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Characterization of Mango Peel Mangiferin to Elucidate Its Nutraceutical Potential

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Characterization of Mango Peel Mangiferin to Elucidate Its Nutraceutical Potential Characterization of mango peel mangiferin to elucidate its nutraceutical potential By Muhammad Imran M.Sc. (Hons.) Food Technology A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN 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 i 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 ii 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 iv 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 v 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 vi 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.
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