna unguiculata L.) see extracts. Food Chem. 101:10-19. Siddhuraju, P., K. Vijayakumari and K. Janardhanan. 1996. Chemical composition and protein quality of the little-known legume, velvet bean (Mucuna pruriens L.). J. Agric. Food Chem. 44:2636-2641. Singh, B.B., S.P Vinjamury, C. Der-Martirosian, E. Kubik, L.C. Mishra, N. Shepard, V.J. Singh, M. Meier and S.G. Madhu. 2007. Ayurvedic and collateral herbal treatments for hyperlipidemia: a systematic review of randomized controlled trials and quasi-experimental designs. Altern. Ther. Health Med. 13:22-28. Singh, U., A. Kunwar, R. Srinjivasan, M.J. Nanjan and K.I. Priyadarsini. 2009. Differential free radical scavenging activity and radioprotection of Caesalpinia Digyna extracts and its active constituent. J. Rad. Res. 50:425-433. Singh, U.P., D.P. Singh, M. Singh, S. Maurya, J.S. Srivastava, R.B. Singh and S.P. Singh. 2004. Characterization of phenolic compounds in some Indian mango cultivars. Int. J. Food Sci. Nutr. 55(2):163-169. Singleton, V.L., R. Orthofer and R.M. Lamuela-Raventos. 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Enzymol. 299:152-178.

164

Siro, I., E. Kapolna, B. Kapolna and A. Lugasi. 2008. Functional food. Product development, marketing and consumer acceptance-A review. Appetite. 51:456- 467. Sistrunk, W.A. and J.R. Morris. 1985. Quality acceptance of fruit juices of two cultivars of muscadinac grapes mixed with other juices. J. Amer. Soc. Hort. Sci. 110:328- 332. Soong, Y.Y. and Barlow P.J. 2004. Antioxidant activity and phenolic content of selected fruit seeds. Food Chem. 88:411-417. Sroka, Z. and W. Cisowski. 2003. Hydrogen peroxide scavenging, antioxidant and antiradical activity of some phenolic acids. Food chem. Toxicol. 44:753-758. Steel, R.G.D., J.H. Torrie and D. Dickey. 1997. Principles and procedures of statistics: a biometrical approach, 3rd ed. McGraw Hill Book Co., Inc., New York. Steyn, N.P., P. Wolmarans, J.H. Nel and L.T. Bourne. 2008. National fortification of staple foods can make a significant contribution to micronutrient intake of South African adults. Public Health Nutr. 11(3):307-13. Stoilova, I., S. Gargova, A. Stoyanova and L. Ho. 2005. Antimicrobial and antioxidant activity of the polyphenol mangiferin. Herba. Polonica. 51:37-44. Sultan, M.T., M.S. Butt, F.M. Anjum, A. Jamil, S. Akhtar and M. Nasir. 2009. Nutritional profile of indigenous cultivar of black cumin seeds and antioxidant potential of its fixed and essential oil. Pak. J. Botany. 41(3):1321-1330. Sun, C., J. Fu, J. Chen, L. Jiang and Y. Pan. 2010. On-line HPLC method for screening of antioxidants against superoxide anion radical from complex mixtures. J. Sci. 33:1018-1023. Supraditareporn, W. and R. Pinthong. 2012. Physical, chemical and microbiological changes during storage of orange juices cv. Sai Nam Pung and cv. Khieo Waan in Northern Thailand. Int. J. Agri. Biol. 9(5):726-730. Sushamakumari, S., A. Varghese, D. Muraleedharan and V.P. Menon. 1990. Protective action of aspirin in experimental myocardial infarction induced by isoproterenol in rats and its effect on lipid peroxidation. Ind. J. Exp. Biol. 28(5):480-485. Swamy, M.S., N. Sivanna, A. Tamatam and F. Khanum. 2011. Effect of Poly Phenols in Enhancing the Swimming Capacity of Rats. Functional Foods in Health and Disease. 1(11):482-491.

165

Taga, M.S., E.E. Miller and D.E. Pratt. 1984. China seeds as source of natural lipid antioxidation. J. Am. Oil Chem. Soc. 61:928-931. Tagliazucchi, D., E. Verzelloni, D. Bertolini and A. Conte. 2010. In vitro bio-accessibility and antioxidant activity of grape polyphenols. Food Chem. 120:599-606. Tavarini, S., E.D. Innocenti, D. Remorini, R. Massai and L. Guidi. 2008. Antioxidant capacity, ascorbic acid, total phenols and carotenoids changes during harvest and after storage of Hayward kiwifruit. Food Chem. 107:282-288. Thomas, L. 1998. Clinical laboratory diagnostics, 1st ed. Frankfurt: TH-Books Verlagsgesellschaft. pp 241-247. Toda. M., S. Okubo, H. Ikigai, T. Suzuki, Y. Suzuki and T. Shimamura. 1991. The protective activity of tea against infection by Vibrio cholera O1. J. Appl. Bacteriol. 70:109-112. Torres, J.L., B. Varela and M.T. García. 2002. Valorization of grape (Vitis vinifera) byproducts. Antioxidant and biological properties of polyphenolic fractions differing in procyanidin composition and flavonol content. J. Agric. Food Chem. 50:7548. Torunn, S., F.R. Siv and A.L. Kare. 2009. Total antioxidant activity in 35 Ugandan fruits and vegetables. Food Chem. 113:85-91. Toutain, P.L., A. Ferran and A. Bousquet-Melou. 2010. Species differences in pharmacokinetics and pharmacodynamics. Handbook Exp. Pharmacol. 199:19-48. Trojan-Rodrigues, M., T.L.S. Alves, G.L.G. Soares and M.R. Ritter. 2011. Plants used as antidiabetics in popular medicine in Rio Grande do Sul, southern Brazil. J. Ethnopharmacol. 139(1):155-63. Turkben, C., E. Sariburun, C. Demir and V. Uylaser. 2010. Effect of freezing and frozen storage on phenolic compounds of raspberry and blackberry cultivars. Food Anal. Methods. 3:144-153. Uchida, K., T. Satoh, Y. Ogura, N. Yamaga and K. Yamada, 2001. Effect of partial ileal bypass on cholesterol and bile acid metabolism in rats, Yanago Acta. Medica. 44:69-77. Uchiyama, S., Y. Taniguchi, A. Saka, A. Yoshida and H. Yajima. 2011. Prevention of diet-induced obesity by dietary black tea polyphenols extract in vitro and in vivo. Nutr. 27:287-292.

166

Ueda, M., K.S. Sasaki, N. Utsunimiya, K. Inaba and Y.S. Bayashi. 2000. Variation of total polyphenol and polyphenol oxidase activity during maturation of mango fruit (Mangifera Indica L. Irwin) cultured in plastic green house. Food Sci. Technol. Res. 6:299-305. Vaghasiya, Y. and S. Chanda. 2010. Antimicrobial and free radical scavenging activity of different solvent extracts of Mangifera indica L. seeds. Res. J. Microbiol. 5:1207- 1212. Van Dam, R.M., W.C. Willett, E.B. Rimm and F.B. Hu. 2002. Dietary fat and meat intake in relation to risk of type 2 diabetes in men. Diabetes Care. 25:417-424. Van Langendonckt, A., F. Casanas-Roux and J. Donnez. 2002. Oxidative stress and peritoneal endometriosis. Fertil. Steril. 77:861-870. Vasant, R.A. and A.V.R.L. Narasimhacharya. 2011. Antihyperglycemic and antihyperlipemic effects of Mangifera indica L. in fluoride induced toxicity. Pharmacol. 3:265-274. Vercesi, A.E., R.F. Castilho, A.J. Kowaltowski and H.C. Oliveira. 2007. Mitochondrial energy metabolism and redox state in dyslipidemias. IUBMB Life. 59:263-8. Verrijn, S.A.A., H.S. Schipper, I. Tasdelen, D.A. Egan, B.J. Prakken, E. Kalkhoven and W. de Jager. 2012. Altered plasma adipokine levels and in vitro adipocyte differentiation in pediatric type 1 diabetes. J. Clin. Endocrinol. Metab. 97:463- 472. Vinson, J.A. and J. Jang. 2001. In vitro and in vivo Lipoprotein antioxidant effect of a citrus extract and ascorbic acid on normal and hypercholesterolemic human subjects. J. Med. Food. Winter. 4(4):187-192. Volek, J.S., A.L. Gomez, D.M. Love and N.G. Avery. 2001. Effects of a high-fat diet on postabsorptive and postprandial testosterone responses to a fat-rich meal. Metabol. 50:1351-1355. Wang, S.Y. and H. Jiao. 2000. Scavenging capacity of berry crops on superoxide radicals, hydrogen peroxide, hydroxyl radicals, and singlet oxygen. J. Agri. Food Chem. 48:5677-5684. Wang, T.J., M.G. Larson, R.S. Vasan, S. Cheng, E.P. Rhee, E. McCabe, G.D. Lewis, C.S. Fox, P.F. Jacques, C. Fernandez, C.J. O’Donnell, S.A. V.K. Carr, J.C. Mootha Florez, A. Souza, O. Melander, C.B. Clish and R.E. Gerszten.2011. Metabolite profiles and the risk of developing diabetes. Nat. Med. 17:448-453. 167

Wang, Z., T. Yao and Z. Song. 2010. Involvement and mechanism of DGAT2 upregulation in the pathogenesis of alcoholic fatty liver disease. J. Lipid Res. 51:3158-3165. Wauthoz, N., A. Balde, E.S. Balde, M.V. Damme and P. Duez. 2007. Ethnopharmacology of Mangifera indica L bark and pharmacological studies of its main glucosylxanthone, mangiferin. Int. J. Biomed. Pharma. Sci. 1(2):112-119. Wei, M., L. Ong, M.T. Smith and F.B. Ross. 2003. The streptozotocin-diabetic rat as a model of the chronic complications of human diabetes. Heart Lung Circ. 12:44- 50. Weichman, J. 1987. Post harvest physiology of vegetables. Marcel Becker, Inc New York. pp. 145. Wild, S., G. Rolglic, A. Green, R. Sicress and H. King. 2004. Global prevalence of diabetes. Diabetes Care. 27:1047-1053. Wildman, R.E.C. and M. Kelley. 2007. Nutraceuticals and Functional Foods. In: Wildman, R.E.C. (Ed.), Hand book of neutraceutical and functional foods (2nd ed.). CRC press, Taylor and Francis group, New York. pp. 1-21. Wilkinson, D., C. Holmes, D. Boche, G. Yadegarfar, V. Hopkins, A. Bayer, R.W. Jones, R. Bullock, S. Love and J.W. Neal. 2008. Long-term effects of A [beta] 42 immunisation in Alzheimer’s disease: follow-up of a randomised, placebocontrolled phase I trial. Lancet. 372:216-223. Witztum, J.L. and Steinberg, D. 1991. Role of oxidized low density lipoprotein in atherogenesis. J. Clin. Invest. 88:1785-1792. Wolf, B.W. and S.E. Weisbrode. 2003. Safety evaluation of an extract from Salacia oblonga. Food Chem. Toxicol. 41:867-874. Wong, S.P., L.P. Leong and J.H. William-Koh. 2006. Antioxidant activities of aqueous extracts of selected plants. Food Chem. 99:775-783. Wright, J.S., E.R. Johnson and G.A. DiLabio. 2001. Predicting the activity of phenolic antioxidants: theoretical method, analysis of substituent effects, and application to major families of antioxidants. J. Am. Chem. Soc. 123:1173-1183. Xiao, W., L. Han and B. Shi. 2008. Microwave-assisted extraction of flavonoids from Radix astragali. Sep. Purif. Technol. 62:614-618. Yadav, N.P. and V.K. Dixit. 2003. Hepatoprotective activity of leaves of Kalanchoe pinnata Pers. J. Ethnopharmacol. 86(23):197-202. 168

Yadav, S.R., G.G. Potdar, A.A. Kumar, M. Otaghvari and A. Sonkar 2008. Eriocaulon epedunculatum, a new species of Eriocaulaceae from Western Ghats of India. Kew Bull. 63: 503-505. Yaman, I. and E. Balikci, 2010. Protective effects of Nigella sativa against gentamicin- induced nephrotoxicity in rats. Experim. Toxicol. Pathol. 62(2):183-190. Yang, B., Y.M. Jiang, J. Shi, F.Chen and M. Ashraf. 2011. Extraction and pharmacological properties of bioactive compounds from longan (Dimocarpus longan L.) fruit - a review. Food Res. Int. 44:1837-1842. Yang, J., E.W.N. Lam, H.M. Hammad, T.D. Oberley and L.W. Oberley. 2002. Antioxidant enzyme levels in oral squamous cell carcinoma and normal human oral epithelium. J. Oral. Patho.l Med. 31:71-70. Yang, J., L. Zhong, B. Zou, Y. Tian, S.F. Xu and P. Yao. 2012. Spectroscopic investigations on the binding of persimmon tannin to phospholipase A2 from Chinese cobra (Naja naja atra). J. Mol. Str. 1008(0):42-48. Yao, Q., H.H. Zhu and R.S. Zeng. 2007. Role of phenolic compounds in plant defence: Induced by arbuscular mycorrhizal fungi. Allelopathy J. 20(1):1-13. Yoshikawa, M., N. Nishida, H. Shimoda, M. Takada, Y. Kawahara and H. Matsuda. 2001. Polyphenol constituents from salacia species: quantitative analysis of mangiferin with glucosidase and aldose reductase inhibitory activities. J. Pharmac. Soc. Jap. 121:371-378. Yue, Y.Y., X. Chen, J. Qin and J. Yao. 2009. Characterization of the mangiferin-human serum albumin complex by spectroscopic and molecular modeling approaches. Pharm. Biomed. Anal. 49 756-759. Zamorano, M., J.I. Pérez, I.A. Pavés and Á.R. Ridao. 2007. Study of the energy potential of the biogas produced by an urban waste landfill in Southern Spain. Renewable and Sustainable Energy Reviews. 11:909-922. Zasada, I., D. Zajac, M. Pozdzik and M. Pokorski. 2011. Influence of magiferin on lipid peroxidation. International Conference ’Advances in Pneumology’ Bonn. 17-18. Zein, R.E., A.A. El-Bagoury and H.E. Kassab. 2005. Chemical and nutritional studies on mango seed kernels. J.Agric. Sci. 30(6):3285-3299. Zern, T.L., K.L. West and M.L. Fernandez. 2003. Grape polyphenols decrease plasma triglycerides and cholesterol accumulation in the aorta of ovariectomized guinea pigs. J. Nutr. 133(7):2268-2272. 169

Zhang, Z., C.W. Liew, D.E. Handy, Y. Zhang, J.A. Leopold, J. Hu, L. Guo, R.N. Kulkarni, J. Loscalzo and R.C. Stanton. 2010. High glucose inhibits glucose-6- phosphate dehydrogenase, leading to increased oxidative stress and β-cell apoptosis. FASEB J. 24:1497-1505. Zhao, D., J. Feng, Q. Huo, N. Melosh, G.H. Fredrickson, B.F. Chmelka and G.D. Stucky. 1998. Triblock Copolymer Syntheses of Mesoporous Silica with Periodic 50 to 300 Angstrom Pores. Sci. 279(5350):548-552. Zhao, T. and Z.N. Zhang. 2011. Immunogenicity of induced pluripotent stem cells. Nature. 2(1):09-34. Zhaoyun, Z., C.W. Liew, D.E. Handy, Y. Zhang, J. A. Leopold, J. Hu, L. Guo, R.N. Kulkarni, J. Loscalzo and R.C. Stanton.2010. High glucose inhibits glucose-6- phosphate dehydrogenase, leading to increased oxidative stress and β-cell apoptosis. FASEB J. 24:1497-1505. Zheng, W., and Wang, S.Y. 2001. Antioxidant activity and phenolic compounds in selected herbs. J. Agric. Food Chem. 49(11):5165-70.

170

APPENDICES

Appendix I Performa for sensory evaluation of functional drink Name of the judge………………….. Date…………….. Character T0 T1 T2

Color

Flavor

Sweetness

Sourness

Acceptability

Signature……………. INSTRUCTION Take a sample of functional drink and score for color, flavor, sweetness, sourness and acceptability using the following 9-point Hedonic Scale. 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

Note: 1. Take a sample of functional drink and score for color, flavor etc. 2. Before proceeding to the next sample, rinse mouth with water. 3. Make inter comparison of the sample and record the score. 4. Don’t disturb the order of samples.

171

Appendix II Composition of experimental diets

Ingredients Normal rats Hypercholestrolemic Diabetic rats (%) rats Corn oil 10 10 10 Corn starch 66 64.5 66 Casein 10 10 10 Cellulose 10 10 10 Salt mixture 3 3 3 Vitamins 1 1 1 Cholesterol - 1.5 - Sucrose - - -

172

Appendix III

Composition of salt mixture

Calcium citrate 308.2

Ca (H2PO4)2 H2O 112.8

H2HPO4 218.7

HCl 124.7

NaCl 77.0

CaCO3 68.5

3MgCO3. Mg (OH) 2. 3H2O 35.1

MgSO4 anhydrous 38.3

Ferric ammonium citrate 91.41

CuSO4. 5H2O 5.98

NaF 0.76

MnSO4. 2H2O 1.07 16.7

KAl (SO4)2. 12H2O 0.54

KI 0.24

100.00 1000.00

173

Appendix IV

Composition of vitamin mixture

Thiamin hydrochloride 0.060

Riboflavin 0.200

Pyridoxin hydrochloride 0.040

Calcium pentothenate 1.200

Nicotinic acid 4.000

Inositol 4.000 p-aminobenzoic acid 12.000

Biotin 0.040

Folic acid 0.040

Cyanocobalamin 0.001

Choline chloride 12.000

Maize starch 966.419

1000.00

174