BIOCHEMICAL PORTRAYAL OF DIFFERENT BITTER GOURD (Momordica charantia L.) CULTIVARS WITH SPECIAL REFERENCE TO THEIR THERAPEUTIC POTENTIAL By Mahwish 2006-GCUF-557-524 A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN FOOD & NUTRITION DEPARTMENT OF FOOD SCIENCE, NUTRITION & HOME ECONOMICS INSTITUTE OF HOME & FOOD SCIENCES, GOVERNMENT COLLEGE UNIVERSITY, FAISALABAD. 2017 2 DEDICATED TO HOLY PROPHET MUHAMMAD (Peace be upon Him) & My Loving Family 3 DECLARATION The work reported in this thesis was carried out by me under the supervision of Dr. Farhan Saeed, Assistant Professor, Institute of Home & Food Sciences, GC University, Faisalabad, Pakistan. I hereby declare that the title of thesis, “BIOCHEMICAL PORTRAYAL OF DIFFERENT BITTER GOURD (Momordica charantia L.) CULTIVARS WITH SPECIAL REFERENCE TO THEIR THERAPEUTIC POTENTIAL” and the contents of this thesis 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. Mahwish 2006-GCUF-557-524 4 5 CONTENTS Sr. No. Title Page # 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 6 3 MATERIALS AND METHODS 27 4 RESULTS AND DISCUSSION 44 5 SUMMARY 162 6 REFERENCES 169 6 LIST OF CONTENTS Acknowledgments Abstract 1. INTRODUCTION 1 2. REVIEW OF LITERATURE 6 2.1. Concept of functional and nutraceutical foods 6 2.2. Bitter gourd (Momordica charantia); an overview 7 2.3. Chemistry 9 2.3.1. Chemical composition and Mineral analysis 9 2.3.2. Ascorbic acid contents 11 2.4. Bitter gourd-a source of natural antioxidants 11 2.5. Preparation of bitter gourd extract 16 2.6 Bioactive molecules in bitter gourd 16 2.6.1. Polypeptide-p 18 2.6.2. Charantin 18 2.6.3. Other bioactive molecules 18 2.7. Health claims of bitter gourd 19 2.7.1 Role of bitter gourd in diabetes mellitus 20 2.7.2 Lipid lowering potential of bitter gourd 23 3. MATERIALS AND METHODS 27 3.1. Procurement of raw material 27 3.2. Raw material handling 27 3.3. Chemical analysis of raw material 28 3.3.1. Moisture content 28 3.3.2. Ash content 28 3.3.3. Crude protein 29 3.3.4. Crude fat 29 3.3.5. Crude fiber 30 3.3.6. Nitrogen free extracts (NFE) 30 3.4. Minerals analysis 30 3.5. Quantification of Ascorbic acid 30 3.6. Preparation of extracts 31 3.7. Antioxidative profiling of bitter gourd extracts 31 3.7.1. Determination of total polyphenols 31 3.7.2. Determination of total flavonoid content 31 3.7.3. Free radical scavenging activity (DPPH assay) 32 3.7.4. Ferric reducing antioxidant power (FRAP) 32 3.7.5. β Carotene bleaching assay 33 3.7.6. ABTS assay 33 3.8. Bioactive compounds or phytochemicals 34 7 3.8.1. Total saponin contents 34 3.8.2. Charantin 34 3.8.3. Total alkaloid 35 3.8.4. Momordicin I & II 35 3.8.5. Vicine 36 3.8.6. Polypeptide P 36 3.9. Selection of best treatment 36 3.10. Product development (Functional drink) 36 3.11. Physicochemical assay of bitter gourd juice 37 3.11.1. Total soluble solids 37 3.11.2. pH 38 3.11.3. Acidity 38 3.11.4. Colour 38 3.11.5. Sensory evaluation 38 3.12. Bioevaluation studies 39 3.12.1. Study I: Rats fed with normal diet 39 3.12.2. Study II: Hyperglycemic rats 40 3.12.3. Study III: Hyperlipidemic rats 40 3.13. Feed plans for experimental rats 40 3.14. Feed and water intake 41 3.15. Body weight gain 41 3.16. Hypoglycemic perspectives 41 3.17. Serum lipid profile 42 3.17.1. Cholesterol 42 3.17.2. Low density & high density lipoproteins 42 3.17.3. Triglycerides 42 3.18. Liver functioning tests 42 3.19. Kidney functioning tests 42 3.20. Weight of body organ 43 3.21. Statistical analysis 43 4. RESULTS AND DISSCUSSION 44 4.1. Chemical composition 44 4.2. Macro and micro mineral analysis 52 4.3. Antioxidant indices of bitter gourd extracts 65 4.4. Phytochemical or bioactive molecules in bitter gourd 80 4.5. Product development 93 4.5.1. Physical attributes of bitter gourd functional drink 93 4.5.2. Sensory evaluation of bitter gourd functional drink 97 4.6. Bio-evaluation studies 105 4.6.1. Feed intake 105 4.6.2. Water intake 106 4.6.3. Body weight gain 112 4.6.4. Glucose 115 4.6.5. Insulin 120 8 4.6.6. Cholesterol 125 4.6.7. Low Density Lipoproteins (LDL) 130 4.6.8. High Density Lipoproteins (HDL) 131 4.6.9. Triglycerides 139 4.6.10. Alkaline phosphatase (ALP) 143 4.6.11. Alanine Transferase (ALT) 144 4.6.12. Aspartate Transferase (AST) 147 4.6.13. Serum creatinine 150 4.6.14. Serum urea 153 4.6.15. Effect on different organs 156 5. SUMMARY 162 CONCLUSIONS 167 RECOMMENDATIONS 168 REFERENCES 169 APPENDICES 200 9 LIST OF TABLES Sr. No Title Page # 3.1 Composition of different bitter gourd functional drinks (100g) 37 3.2 Diet plan used in the studies 41 4.1 Mean squares for proximate composition of different cultivars and parts 47 4.2 Mean values for moisture contents (%) in different cultivars and parts 47 4.3 Mean values for ash (%) in different cultivars and parts 48 4.4 Mean values for crude protein (%) in different cultivars and parts 48 4.5 Mean values for crude fat (%) in different cultivars and parts 51 4.6 Mean values for crude fiber (%) in different cultivars and parts 51 4.7 Mean values for crude NFE (%) in different cultivars and parts 53 Mean squares for macro and micro mineral analysis of different cultivars 4.8 56 and parts 4.9 Mean values for K (mg/100g) in different cultivars and parts 57 4.10 Mean values for P (mg/100g) in different cultivars and parts 57 4.11 Mean values for Mg (mg/100g) in different cultivars and parts 60 4.12 Mean values for Na (mg/100g) in different cultivars and parts 60 4.13 Mean values for Ca (mg/100g) in different cultivars and parts 61 4.14 Mean values for Fe (mg/100g) in different cultivars and parts 61 4.15 Mean values for Zn (mg/100g) in different cultivars and parts 64 4.16 Mean squares for antioxidant indices of different treatments and parts 66 Mean values for total phenolic contents (mg GAE/100g) of water and 4.17 67 methanolic extract of BG cultivars and parts Mean values for total flavonoid contents (mg RuE/100g) of water and 4.18 70 methanolic extract of BG cultivars and parts 10 Mean values for DPPH (%) assay of water and methanolic extract of BG 4.19 74 cultivars and parts Mean values for FRAP (μg FE/g) assay of water and methanolic extract 4.20 75 of BG cultivars and parts Mean values for β-carotene bleaching assay (%) of water and methanolic 4.21 78 extract of BG cultivars and parts Mean values for ABTS (μmol TE/g) assay of water and methanolic 4.22 79 extract of BG cultivars and parts 4.23 Mean square for Ascorbic acid in different cultivars and parts 81 4.24 Mean values for Ascorbic acid (mg/100g) in different cultivars and parts 81 Mean squares for total saponin and charantin in different cultivars and 4.25 84 parts 4.26 Mean values for total saponin (%) in different cultivars and parts 84 4.27 Mean values for charantin (mg/g) in different cultivars and parts 85 4.28 Mean squares for alkaloids in different cultivars and parts 87 4.29 Mean values for alkaloids (%) in different cultivars and parts 87 Mean squares for momordicin I, II & vicine in different cultivars and 4.30 88 parts 4.31 Mean values for Momordicine I (mg/100g) in different cultivars and parts 88 Mean values for Momordicine II (mg/100g) in different cultivars and 4.32 90 parts 4.33 Mean values for Vicine (μg/100μg) in different cultivars and parts 90 4.34 Mean square for Polypeptide P in different cultivars and parts 92 4.35 Mean values for Polypeptide P (mg/g) in different cultivars and parts 92 4.36 Mean squares for physical attributes of bitter gourd drink 94 4.37 Effect of treatments and storage on TSS of bitter gourd functional drink 94 4.38 Effect of treatments and storage on pH of bitter gourd functional drink 96 4.39 Effect of treatments and storage on acidity of bitter gourd functional drink 96 Effect of treatments and storage on L* value of bitter gourd functional 4.40 98 drink 11 Effect of treatments and storage on a* value of bitter gourd functional 4.41 98 drink Effect of treatments and storage on b* value of bitter gourd functional 4.42 99 drink 4.43 Mean squares showing treatments and storage effect on sensory attributes 101 4.44 Effect of treatments and storage on Colour scores of drink 101 4.45 Effect of treatments and storage on Aroma scores of drink 102 4.46 Effect of treatments and storage on Flavour scores of drink 102 4.47 Effect of treatments and storage on Taste scores of drink 103 4.48 Effect of treatments and storage on Overall acceptability scores of drink 103 4.49 Mean squares for effect of diet and study weeks on feed intake 107 4.50 Mean squares for effect of diet and study weeks on water intake 110 4.51 Mean squares for effect of diet and study weeks on body weight gain 113 4.52 Mean squares for effect of diet and study intervals on Glucose of rats 117 4.53 Effect of diet and study intervals on Glucose (mg/dL) 118 4.54 Mean squares for effect of diet and study intervals on Insulin of rats 122 4.55 Effect of diet and study intervals on Insulin (μIU/mL) 123 4.56 Mean squares for effect of diet and study intervals on Cholesterol of rats 127 4.57 Effect of diet and study intervals on Cholesterol (mg/dL) 128 4.58 Mean squares for effect of diet and study intervals on LDL of rats 132 4.59 Effect of diet and study intervals on LDL (mg/dL) 133 4.60 Mean squares for effect of diet and study