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
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DEDICATED
TO
HOLY PROPHET MUHAMMAD (Peace be upon Him)
&
My Loving Family
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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
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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
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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
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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
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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
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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
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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
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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 intervals on HDL of rats 136
4.61 Effect of diet and study intervals on HDL (mg/dL) 137
4.62 Mean squares for effect of diet and study intervals on Triglycerides of rats 140
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4.63 Effect of diet and study intervals on Triglycerides (mg/dL) 141
4.64 Mean squares for effect of diet and study intervals on ALP of rats 145
4.65 Effect of diet and study intervals on Alkaline Phosphatase (IU/L) 146
4.66 Mean squares for effect of diet and study intervals on ALT of rats 148
4.67 Effect of diet and study intervals on Alanine Transferase (IU/L) 149
4.68 Mean squares for effect of diet and study intervals on AST of rats 151
4.69 Effect of diet and study intervals on Aspartate Transferase (IU/L) 152
4.70 Mean squares for effect of diet and study intervals on creatinine of rats 154
4.71 Effect of diet and study intervals on Serum creatinine (mg/dL) 155
4.72 Mean squares for effect of diet and study intervals on urea of rats 157
4.73 Effect of diet and study intervals on Serum Urea (mg/dL) 158
4.74 Mean squares showing effect of bitter gourd on Heart, Lungs and Kidneys 159 Mean squares showing effect of bitter gourd on Liver, Pancreas and 4.75 160 Spleen 4.76 Means for organ weight in different studies 161
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LIST OF FIGURES
Sr. No Title Page #
4.1. Feed intake in Study I (g/rat/day) 108
4.2. Feed intake in Study II (g/rat/day) 108
4.3. Feed intake in Study III (g/rat/day) 108
4.4. Water intake in Study I (mL/rat/day) 111
4.5. Water intake in Study II (mL/rat/day) 111
4.6. Water intake in Study III (mL/rat/day) 111
4.7. Body weight gain in Study I (g/rat/day) 114
4.8. Body weight gain in Study II (g/rat/day) 114
4.9. Body weight gain in Study III (g/rat/day) 114
4.10. Percent decrease in glucose level 119
4.11. Percent increase in insulin level 124
4.12. Percent decrease in cholesterol level 129
4.13. Percent decrease in LDL level 134
4.14. Percent increase in HDL level 138
4.15. Percent decrease in triglycerides 142
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LIST OF APPENDICES
Sr. No Title Page #
I Performa for sensory evolution of functional drink 200
II Composition of experimental diets 201
III Composition of salt mixture 202
IV Composition of vitamin mixture 203
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ACKNOWLEDGEMENT
I bow my head in gratitude to Almighty ALLAH, The Lords of the Worlds, The omnipotent, The omnipresent and The omniscient, The merciful and compassionate to whom every praise is due. Trembling lips and wet eyes praise for PROPHET MUHAMMAD (Salallaho Alaih Wassalam) who is forever a torch of knowledge and guidance for humanity.
I deem it a great honor and privilege to record profound sense of gratitude to Dr. Farhan Saeed, Assistant Professor, Institute of Home and Food Sciences, GC University, Faisalabad, for their consistent, sincere and inspiring guidance, keen interest, esoteric attention, philanthropic behavior, constructive criticism and unstinted help in the planning, execution, writing and successful completion of this manuscript.
I deem it my utmost pleasure in expressing my gratitude to Dr. Mahr un Nisa, Associate Professor, Institute of Home and Food Sciences, GC University, Faisalabad. Her sympathetic attitude and parental guidance is worth appreciating. Her suggestions and criticism are indeed incalculable wealth for me. I am also very thankful to Dr. Muhammad Tahir Nadeem, Assistant Professor, Institute of Home and Food Sciences, GC University, Faisalabad, for his valuable advices and suggestions in the planning and execution of the research project. Special appreciation is extended to Director, Dr. Faqir Muhammad Anjum and all the staff members, Institute of Home and Food Sciences, GC University, Faisalabad, for their general guidance during my studies.
I have no words to express my gratitude to Dr. Muhammad Umair Arshad, Assistant Professor, Institute of Home and Food Sciences, GC University, Faisalabad, for his kind support, guidance and valuable suggestions. I would also like to thanks Higher Education Commission, Islamabad, Pakistan for the financial support. Words are lacking to express my humble obligation to my ideal personality, my respectable father Muhammad Younas, for their eternal devotion, immortal sacrifices, lavishing efforts, countless prays for me, without which present destination would have been a mere dream. May he live long and enjoy glorious and prosperous life. Thanks are also extended to my loving mother, brothers and sister for helping me in my academic as well as in my research work. I am especially thankful to my husband and sweet daughter for remaining cooperative during my research work.
Very special gratitude is due to my ever best friend Saleha Hameed with whom I have passed many memorable years of my life. Her ever smiling face and her words console me on tough hours of my life and giving me strength to reach for the stars and chase my dreams. Her exemplary cooperation, supportive attitude, warm-heartedness, altruistic behavior, philanthropic, caring and ever-ready helping attitude is an unending asset of my life. My sincere thanks to all my friends with whom I share joyful moments during my studies.
May God bless them. Mahwish
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ABSTRACT
In current study, the core objective is to elucidate the nutritional composition and bioactive profile of bitter gourd (Momordica charantia L.) cultivars in relation to their antidiabetic and antihypercholesterolemic effects. For the purpose, six cultivars viz., BG
20, Black King, FSD Long, KHBG-1, GHBG-1 & Noor were procured from Vegetable
Research Section, Ayub Agriculture Research Institute, Faisalabad. In first phase, bitter gourd cultivars and their parts (skin, flesh, seeds and whole fruit) were analyzed for biochemical and nutritional aspects. Results showed that bitter gourd cultivars are good source of carbohydrate, protein and minerals. Water extracts exhibited higher polyphenols, flavonoids, DPPH, FRAP, β carotene and ABTS values than methanol. In addition, bitter gourd was found to be rich source of charantin, momordicin I & II, vicine and polypeptide P. Black King was selected, owing to rich chemistry for the development of functional drink and subsequent bioevaluation trial. Efficacy trials were carried containing bitter gourd skin, flesh, seeds and whole fruit powder @ 100 and 300 mg/kg body weight against hyperglycemia and hypercholesterolemia. In this regard, rats were given normal diet (Study I), high sucrose diet (Study II) and high cholesterol diet (Study
III). Bitter gourd supplementation in these studies showed significant reduction in body weight, serum glucose, cholesterol, triglycerides & LDL and marked increase in insulin and HDL level of rats indicating their effectiveness against life style oriented disorders.
The whole bitter gourd fruit powder (300 mg/kg BW) performed better results than rest of the treatments. Conclusively, bitter gourd supplementation is effective to attenuate lifestyle related maladies.
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Chapter 1 INTRODUCTION
During the last few decades, the interests of the consumers have burgeoned in the natural products owing to the raised awareness. In this context, the therapeutic benefits are mostly considered to be associated with consumption of proper food (Xutian et al., 2009). However, the philosophy of “food as medicine” becomes oblivion with the development of industrialization, urbanization and globalization in the nineteenth century. Modern and materialistic lifestyle with less focus on healthy diet has resulted in the development of various life threatening ailments (Ashakiran & Deepthi, 2012).
The role of dietary components in controlling the diseases and promoting the health has been come to forefront once again in past few decades. The identification of food with rich supply of physiologically active compounds has been gaining immense attention in recent years (Malla et al., 2013a, 2013b).
The literature is available regarding different plants, however, some horizons still need to be elucidated and bitter gourd (Momordica charantia L.) is one of such example. It a climbing perennial, tendril-bearing vine belongs to family Cucurbitaceae. Bitter gourd is also called as balsam-pear, karella, bitter melon, bitter squash or wild cucumber (Krawinkel & Keding, 2006) is characterized by elongated, warty fruit-like gourds or cumbers which are extensively consumed as vegetable (Assubaie & El-Garawany, 2004). In ancient times, utilization of bitter gourd as a folk medicine is common to cure large number of maladies. It was frequently used as antidote for diabetes, stomach pain, wounds, tumors, malaria, rheumatism, colic, inflammation, measles and fevers (Grover & Yadav, 2004; Subratty et al., 2005). This versatile plant is considered to be commendable for treatment of any disease wreaked on human beings. This is due to presence of hundreds of different chemical constituents of medicinal importance in this plant (Taylor, 2002).
It has been observed that bitter gourd is rich in protein, lipid, water and carbohydrate in addition to presence of phytonutrients like dietary fiber, minerals, vitamins and anti-oxidants. Around 228 different compounds were identified from different parts of bitter gourd (Singh et al., 2011). Dietary fiber in bitter melon helps in stimulation of peristalsis movements and
1 increases the production of gastric juice in stomach, which improves digestion and reduces the chances of constipation, and hence gives protection from many severe disorders like colo- rectal cancer. Fiber also play role in reducing cholesterol level from the blood vessels. Many essential and valuable minerals like calcium, sodium, potassium magnesium, manganese, iron, and zinc found in appreciable quantity in bitter gourd (Bakare et al., 2010). These minerals are vital in proper function of body as calcium have a pivotal role in contraction of muscle, in the formation of bone and teeth and in clotting of blood in trauma (Ahmed & Chaudhary, 2009; Peters & Martini, 2010). Zinc and magnesium are helpful as cofactor in many enzymetic controlled catalytic reactions within the body (Ahmed & Chaudhary, 2009). Intracellular and extracellular fluid has sodium and potassium which has a role in maintaining electrolytic balance and fluidity across membranes (Ahmed & Chaudhary, 2009).
Iron is a basic constituent of haemoglobin, myoglobin and many metalloenzymes (Ahmed &
Chaudhary, 2009), which is required for oxygen and CO2 transport during respiratory activity and cellular metabolism. To regulate changes in blood pH, this iron containing heamoglobin also serves as buffer (Naik, 2016). Potassium, calcium and zinc play important roles in stimulation of beta cells of islets of Langerhans to release insulin (Kar et al., 1999). Potassium is also valuable for cardiovascular health and regulation of water within the body. Zinc in bitter gourd regulates normal functioning of immune system. Manganese which is essential mineral also found in bitter gourd is an important element for a number of enzymes that are involved in production of energy and antioxidant defenses. Mitochondria require manganese for many enzymes to work against free radicals activities.
Bitter gourd is also a huge reservoir of vitamin A, B-Complex, C, niacin and folate essential for many metabolic pathways. The protein in bitter gourd is an excellent source of essential amino acids i.e., Tyrosine, Cystine, Phenylalanine, Methionine, Isoleucine, and Lysine and as far as functional ingredients in a food system are concerned, it could be a decent protein source as well.
Bitter melon is thought to beneficial for health and prevent from many diseases due to presence of chemicals i.e., flavonoids, phenols, terpenes, anthroquinones, glucosinolates and isoflavones having antioxidant potential (Drewnowski & Gomez-Carneros, 2000). Many ill
2 effects like cancer, diabetes and cardiovascular diseases are due to accumulation of harmful particles known as free radicals which are building up within the body with age. Consuming bitter gourd may overcome these damages on regular basis as it possess those antioxidants that are crucial to fight off free radicals, and may reduce the development of many types of maladies (Kubola & Siriamornpun, 2008). Huge quantity of antioxidants scavenges free radicals from the body cells, and prevents or reduces the damage caused by oxidation.
Recently, many researchers evaluated the role of bitter gourd for lowering blood glucose level (Paul & Raychaudhuri, 2010; Fuangchana et al., 2011; Bano et al., 2011; Wehash et al., 2012; Tayyab et al, 2012; Tayyab & Lal, 2013), cholesterol (Bano et al., 2011, Tayyab et al, 2012; Tayyab & Lal, 2013), visceral fat mass (Chen et al., 2012) and proliferation of cancerous cells (Ray et al., 2010; Manoharan et al., 2014), as well as for the prevention from Alzheimer's disease and HIV growth (Fang & Ng, 2011). In addition, many chemical constituents present in bitter gourd have anti-diabetic (Joseph & Jini, 2013; Panara, 2013), anti-hyperlipidemic (Hossain et al., 2012; Mohammady et al., 2012), anti-oxidative (Wu & Ng, 2008; Lu et al., 2012), anti-tumour (Fang & Ng, 2011), anti-ulcerogenic (Alam et al., 2009; Gill et al., 2012), anti-inflammatory activities (Kobori et al., 2008; Lii et al., 2009; Gill et al., 2012; Chao et al., 2014) anti-mutagenic (Islam et al., 2011) and immune-modulatory activities (Deng et al., 2014; Majumda & Debnath, 2014) etc.
In addition, chemical constituents in bitter melon possess broad-spectrum antimicrobial activity (Braca, 2008) and protect the individuals from many disorders of digestive tract. Several phytochemicals isolated from bitter gourd have been reported to have in vitro antiviral activity against herpes, Epstein-Barr, Coxsackie virus B3, HIV and polio viruses (Palamthodi & Lele, 2014).
Owing to the presence of enormous quantity of valuable biologically active molecules particularly charantin, vicine, polypeptide p, momordicine and momordine bitter gourd can be considered as valuable for diabetic and prediabetic patients and can be used as component of the diet or a dietary supplement under these conditions (Basch et al., 2003). These moieties are structurally similar to human insulin hence considered valuable for controlling diabetes. Diabetes mellitus is thought to be lethal and among the main reason of death in the world (Joseph & Jini, 2011). Diabetes mellitus pose a continuous threat to health globally
3 and placed third major lethal disease which poses death to humanity and also escalating hastily (Ogbonnia et al., 2010). According to one estimate it was present in 285 million individuals in 2010 which may rise to 439 million in 2030 (Shaw et al., 2010). Diabetes is a syndrome, whose basis might be hereditary or environmental or combination of both, which result in high sugar level in blood due to abnormal functioning in metabolism (Patel et al., 2012). Diabetes mellitus treatment often results in some late complex abnormalities including nephropathy and retinopathy etc.
Among various types of diabetes, type 2 diabetes (non-insulin dependent) makes up about 90% of cases of diabetes, with the other 10% due primarily to diabetes mellitus type 1 (insulin dependent) and gestational diabetes. Many lifestyle factors are associated in the onset of type 2 diabetes, including sedentary life that leads to overweight and hence obesity, diet deficient in healthy components, lack of exercise, stress conditions and utilization of junk food (Cecchini et al., 2010; Hu, 2011).
Due to adverse responses and rely upon low cost therapeutic ways, about 30% of diabetic patients use alternative therapeutic ways (Raman et al., 2012). Among these, bitter gourd and its various formulations and components can be used due to their sugar lowering effects via biochemical, pharmacological and physiological modes (Taylor, 2002; Bhushan et al., 2010). This hypoglycemic role is due to ability of components in this plant to stimulate utilization of glucose from skeletal as well as peripheral muscles (Cummings et al., 2007) minimize the uptake of glucose from intestine (Hui et al., 2009), reduction in differentiation of adipocyte (Nerurkar et al., 2010), inhibition in activity of many enzymes involved in gluconeogenesis (Singh et al., 2011) and by increasing endurance of islet β cells and enhancing their functional abilities (Singh & Gupta, 2007). Number of studies on bitter gourd for hypoglycemic effects and anti-hyperglycemic activity in animals has been reported recently (Fuangchana et al., 2011; Wehash et al., 2012; Rahman et al., 2015).
Recent researches also suggested that bitter gourd extracts may ameliorate high fat diet induced obesity and hyperlipidemia in animal models. Bitter gourd supplements in food result in lowering weight gain and visceral fat mass (Chen & Li, 2005). This might be due to increase in the level of oxidation of fatty acid and ultimately reduction in weight and peritoneal fat deposition (Chen & Li, 2005). Weights of visceral fat, epididymal white
4 adipose tissue and the adipose leptin and resist in mRNA levels also decreased significantly by utilizing bitter gourd extract (Shih et al., 2008). Not only fruit but seed oil of this plant is also helpful in reduction of fat and ultimately weight (Chen et al., 2012)
Due to diabetes and high fat diet feeding, concentrations of plasma lipids such as triglycerides, fatty acids and cholesterol are increased (Panchal et al., 2011). Extracts from bitter gourd remarkably lower the amount of lipid both in diabetic and high fat diet utilization. Pronounced reduction in the hepatic total cholesterol and triglycerides was noticed by employing bitter gourd extract (Jayasooriya et al., 2000).
Bitter gourd extracts, despite its bitter taste attract the attention of nutritionists and health co- workers due to its glucose and lipid lowering effects. The previous literature suggested that the clinical studies with bitter gourd and its formulations have limitations due to poor methodology in study design and discrepencies in statistical approach (Leung et al., 2009).
Bitter gourds are the richest source of bioactive compounds needed to control diabetes and hyperlipidemia but presence of such compounds varies in different cultivars although very little information is available on this aspect. Moreover, consumption of bitter gourd is often limited due to its characteristic bitter and astringent taste. So there is continuously a need to improve the palatability of bitter gourd in a more desirable food product. Thus, keeping in view the health claims of bitter gourd, present project has been designed with following objectives;
To characterize different parts of bitter gourd cultivars for their nutritional and bioactive profile To develop bitter gourd juice and to explore its nutritional & sensorial attributes To elucidate the health claims of bitter gourd using rodent experimental modeling
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Chapter 2 REVIEW OF LITERATURE
Functional foods and nutraceuticals have captured the global market owing to trends and perception of global market. Health promoting potential of such foods is due to the presence of bioactive moieties present in them. Plants contain significant nutritional sources with the presence of such bioactive molecules. Investigations on many plants elucidate their persuasive role to lessen many life-style related illnesses. However; several aspects of these plants are not revealed yet. The present research was designed to use bitter gourd as functional food. A wide-ranging discussion regarding different avenues of the present research has been given in following sections:
2.1. Concept of Functional Foods and Nutraceuticals 2.2. Bitter Gourd (Momordica charantia); An Overview 2.3. Chemistry of Bitter Gourd 2.4. Bitter gourd – A Source of Natural Antioxidants 2.5. Preparation of Bitter Gourd Extract 2.6. Bioactive Molecules 2.7. Health Claims
2.1. Concept of Functional Foods and Nutraceuticals
Food and medicine has the same source with the purpose of similar function in living body but medicines have side effects and are not as important as food in life of a healthy person on daily life. Thus, a diet rich in essential components not only boost the health but also impede the development of certain diseases. Sedentary life style and higher medicinal cost have enforced researchers to find out diet based therapies that are cost effective and safe. In this respect, functional foods and nutraceuticals not only fulfill nutritional requirements but also provide medicinal benefits. Now a days, functional foods and nutraceuticals have become an integral component of diet due to its therapeutic effects against various life style related disorders such as diabetes, hypercholesterolemia, cardiovascular diseases, cancer, etc. Diet
6 and health are related to each other and considered important as consumers are sentient toward foods to be nutritional as well as possess functional properties (Urala & Lähteenmäki, 2007). However, many strategies are in utilization to explore nutrients in plants as nutraceutical, their prime use, appliances and importantly ways of activity (Butt et al., 2009). Foods containing functional ingredients are unique due to ease in access, economical and allied health claims. In this perspective, various vegetables & fruits, cereals, nuts and pulses are found to be valueable due to presence of an array of bioactive ingredients. Among them vegetables attain prime importance in human diet since ancient times and hold inimitable place many dietary approaches. To fulfill the requirements of desired nutrients, The American Dietetic Association (ADA) recommended that four or five servings of vegetables are required per day. Bioactive components particularly presence of phytochemicals in plant foods especially inside many vegetables are crucial in maintaining metabolism, biochemical pathways like scavenging activity by free radicals, micobicidal activity and protecting from many malignant situations (Gupta et al., 2011). Among other components, carotenoids, flavonoids and anthocyanins etc. are the phytonutrients present abundantly in vegetables. (Andersen & Jordheim, 2006). These act at molecular level and not only alter the enzyme kinetics but also have effect cytokines release and signal transduction (Barta et al., 2006). Critical relationship has been founded between valueable dietary components and human health perspectives (Schwager et al., 2008; Ares et al., 2009). There are a number of studies that highlighted better human health and its correlation with consumption of vegetables owing to abundancy in dietary fibers, minerals, vitamins, anti-oxidants, and carotenoids (Tapsell et al., 2006). Many epidemiological studies have revealed that utilization of vegetables in daily diet especially having functional components is associated with reducing risk of chronic disorders (Visioli & Hagen, 2007).
2.2. Bitter Gourd (Momordica charantia); An Overview
Among vegetables, Bitter gourd (Momordica charantia L.), belongs to family cucurbitaceae commonly known as karala, bitter melon or balsam pear (Marr et al., 2004; Krawinkel & Kedig, 2006). Morphologically, the bitter gourd is a tendrils bearing herbaceous vine which creeps along supports. There are well-known for the bitter taste due to the presence of anti-
7 nutritional compounds alkaloid and have a wide range of medicinal values. Although the exact origin of Momordica genus is not clear from past records, yet according to most experts, the domestication of bitter gourd lies in east part of Asia, most possibly eastern part of India and southern part of China (Miniraj, 1993). However, it is stated in Ayurvedic texts, written by members of the Indo-Aryan culture, that bitter gourd emerged in India from 2000 to 200 BCE (Decker-Walters, 1999). Traditionally, people in tropical and subtropical regions commonly used it to cure many ailing effects and number of dazzling array of conditions within the body. Many Asian countries are popular now for its cultivation including Pakistan, India, China, Bangladesh and Korea. Amazon, East Africa, and the Caribbean are also familiar regions for the growth of this valuable nutrient rich plant (Basch et al., 2003). It is not only used as culinary vegetable but also widely consumed as medicine in developing countries for curing different diseases (Grover & Yadav, 2004). The whole fruit of bitter gourd including the seeds and pith can be used to boost health. It has long history of utilization as food and medicine (El-Batran et al., 2006). All parts of this plant have a tremendous potential in controlling blood glucose level and lowering cholesterol level. This plant can also be used for treatment of many pathological conditions like pneumonia, piles, gout, jaundice, scabies, rheumatism, eczema, leprosy dysmenorrhea, psoriasis and kidney stone. These disorders can effectively be controlled by consuming of its fruit on regular basis. It possesses antimalarial, anthelmintic, contraceptive, abortifacient and laxative properties (Grover & Yadav, 2004). This versatile plant is considered being potent enough that it can be used for treatment of any disease affecting man. This reason behind this norm is that the plant possesses hundreds of novel chemicals which no other sole vegetable possess by nature. Bitter gourd possessed some medicinal properties like anti-diabetic, anti-tumor (Budrat & Shotipruk, 2009; Fang & Ng, 2011), anti-inflammatory, antiviral, anticancer and cholesterol lowering effects (Budrat and Shotipruk, 2009). The anti-diabetic properties of bitter gourd are due to presence of glucose lowering agents such as polypeptide-p, charantin, vicine, and other bioactive components possessing strong antioxidant activity (Krawinkel & Kedig, 2006). It is used to treat infections incurred by worms and parasites and also helpful in external and internal treatment of wounds (Grover & Yadav, 2004; Wu & Ng, 2008). The extract of this medicinal plant has been found to be suitable as bactericidal particularly
8 against infections caused by Pseudomonas, Staphylococcus, S. aureus, Escherichia coli, Streptobaccilus and Salmonella (Saeed and Tariq, 2005). In addition to rich source of essential nutrients and minerals; it is reservoir of several biologically active chemical constituents. These biologically active chemical moieties have been isolated from all parts of this plant, particularly from leaves, fruit pulp, and seeds. In traditional medicinal system, many parts of cucurbit plants have been used particularly in the Chinese (Fu, et al., 2006) and Ayurvedic systems (Chaturvedi, 2012), including the flesh and seeds portion of bitter gourd (Dhiman, et al., 2012). Among all these plants bitter gourd is actually giving life longevity plant but all parts of this plant like leaves, young stem and fruit are bitter in taste and as such consumption is quite difficult because of its bitter taste. It needs to be processed and fortified to make it palatable and acceptable. This bitter taste can be minimized by giving salt treatment, dehydration and mixing with some other products. To reduce the bitter taste of bitter gourd, the fruits are commonly preboiled or dip in salted water before cooking (Behera et al., 2011). To lower blood glucose level, it can be used as salads or vegetable dishes. Bitter gourd can be made more organoleptic by consuming it in more palatable forms i.e., in the form of chips, sweets, tea, beverages and burfi etc. In this way bitter gourd can be used to get maximum health boosting effects.
2.3. Chemistry of Bitter Gourd
2.3.1. Chemical Composition and Mineral Analysis
Chemical and nutrient analysis of different vegetables plays a fundamental role in assessment of nutritional implications (Pandey et al., 2006). According to USDA National Nutrient Database (2008), the chemical composition of bitter gourd was water 88.69 g/100g, protein 3.60 g/100g, total lipids 0.20 g/100g, ash 1.35 g/100g, fibers 1.9 g/100g, carbohydrates 6.16 g/100g, energy 32 kcal, calcium 42 mg/100g, magnesium 94 mg/100g, phosphorus 77 mg/100g, sodium 249 mg/100g, potassium 6.2 mg/100g, iron 1.02 mg/100g, zinc 0.30 mg/100g, manganese 0.536 mg/100g, copper 0.201 mg/100g, , selenium 0.9 mg/100g and vitamin C 55.6 mg/100g. In addition, riboflavin, thiamin, niacin, vitamin A, vitamin B-6, vitamin E and vitamin K were also reported in bitter gourd.
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The nutritional composition of bitter gourd elucidated by Gayathri (2014) is moisture 6.14%, crude fat 2.38%, crude protein 27.88 mg/100g, ash content 2.76%, crude fibre 2.31% and carbohydrates 85.41 mg/100g. Saeed et al. (2010) evaluated nutritional analysis on bitter gourd‟s peel, seed and flakes. According to them, peel, seed and flakes holds moisture content 4.15%, 4.09%, 4.72%; ash 14.99%, 4.56%, 6.43%; fat 0.18%, 5.24%, 0.25%; protein 20.37%, 19.01%, 20.66%; fibre 17.77%, 22.46%, 17.08% and carbohydrates 42.54%, 44.64%, 50.86%, respectively. The peel, seed and flakes, when consume 100g, provide energy of 253.26 Kcal, 301.76 Kcal and 283.33 Kcal, respectively (Saeed et al., 2010). The nutritional composition of bitter gourd seeds were determined which were 7.0, 19.50, 34.0, 4.0, 12, 23.50% moisture, crude protein, crude fat, ash, crude fiber and carbohydrate, respectively (Mathew et al., 2014). Islam et al. (2010) analyzed flesh and seeds of four bitter gourd varieties. The moisture contents were ranged between 92.4- 93.5% for flesh and between 53.3-75.9% for seeds. Protein contents for flesh part were in the range of 9.8% to 8.4%. Seed protein contents were the 31.3% to 27.0%. Overall, varieties containing whiter colour fruit have higher protein contents than the varieties having green colour of fruit (Islam et al., 2010). However, the variations in nutritive value depend in part on the type of soil, climate, time of harvest and varietal differences. Bitter gourd is also rich in macro and micro mineral contents (mg/100 g) including calcium (45 ± 0.12), sodium (31 ± 0.03), potassium (390 ± 0.08), magnesium (31 ± 0.10), iron (7 ± 0.07), copper (0.08 ± 0.01), zinc (0.85 ± 0.08), manganese (0.26 ± 0.04), chromium (0.11 ± 0.05) (Bangash et al., 2011). The data shows presence of appreciable amount of minerals, nevertheless, their level varied due to soil, climatic conditions, cropping practices and variety (Ullah et al., 2011). Similarly, Bakare et al. (2010) examined mineral profile of bitter gourd and found calcium was abundantly present in the fruit of bitter gourd along with other minerals like Mg, Na, K, Fe, Zn, Mn and Cu. Bangash et al. (2011) also illuminated high quantity of K and Ca contents as compared to Na. Other minerals like Mg, Fe, Cu, Zn, Mn, Cr were also present in reasonable magnitudes.
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2.3.2. Ascorbic Acid Contents
Ascorbic acid (Vitamin C) is required in large amount as it is a water soluble vitamin and frequently loss from body. Reversible oxidation-reduction reactions depend upon the presence of ascorbic acid to accomplish the conversion of reactants into products. Vitamin C plays a vital role in the prevention from scurvy disease and also helpful in the folic acid derivatives formation which are needed for synthesis of DNA. Numerous studies directed that bitter gourd is a rich source of vitamin C (Bangash et al., 2011; Gayathri, 2014. The intake of ascorbic acid is helpful in minimizing the oxidative stress caused by free radicals.
2.4. Bitter gourd – A Source of Natural Antioxidants
Bitter gourd is considered as one of the most promising plant source for natural antioxidants (Kubola & Siriamornpun, 2008). Its fractions are rich in phenolics and due to this reason it has a strong antioxidant and free radical scavenging activity (Kubola & Siriamornpun, 2008). Not only phenols, but flavonoids, terpenes, isoflavones, glucosinolates and anthroquinones all of which confer a bitter taste, has been attributed to be the cause of high antioxidant potential (Snee et al., 2011). Different parts of plant body possess different antioxidant potential and this also varies with reference to cultivars. Various factors are involved in variation of antioxidant potential such as the genetic makeup of cultivar, climatic factors, developmental stages, agronomic practices, time of harvest, storing conditions, and management after harvesting. Due to presence of abundant natural antioxidants, bitter gourd can retard or temporarily stop the lipid and other molecules oxidation. Bitter gourd extracts inhibit the instigation or promulgation of oxidative chain reaction and hence helpful in prevention and repairing of damage done by oxygen radicals to the cells of body. It works effectively as reducing agent, free radical scavenger, potential complexers of pro-oxidant metal and/or quencher of singlet oxygen against the free radicals that are known to harm healthy cells and are involved in creation of harmful molecules and involved in onset of many degenerative processes (Chanwitheesuk et al., 2005). The antioxidants of bitter gourd fruit come from both hydrophilic and lipophilic fractions. The fruit of bitter gourd contains carotenes, vitamin C, vitamin E, xanthophylls
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(Chanwitheesuk et al., 2005) vanilic acid, syringic acid, chlorogenic acid (Horax et al., 2005), (+)-catechin, gallic acid, p-coumaric acid, caffeic acid, tannic acid, benzoic acid (Kubola & Siriamornopun, 2008), zeaxanthin, lycopene (Saeed et al., 2010). The total antioxidant activities of bitter gourd fruit is due to the presence of these compounds which give protection from degenerative diseases of aging, including diabetes (Hasan & Khatoon, 2012), cardiovascular diseases, hypertension, cancer (Fang et al., 2012; Yung et al., 2015) and so on. Bitter gourd fruit has known to be a good source of antioxidants than some other common vegetables (Raghu et al., 2011). The reported values of FRAP, TEAC, TPC and ascorbic acid content of bitter gourd fruit were 6.90±0.04, μmol Fe(II)/g FW, 13.43±0.11 μmol Trolox/g FW, 7.24±0.10 mg GAE/g FW and 29 μg AA/mg extract, respectively (Raghu et al., 2011). Kubola & Siriamornpun (2008) demonstrated that the fruit of bitter gourd was not the only part of the plant which contained antioxidant activities. Their results indicated that the leaf and stem of bitter gourd also had significant antioxidant activities where the total antioxidant capacity was greatest in the leaf followed by green fruit, the stem and the ripe fruit. The antioxidant potential is also affected by the type of extraction method and the nature of solvent used. Amira et al. (2013) had drawn conclusion that the pure acetonic extracts of bitter gourd fruit possessed the high correlation (r2=0.999) for FRAP and TPC assays. The correlation of the antioxidant assays carried out for hydrophilic extracts was very much depending on the polarity of hydrophilic solvent chosen (Amira et al., 2013). By contrast, Kubola & Siriamornpun (2008) found a strong positive correlation between DPPH and TPC (r2=0.711), FRAP and TPC (r2=0.948) for aqueous extracts of bitter gourd leaf, stem and fruit samples. However, they elucidated that total antioxidant activity of bitter gourd was high not only because of phenolic contents but many other antioxidant compounds in different fractions are responsible within this plant. In addition, Chanwitheesuk et al. (2005) reported high antioxidant index of bitter gourd was strongly related to the presence of vitamin E and total carotenes (lipophilic compounds) of the fruit. In some cases, it was noted that lipophilic antioxidant compounds possess stronger antioxidant activity than the hydrophilic antioxidant compounds. The extracts of all parts of bitter gourd possess strong antioxidant and free radical scavenging activities that is due to many compounds such as phenols, flavonoids, ascorbic acid,
12 carotenes, vitamin E, and many other compounds. The therapeutic benefits associated with this plant are mainly due to such compounds having potent antioxidant activities. The free radicals have been claimed to play a key role in development of many diseases such as diabetes, cardiovascular diseases and cancer by degradation of cells. These free radicals can be generated during many metabolic and physiological activities and can be acquired through environment. The cellular and metabolic injury is also due to oxygen radicals. These also involved in accelerating aging, cardiovascular diseases, neurodegenerative diseases, inflammation and cancer (Dasgupta & De, 2006). In recent years, research on the relationships between antioxidants and their preventing effects in controlling many non-communicable diseases has been increasing sharply. The intake of dietary antioxidants has a vital role in prevention and reduction in the oxidative damage incurred due to these radicals (Dasgupta & De, 2006). Many synthetic antioxidants such as butylated hydroxyl anisole (BHA) and butylated hydroxyl toluene (BHT) are extensively used but there are not suggested in many cases due to being responsible for adverse effect on liver damage and development of cancerous cells in the body (Jayalekshmy & Arumughan, 2005; Senevirathne et al., 2006). It has been suggested in number of epidemiological and in vitro studies that phytochemicals in food with strong antioxidant activity have potentially positive effect against development of many malaises including diabetes, cardiovascular diseases and cancer (Senevirathne et al., 2006). Many degenerative processes can be reduced by consuming more fruits and vegetables (Kaur & Kapoor, 2001; Vinson et al., 2001). These foods are a reservoir of antioxidant components particularly contain plethora of phytochemicals. A large number of phytochemicals like phenolic compounds are considered beneficial as there are helpful in boosting human health as well as decrease the risk of degenerative diseases by reduction of oxidative stress and inhibition of macromolecular oxidation (Pulido et al., 2000; Silva et al., 2004; Pereira et al., 2007). These bioactive moieties increase the antioxidant potential to a reasonable extent to fight of many ill effects (Katalinic et al., 2004). Among all the vegetables, bitter gourd extracts was found to have strong antioxidant activity and reduce scavenging activities by free radicals (Wu & Ng, 2007). El-Batran et al. (2006) revealed that extracts of bitter gourd when given to alloxan-induced diabetic rats showed hypolipidemic, anti-diabetic effects alongwith hepatorenal protection. The whole fruit
13 powder extracts of bitter gourd in fresh and dried form showed reduction in blood glucose level in diabetic rats (Virdi et al., 2003). Some studies also claimed that bitter gourd fruit is enclosed with chemopreventive agents or anticarcinogens (Yasui et al., 2005). It was examined that TPC, free radical scavenging activity by DPPH, and FRAP values were dependent on solvents used in extraction whereby in pure solvents, extraction efficiency was highest in water extract of bitter gourd fruit followed by acetone, ethanol and methanol, respectively. Highest values were obtained in aqueous organic solvents. Higher TPC activities in bitter gourd fruit was obtained in extracts of both water and 100% acetone (Amira et al., 2013). Moon et al. (2014) studied on eight cultivars of bitter gourd grown in plastic green house and assessed them for total vitamin C content, total phenolic content. Antioxidant activity DPPH, ABTS and FRAP were investigated to determine antioxidant potential in these cultivars. They examined that some cultivars particularly that have large fusiform fruits has more vitamin C and phenolic contents (112.4±12.0 mg/100 g and 3.81±0.3 mg/g, respectively) than the rest of the cultivars having long and small fruit. Two cultivars namely Nakanokoya and Kanzyukoya showed high antioxidant potential than the other cultivars of bitter gourd according to DPPH, ABTS and FRAP assays. Nakanokoya, a large fusiform fruits bearing variety, reported to be most potent cultivar as it possessed higher amount of vitamin C and also showed maximum antioxidant activities among all the experimented cultivars. Zulkifli (2012) determined variations in antioxidant activities of bitter gourd with reference to different maturity stages. Immature, mature and ripe stages were chosen to assess the possible variations in antioxidant ability. Extraction for all stages was done by water extraction method. Total flavonoid content, total phenolic content, DPPH, beta-carotene bleaching assay and FRAP techniques was employed for all extracted samples. Analysis showed that with reference to total flavonoid content, ripe stage showed the highest value (324.89 ± 23.82 mg RuE/100g), followed by mature stage (154.39 ± 8.04 mg RuE/100g) and the least value was noted for immature stage (114.04 ± 3.04 mg RuE/100g). Significant differences existed in total flavonoid content in all maturity stages. It was also noted that ripe stage possessed abundant total phenolic content (558.56 ± 27.03 mg GAE/100g) followed by immature (351.35 ± 27.03 mg GAE/100g) and mature stage (351.3 ± 31.21 mg GAE/100g). Antioxidant activity determination via DPPH demonstrated that there is an inverse
14 relationship between ripening and DPPH value. More ripened of bitter gourd has lower DPPH value. The DPPH assay highlighted that bitter gourd extract has strong antioxidant activities to trap free radical compounds. The ranges for scavenging activity were between 37% - 64.48%. However, for FRAP assay, mature stage had the highest FRAP value (101.33 ± 5.00 μg FE/g) as comparison with ripe and immature stages which possessed lower FRAP values i.e., 63.00 ± 2.89 μg FE/g and 54.67 ± 2.89 μg FE/g, respectively. The highest antioxidant activity was also noted for ripe stage (41.52 ± 1.23 %) in beta-carotene bleaching assay followed by immature and mature stages with values of 38.15 ± 0.86 % and 27.43 ± 0.55 %, respectively. Antioxidant and radical-scavenging activities is possessed by various crude extracts and absolute natural compounds from plants (Rohman et al., 2010). Phenolics or polyphenols, including flavonoids were considered more beneficial among several other natural compounds as these have potent antioxidant activities which result in retardation or inhibition of oxidation possibly by prohibiting reactive radicals including reactive oxygen species in a biological system (Maltas & Yildiz, 2012). In free radical mediated diseases, like cardiovascular diseases, cancer, diabetes, neurodegenerative diseases, arthritis, gastrointestinal diseases and aging, antioxidants have great therapeutic importance (Sharma & Kumar, 2011). Hence, regular intake of vegetables rich in compounds having strong antioxidant potential is related with reduction in the onset of many types of chronic diseases. Many types of synthetic antioxidants that are currently available like gallic acid, esters, butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT) and tertiary butylated hydroquinone have been noted to cause certain types of adverse health effects. Hence, application of such synthetic antioxidants in different conditions has strongly restricted now a day and there is a continuous need to found naturally occurring antioxidants that may substitute them. In addition, these antioxidants exhibited moderate antioxidant activity and also poorly soluble within the body. In recent years, there has been a continuous rise of attention in reducing free radicals induced tissue injury by utilizing medicinal plants having strong antioxidant potential and therapeutic benefits (El-Hela & Abdullah, 2010).
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2.5. Preparation of Bitter Gourd Extract
Different solvents such as water, methanol, ethanol and acetone are mostly used in extraction of bioactive molecules. Lu et al. (2012) conducted a trial to analyze antioxidant activity using water and methanol for extraction in sixteen cultivars of indigenous wild bitter gourd. Significant differences were noted among different cultivars with reference to scavenging activities against DPPH and hydroxyl radicals. The concentrations of 50% scavenging activity (IC50) for the effective cultivar was 181 μg/mL for water and 246 μg/mL for methanol in DPPH assay while for hydroxyl radicals, higher value was noted in case of water i.e., 148 μg/mL and lower (37 μg/mL) for methanol. Most bitter gourd cultivars showed protective activities at 4000 μg/mL particularly for methanol extract against Cu2+ induced low-density-lipoprotein peroxidation. By thiobarbituric acid reactive substance assays, it was comparable to 0.8 mM Trolox. Some radical particularly reactive oxygen species, abbreviated as ROS, exert oxidative stress towards the human body cells and each cell has to face about 10,000 oxidative hits just in one second (Mondal et al., 2006). The natural antioxidants may have free-radical scavengers, reducing agents, potential complexers of pro-oxidant metals, quenches of singlet oxygen etc., (Ebadi, 2002). These naturally occurring antioxidants hinders the oxidation process by reducing the activity of free radicals after reacting with them (Gupta et al., 2004).
2.6. Bioactive Molecules in Bitter Gourd
Different parts of the world cultivated bitter gourd; as it is a multipurpose herb and mostly used for its edible fruit (Alam et al., 2009). Its utilization as medical herb is also common in Asia, Africa, and South America. The whole plant possess antidiabetic, hypocholesterolemic, hypotriglyceridemic, antiulcer, anti-inflammatory antitumor, antileukemic, hypotensive, antiviral, antibacterial, antimycobacterial, anthelmintic, antioxidant, antimutagenic immunostimulant and insecticidal properties (Fang & Ng, 2011). Bitter gourd attenuates oxidative stress and neuro-inflammation realated to high-fat diet (Nerurkar et al., 2011). Bitter gourd can be used in multitude of medical conditions. So, scientists are continuously struggling to conduct in depth study on bioactive compounds and their actions on the body.
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The bioactivities present in bitter gourd are partially attributable to the antioxygenic and free radical scavenging activities (Pratheepa et al., 2011). It was also well-known phenomena that all the bitter gourd cultivars possess antioxidant activity (Pratheepa et al., 2011) although there extent may vary that is due to variation in genetic makeup and environmental factors. The well-known bioactive compounds from bitter gourd are momordicins, momordicinin, charantin, cucurbitins, cucurbitanes, cucurbitacins, momordin, momordenol, momorcharins momordicilin, charine, momordolol, diosgenin, cycloartenols, gentisic acid, cryptoxanthin, goyaglycosides, elaeostearic acids, galacturonic acids, goyasaponins and erythrodiol (Grover & Yadav, 2004), ferulic acid and caffeic acid (Raj et al., 2005), isorhamnetin and fisetin (Lako et al., 2007), 3b, 25-dihydroxy-7b-methoxycucurbita-5, 23(E)- diene, 3b-hydroxy-7, 25- dimethoxycucurbita-5, 23 (E) -diene and 3 - O - b - D - allopyranosyl - 7b, 25- dihydroxycucurbita- 5, 23 (E) - dien-19-al (Harinantenaina et al., 2006). Anti-tumor and anti- HIV activities are reported to be due to its MAP30 protein (Lee-Huang et al., 1995). Some bioactive molecules like momorcharins and momordins inactivate ribosomes and diminish extra proliferation of cells and momordin 1c and oleanolic acid glycoside play their role in altering gastrointestinal transit time and blood glucose (Chao & Huang, 2003; Grover & Yadav, 2004). Many clinical studies on bitter gourd had reported that extract from different parts of plant including leaves, fruit and seeds shown that all parts are a rich source of bioactive compounds that have wide ranging effects particularly hypoglycemic activity in both diabetic humans and animals (Fuangchana et al., 2011; Wehash et al., 2012). However, with the passage of time more and more emphasis was given on the discovery of such compounds that play substantial role in controlling diseases mainly diabetes and hypoglycemic properties (Islam et al., 2011; Hazarika et al., 2012). Saponins like 3-hydroxycucurbita-5, 24-dien-19-al-7, 23- di-O-β-glucopyranoside and momordicine II were isolated from bitter gourd. Appropriate concentration (10 and 25 µg/mL) of these two compounds showed significant insulin releasing activity in MIN6 β- cells (Keller et al., 2011). The most prominent compounds that have been isolated from bitter gourd are polypeptide-p, charantin and vicine. These compounds have been identified as hypoglycemic agents and have a great potential in controlling diabetes.
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2.6.1. Polypeptide-p
Bitter gourd contains polypeptide-p in such abundant quantity that no other vegetable is comparable to it in this respect. It is very similar to normal human insulin in its function and hence known as p-insulin. A number of studies indicated its role in lowering blood glucose level in human and animals (Paul et al., 2010; Wang et al., 2011; Tayyab et al., 2012). Polypeptide-p or p-insulin is glucose lowering protein and its action in the body is mimicry of human insulin. In some reports, oral intake of seeds of bitter gourd does not show any glucose lowering effect (Wehash et al., 2012) which emphasis that some other components may also present which might have their role in controlling diabetes in some cases.
2.6.2. Charantin
Another important component which has hypoglycemic activity is charantin which is a mixture of two compounds viz., sitosteryl glucoside and stigmasteryl glucoside (Pitipanpong et al., 2007). It is cucurbitane-type triterpenoid and possesses glucose lowering effect in diabetic conditions (Patel et al., 2010). Charantin could be used for the treatment of diabetes and well suited in replacement therapies (Pitipanpong et al., 2007). Thomas et al., (2012) extracted charantin from fruit of bitter gourd and used high performance thin layer chromatographic technique to estimate extent of this valuable compound. In some reports, it was noted that charantin is more potent chemical than some other hypoglycemic agents (Cousens, 2008). Harinantenaina et al. (2006) isolated two aglycones of charantin and these were identified as stigmastadienol glycosides and sitosterol. They analyzed them in vivo for their hypoglycemic effects and surprisingly no change in blood glucose level was observed. From their conclusion, a strong belief is developed regarding hypoglycemic activity that charantin might contain other components which are responsible for hypoglycemic activity.
2.6.3 Other Bioactive Molecules
Vicine is another major component isolated from bitter gourd that has anti-diabetic properties (Haixia et al., 2004). Many other constituents including momordicine and momordin having
18 potent biological activity have be isolated from bitter gourd and identified. A group of Japanese scientists isolated eleven compounds from bitter gourd fruits by fractionation using methanol. They noted that some of these compounds are helpful in lowering blood glucose level (Lee et al., 2009). Abundance of phytochemicals in plants have ability to scavenge free radicals and ultimately helpful in ameliorate oxidative stress (Seifried et al., 2007). Phytochemicals play a vital role in maintenance of many physiological processes and preventing from many types of ill effects. Among the phytochemicals, phytosterols, antioxidants and flavonoids have proven hypoglycemic and hypocholesterolemic potential (Colonna et al., 2008). Bitter gourd contains a number of valuable chemical substances that have medicinal attributes. These include minerals, vitamins, reducing sugars, antioxidants and many other phytochemicals, that is phenolic constituents, saponins, glycosides, alkaloids, fixed oils, free acids and resins. Due to rich in novel chemical constituents and presence of dietary and therapeutic components, many malaises can be well treated by using bitter gourd formulations.
2.7. Health Claims of Bitter Gourd
Various physiological threats like cardiovascular complications, cancer and immune dysfunctions are directly related with poor dietary habits and sedentary lifestyle (Barta et al., 2006). Now, in the modern era, it is common perception that a number of diseases are curable with just brought some changes in lifestyle and dietary modules (Barta et al., 2006; Divisi et al., 2006; Nies et al., 2006). Disease prevention strategy based on changing in diet should be included within nutritional guidelines; predominantly dietary components having desirable role could be a part of this innovative approach. Antioxidant potential has greatly increased by consuming diet rich in nutrients like polyphenols and tocopherols that further minimize the chances of occurrence of various life threatening maladies like diabetes mellitus and atherosclerosis (Sen et al., 2006). The bioactive moieties present in functional foods have therapeutic perspective against various life threats including immune dysfunction, oxidative stress and cancer insurgence (Wong et al., 2009).
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Many macromolecules are frequently damaged by free radicals (Davies, 2000). The abnormally high amount of free radicals particularly reactive oxygen species has resulted profound oxidative stress to cells and tissues (Valko et al., 2007). Oxidative stress is actually a disruption between the antioxidant defense and amount of reactive oxygen species to be produced and intensity of injury to tissues (Betteridge, 2000). It may also cause damage to DNA, generating neuropathy disorders, onset of cardiovascular abnormalities and uncontrolled proliferation of cells leading to cancer (Gackowski et al., 2008; Migliore & Coppedè, 2008). There is extreme necessity to use plant products in their natural form for improved efficacy and safety along with effectual source of antioxidants, immune- modulatory activities and anti-viral viewpoints. Many ill-effects have shown to minimize by consuming bitter gourd in diet including diabetic conditions, cardio-vascular diseases and insurgence of cancer etc. (Steinmetz et al., 1994).
2.7.1. Role of Bitter Gourd in Diabetes Mellitus
Diabetes mellitus is a syndrome in which metabolic rate become disordered and resulted in abnormally high blood sugar levels also known as hyperglycemia (Patel et al., 2012). The insurgence of this disease is usually due to hereditary and environmental factors or combination of both in some cases. In the leading factors worldwide that causes death, one of them is diabetes mellitus (Joseph & Jini, 2011). Diabetes mellitus is continuously rising particularly among individuals of developing countries and its prevalence increases with the passage of time. According to one estimate it was present in 285 million individuals in 2010 which may rise to 439 million in 2030 (Shaw et al., 2010). Diabetes mellitus is also associated with many late problems like nephropathy, retinopathy and others. Diabetes is of two types i.e., insulin dependent diabetes or type 1 diabetes and the other is non-insulin dependent or type 2 diabetes. About 90% population is affected by type 2 diabetes that may arise due to sedentary life style, diet deficient in healthy components, lack of exercise, stress conditions and utilization of junk food. Due to adverse responses and rely upon low cost therapeutic ways, about 30 % of diabetic patients use alternative therapeutic ways (Raman et al., 2012). Many traditional herbs and plants extract have been used for
20 remedy of diabetes in developing countries (Malviya et al., 2010) and among these, bitter gourd is the most popular plant in controlling hyperglycemia (Rahman et al., 2011). It has been thoroughly reported to be used worldwide in treating diabetes (Hasan & Khatoon, 2012). Historically, bitter gourd is extensively used for its glucose lowering effects in Asia, Africa, and parts of America (Basch et al., 2003). Bioactive components isolated from bitter gourd such as polypeptide-p charantin, and vicine are particularly anti-diabetic agents along with some other bioactive components including antioxidants. A number of studies on cell culture, animal models and humans proved that bitter gourd has hypoglycemic effects (McCarty, 2004; Krawinkel & Keding, 2006). Various components in bitter gourd exert their glucose lowering effects through different physiological, pharmacological and biochemical modes (Taylor, 2002; Garau et al., 2003; Bhushan et al., 2010). The possible modes of the hypoglycemic actions are due to its compounds that possess hypoglycemic effects (Ragasa et al., 2011). In addition, other possible modes incurred by bitter gourd is increase in glucose utilization by stimulating skeletal and peripheral muscles (Akhtar et al., 2011), controlling differentiation of adipocytes (Nerurkar et al., 2010), inhibition of uptake of glucose by intestine (Abdollah et al., 2010), inhibition of activity of gluconeogenic enzymes (Singh et al., 2011) and preservation of β cells and their functions (Gadang et al., 2011). In some studies, it was noted that bitter gourd adjusts serum glucose levels by modulating certain gene expression (Gadang et al., 2011). Some sterols also possess strong glucose lowering effect (Ragasa et al., 2011). The extract of bitter gourd plays its role in regulation of uptake of glucose into the blood stream from the gut. In addition, some molecules induce insulin like effect by increasing the uptake of glucose into the skeletal muscle cells. Bitter gourd when orally administered in STZ-induced diabetic rats has resulted in modification in the activity of α, β and δ cells in the pancreas (Ahmed et al., 2004). The islets of Langerhans also showed improvement in their activity by taking alcoholic extract of bitter gourd. It was also observed that extract of bitter gourd is involved in stimulation of endocrine glands in pancreas and also stimulate liver cells to increase the uptake of glucose (Jeong et al., 2008)
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Moreover, bitter gourd and its various formulations involved in rapid proliferation of cells and enhancing the growth parameters just like insulin (Parmar et al., 2011). The chemical constituents in bitter gourd play a vital role in lowering glucose level and hence in controlling diabetes but the exact mechanism is still unknown. Lot of investigation is still required to find proper mechanism by which bitter gourd has played the most potent role in controlling diabetic condition either by repairing damaged β cells or prevention of these cells from death. Not only the chemicals but the dosage of bitter gourd used is important in controlling level of glucose. Daily oral intake of bitter gourd fruit juice over a period of 10 weeks was found to be helpful in reducing amount of glucose (Ahmed et al., 2004). Not only the fruit, but seeds, leaves and even every part of this plant show hypoglycemic activity in normal animals (Ismail et al., 2012; Mohammady et al., 2012). Hypoglycemic activity in some studies is comparable with oral medications such as tolbutamide (Sarandan et al., 2010), chlorpropamide and glibenclamide (Ojewole et al., 2006). It was also worthwhile to note that its hypoglycemic effects are long lasting. Singh et al. (2008) found that lowered blood sugar level and improvement in histology of islet cells remained as such even after discontinuation of extract feeding. Acetone extract of whole fruit powder of bitter gourd lowered the blood glucose level from 13.3% to 50.0% by giving doses in the range of 0.25, 0.50 and 0.75 mg/kg of body weight after 8 to 30 days treatment in alloxan diabetic albino rats. The result, thus, confirmed hypoglycemic activity of bitter gourd in diabetic animals as well as humans (Singh & Gupta, 2007). Tongia et al. (2004) reported that intake of capsule (200mg) of bitter gourd twice on daily basis synergistically improved efficacy of glibenclamide and metformin by meritoriously lowering level of sugar in blood. But there are some limitations in the research plan as these trials are of short span, improper in study designs and conducted on very few patients. Fuangchana et al. (2011) reported that a dose of 2000mg/day significantly reduced fructosamine level at week four in diabetic patients while other doses does have and profound effect. Tsai et al. (2012) reported declining in incident of metabolic syndrome and reduction in waist size of patients compared to control. Trakoon-osot et al. (2013) also reported declining in blood sugar level after treated diabetic patients with bitter gourd and also observed dwindling trend after sixteen weeks of the intervention in serum advanced
22 glycation end products. However, in some cases bitter gourd intake failed to produce any betterment in diabetic conditions (Dans et al., 2007). Almost all authors reported no serious side effects during the study period (Ooi et al., 2012). The formation of extract in different solvents showed hypoglycemic activity of bitter gourd in animal models. Reduction in blood sugar level was observed in streptozotocin treated diabetic rats after oral administrations of extract of whole plant (Ojewole et al., 2006). Glucose tolerance, insulin sensitivity was also improved by administration of these extracts in rats having high fat diet-induced insulin resistance (Sridhar et al., 2008). Normal concentration of glucose was also maintained by bitter gourd in chronic sucrose loaded rats (Chaturvedi & George, 2010). Ahmed et al. (1998) use bitter gourd fruit juice to evaluate its effects on the distribution and number of α, β, and δ cells of pancreas in streptozotocin induced diabetic rats and observed that number of β cells increased significantly after giving the juice in comparison with untreated diabetic rats. seed extracts is also beneficial in prevention of degeneration of pancreatic islets when dosage of 150 mg/kg body weight were given for 30 days and it was also found to be helpful in restoring functions of islets (Sathishsekar & Subramanian, 2005). Extract prepared with powder form of fruit of plant in acetone and giving at doses of 25, 50, and 100 mg/kg body weight influenced islets of Langerhans cells particularly recovery of β-cells of the and normalizes their functioning (Singh & Gupta, 2007). The extract also promotes formation of islets from already existing islet cells along acinar tissues (Singh & Gupta, 2007). It was also observed that extract of bitter gourd fruit pulp in ethanol greatly increased the amount of β- cells, total β-cell area, islet size and level of insulin compared with normal rats (Hafizur et al., 2011). Increase in insulin secretion was also observed by administering fruit pulp protein extract in pancreas of experimental rats (Yibchok et al., 2006).
2.7.2. Lipid Lowering Potential of Bitter Gourd
Dyslipidemia are disorders in which there is abnormal increase in the synthesis of cholesterol and malfunctioning in the metabolism of lipoprotein metabolism. This may result in increase in accumulation of lipoprotein which is probably the first sign of obesity. Plasma lipids particularly fatty acids, cholesterol and triglycerides concentrations are increased due to high
23 fat diet and diabetes in laboratory animal and human study (Panchal et al., 2011). Increased in concentrations of cholesterol and triglycerides in diabetic dyslipidemia is due to increase in the amount of free fatty acids that are released from fat cells (Chahil & Ginsberg, 2006). These free fatty acids accumulated in the liver and increase glycogen stores which ultimately increase the production of triglycerides which result in production of apolipoprotein B and very low density lipoprotein cholesterol (Mooradian, 2009). Dyslipidemia is associated with resistance to insulin in case of type 2 diabetes and also poses other threats like coronary heart disease (Mooradian, 2009). High body fat increases the risk of several other diseases such as arteriosclerotic, hypertension, hyperlipidemia and metabolic syndrome (Guh et al., 2009). The role of bitter gourd in lowering of lipids was more pronounced in rats fed with diet rich in fat. Hepatic total cholesterol as well as triglycerides is markedly reduced in cholesterol fed rats (Jayasooriya et al., 2000). Ahmed et al. (2001) used bitter gourd extract for the period of 10 weeks in streptozotocin induced diabetic rats and observed normal ranges for plasma cholesterol, phospholipids and triglycerides. Utilization of 0.75% fruit extract of bitter gourd for 30 days considerably reduced the level of triglycerides and low density lipoprotein and increase high density lipoproteins in diabetic rats (Chaturvedi et al., 2004). In another study, plasma cholesterol level was abridged noticeably after supplementation of bitter gourd in rats fed with high fat diet (Chen & Li, 2005). Similar results were also noted in high sucrose fed rats which were given bitter gourd extract that also resulted in lowering of triglycerides and low density lipoprotein (Chaturvedi & George, 2010). Not only the fruit extract but seeds extract was also proved effective in lowering cholesterol, low density lipoprotein and prominently increase the amount of high density lipoprotein in female Zucker rats (Gadang et al., 2011). The exact mechanism of lipid lowering effect due to bitter gourd was not clear yet. Molecular mechanism behind the beneficial role of bitter gourd extracts was decreased pancreatic lipase activity. The reduction of pancreatic lipase activity is important for absorption of fat from intestine because it not only involves in breakdown of fat into fatty acids but also enhances fatty acid level of plasma after intake of fat. Thus pancreatic lipase reduction is important in lowering circulating free fatty acids. Serum total cholesterol level and fatty acids concentration were observed considerably lower in mice fed with high fat diet after appended with 45% bitter gourd (Shih et al., 2014). This
24 lipid lowering effect is attributed to its ability to increase adenosine monophosphate- activated protein kinase (AMPK) phosphorylation and peroxisome proliferator-activated receptor (PPARγ) mediated lipid metabolism in liver (Shih et al., 2014). Liver X receptors (LXR) also plays an important role in metabolism of lipid as well as cholesterol. LXRα knockout mice develop high cholesterol levels, liver cells degeneration, fatty livers enlargement and impaired functioning of liver in high cholesterol diet fed rats (Peet et al., 1998). Down regulation of hepatic LXRα due to bitter gourd also involve in declining serum total cholesterol and low density lipoprotein in high cholesterol fed Wistar rats (Matsui et al., 2013). Elevated concentrations of hydroperoxides, AST, ALT and ALP reached to normal level by adding bitter gourd extracts in diet of rats while reversing the oxidant-antioxidant imbalance (Thenmozhi & Subramanian, 2011). This normalization of liver markers is mainly due to stimulation of superoxide dismutase and catalase production and functional responses and stabilizes the level of glutathione in liver and brain tissues (Thenmozhi & Subramanian, 2011). The extract of bitter gourd is also helpful in prevention of lipid peroxidation in sucrose fed rats and normalizes the glutathione level in liver (Chaturvedi & George, 2010). Nagy et al. (2012) evaluated effects of bitter gourd on hypoglycemic, hypolipedimic and antioxidant status in streptozotocin induced diabetic male rats. They used aqueous extract of bitter gourd for exploring its possible effect on insulin and nitric oxide release. Intraperitoneal dose of streptozotocin was given in a dose of 30 mg/kg. Significant increase in serum glucose, total cholesterol, triglyceride, low density lipoprotein cholesterol, creatinine, urea and renal reduced glutathione, nitric oxide and decrease in insulin levels, myeloperoxidase and erythrocyte glutathione–S-transferase activities after giving streptozotocin was noted. Oral intake of extract of bitter gourd (400 mg/kg/day) up to 8 weeks markedly ameliorated these effects. Parmar et al. (2011) evaluated glucose tolerance level and lipid profile by providing fruit juice of bitter gourd in streptozotocin (STZ) induced type-II diabetic rat. Pups of just two days having weight of 7 to10 g were picked and made them diabetic with 90 mg/kg STZ in citrate buffer solution. 25% and 50% bitter gourd fruit juice was given for a period of 8 weeks. Biochemical analysis after the experiment clearly highlighted significant differences of glucose, cholesterol, HDL, LDL, triglyceride in group treated with 50% bitter gourd juice compared to diabetic control group.
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Sedentary life style and high fat diet like cafeteria type diet are two major factors for increasing number of obese people worldwide (Power & Schulkin, 2008). Utilization of junk food is also a major cause of obesity. Increase in body weight and accumulation of abdominal fat are the onset of obesity. Consumption of bitter gourd showed expedient benefit in reducing body weight gain and deposition of fat. A number of reports suggested that bitter gourd utilization is helpful in reduction of body mass and hence inception of obesity. In an experiment on rats that fed with diet rich in fat and subsequent supplementation of bitter gourd (0.75% of diet) resulted in controlling body weight and reduction in visceral fat mass (Chauhan et al., 2010). The probable reason for this reduction might be due to increase rate of oxidation of fatty acids in the body which further played its role in weight reduction and peritoneal fat deposition (Chauhan et al., 2010). Singh, (2011) also noted that significant reduction in epididymal white adipose tissues, visceral fat and levels of adipose resistin and leptin mRNA in mice. Bano et al. (2011) reported reduction in body weight by giving dose of 2mL/day of aqueous extract of bitter gourd. Not only the fruit, but seed oil supplementation also helpful in reduction of weight and fat mass in mice fed a high fat diet (Chen et al., 2012).
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Chapter 3 MATERIALS AND METHODS
The present research project was carried out in the Institute of Home and Food Sciences at Government College University, Faisalabad. In this study, biochemical and nutritional portrayal of different cultivars of bitter gourd (Momordica charantia L.) were analyzed. Moreover, in vivo and in vitro study was also conducted to determine therapeutic potential. Materials to be used and protocols to be followed are described below:
3.1. Procurement of Raw Material
Fruits of six promising bitter gourd cultivars i.e., BG 20, Black King, FSD Long, KHBG-1, GHBG-1 and Noor were procured from Vegetable Research Section, Ayub Agriculture Research Institute (AARI), Faisalabad. Selection of these varieties was conceded on the basis of their yield, quality and freshness.
Various reagents of analytical and HPLC grade including differeent standards were purchased from Sigma-Aldrich (Sigma-Aldrich Tokyo, Japan) and Merck (Merck KGaA, Darmstadt, Germany). For bio-evaluation study, Sprague-Dawley rats were kept in the Animal Room of College of Pharmacy, Govt. College University Faisalabad. For efficacy trial, diagnostic kits were purchased from Cayman Chemicals (Europe) and Sigma Aldrich, Bioassay (Bioassays Chemical Coorporation, Germany) and.
3.2. Raw Material Handling
The bitter gourd fruits were washed thoroughly under running tap water so as all the dust, dirt and other foreign debris removed. After washing, the fruits were peeled and seeds were isolated and pulp was cut into small pieces. Some fruits of each cultivar were cut to small pieces as a whole. In this way, skin, flesh, seeds and whole fruit of each cultivar were obtained. These were dried at room temperature till dried completely. For each category, the dried materials were ground further by using a small laboratory grinder to very fine powder (National, Japan, Model MT-W166A) and for further refining; the fine powder was passed
27 through a sieve. After obtaining very fine quality fine powder for each category, it was packed separately for further analysis in plastic jars which are absolutely air tight.
3.3. Chemical Analysis of Raw Material
The skin, flesh, seeds and whole fruit of six selected cultivars of bitter gourd were scrutinized for moisture contents, total ash contents, crude fiber contents, crude fat, crude protein and nitrogen free extract in accordance with their respective protocols stated accordingly in AACC (2000). The protocol for each method is described briefly underneath. 3.3.1. Moisture Content
The estimation of moisture contents was determined according to the AACC Method No. 44- 01 (AACC, 2000). Dry 3g of the sample in a hot air oven (Model: 202-0A, China) at 105±5 ºC till the constant weight of sample achieved. To calculate moisture contents in the sample, following formula is used:
3.3.2. Ash Content
Ash was determined according to AACC Method No. 08-01 (AACC, 2000) by direct burning of sample in crucible. The oxidizing flame was used to heat crucible up till no fumes were produced and then inflamed in muffle furnace (MF 1 / 02, PCSIR-Pakistan) at 550 ºC. The experiment was continued till residues of grayish white colour appeared. Ash will be calculated according to the formula:
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3.3.3. Crude Protein
The AACC (2000) Method No. 46-13 was used to determine the nitrogen percentage in the sample using Kjeltech Apparatus ((Model: Serin-Nr: 808-100, Behr Dusseldort). The concentrated H2SO4 was used to digest the sample along with digestion mixture i.e., K2SO4:
FeSO4: CuSO4 10: 5: 85 for 2 to 3 hours till appearance of light green colour or becoming transparent. The dilution of digested material was undertaken (250 mL). 10mL of diluted material and 10mL of 40 % Sodium Hydroxide solution was used in distillation apparatus for distillation. 4% boric acid solution was used to estimate the ammonia liberated by pouring indicator viz., methyl red. At the end, titration for distillate was carried out against 0.1 N
H2SO4 till colour changes to golden brown. The percentage of crude protein was determined by following formula and multiplying N with a factor (6.25) as:
Nitrogen (%) 6.25
3.3.4. Crude Fat
3g sample in a solvent i.e., n-hexane was taken in Soxhlet Apparatus (PCSIR, Model: SE- 1A/02, Pakistan) to determine crude fat contents according to the AACC Method number 30- 10 (AACC, 2000).
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3.3.5. Crude Fiber
Fat free sample was taken to estimate crude fiber and firstly digest it with H2SO4 (1.25%), followed by solution of NaOH (1.25%). Labconco Fibertech (Labconco Corporation Kansas, USA) was used to determine crude fiber of the samples. The amount of residues produced were noted and kindled at 550 oC till colour of residues turned to white in a muffle furnace. AACC Method No. 32-10 (AACC, 2000) was used to measure the extent of percentage of fiber. The crude fiber was calculated as per expression given below:
3.3.6. Nitrogen Free Extracts (NFE)
The nitrogen free extract (NFE) on dry basis was calculated according to the following expression:
NFE (%) = 100 – (Total ash % + crude fat % + crude fiber % + crude protein %)
3.4. Minerals Analysis
The skin, flesh, seeds and whole fruit of all the cultivars were scrutinized to note the variations in minerals contents in these parts according to the procedures of AOAC (2006). Flame Photometer-410 (Sherwood Scientific Ltd., Cambridge) was used to assess the sodium (Na) and potassium (K) concentrations whilst, Atomic Absorption Spectrophotometer (Varian AA240, Australia) was utilized to determine amounts of magnesium (Mg), calcium (Ca), zinc (Zn) and iron (Fe) in the aforesaid parts.
3.5. Quantification of Ascorbic Acid
The quantification of Ascorbic acid (Vitamin C) was done by titrating it against phenol indo- 2, 6 - dichlorophenol (DPIP) (Papuc et al., 2001). The samples (0.2 g) were separately homogenized with 40 ml of a buffer solution. The buffer solution is prepared by using 4 g/L sodium acetate anhydrous and 1 g/L oxalic acid. The titration of the said solution was performed against 100 mg/L sodium bicarbonate and 295 mg/L DPIP solution. The results were expressed as mg/100 g dry weight.
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3.6. Preparation of Extracts
Extracts from different parts of respective cultivars were made and obtained using two solvents i.e. water and 80% aqueous CH3OH. The solids and other unnecessary components were isolated from the extracts through filteration using Whatman No. 1 filter paper. Afterwards, the extracts were moved to Rotary Evaporator (Eyela, Japan). The extracts in concentrated form were stored at 4 °C for further analysis and evaluation.
3.7. Antioxidative Profiling of Bitter Gourd Extracts
To determine antioxidant potential of different extracts, respective procedures were used:
3.7.1. Determination of Total Polyphenols
Folin-Ciocalteu method was used to determine Total polyphenols (TP) according to method of Singleton et al. (1999). Firstly, addition of extract (50 μL) was carried out in Folin-
Ciocalteu reagent (250 μL) with 20% Na2CO3 solution (750 μL) and further distilled water was added till the final volume reached to 5 mL. Absorbance was observed and noted against control after a period of 2 hours, with ultraviolet / visible Spectrophotometer (IRMECO, U2020) at 765 nm. Following expression was used to determine total polyphenols [gallic acid equivalent (mg gallic acid/g)]: C = c × V / m C = Total phenolic contents (GAE) of mg/g plant extract c = Gallic acid (mg/mL) concentration V = Volume (mL) of extract m = Extract weight in grams (g)
3.7.2. Determination of Total Flavonoid Content
The total flavonoid contents of bitter gourd were determined by the method proposed by Zou et al. (2004) using colorimetre. A small quantity (0.5ml) of sample solution was taken and interspersed in 2ml of H2O (distilled) and further add 5% sodium nitrate solution (0.15ml).
10% AlCl3 solution (0.15ml) was added after incubation of 6 min. The whole apparartus and
31 ingredients remain in constant position for 6 min. Solution of 4% NaOH (2ml) was mixed after some time. In addition, distilled water was poured into the solution to attain the final volume upto 5mL. The whole apparatus again place in a constant state for about 15 minutes. The absorbance of the resultant mixture was ascertained at a wavelength of 510 nm. The bitter gourd extract for total flavonoid content (in mg) was represented against rutin equivalents per gram of sample.
3.7.3. Free Radical Scavenging Activity (DPPH Assay)
The protocol of Muller et al. (2011) was used to measure the free-radical scavenging activity of bitter gourd extract by DPPH (1, 1- diphenyl - 2 - picrylhydrazyl). For this purpose, 4 mL of sample was taken and DPPH (1 mL) was added gradually to it. After that the solution was incubated at room temperature for 30 minutes. Ultraviolet / Visible Spectrophotometer (IRMECO, U2020) was used to note absorbance with wavelength set to 520 nm. Following expression was applied to calculate the DPPH free radical scavenging activity:
Reduction of absorbance (%) = [(AB - AA) / AB] × 100
AB = absorbance of blank sample (t = 0 min) AA = absorbance of tested extract solution (t = 15 min)
3.7.4. Ferric Reducing Antioxidant Power (FRAP)
The procedure of Benzie & Strain (1996) with slight modifications was applied to calculate
FRAP assay. Concisely, acetate buffer with pH maintained to 3.6 alongwith 20 mmol FeCl3 solution and TPTZ solution (10 mmol) in HCl (40 mmol) was used in ratios of 10:1:1 (v/v), respectively for preparation of FRAP reagent. For each sample, freshly prepared FRAP reagent was used and prior to its utilization in experimental work, warm it slightly in water bath maintain temperature to about 37oC. In the next step, 0.1 mL of sample was added to FRAP reagent (2.9 mL). After that, the sample was incubated for one hour. The absorbance was noted at wavelength of 593 nm of the mixture. Fe2(SO4)3 solution in amount of 200-1000 ppm was used to make standard curve and the results were expressed as mM/g crude extract. All measurements were carried out in triplicate and the readings were averaged.
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3.7.5. β Carotene Bleaching Assay
Taga et al., (1984) used coupled oxidations of linoleic acid and β - carotene through spectrophotometer. Bitter gourd extracts for their antioxidant activity was estimated based on the above procedure. The wavelength of the spectrophotometer was controlled to 470 nm.
Degradation rate of sample = In ( a / b ) × 1 / t Where, a = initial absorbance at wavelength 470 nm and zero time b = absorbance at wavelength 470 nm after interval of 40 minutes ln = natural logaritham t = time in minutes Antioxidant activity (AA) was expressed as % inhibition relative to control
3.7.6. ABTS (2,2‟-azino-bis, 3-ethylbenzothiazoline-6-sulfonic acid) Assay
The preparation of ABTS stock solution was performed by taking ABTS solution and pass it through filter paper on which MnO2 was coated. Further refine solution was obtained by using membrane filtration with pore size of 0.2 μm. Prior to analysis of the samples, the dilution of stock solution was carried out using phosphate buffered saline (PBS). This help to maintain the absorbance at wavelength of 734 nm to 0.70 ± 0.05. In ABTS assay, vortexes for 30 second in a reaction tube was performed using 100 μL carotenoid solution or sample extract and 600 μL ABTS working solution. Standard (4.5 – 114μmol α - T / L) or blank (n - hexane) were balso used in place of carotenoid solution. After that, transfer of mixture to the half micro-cuvettes was under taken. To separate the layers, centrifugation was performed for 30 seconds at 1000 g. Exactly two minutes after shaking, the lower layer absorbance was observed at wavelength of 734 nm in accordance with protocol of Böhm et al., (2002).
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3.8. Bioactive Compounds or Phytochemicals
Different preparation of bitter gourd extracts was analyzed to estimate the comparative abundance of bioactive molecules.
3.8.1. Total Saponin Contents
The total saponin contents were determined by the procedure Obadoni & Ochuko (2001). The 100 ml of 20% aqueous ethanol was taken and 1 g of sample has been added in it. Stir it thoroughly and then heated for about four hours at 45 °C with continuous stirring. The whatman filter paper was used to filter the mixture and the residue obtained was again extracted with fresh solution of 100 ml of 25% aqueous ethanol. The rotary evaporator at 40 °C was used to concentrate the sample to get 40 ml extract approximately. The concentrate was extracted twice with 20 ml diethyl ether by transferring into separator funnel. Then, the ether layer was removed and the aqueous layer was used to re-extract by adding 30 ml n- butanol. This extract was washed two times using 5% aqueous sodium chloride (10 ml). After that, evaporating of the remaining solution was done and constant weight was obtained after drying the samples in oven at temperature of 40°C till constant weight. Following epression was used to assess the saponin contents:
3.8.2. Charantin
The quantification of charantin was done by HPLC, following the procedure used by Pitipanapong et al. (2008). Initially, about 1.0g powder of each sample was extracted through ethanol and then to obtain charantin in the residue was extracted repeatedly with methanol. To obtain viscous crude extract, filtration and subsequent evaporation was done. The samples were purified before HPLC analysis. For this purpose, 5mL of methanol and water solution in ratio of 50 : 50 v/v was added and mixture was vertexed and centrifuged for 15 minutes at 3500 rpm. Later, precipitate was separated and the residues were again filtered with 5 mL of
34 methanol and water solution (70 : 30 v / v) followed by centrifugation. The precipitates thus obtained were added with hexane. The precipitates from this step were dissolved in methanol and chloroform mixture (1 : 1 v / v) and volume was made 2 mL. The filtration was carried out prior the HPLC analysis. HPLC analysis was performed with column (250 mm × 4.6 mm, 5.0 μm particle size). Mobile phase used was methanol-water (100:2 v/v). The UV injection detector was 204nm and sample injection volume was 200μl.
3.8.3. Total Alkaloid
To quantify alkaloids in bitter gourd samples, the method of Harborne (2005) was used with slight modifications. One gram dry powder of the sample was taken and 100 ml of 10% acetic acid in ethanol was mixed into it. The mixture was covered and kept for 4 hours. After that, filtration of extract was done with subsequent concentration on a water bath to 25 ml of its original volume. The concentrated ammonium hydroxide in the form of droplets was added to the extract till the whole precipitation in the solution settled. The dilute ammonium hydroxide was used to wash the precipitates and meanwhile filteration was done using whatman filter paper. The residues were then dried using oven at 40 °C and weight was determined. The total alkaloids in each sample were calculated using the following formula:
3.8.4. Momordicin I & II
The determination of momordicin contents were based upon the method of Beloin et al. (2005). The samples of different parts of bitter gourd were kept in 125 ml of 95% ethanol. After some hour‟s interval, the filteration and subsequent evaporation of the ethanolic extracts were carried out at 40 ◦C. From the samples, residual water contents were removed by freeze drying method. These extracts were dissolved in acetonitrile:MeOH (1:1) and filtered. Momordicins I and II contents of these extracts were analyzed by HPLC with column (125mm × 4 mm, 3μm).
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The water and MeCN were used as mobile phase with 1.5 ml per min of flow rate. The momordicins were eluted with a gradient of 30% changing to 50% MeCN in 10 min, 50% MeCN changing to 80% MeCN in 5 min and held at 80% for 5 min, recycled to 30% MeCN in 1 min and re-equilibrated for 9 min. The detection was achieved at 205 nm.
3.8.5. Vicine
The powdered samples of bitter gourd were extracted with 10 – 25 mL of water by ultra- sonification for 10 minutes, centrifugation of the mixture was done and supernatant was put into volumetric flask. All the samples were filtered before HPLC quantification. The column (250mm×4.6mm.) was packed with 5 mm mean particle size. Absorbance of the samples was monitored at 280 nm and 0.025 mol / L phosphate buffer having pH value of 3.0. The mobile phase used was methanol (90 : 10, v / v) (Zhang et al., 2003).
3.8.6. Polypeptide P
The bitter gourd samples were freezed and the freezed form of the samples was crushed using distilled water (10mL). Then, 95% ethanol (45 mL) and H2SO4 (3.6 mL) was added and mix them thoroughly after vigourous stirring for 15 to 20 minutes at temperature 25 °C to 28 °C. Distilled water (60 ml) was added further to obtain homogenized solution. After that, 95% ethanol separately added (20 mL), then filtered and ammonium hydroxide (28% v/v) was used to maintain the pH 3.0. 1.5 L of acetone was added to the flask and placed it in a steady state for 8 to 10 hours at temperature 5 °C (Khana et al., 1981).
3.9. Selection of Best Treatment
Considering the results of mineral analysis, antioxidant assays and bioactives concentrations, best form (Black King) from all cultivars were selected for product development.
3.10. Product Development (Functional Drink)
Bitter gourd fruits of best cultivar were selected in formation of bitter gourd juice. The fully matured, healthy and uniform size fruits were carefully selected for further experimentation.
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A known quantity of freshly harvested, fully matured bitter gourd fruits were washed and cleaned. The pulp was obtained by grinding and juice was extracted and filtered through muslin cloth. For the preparation of functional bitter gourd juice, six treatments were prepared (Table 3.1).
Table 3.1. Composition of different bitter gourd functional drinks (100g)
Formulations Ingredients
T0 T1 T2 T3 T4 T5
Bitter gourd extract (%) 0 5 7.5 10 12.5 15
Citric acid (%) 0.05 0.05 0.05 0.05 0.05 0.05
Aspartame (%) 0.07 0.07 0.07 0.07 0.07 0.07
Water (%) 99.88 94.88 92.5 89.88 87.38 84.88
Sodium Benzoate (%) 0.05 0.05 0.05 0.05 0.05 0.05
3.11. Physicochemical Assay of Bitter Gourd Juice
The resultant bitter gourd juice was analyzed for physic-chemical parameters on different storage days (0, 15, 30 and 45 days) in accordance with respective protocols described below.
3.11.1. Total Soluble Solids
Estimation about Total soluble solids (TSS) of bitter gourd juice was done with the help of Hand Refractometer (TAMCO, Model No. 90021, Japan) at different storage periods and written as per cent soluble solids.
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3.11.2. pH pH of the bitter gourd juice was made by pH meter (Ino Lab 720, Germany) after putting the juice in 50 mL beaker according to the method of AOAC (AOAC, 2006).
3.11.3. Acidity
AOAC (2006) guidelines were followed to determine the acidity of bitter gourd juice during storage days of 0, 15, 30 and 45 days. 0.1 N sodium hydroxide (NaOH) solution was used for titration against solution till arrival of persistent pink color.
3.11.4. Colour
CIE-Lab Color Meter (CIELAB SPACE, Color Tech - PCM, United States of America) was used to estimate the the colour of bitter gourd juices. For this purpose, color values like a* (– a greenness; + a redness), b* (– b blueness; + b yellowness) and L* (lightness) were recorded by taking 5 mL of each respective juice in a small laboratory beaker. The method of Duangmal et al. (2008) was used to compute chroma (C*) and hue-angle of the juices.
Chroma (C*) = [(a*) 2 + (b*) 2] 1/2
Hue angle (h) = tan - 1 (b*/a*)
3.11.5. Sensory Evaluation
Bitter gourd juices (T0, T1, T2, T3, T4 and T5) were sensory evaluated by trained experts, following the guidelines of Meilgaard et al. (2007) using 9 point hedonic scale considering 9 for “like extremely” and 1 for “dislike extremely”. Consequently, various quality traits of juices like color, aroma, taste, flavor and overall acceptability was documented considering sensory responses by the panel. Panelists were positioned in separate booths under suitable light and environmental conditions for evaluation in the Sensory Evaluation Laboratory, Institute of Home and Food Sciences at Government College University, Faisalabad. Random codes were assigned to panelists and each of them was served with chilled juices of bitter gourd in goblets for evaluation. Unsalted crackers and mineral water were also
38 presented to panelists during the process of evaluationso that they neutralize and rinsize their receptors associated with taste so as to complete assessment without any biased. Each panelist was asked to rate the quality of the product by writing score for each parameter.
3.12. Bioevaluation Studies
For bio-evaluation, bitter gourd skin, flesh and whole fruit powder were investigated for their anti-diabetic and anti-lipidemic perspective in 8 weeks study on rats feeding trial. Sprague Dawley rats (210) were procured from National Institute of Health, Islamabad and housed in the Animal Room of College of Pharmacy at Government College University, Faisalabad. Firstly, the rats were fed with basal diet for a period of one week so as to acclimatize the rats to the surrounding environment. The environmental conditions were maintained during the course of experiment, i.e. temperature 25 ± 3 ºC and relative humidity 52 ± 5 % with twelve hour light / dark spell. Some rats were euthanized at the commencement of trial to get the base-line values for the selected traits. In the bioevaluation study, seven groups of rats were formed in three different studies assigning 10 rats in each group to determine effect of bitter gourd against selected maladies. During the entire trial, bitter gourd formulations based feed was given to the respective groups.
3.12.1. Study I: Rats Fed with Normal Diet
Initially, efficacy trial was conducted in rats given normal diet. Throughout the trial, the rats were continuously observed for water intake, feed intake and gain in body weight. At the middle of study (28th day) half number of rats for each group were dissected by keeping fasting condition for night. The remaining rats for each group were decapitated at the end of trial (56th day). EDTA coated tubes were used for blood collection. Centrifugation of the blood was performed using centrifuge machine (Centrifugal Machine, Rotrofix 32-A Heltich, Germany) @ 4000 rpm for 6 min to separate the serum. Microlab (Rendox Toerauta RX- Monza, Republic of Ireland) was used to collect sera for biochemical evaluation. Respective commercial kits were used for determining glucose and insulin levels; total cholesterol, LDL, HDL and triglycerides in different samples.
Moreover, liver soundness tests including alkaline phosphatase (ALP), aspartate transferase (AST) and alanine transferase (ALT), and kidney functioning parameters like urea and
39 creatinine were determined for safety assessment. The results were subjected to statistical analysis to establish a sound conclusion.
Two other studies were conducted simultaneously, following similar approach, to determine the impact of bitter gourd against sucrose and cholesterol diet in separate rat modeling.
3.12.2. Study II: Hyperglycemic Rats
In study II, high sucrose diet containing 40% sucrose was given to the normal rats to induce diabetes and determine its effect on serum glucose and insulin level. At the same time, effect of bitter gourd on the induced trait in respective groups of rats was assessed.
3.12.3. Study III: Hyperlipidemic Rats
In study III, high fat diet containing 1.5% cholesterol was given to rats to raise their lipid profile and the effect of bitter gourd was noted on the induced trait.
3.13. Feed Plans for Experimental Rats
During the efficacy study, rats were divided into seven homogenous groups with ten rats in each group. For control group, experimental diet was prepared by using 10% cellulose, 10% protein, 10% corn oil, 66 % corn starch, 3% mineral and 1% vitamin mixture. Whereas, for
D1, D2, D3, D4, D5, D6, bitter gourd skin (150 and 300 mg/kg body weight), bitter gourd flesh (150 and 300 mg/kg body weight) and bitter gourd whole fruit (150 and 300 mg/kg body weight) were added, respectively in the aforementioned diet formulation (Table 3.2).
40
Table 3.2. Diet plan used in the studies Study I Study II Study III
(Normal diet) (High sucrose diet) (High cholesterol diet)
Groups 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7
Diet D0 D1 D2 D3 D4 D5 D6 D0 D1 D2 D3 D4 D5 D6 D0 D1 D2 D3 D4 D5 D6
D0: Control
D1: Diet containing bitter gourd skin powder 150 mg / kg of body weight
D2: Diet containing bitter gourd skin powder 300 mg / kg of body weight
D3: Diet containing bitter gourd flesh powder 150 mg / kg of body weight
D4: Diet containing bitter gourd flesh powder 300 mg / kg of body weight
D5: Diet containing bitter gourd whole fruit (WF) powder 150 mg / kg of body weight
D6: Diet containing bitter gourd whole fruit (WF) powder 300 mg / kg of body weight
3.14. Feed and Water Intake
Throughout the study period, the gross intake of feed for each group was monitored on daily basis. However, the spilled diet was excluded from diet during study period. The intake of water was also recorded on each day by observing the variations using graduated bottles.
3.15. Body Weight Gain
To note any impact after feeding bitter gourd formulations, the change in body weight was also monitored for each group on weekly basis to assess any positive or negative impact.
3.16. Hypoglycemic Perspectives
The concentration of glucose was determined by GOD-PAP method (Katz et al. 2000) for each group of rats at 4th and 8th week study interval. Similarly, the level of insulin in each group of rat was observed as described by Ahn et al. (2011).
41
3.17. Serum Lipid Profile
The lipid contents in the sera of each group with particular focus cholesterol, low density lipoproteins (LDL), high density lipoproteins (HDL) and triglycerides (TG) was performed at 4th & 8th week study interval as well. The detail about these parameters is described below.
3.17.1. Cholesterol
The procedure of Kim et al. (2011) was used to observe level of cholesterol in collected sera of different rat groups using liquid cholesterol CHOD–PAP method.
3.17.2. Low Density & High Density Lipoproteins (LDL & HDL)
Serum low density lipoproteins (LDL) were assessed by thye procedure of Kim et al. (2011). Accordingly, Cholesterol Precipitant method was followed to determine high density lipoproteins (HDL) (Alshatwi et al., 2010).
3.17.3. Triglycerides
GPO–PAP method was used to determine triglycerides in the collected sera by following the procedure of Kim et al. (2011).
3.18. Liver Functioning Tests
Liver soundness tests (ALP, AST and ALT) were assessed following the layout given by Basuny et al. (2009). Dinitrophenylhydrazene (DNPH) was used for determination of ALT and AST levels through Sigma Kits 58-50 and 59-50, respectively whereas; Alkaline Phosphatas-DGKC was used for assessment of ALP.
3.19. Kidney Functioning Tests
The serum samples were analyzed for urea by GLDH-method, whilst creatinine by Jaffe procedure via commercial kits to evaluate the kidney functioning (Jacobs et al., 1996; Thomas, 1998).
42
3.20. Weight of Body Organ
After the trial period, the mice were slaughtered and body organs like heart, lungs, kidney, liver, pancreas and spleen were also collected and weighed.
3.21. Statistical Analysis
The data were obtained by applying completely randomized design (CRD) and further subjected to statistical analysis using Statistical Package (Microsoft Excel 2010 and SPSS v20.). Level of significance was determined (ANOVA, LSD for comparison) using 2-factor factorial CRD where applicable following the principles outlined by Steel et al. (1997).
43
Chapter 4 RESULTS AND DISCUSSION
Vegetables intake is important for the human health owing to their nutritional and medicinal properties. Dietary intervention through the consumption of specialty foods may assuage the nutrition related disorders. Bitter gourd is one of such plants with indications that it can alleviate various lifestyle-related disorders including diabetes, hypercholesterolemia and obesity. There is a growing area of research to analyze genetically diverse cultivars of this plant with the purpose to explore the desirable variety with abundant nutritional, bioactive and functional compounds and appealing physiological properties. For this purpose, present research project was planned to explore the biochemical portrayal of different cultivars of bitter gourd and functional/nutraceutical worth of this plant against different lifestyle oriented disorders in experimental rat modeling. Different statistical analysis was performed on collected data to determine significant relationship. The results of different aspects of study alongwith discussion are described underneath.
4.1. Chemical Composition
Chemical assay is an important criterion to assess the overall composition and nutritional status of any ingredient intended for food utilization. In addition, development of designer foods and their acceptance rely on this aspect. In this context, skin, flesh, seeds and whole fruit of selected bitter gourd cultivars were analyzed for different quality attributes such as moisture, ash, crude protein, crude fat, crude fiber and nitrogen free extract (NFE).
Mean squares for chemical composition (Table 4.1) showed highly significant variations. Mean squares for moisture contents expounded highly significant differences among cultivars and parts. The mean values regarding moisture contents have been depicted in Table 4.2. The results indicated that BG 20 exhibited relatively high moisture content (7.82%) followed by KHBG-1 (7.03%), Black King (6.86%), FSD Long (6.75%), Noor (6.88%) and GHBG-1 (6.46%). The moisture contents were also significantly differed in various parts (Table 4.2). Considering the overall percentage of moisture composition in parts, it was the
44 highest in flesh (7.91%) followed by whole fruit (7.23%), skin (6.92%) and seed (5.82%). The moisture contents were in the range of 9.07±0.55% for flesh of BG 20 to 5.20±0.30% for seeds of GHBG-1.
The results are in accordance with the earlier findings of Saeed et al. (2010) who also reported low values for moisture contents in the peel, seed and flakes as 4.15±0.9%, 4.09±0.8% and 4.72±1.1%, respectively. Similarly, Gayathri, (2014) probed 6.14±0.03% moisture contents in the fruit of bitter gourd. Aslam et al. (2013) reported that bitter gourd contained moisture contents with value of 4.71±0.16%. Earlier, Hussain et al. (2009) observed 8.06±0.05% moisture contents in the fruit of bitter gourd on dry weight basis. In another study, moisture contents in different parts of bitter gourd plant were assessed by Bakare et al. (2010) and narrated that seeds possessed higher moisture contents (20.69 ± 5.85) than the fruit (10.74 ± 2.29). However, Mathew et al. (2014) reported 7.00% moisture contents in the seeds of bitter gourd. Ali et al. (2008) reported moisture contents ranged from 7.62 to 8.20% in the seeds of three selected cultivars. Significant differences were observed by Anjum et al. (2013) between seeds of two varieties in their contents of moisture i.e., 22.91 and 29.32%, respectively. On the other hand, Ullah et al. (2011) found out very high amount of moisture contents (91.6% to 92.92%) in four varieties of bitter gourd. In another study on different cultivars of bitter gourd, Islam et al. (2011) determined that flesh contained higher amount of moisture (92.4±0.2% to 93.5±0.2%). Whereas, the moisture content of seeds varied from 53% to 75%.
The low value of moisture contents in the present study was an indication that these samples can be preserved for a reasonable period of time without the risk of microbial deterioration and spoilage. The higher moisture contents in some studies are due to their determination on fresh weight basis.
Mean squares for ash illustrated that ash contents were significantly affected with reference to cultivars and their parts. The mean values of ash contents in different parts of bitter gourd cultivars are presented in Table 4.3. It is obvious from results that the Black King possessed higher amount of ash (3.18%) while the least amount was observed in Noor (1.85%). The ash contents for FSD Long, KHBG-1, BG 20 and GHBG-1 were 2.95%, 2.72%, 2.32% and 1.94%, respectively. The results revealed statistical variations in different parts as well. The
45 mean values concerning ash contents in parts elucidated higher amount for flesh (3.16%) followed by skin (2.71%), whole fruit (2.66%) and seed (1.45%).
The ash content of the plant materials is the reflection of total mineral elements in the plant. All the cultivars and parts possessed adequate amount of ash contents. The results of present study regarding ash contents are in close conformity with the findings of Gayathri (2014) who found 2.76±0.11% ash contents in bitter gourd. Saeed et al. (2010) found higher amount of ash than the present findings in peel (14.99±1.8%), seed (4.56±0.9%) and flakes (6.43±1.4%). They highlighted that different parts of bitter gourd possessed variable amount of ash. Similarly, Hussain et al. (2009) also reported higher amount of ash in bitter gourd (8.96±0.01). While Bangash et al. (2011) reported very low amount of ash contents (0.9±0.09%) in bitter gourd fruit than the current findings. Similarly Ullah et al. (2011) also reported low amount of ash in four selected cultivars ranged between 0.75-1.20 percent. In their study, varietal differences were more obvious regarding ash contents. According to them, variety Muricata contained higher amount of ash that the rest of the cultivars. Ali et al. (2008) estimated ash contents were to be in the range of 2.25–2.73% in the seeds of bitter gourd while in another study Mathew et al. (2014) reported 4.00% ash contents in the seeds of bitter gourd. Bakare et al. (2010) showed variation in amount of ash in fruit (7.36±0.52%) and seed (9.73±2.34%).
The statistical analysis revealed that crude protein composition was significantly influenced by variations in parts and cultivars. The mean values concerning amount of protein in selected bitter gourd cultivars were 14.90%, 16.02%, 24.84%, 15.45%, 24.48% and 25.63% for BG 20, Black King, FSD Long, KHBG-1 GHBG-1 and Noor, respectively (Table 4.4). Among parts the maximum value for protein was measured in flesh (24.40%) followed by skin (23.59%) and whole fruit (21.27%), whereas, minimum protein contents were observed in seeds (11.62%).
The present results revealed that bitter gourd is a potential rich source of protein and could be used in various food applications. The current results are in agreement with the findings
46
Table 4.1. Mean squares for proximate composition of different cultivars and parts
Crude Crude Crude S.O.V. df Moisture Ash NFE Protein Fat Fiber
Cultivars (C) 5 2.51** 3.57** 329.51** 7.15** 8.02** 184.17**
Parts (P) 3 13.71** 9.61** 623.86** 95.09** 1058.09** 215.77**
C × P 15 0.19N.S 0.25** 38.22** 4.27** 2.45** 62.56**
Error 48 0.14 0.025 0.21 0.08 0.05 0.44
Total 71
** = Highly significant N.S = Non-significant
Table 4.2. Mean values for moisture contents (%) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 7.97±0.31bcd 9.07±0.55a 6.23±0.35jkl 8.00±0.26bc 7.82a
Black King 6.90±0.20fghi 7.60±0.26bcde 5.93±0.32kl 7.03±0.38efgh 6.86b
FSD Long 6.40±0.56ijk 7.63±0.35bcd 5.63±0.42lm 7.37±0.15defg 6.75bc
KHBG-1 6.77±0.45ghij 8.10±0.30b 6.17±0.42jkl 7.10±0.26efgh 7.03b
GHBG-1 6.60±0.56hij 7.43±0.42cdef 5.20±0.30m 6.60±0.20hij 6.46c
Noor 6.87±0.31fghi 7.63±0.45bcd 5.73±0.59lm 7.27±0.32efg 6.88b
c a d b Means 6.92 7.91 5.82 7.23
47
Table 4.3. Mean values for ash (%) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 2.72±0.19f 2.86±0.01ef 1.31±0.13kl 2.39±0.06g 2.32d
Black King 3.18±0.18cd 4.39±0.07a 1.83±0.25j 3.33±0.13c 3.18a
FSD Long 3.10±0.12cde 3.60±0.18b 1.87±0.09ij 3.23±0.20cd 2.95b
KHBG-1 2.81±0.20f 3.71±0.27b 1.41±0.25k 2.98±0.15def 2.72c
GHBG-1 2.09±0.22hij 2.37±0.21g 1.19±0.07kl 2.13±0.09ghi 1.94e
Noor 2.34±0.12gh 2.06±0.07ij 1.11±0.07l 1.88±0.13ij 1.85e
Means 2.71b 3.16a 1.45c 2.66b
Table 4.4. Mean values for crude protein (%) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 16.11±0.71g 16.52±0.27g 12.04±0.70i 14.95±0.24h 14.90e
Black King 17.71±0.34f 17.87±0.62f 12.48±0.29i 16.03±0.25g 16.02c
FSD Long 27.01±0.59de 30.53±0.38c 14.47±0.34h 27.34±0.47d 24.84b
KHBG-1 18.19±0.51f 18.29±0.68f 9.06±0.35k 16.26±0.32g 15.45d
GHBG-1 30.40±0.62c 30.07±0.38c 11.14±0.04j 26.30±0.28e 24.48b
Noor 32.11±0.29b 33.14±0.48a 10.51±0.45j 26.76±0.70de 25.63a Means 23.59b 24.40a 11.62d 21.27c
48 of Hussain et al. (2009) reported comparable protein contents (21.12±0.08%) in bitter gourd. Almost similar result was reported for crude protein (15.56±0.18%) by Aslam et al. (2013) in bitter gourd. Likewise, Saeed et al. (2010) also observed similar amount of protein in peel and flakes (20.37±1.9% and 20.66±2.0%, respectively) while higher amount was observed in case of seed (19.01±1.8%). Mathew et al. (2014) also reported higher amount of crude protein (19.50%) in bitter gourd seed. Substantial differences were observed by Anjum et al. (2013) between seeds of two varieties in their total protein values i.e., 14.92±0.61 and 19.17±0.55%. Contrary to this, Ullah et al. (2011) assessed four varieties for nutritional analysis and found total protein in the range of 1.17-2.4%. Among the different varieties examined, the highest amount of protein was recorded in Muricata (2.4±0.02%) followed by variety charantia (1.58±0.02%).
Gayathri (2014) reported crude proteins in bitter gourd with value of 27.88±0.19 mg/100g. In most of the studies, flesh and edible portion is rich in protein but Islam et al. (2011), explicated that the protein contents are higher in the seeds as compared to flesh. Some of the varieties showed higher protein contents than the others in the current study. That might be due to varietal differences as Islam et al. (2011) reported that white varieties has higher protein contents than the green varieties.
Mean squares regarding crude fat indicated highly significant differences in different cultivars and parts. The results pertaining to mean values for crude fat in different cultivars and parts of bitter gourd are depicted in Table 4.5. It is obvious from the results that the maximum amount of fat was found in Black King (10.13%) and minimum in Noor with value of 7.86%. The mean values for crude fat in other cultivars were 8.89% (BG 20), 8.77% (FSD Long), 9.46% (KHBG-1) and 8.63% (GHBG-1). Among parts, crude fat was found significantly higher in seed (12.14%) followed by whole fruit (9.15%), flesh (7.35%) and skin (7.19%).
The results regarding crude fat of present research work are corroborated with the work of Bakare et al. (2010) who analyzed different parts of bitter gourd and found almost similar amounts of crude fat in flesh (6.11±0.42%) and the seed (11.50±1.77%). However, Saeed et al. (2010) evaluated bitter gourd parts and found crude fat in lesser quantity in all parts including peel (0.18±0.2%), seed (5.24±1.2%) and flakes (0.25±0.2%). Mathew et al. (2014)
49 reported very high crude fat percentage (34.00%) in the seeds of bitter gourd. Higher amount of fat was also noted by Aslam et al. (2013) with value of 26.67±0.15 in the fruit of bitter gourd. Ali et al. (2008) reported nutritive compositions in seeds of three selected cultivars. They reported significant differences in the presence of lipids among the cultivars.
The data pertaining to mean squares for crude fiber elucidated that fiber contents differed significantly among different parts and cultivars. It is evident from the data that crude fibers were present in variable concentrations among different cultivars (Table 4.6). The maximum amount of crude fiber i.e., 8.47% was observed in Black King, followed by 8.42%, 7.09%, 7.07%, 6.96%, and 6.53% for BG 20, Noor, KHBG-1, GHBG-1 and FSD Long, respectively. It is also clear from the data that maximum fiber contents were present in seed part that was 18.61% followed by whole fruit (6.17%) and flesh (2.74%), while, the lowest amount of 2.17 % was recorded for the skin part of bitter gourd fruit.
The crude fiber in bitter gourd is valuable and this could be beneficial when consumed in diet. It is known to reduce the risk of diseases such as obesity, diabetes, breast cancer, hypertension and gastrointestinal disorder (Saldanha, 1995). It was also noted that seed part possessed higher amount of fiber contents than the others. These results are in accordance with the results of Saeed et al. (2010). They depicted higher amount of crude fiber in seeds (22.46±2.3%) followed by peel (17.77±1.8%) and flakes (17.08±1.9%). In another study, Mathew et al. (2014) determined higher amount of crude fiber (12.00%) in the seeds of bitter gourd. The crude fiber contents in the present research are in close conformity in a recent research work of Gayathri (2014) with reported value for bitter gourd fruit as 2.31± 0.23. In another study on different cultivars of bitter gourd seeds, very low fiber contents (1.02±0.07- 1.20±0.15) were observed. Bangash et al. (2011) determined 1.4±0.07g/100g crude fiber contents in bitter gourd. Earlier, Hussain et al. (2009) analyzed bitter gourd for fiber contents and found higher amount in the fruit of bitter gourd (16.62±0.02%). In another study, fiber contents were found lesser in seeds of two bitter gourd varieties with the values of 10.03±0.51 and 11.07±0.61.
50
Table 4.5. Mean values for crude fat (%) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 6.60±0.17lm 6.50±0.12lm 14.19±0.42a 8.31±0.36hi 8.89c
Black King 7.70±0.18jk 7.79±0.18jk 14.28±0.25a 10.76±0.47d 10.13a
FSD Long 7.60±0.08jk 7.41±0.24k 11.48±0.26c 8.60±0.41gh 8.77cd
KHBG-1 6.89±0.45l 7.47±0.27k 13.50±0.03b 9.97±0.40e 9.46b
GHBG-1 7.95±0.09ij 8.42±0.23h 9.23±0.09f 8.93±0.35fg 8.63d
Noor 6.42±0.32m 6.52±0.21lm 10.15±0.14e 8.34±0.30hi 7.86e
Means 7.19c 7.35c 12.14a 9.15b
Table 4.6. Mean values for crude fiber (%) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 2.24±0.11j 3.83±0.09i 21.42±0.17a 6.20±0.58g 8.42a
Black King 3.81±0.13i 4.02±0.07i 19.07±0.20b 6.99±0.21f 8.47a
FSD Long 1.21±0.11m 1.84±0.06kl 16.51±0.28e 6.55±0.16g 6.53c
KHBG-1 2.34±0.11j 2.28±0.11j 17.11±0.37d 6.56±0.12g 7.07b
GHBG-1 1.55±0.12lm 2.33±0.21j 18.70±0.30c 5.25±0.11h 6.96b
Noor 1.86±0.11kl 2.15±0.08jk 18.89±0.20bc 5.49±0.43h 7.09b
Means 2.17d 2.74c 18.61a 6.17b
51
Mean squares for NFE exhibited highly significant differences regarding cultivars and parts. The mean values explicated that different cultivars showed variations regarding NFE (Table 4.7). It is clear from the results that among selected bitter gourd cultivars, BG 20 possessed the highest amount of NFE (65.45%) followed by KHBG-1 (65.29%), Black King (62.03%), GHBG-1(57.99%), Noor (57.57%) and FSD Long (56.91%). The results regarding NFE in different parts of bitter gourd explicated that maximum amount was present in skin (64.34%) followed by flesh (62.23%), whole fruit (60.74%) and seed (56.18%).
The nitrogen free extract is basically the indication of presence of carbohydrates. All the parts possessed higher amount of NFE. The results of present research are in accordance with the earlier findings of Saeed et al. (2010). According to them, peel, seed and flakes contained higher values for carbohydrates (42.54±2.7%, 44.64±2.8%, 50.86±2.9%, respectively). The higher amount of carbohydrate in bitter gourd (85.41mg/100g) was also determined by Gayathri et al. (2014). Hussain et al. (2009) also found similar amount of carbohydrate in bitter gourd with value of 56.02±0.09. Slightly lesser amount of carbohydrates (43.20±0.35%) in fruits of bitter gourd was reported by Aslam et al. (2013). The variations in presence of carbohydrates in different cultivars were also reported in a study by Ali et al. (2008). They reported carbohydrates in the range of 32.51-35.52 in different cultivars.
The results for chemical composition in the present research work showed differences from some past literature. The probable reasons for these variations might be due to selection of different cultivars, environmental factors, soil conditions, agricultural practices, time of harvest and postharvest storage conditions.
4.2. Macro and Micro Mineral Analysis
Vegetables are very rich source of valuable minerals including palpable concentrations of trace minerals. The assessment of these minerals is often become essential in many diet plan strategies. In this regard, selected bitter gourd cultivars and their different parts were analyzed for assessing these elements. Analysis of variance (ANOVA) regarding macro and micro mineral profile exhibited highly significant results for potassium (K), phosphorous (P), magnesium (Mg), sodium (Na), calcium (Ca), iron (Fe) and zinc (Zn) in different cultivars of bitter gourd and parts (Table 4.8).
52
Table 4.7. Mean values for crude NFE (%) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 72.34±0.59a 70.29±0.41b 51.04±1.19o 68.15±0.38c 65.45a
Black King 67.60±0.23c 65.27±1.73d 52.34±0.49n 62.90±0.25e 62.03b
FSD Long 61.09±0.63f 56.62±0.78jkl 55.67±0.64l 54.28±0.07m 56.91d
KHBG-1 69.76±0.11b 68.25±1.03c 58.92±0.66gh 64.23±0.18d 65.29a
GHBG-1 58.00±0.29hi 56.82±0.27jk 59.75±0.34g 57.39±0.36ij 57.99c
Noor 57.28±0.13ij 56.13±0.21kl 59.34±0.38g 57.53±1.26ij 57.57c
Means 64.34a 62.23b 56.18d 60.74c
53
The mean values regarding potassium contents in different parts of bitter gourd cultivars have been depicted in Table 4.9. It is obvious from the results that the highest potassium contents were found in Black King (258.83 mg/100g). All the parts of this cultivar are rich source of potassium including skin (326.33±1.53 mg/100g), flesh (397.00±4.58 mg/100g), seed (37.33±1.53 mg/100g) and whole fruit (274.67±4.16 mg/100g) than the rest of the cultivars. The skin, flesh and whole fruit showed non momentous differences in their potassium contents while inconsequentially the lowest amount was measured in the seed. The values for potassium in BG 20, FSD Long, KHBG-1, GHBG-1, and Noor were 217.50 mg/100g, 235.42 mg/100g, 176.83 mg/100g, 158.83 mg/100g and 158.83 mg/100g, respectively. It was also revealed from the data (Table 4.9) that the highest potassium contents (291.89 mg/100g) were observed in flesh followed by skin and whole fruit which were 261.78 mg/100g and 227.11 mg/100g, respectively. Moreover, seed contained the least amount of potassium (25.61 mg/100g).
The results regarding potassium contents in pulp of some varieties were in close conformity with the previous findings of Bangash et al. (2011) in which they reported higher amount of potassium (390±0.08 mg/100g) in bitter gourd. Similarly, Islam et al. (2011) also reported higher potassium contents (260 mg//100g) in the edible portion of bitter gourd. Aslam et al. (2013) explicated that the potassium contents in bitter gourd were 155.6 ppm. In another study, Horax et al. (2010) reported comparable results that potassium contents were much higher in pericarp than the seeds. However, recent investigation by Mathew et al. (2014) reported very low amount of potassium (3.17 mg/100g) in the seeds of bitter gourd than the present results.
The mean values pertaining to phosphorus contents in different cultivars and parts (Table 4.10) unveiled that the highest amount of phosphorus (92.92 mg/100g) was present in Black King followed by FSD Long (84.50 mg/100g), BG 20 (74.74 mg/100g), KHBG-1 (68.50 mg/100g), Noor (59.58 mg/100g) and GHBG-1 (52.08 mg/100g). Among parts, phosphorous contents were significantly higher in flesh (98.22 mg/100g) followed by skin (88.78 mg/100g), whole fruit (80.72 mg/100g) and seed (20.49mg/100g). Black King was found to be dominated variety regarding potassium contents with the highest values of 128.67±0.58 mg/100g and 125.00±2.65 mg/100g for flesh and skin, respectively. The amount of
54 phosphorous is considerably lower in seeds of all cultivars and minimum value (14.33±1.15 mg/100g) was noted for the seeds of Noor.
The results regarding phosphorous contents are slightly different from the values given by Jayasinha et al. (1999) who found 38-70 mg of phosphorous per 100 gram of edible portion in small and normal size fruits of bitter gourd. Mathew et al. (2014) reported lower value of phosphorous (11.09±0.30 mg/100g) in the seeds of bitter gourd than the current findings. This might be due to varietal differences as Ali et al. (2008) also showed varietal differences regarding phosphorous contents in the seeds of three bitter gourd cultivars. According to them, phosphorous contents were in the range of 134.65±1.59 μg/g-142.39±2.39 μg/g in different cultivars. Similar findings were also reported by Anjum et al. (2013) by comparing seeds of two cultivars and found variations regarding phosphorus contents with values of 135.92 ± 3.86 μg/g and 146.19 ± 3.04 μg/g, respectively for seeds of different cultivars.
The statistical analysis regarding means values of magnesium in different bitter gourd cultivars and parts are presented in Table 4.11. The results elaborated that all the cultivars contained adequate amount of magnesium with the highest amount of 46.74 in Black King. The mean values for BG 20, FSD Long, KHBG-1, GHBG-1 and Noor were 40.02 mg/100g, 40.53 mg/100g, 33.61 mg/100g, 35.23 mg/100g and 37.22 mg/100g, respectively. The variation related to magnesium contents were also noted in different parts with the highest value (56.11 mg/100g) in the flesh followed by whole fruit (50.33 mg/100g), skin (45.89 mg/100g) and seed (3.23 mg/100g).
The present results about presence of magnesium in bitter gourd fruits are in harmony with the findings of Bangash et al. (2011) showing 31.00±0.10 mg/100g magnesium in this vegetable. In another report, Islam et al. (2011) described the lower magnesium concentration (16 mg/100g) in bitter gourd. The results are further strengthened by the work of Mathew et al. (2014) portraying lesser amount of magnesium (3.50 mg/100g) in the seeds of this bitterly taste fruit. On the other hand, previous findings by Ullah et al. (2011) on fruits of four cultivars of bitter gourd showed considerably lower magnesium contents with values in the range of 0.99-1.10mg%.
55
Table 4.8. Mean squares for macro and micro mineral analysis of different cultivars and parts
S.O.V. df K P Mg Na Ca Fe Zn
Cultivars (C) 5 20807** 2794.4** 263.2** 173.0** 832.82** 1.13** 0.31**
Parts (P) 3 260378** 22187.9** 10487.4** 24737.4** 9606.67** 7.37** 16.83**
C × P 15 2374** 428.9** 60.8** 79.1** 101.93** 0.48** 0.26**
Error 48 14 8.0 8.2 5.4 5.71 0.03 0.01
Total 71
** = Highly significant
56
Table 4.9. Mean values for K (mg/100g) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 284.33±2.08e 301.67±2.31d 26.67±0.58pq 257.33±3.06g 217.50c
Black King 326.33±1.53c 397.00±4.58a 37.33±1.53o 274.67±4.16f 258.83a
FSD Long 303.67±4.73d 349.67±5.69b 28.00±1.73p 260.33±3.79g 235.42b
KHBG-1 225.67±6.43j 245.67±4.51h 21.67±3.51qr 214.33±3.06k 176.83d
GHBG-1 203.67±5.51l 224.33±4.04j 24.33±1.53pq 183.00±3.61m 158.83f
Noor 227.00±4.36ij 233.00±4.58i 15.67±3.51r 173.00±3.00n 162.17e
Means 261.78b 291.89a 25.61d 227.11c
Table 4.10. Mean values for P (mg/100g) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 96.33±0.58de 104.67±1.15c 17.63±0.42pq 80.33±2.08gh 74.74c
Black King 125.00±2.65a 128.67±0.58a 18.67±2.52opq 99.33±1.53d 92.92a
FSD Long 106.67±4.16c 116.00±2.65b 22.33±2.89o 93.00±2.65ef 84.50b
KHBG-1 76.00±3.61hi 91.33±4.04f 28.67±2.52n 78.00±2.65gh 68.50d
GHBG-1 55.67±3.51m 67.00±4.36kl 21.33±2.08op 64.33±2.31l 52.08f
Noor 73.00±3.00ij 81.67±5.51g 14.33±1.15q 69.33±2.52jk 59.58e
Means 88.78b 98.22a 20.49d 80.72c
57
Means for the sodium contents (Table 4.12) revealed that the maximum amount (61.81 mg/100g) was present in KHBG-1 followed by GHBG-1(58.97 mg/100g), Black King (58.24 mg/100g), BG 20 (57.61 mg/100g), FSD Long (54.22 mg/100g), whilst the minimum value (51.05 mg/100g) was observed in Noor. Among different parts, flesh contained the highest amount of sodium (84.11 mg/100g) than the skin (73.28 mg/100g), whole fruit (68.28 mg/100g) and seed (2.27 mg/100g). Overall, maximum value of sodium was observed in the pulp of Black King with the value of 91.00±1.73 mg/100g and the least amount in the seeds of KHBG-1 i.e., 1.57±0.25 mg/100g.
The instant results are well supported by the findings of Mathew et al. (2014), reported similar amount of sodium in the seeds of bitter gourd (2.10±0.00 mg/100g) but present results showed variations with the findings of Bangash et al. (2011) who reported lesser amount of sodium (31±0.03 mg/100g) in fruit of bitter gourd. According to the results by Horax et al. (2010), seed part contained lower amount of sodium than the flesh part. Aslam et al. (2013) found lesser amount of sodium in bitter gourd with value of 7.4 ppm. Hussain et al. (2009) also depicted lesser amount of sodium 45.47 ppm in fruit part of this plant.
The statistical analysis illustrated that calcium contents were significantly varied in different cultivars and their parts (Table 4.13). The maximum amount of calcium (70.33±1.53 mg/100g) was observed in pulp of Black King followed by peel of this cultivar (62.33±2.52 mg/100g). Significantly higher mean value was also calculated for Black King (48.42 mg/100g) than the rest of the cultivars. The second prominent cultivar was found to be FSD long with regard to possession of calcium content (41.44 mg/100g) followed by GHBG-1 (39.69 mg/100g), KHBG-1 (32.08 mg/100g), BG 20 (30.42 mg/100g) and Noor (25.82 mg/100g). Furthermore, Table 4.13 indicated that the parts also differed significantly with higher values in flesh (53.42 mg/100g), skin (45.56 mg/100g) and whole fruit (44.06 mg/100g) than the seed (2.21 mg/100g).
The results of present mineral analysis regarding calcium are in agreement with the findings of Bangash et al. (2011), finding 45±0.12 mg/100g calcium in bitter gourd. On the other hand, higher amount of calcium (137.69 mg/100g) was assessed by Somroo and Ansari (2005) in whole fruit of bitter gourd. Ullah et al. (2011) quantified calcium in different cultivars and found wide variations regarding calcium contents. They reported calcium
58 contents in the range of 0.540.30-7.00±0.25 mg% in these cultivars. Mathew et al. (2014) found out that calcium in the seeds of bitter gourd were 3.69±0.00 mg/100g which is in close conformity of the present results. Gayathri (2014) reported lesser amount of calcium (22.4 ±0.06 mg/100g) in bitter gourd than the other studies. Aslam et al. (2013) also reported lesser amount of calcium in bitter gourd with value of 35.2ppm. Anjum et al. (2013) studied seeds of two bitter gourd cultivars and reported relatively large quantities of calcium (425.08 and 399.81μg/g) than other minerals. Earlier, Ali et al. (2008) reported calcium contents in the seeds of three cultivars of bitter gourd in the range of 383.45-440.96 μg/g.
Means in Table 4.14 indicated that maximum iron contents were present in GHBG-1 (4.00 mg/100g) followed by Black King (3.90 mg/100g), KHBG-1 (3.50 mg/100g), Noor (3.38 mg/100g), BG 20 (3.34 mg/100g) and FSD Long (3.28 mg/100g). Bitter gourd parts possessed variability regarding iron contents with the highest amount (4.44 mg/100g) in the seed than the skin, flesh and whole fruit with values of 2.94 mg/100g, 3.29 mg/100g and 3.59 mg/100g, respectively.
The present data regarding iron in bitter gourd cultivars are correlated with the study of Jayasinha et al. (1999) who compared normal and small sized fruit of bitter gourd for iron contents and found 1.8 mg/100g and 2.0 mg/100g of iron in large and small fruits, respectively. Slightly higher value was reported by Bangash et al. (2011) while studying bitter gourd fruit and their reported value for iron contents were 7±0.07 mg/100g. However, in a recent study by Gayathri (2014) only 0.45±0.16 mg/100g of iron in the fruit of bitter gourd was observed. Earlier, Islam et al. (2011) also analyzed bitter gourd for iron contents and found 0.9 mg/100g in edible portion of bitter gourd. Higher values for iron contents in the seed portion were highlighted by a number of studies. Mathew et al. (2014) reported 22.44±0.04 mg/100g of iron in the seeds of bitter gourd. Ali et al. (2008) reported iron contents of 41.10±1.13 μg/g, 42.57±1.47 μg/g and 45.03±1.23 μg/g in their analysis on seeds of three cultivars of bitter gourd. Similar findings were reported by Anjum et al. (2013) on the seeds of two bitter gourd cultivars and reported iron contents of 49.21 ± 1.02 μg/g and 43.06 ±1.04 μg/g. Aslam et al. (2013) also reported iron components in bitter gourd with value of 167.6 ppm. In a similar study, Hussain et al. (2009) reported 139 ppm iron value in fruit of bitter gourd.
59
Table 4.11. Mean values for Mg (mg/100g) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 41.67±1.53gh 60.33±2.52ab 3.73±0.15i 54.33±2.52cd 40.02b
Black King 56.33±1.53bc 64.67±3.06a 4.63±0.25i 61.33±2.52a 46.74a
FSD Long 51.00±2.65de 54.33±0.58cd 3.47±0.31i 53.33±6.81cd 40.53b
KHBG-1 45.67±3.06fg 45.67±3.21fg 2.77±0.15i 40.33±1.53h 33.61d
GHBG-1 41.33±6.11gh 51.33±5.03de 2.27±0.06i 46.00±2.65fg 35.23cd
Noor 39.33±2.52h 60.33±1.53ab 2.53±0.21i 46.67±3.06ef 37.22c
Means 45.89c 56.11a 3.23d 50.33b
Table 4.12. Mean values for Na (mg/100g) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 76.67±2.08g 81.67±3.21cd 3.10±0.20j 69.00±2.65h 57.61b
Black King 71.67±2.52h 91.00±1.73a 2.30±0.46j 68.00±2.00h 58.24b
FSD Long 62.67±4.93i 88.33±1.15ab 2.87±0.45j 63.00±2.65i 54.22c
KHBG-1 83.33±4.16cd 85.00±2.65bc 1.57±0.25j 77.33±2.52fg 61.81a
GHBG-1 81.33±0.58cde 81.00±2.65def 1.90±0.20j 71.67±2.52h 58.97b
Noor 64.00±2.65i 77.67±2.52efg 1.87±0.42j 60.67±2.08i 51.05d
b a d c Means 73.28 84.11 2.27 68.28
60
Table 4.13. Mean values for Ca (mg/100g) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 38.67±1.53hi 44.67±0.58g 2.66±0.02k 35.67±3.51i 30.42c
Black King 62.33±2.52b 70.33±1.53a 1.67±0.08k 59.33±2.52bc 48.42a
FSD Long 53.67±2.52de 59.17±1.26bc 2.24±0.11k 50.67±2.52ef 41.44b
KHBG-1 36.67±2.08hi 48.67±2.52f 2.65±0.12k 40.33±2.08h 32.08c
GHBG-1 50.33±4.04ef 57.33±4.16cd 2.09±0.05k 49.00±3.61f 39.69b
Noor 31.67±2.52j 40.33±4.73h 1.93±0.07k 29.33±1.53j 25.82d
Means 45.56b 53.42a 2.21c 44.06b
Table 4.14. Mean values for Fe (mg/100g) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 2.57±0.15n 2.63±0.25mn 4.56±0.08b 3.61±0.20ef 3.34c
Black King 3.27±0.15ghijk 3.30±0.53fghij 5.10±0.18a 3.92±0.09cd 3.90a
FSD Long 2.97±0.25kl 2.93±0.15lm 4.04±0.10c 3.19±0.02ijkl 3.28c
KHBG-1 2.40±0.26n 3.17±0.06ijkl 4.91±0.21a 3.51±0.12efgh 3.50b
GHBG-1 3.43±0.12efghi 4.03±0.23c 4.45±0.09b 4.09±0.02c 4.00a
Noor 3.03±0.21jkl 3.67±0.15de 3.58±0.09efg 3.24±0.09hijkl 3.38bc
Means 2.94d 3.29c 4.44a 3.59b
61
Mean values for zinc in different cultivars of bitter gourd and parts are presented in Table 4.15. Maximum quantity of element zinc was observed in case of Black King (1.78 mg/100g) followed by FSD Long (1.50 mg/100g), BG 20 (1.47 mg/100g), KHBG-1 (1.41 mg/100g), GHBG-1 (1.35 mg/100g) and Noor (1.35 mg/100g). The results regarding zinc contents in different parts showed that seed possessed the highest amount (2.80 mg/100g) than whole fruit (1.58 mg/100g), flesh (0.84 mg/100g) and skin (0.68 mg/100g).
The results of present study regarding zinc contents are in agreement with the findings of Bangash et al. (2011) who reported 0.85±0.08 mg/100g of iron in bitter gourd fruit. However, Ismail et al. (2011) calculated amount of zinc in bitter gourd and found lesser amount (0.4 mg/100g) in fruit of this plant. Very low amount of iron (0.1 mg/100g) was also documented by Islam et al. (2011) while studying fruits of bitter gourd. The present results regarding value of iron in seeds is in accordance with the finding of Mathew et al. (2014) who reported higher amount of zinc (3.45±0.30 mg/100g) in the seeds of bitter gourd. Earlier, Anjum et al. (2013) reported 12.91±0.29 and 10.88±0.31 in the seeds of two cultivars of bitter gourd. Horax et al. (2010) showed higher amount of zinc in pericarp (33-57 μg/g) than the seed (12-22 μg/g).
Bitter gourd was found to be valuable source of macro and micro minerals. Number of studies indicated that a diet rich in potassium favours lowering of blood pressure. Increase in dietary potassium is helpful in reducing blood pressure. A diet lower in sodium and higher in potassium was considered to be suitable against developing of cardio vascular diseases (Umesawa et al., 2008). Deficiency of phosphorous and calcium may lead to abnormalities in the skeleton system including bowlegs, knock knees, deformities in the thoracic and pelvic region, curvature of the spine, hence important for bones (Moe, 2008). Magnesium plays important role in the structure and the function of the human body. Iron is a key element in the metabolism of almost all living organisms. In humans, iron is an essential component of hundreds of proteins and enzymes (Paul et al., 2013). Zinc plays a critical structural role (King & cousins, 2006). The structure and function of cell membranes are also affected by zinc. Loss of zinc from biological membranes increases their susceptibility to oxidative damage and impairs their function. It is needed for the body's defensive system to properly work. It plays a role in cell division, cell growth, wound healing, and the breakdown of
62 carbohydrates. Zn is considered to play a role as cofactor in diabetes and enhancing the activity of insulin (Tang & Shay, 2001). Mineral analysis indicated that bitter gourd contained adequate amount of these minerals. The variations in the amounts of certain minerals from previous findings might be due to varietal differences, environmental factors, agricultural practices, application of fertilizers, ripening stage and postharvest storage.
63
Table 4.15. Mean values for Zn (mg/100g) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 0.86±0.02hij 0.92±0.03hi 2.66±0.04c 1.44±0.10f 1.47bc
Black King 0.94±0.06h 0.97±0.02h 3.52±0.13a 1.69±0.06e 1.78a
FSD Long 0.66±0.03kl 0.74±0.03jk 3.00±0.12b 1.60±0.06e 1.50b
KHBG-1 0.56±0.09lm 0.66±0.07kl 2.95±0.07b 1.46±0.08f 1.41cd
GHBG-1 0.65±0.07kl 0.79±0.04ij 2.69±0.04c 1.27±0.06g 1.35d
Noor 0.44±0.09m 0.96±0.08h 2.00±0.16d 1.26±0.13g 1.35d
Means 0.68d 0.84c 2.80a 1.58b
64
4.3. Antioxidant Indices of Bitter Gourd Extracts
Statistical results of regarding antioxidant assays exhibited highly significant response for extract of different bitter gourd cultivars and parts. In addition, interaction of treatment and solvents also revealed highly significant results (Table 4.16). It is obvious from the results that significant differences existed with regard to total phenolic contents (TPC) in different treatments and parts. Mean values for total phenolic contents (Table 4.17) revealed that T3 (water extract of Black King) showed the highest TPC (349.14 mg GAE/100g) followed by
T5 (water extract of FSD Long) with value of 337.98 mg GAE/100g. TPC for other treatments were in the range of 253.47 mg GAE/100g for T12 (methanolic extract of Noor) to
317.24 mg GAE/100g for T4 (methanolic extract of Black King). Overall, water extracts showed higher TPC than the methanolic extracts. With regard to parts, maximum phenolic contents (364.56 mg GAE/100g) were noted in flesh, followed by skin (363.19 mg GAE/100g), whole fruit (273.77 mg GAE/100g) and seed (183.60 mg GAE/100g) extract.
The results of instant investigation are comparable with the findings of Kubola and Siriamornpun (2008), they illuminated that TPC values for extract of green fruit and ripe fruit of bitter gourd were 324 ± 1.63 and 224 ± 0.86 mg GAE/100g of dry sample, respectively. The results are also in harmony with the findings of Zulkifli (2012), who analyzed total phenolic contents in ripe, immature and mature stages of bitter gourd fruit and found total phenolic contents of 558.56±27.03 mg GAE/100g, 351.35±27.03 mg GAE/100g and 351.3±31.21 mg GAE/100g in these parts, respectively. Moreover, higher TPC value in flesh part in the present study than the seed is in harmony with the work of Islam et al. (2011), they elaborated that the phenolic contents of the flesh were significantly higher than those of the seed coat tissue and seed. They further reported that the phenolic contents of the seed were lesser in amount than the rest of the tissues. The total phenolic contents are variably present in different cultivars and this verdict was also in accordance with the findings of Islam et al. (2011), revealed significant variations in TPC in different cultivars.
Lin and Tang (2007) found low amount (143.6±8.4 mg GAE/100g) of total phenolic contents in bitter gourd while Ng et al. (2011) noticed higher amount (582.9±47.9 mg GAE/100g) of TPC in bitter gourd fruit. The variation in TPC might be due to selection of fruit of different
65
Table 4.16. Mean squares for antioxidant indices of different treatments and parts
S.O.V. df TPC TFC DPPH FRAP β-carotene ABTS
Treatment (T) 11 10987** 2443.2** 855.6** 638.55** 436.29** 209.71**
Parts (P) 3 263808** 57240.5** 12611.1** 4489.51** 2740.46** 2263.98**
T × P 33 1385** 246.4** 78.5** 23.66** 37.03** 11.61**
Error 96 11 8.8 5.2 5.83 4.85 4.05
Total 143
** = Highly significant
66
Table 4.17. Mean values for total phenolic contents (mg GAE/100g) of water and methanolic extract of BG cultivars and parts
Parts Treatments Means Skin Flesh Seed Whole fruit
T1 368.69±6.11g 368.38±4.58g 192.13±2.08wx 286.23±2.52p 303.86e
T2 318.30±2.08jk 323.72±5.51i 174.77±1.53y 256.27±3.00t 268.26i
T3 441.20±1.73a 441.27±2.52a 200.10±1.73u 313.99±4.36lm 349.14a
T4 394.43±4.51e 401.33±2.52d 174.47±2.52yz 298.73±2.52no 317.24c
T5 429.11±4.58a 423.21±2.52b 197.40±2.65vw 302.23±3.61n 337.98b
T6 394.00±6.00e 388.55±4.16f 177.87±2.31y 276.78±3.06q 309.03d
T7 386.57±5.51f 388.20±2.52f 195.93±4.16vw 269.97±4.62r 310.17d
T8 370.01±4.00g 372.28±3.21g 174.21±2.52y 252.23±3.61tu 292.18f
T9 332.62±3.06h 330.17±2.08h 193.00±1.73wx 269.10±2.65r 281.22g
T10 309.07±2.00m 316.28±1.53kl 162.27±2.89z 249.02±1.53u 259.16j ijk ij x s h T11 319.12±1.73 323.39±2.31 189.45±1.53 261.61±2.31 273.39
T12 295.21±2.65o 298.00±2.65no 171.62±2.08z 249.04±3.46u 253.47k Means 363.19a 364.56a 183.60c 273.77b
T1 = Water extract of BG 20 T7 = Water extract of KHBG-1 T2 = Methanolic extract of BG 20 T8 = Methanolic extract of KHBG-1 T3 = Water extract of Black King T9 = Water extract of GHBG-1 T4 = Methanolic extract of Black King T10 = Methanolic extract of GHBG-1 T5 = Water extract of FSD Long T11 = Water extract of Noor T6 = Methanolic extract of FSD Long T12= Methanolic extract of Noor
67 variety by these authors. In addition, sample preparation method by these authors also differed from the present work. In addition, the solvent used for extraction also played contributing role in assessment of TPC as in the present work, water extract exhibited higher TPC values than the methanolic extract. These results are well supported by the work of Amira et al. (2013) demonstrated that total phenolic contents were sensitive to solvents used for extraction. They reported that water extract was the best solvent with the highest TPC than acetone, ethanol and methanol. Later, Tan et al. (2014) also recommended water to be used as solvent of choice for extraction of phenolic compounds from bitter gourd. Earlier, Horax et al. (2010) suggested 80% ethanol to be the best solvent to extract phenolic compounds from bitter gourd. Not only the solvent, but many other factors are also involved in this regard including temperature, time, particle size etc. (Tan et al., 2014). Drying techniques are also influenced on the occurrence of total phenolic contents as described by Tan et al. (2013). They analyzed TPC of hydrophilic and lipophilic extracts in oven dried, microwave dried and freeze dried extracts and observed considerable variations in the presence of total phenolic contents by these techniques.
Li et al. (2015) showed that total phenolic contents of different extracts varied considerably and ranged from 0.69 to 10.80 mg GAE/g, respectively. The methanol extract was found to be the best solvent than ethanol, acetone, ethyl acetate and hexane. Pratheepa et al. (2011) assessed different bitter gourd cultivars and observed that total phenolic contents were in the range of 12.39±0.79 and 27.66±1.84 mg gallic acid equivalents per 100 g of fresh fruit. Similarly, Hamissou et al. (2013) recorded 13.28 GAE/g of total phenolic compounds in bitter gourd on fresh weight basis. Fidrianny et al. (2015) determined TPC in various parts of bitter gourd using different solvent and observed that ethyl acetate extracts was suitable with higher value of TPC in fruit than the n-hexane and ethanolic extracts.
In the nutshell, bitter gourd is one of the rich sources of phenolic compounds although the amount may vary due to varietal differences (Islam et al., 2011), use the sample in powder (Lin and Tang, 2007), lyophilized (Ng et al., 2011) and liquid form (Choo et al., 2014), drying methods (Tan et al., 2013) and difference in methodology of extraction (Anjum et al., 2013) and solvent used (Tan et al., 2014; Li et al., 2015).
68
Mean squares expounded highly significant differences due to treatments and parts on total flavonoid contents (TFC). Mean values (Table 4.18) indicate that higher amount of TFC (208.68 mg RuE/100g) were present in T3 (water extract of Black King). On the other hand, the TFC was found low in T4, the mehanolic extract of this cultivar, with value of 178.02 mg RuE/100g. The TFC for water extract of other cultivars were also observed to be higher with values of 184.06 mg RuE/100g, 193.22 mg RuE/100g, 184.42 mg RuE/100g, 172.23 mg RuE/100g and 172.67 mg RuE/100g as compare to their methanolic extracts with values of 173.88 mg RuE/100g,178.80 mg RuE/100g, 171.48 mg RuE/100g, 163.53 mg RuE/100g and 151.98 mg RuE/100g for BG 20, FSD Long, KHBG-1, GHBG-1 and Noor, respectively. It is also obvious from the results that considerably the highest total flavonoid contents (208.61 mg RuE/100g) were found in flesh followed by skin (193.36 mg RuE/100g), whole fruit (189.11 mg RuE/100g) and seed (119.06 mg RuE/100g). The highest value from all treatments and parts were observed in flesh part of T3 (water extract of Black King) with the value of 257.17±5.51 mg RuE/100g and the least value (108.21±1.53 mg RuE/100g) was observed in T2 (methanolic seed extract of BG 20).
The observations of Zulkifli (2012) confirmed the current findings for total flavonoid contents and revealed that at maturity stage the flavonoid contents in bitter gourd was 154.39±8.04 mg RuE/100g while immature stage contained lower flavonoid contents (114.04±3.04 mg RuE/100g). They observed higher amount of flavonoid (324.89±23.82 mg RuE/100g) at the ripe stage of bitter gourd. Water extract showed higher amount of flavonoid in current research which is in conformity with the findings of Wu and Ng (2007). They reported that lesser amount of total flavonoid (44.0 mg/g) were obtained in ethanol extract while higher concentration (62.0 mg/g) with water extraction.
Later, Anjum et al. (2013) compared different solvents to assess total flavonoid contents in bitter gourd seeds and found the highest value for TFC (69.01 and 82.01 mg CE/g in aqueous ethanol extract, followed by water extract (66.03 and 77.48 mg CE/g) and absolute ethanol (41.25 and 37.52 mg CE/g). they further reported that not only the solvents affect total flavonoid contents but extraction techniques are also important in determination of flavonoids. Wen & Liu (2007) also reported that selection of solvent imparted profound
69
Table 4.18. Mean values for total flavonoid contents (mg RuE/100g) of water and methanolic extract of BG cultivars and parts
Parts Treatments Means Skin Flesh Seed Whole fruit
h de yz jkl d T1 204.09±5.51 217.09±1.73 118.16±1.00 196.91±3.79 184.06
lm gh B no f T2 194.72±4.16 206.38±1.53 108.21±1.53 186.23±1.53 173.88 b a v fg a T3 237.09±4.58 257.17±5.51 130.39±2.31 210.08±3.21 208.68
hi f zA op e T4 202.07±3.61 211.78±2.08 113.28±1.15 184.95±3.79 178.02 def c v ij b T5 214.64±2.89 224.23±2.89 132.90±2.65 201.12±2.52 193.22
ijkl d zA nop e T6 198.38±3.06 217.17±0.58 114.47±1.53 185.18±1.53 178.80 ijk ef vw ijkl c T7 199.43±1.53 212.24±4.16 127.83±1.53 198.19±4.04 184.42 n ijkl z pq g T8 189.84±2.08 199.10±2.65 115.37±0.58 181.60±2.65 171.48 rs kl wx lm fg T9 174.13±3.21 196.63±4.51 123.89±1.53 194.29±2.31 172.23
st mn zA qr h T10 171.20±3.79 190.79±1.53 114.93±1.53 177.19±3.21 163.53 op ijk xy op fg T11 184.76±4.51 200.11±2.65 121.00±2.00 184.83±3.21 172.67
u st AB t i T12 159.17±4.04 170.16±3.51 110.11±1.53 168.49±2.31 151.98 Means 193.36b 208.61a 119.06d 189.11c
T1 = Water extract of BG 20 T7 = Water extract of KHBG-1 T2 = Methanolic extract of BG 20 T8 = Methanolic extract of KHBG-1 T3 = Water extract of Black King T9 = Water extract of GHBG-1 T4 = Methanolic extract of Black King T10 = Methanolic extract of GHBG-1 T5 = Water extract of FSD Long T11 = Water extract of Noor T6 = Methanolic extract of FSD Long T12= Methanolic extract of Noor
70 impact on flavonoid contents and found ethanol as the best solvent than methanol, acetone and hexane. In a recent study, Li et al. (2015) found variability in flavonoid contents from 0.94 mg RE/g for hexane to 18.69 mg RE/g for methanol. On the other hand, Tan et al. (2014) delineated that maximum TFC (23.17 mg RE/g) was obtained with acetone and declared the best solvent for extraction of flavonoids than methanol, ethanol, n-butanol and water with values of 7.67 mg RE/g, 5.38 mg RE/g, 1.81 mg RE/g and 0.66 mg RE/g, respectively. These variations in total flavonoid contents are due to variation in solubility of flavonoids and nature of extraction solvent (Chebil et al., 2007). Moreover, total flavonoid contents might also influenced by dielectric constant and variations in chemical nature of solvents, chemical properties of plant phytochemicals, drying methods, maturity stages (Zhang et al., 2009) and bitter gourd varieties (Huang et al., 2012).
The statistical analysis revealed that DPPH assay was significantly varied with regard to cultivars and parts. The mean values for DPPH free radical scavenging activity (Table 4.19) was recorded maximum (72.55%) in water extract of Black King (T3) followed by 70.25%,
68.47%, 65.79%, 64.32%, 60.42%, 59.22%, 56.73%, 52.54%, 52.48% and 50.65% in T5
(water extract of FSD Long), T4 (methanolic extract of Black King), T1 (water extract of BG
20), T6 (methanolic extract of FSD Long), T2 (methanolic extract of BG 20), T7 (water extract of KHBG-1), T9 (water extract of GHBG-1), T8 (methanolic extract of KHBG-1), T11
(water extract of Noor) T10 (methanolic extract of GHBG-1), respectively and the lowest
DPPH free radical scavenging activity was observed in case of T12 (methanolic extract of Noor) with value of 46.00%. The results also revealed that maximum free radical scavenging activity was possessed by the whole fruit (84.25%) followed by flesh (60.81%), skin (55.95%) and the seed (38.78%). The highest DPPH activity (93.95±3.21%) was observed in the whole fruit water extract of Black King and the least activity (32.13±0.58%) was noted in methanolic seed extract of Noor.
In assessment of free radical scavenging activity, DPPH assay is widely used due to easy in approach (Scherer & Godoy, 2009) and relatively quick and precise method for testing scavenging activity for various plant extracts. Wu and Ng (2008) reported that phenolic compounds were mainly responsible for the free radical scavenging activity in bitter gourd. Kubola and Siriamornpun (2008) also established positive correlation between phenolic
71 contents and free radical scavenging activity in bitter gourd. The number and position of hydroxyl group in phenolic compounds played a contributing role in antioxidant activity and free radical scavenging activity (Balasundram et al., 2006).
The present findings are in accordance with the results of Saeed et al. (2010) who used different parts of bitter gourd to assess DPPH radical scavenging effect and found that flakes owned higher DPPH scavenging activity 63.20%, than seeds 33.05%. The results of present research are also in line with Amira et al. (2013). They used different solvents to evaluate antioxidant activity of bitter gourd by DPPH. The results indicated that water is the best solvent with the highest DPPH value (98.29±2.02) followed by 70% acetone (83.10±0.29), 70% methanol (82.84±0.32) and 70% ethanol (82.48±0.59) while lower value was observed in 50% ethanol extract (34.22±0.50). The results of present study are in close conformity with the findings of Gupta and Verma (2011) who reported that bitter gourd possessed 88% radical scavenging activity. The results of present research also supported by the work of Ansari et al. (2005) who reported that, water extract of bitter gourd showed higher free radical antioxidant activities. Similarly, Wu and Ng (2008) showed a high scavenging rate for water extract (36.6% to 75.8%) than for ethanol extract (28.1% to 71.1%). Moreover, stage of maturity also influenced on the DPPH free radical scavenging activity. Zulkifli (2012) assessed DPPH free radical scavenging activities in bitter gourd at different maturity stages and found that mature stage has higher value (66.45±1.99%) than immature (22.05 %) and ripe stages (12.02%). The results of scavenging activity by Aminah and Anna (2011) ranged from 37%-64.48% that is lower than the current findings. They demonstrated that more ripened fruit had a lower value for DPPH. Previously, Kubola and Siriamornpun (2008) also reported lower free radical scavenging activity in green and ripe fruit with value of 11.0% and 27.6%, respectively.
DPPH scavenging activity of different plant materials is also affected by extracting methods (Anjum et al., 2013). It was also reported that seeds are also of worth consideration regarding scavenging activity (Anjum et al., 2013). Fidrianny et al., (2015) exhibited that IC50 of DPPH scavenging activities of ethanolic extract of fruit of bitter gourd was lower (58.62 μg/ml) and hence designated as strong antioxidant while ethyl acetate fruit extract of bitter gourd had higher IC50 of DPPH (102.46 μg/ml) and categorized as medium antioxidant. The
72 free radical scavenging activity of whole fruit was the highest than the other parts suggesting that many compounds are actively involved in free radical scavenging activity. In another study by Tan et al. (2013), it was observed that drying techniques also played an important role in DPPH scavenging activity. The percentage of freeze dried bitter gourd fruit extracts was found to be the highest than other drying methods. Moreover, the overall percentage of DPPH scavenging activity of lipophilic bitter gourd fruit extracts was better compared to the hydrophilic one.
Analysis of variance illustrated highly significant results for FRAP assay in different cultivars of bitter gourd and parts. The FRAP values (Table 4.20) were recorded maximum (94.90 μg FE/g) in water extract of Black King as compared to methanolic extract of this cultivar (86.12 μg FE/g). The water extract of other cultivars also exhibited higher output with values of 88.77 μg FE/g, 94.30 μg FE/g, 87.79 μg FE/g, 83.35 μg FE/g and 81.95 μg
FE/g for T1, T5, T7, T9 and T11, respectively than the methanolic extracts with values of 77.61
μg FE/g, 84.38 μg FE/g, 77.32 μg FE/g, 74.21 μg FE/g and 72.73 μg FE/g for T2, T6, T8, T10,
T12, respectively. Mean values also probed that FRAP value was higher in flesh (94.56 μg FE/g) followed by whole fruit (89.67 μg FE/g), skin (80.94 μg FE/g) and seed (69.30 μg FE/g).
The results of present research are in harmony with the earlier work of Zulkifli (2012) demonstrated FRAP values in the range of 54.67±2.89 μg FE/g-101.33±5.00 μg FE/g in different maturity stages. Kubola and Siriamornpun (2008) studied different parts of bitter gourd and highlighted that the FRAP value for green fruit is 43.8±0.008 which is low than the leaf extract (433±0.007) but higher than the stem (39.0±0.008) and ripe fruit (9.41±0.007). In another study, Aminah and Anna (2011) reported that the ripe fruit possessed the highest FRAP value (49.50 μg FE/g) and unripe fruit gave the lowest FRAP value (43 μg FE/g). The current results are also in line with Li et al. (2015) demonstrated that different bitter gourd fruits extracts exhibited different ferric reducing power. According to them, the reducing power of extracts ranged from 71.24 to 183.37 μmol Fe (II)/g.
Tan et al. (2013) determined effect of drying methodology on ferric reducing power in bitter gourd fruit and noticed that the highest ferric reduction ability (7627.59 mg TE/100 g of DW) was in hydrophilic microwave dried samples on medium power. Amira et al. (2013) analyzed
73
Table 4.19. Mean values for DPPH (%) assay of water and methanolic extract of BG cultivars and parts
Parts Treatments Means Skin Flesh Seed Whole fruit
lm j qr bc c T1 58.21±1.15 69.67±1.15 46.29±0.58 89.01±3.00 65.79 mn kl uvw cde d T2 56.93±0.58 62.13±1.53 36.74±1.53 85.89±2.52 60.42 ij fg r a a T3 72.65±1.53 79.00±2.00 44.62±2.08 93.95±3.21 72.55 j gh tuv cd b T4 70.28±1.53 76.39±1.53 39.90±2.52 87.30±1.53 68.47 ij hi rs ab a T5 71.03±2.31 74.49±0.58 44.41±1.53 91.07±1.53 70.25 k j uvw cd c T6 64.27±3.06 70.60±3.21 36.29±1.15 86.13±2.08 64.32 no m tuv de d T7 54.56±0.58 58.83±1.53 38.40±2.08 85.10±4.36 59.22 qr op wx fg f T8 46.64±1.53 50.78±1.15 33.10±4.73 79.66±3.21 52.54 pq mn st ef e T9 48.97±0.58 55.04±0.58 40.07±4.04 82.83±1.00 56.73 rs qr vwx gh g T10 44.10±2.00 45.58±4.73 36.80±2.65 76.13±1.53 50.65 rs pqr uvw f fg T11 44.08±2.00 47.30±2.08 36.64±2.08 81.90±3.00 52.48 tuv tu x hij h T12 39.79±2.65 40.00±1.73 32.13±0.58 72.07±3.06 46.00 Means 55.95c 60.81b 38.78d 84.25a
T1 = Water extract of BG 20 T7 = Water extract of KHBG-1 T2 = Methanolic extract of BG 20 T8 = Methanolic extract of KHBG-1 T3 = Water extract of Black King T9 = Water extract of GHBG-1 T4 = Methanolic extract of Black King T10 = Methanolic extract of GHBG-1 T5 = Water extract of FSD Long T11 = Water extract of Noor T6 = Methanolic extract of FSD Long T12= Methanolic extract of Noor
74
Table 4.20. Mean values for FRAP (μg FE/g) assay of water and methanolic extract of BG cultivars and parts
Parts Treatments Means Skin Flesh Seed Whole fruit
klmn cd stu efg b T1 86.21±2.00 99.39±1.53 74.21±3.21 95.28±3.06 88.77 rst klmn wx opq g T2 75.18±2.08 85.60±2.08 68.65±2.00 81.03±1.53 77.61 ijk a pq ab a T3 89.13±1.53 107.00±2.00 79.90±2.08 103.56±2.52 94.90 qr bcd vw fgh cd T4 78.03±1.53 101.00±2.00 70.67±1.53 94.77±3.06 86.12 ijk ab nop bc a T5 89.34±2.08 103.33±1.53 82.34±2.08 102.21±1.53 94.30 qrs def uvw hij de T6 78.78±1.73 97.13±2.08 70.60±1.53 91.00±4.36 84.38 klm de tuv ghi bc T7 86.18±2.52 98.12±2.52 74.72±2.00 92.13±1.53 87.79 tuv ijk yz nop g T8 74.29±1.73 89.25±1.53 62.81±3.06 82.95±4.04 77.32 mnop fgh xy ij ef T9 83.12±1.53 94.78±2.08 64.88±3.06 90.61±1.53 83.35
tuv klmn zA pq h T10 74.29±2.08 86.10±1.00 56.72±2.00 79.73±2.31 74.21 mno ijk wx jkl f T11 84.00±2.00l 89.09±3.21 67.01±3.61 87.72±3.06 81.95 tuv mnop z rst h T12 72.76±3.06 83.96±4.16 59.19±2.89 75.01±2.65 72.73 Means 80.94c 94.56a 69.30d 89.67b
T1 = Water extract of BG 20 T7 = Water extract of KHBG-1 T2 = Methanolic extract of BG 20 T8 = Methanolic extract of KHBG-1 T3 = Water extract of Black King T9 = Water extract of GHBG-1 T4 = Methanolic extract of Black King T10 = Methanolic extract of GHBG-1 T5 = Water extract of FSD Long T11 = Water extract of Noor T6 = Methanolic extract of FSD Long T12= Methanolic extract of Noor
75 different solvent to assess ferric reducing antioxidant power and found that water is excellent medium for extraction the with the highest FRAP value of 38.92±2.05 followed by ethanol, acetone and methanol. This is in accordance with the present findings that all cultivars showed higher FRAP values in water extract compared to respective methanolic extract. However, Tan et al. (2014) showed that methanol is more suitable solvent for FRAP analysis than the water. The variation in ferric reducing power is due to varietal differences, maturity stages and selection of different solvents for extraction (Amira et al., 2013).
Mean squares revealed highly significant results among different treatments and also for parts. The results exhibited that value for β- carotene (Table 4.21) was higher in water extract of Black King (67.13%) trailed by water extract of FSD Long (62.92%). The water extract of BG 20, KHBG-1, GHBG-1 and Noor exhibited β-carotene values as 59.84%, 57.39%, 55.10% and 51.80%, respectively whilst the methanolic extract of these cultivars showed lesser output of 56.13%, 61.74%, 58.51%, 51.98%, 49.16% and 46.31% for BG 20, Black King, FSD Long, KHBG-1, GHBG-1 and Noor, respectively. β-carotene bleaching assay of water and methanolic extract showed variations in different parts with the highest value of 64.10% for flesh followed by 61.48%, 56.03% and 44.40% for skin, whole fruit and seed, respectively.
The current results are well supported by Kubola and Siriamornpun (2008) who measured the antioxidant activity of bitter gourd extracts and noticed that green fruit possessed the highest antioxidant activity (79.9±0.70) followed by leaf (63.9±0.71), ripe fruit (59.0±0.44) and stem (36.2±0.59). They observed significant differences in antioxidant activities in different parts. The present results are further supported by work of Sharma and Dikshit (2012) who reported that antioxidant activity of whole bitter gourd fruit was 63.33%. The antioxidant activity is directly related with phenolic contents. The higher antioxidant activity of flesh and skin may be due to higher amount of phenolic compounds in these parts.
Mean squares explicated highly significant differences due to treatments and parts in ABTS assay. The cultivars and their parts imparted significant differences in ABTS mean values (Table 4.22). The highest ABTS value was exhibited by Black King water
76 extract (69.02 μmol TE/g) followed by water extract of FSD Long (66.84 μmol TE/g). The recorded values for methanolic extract were lesser in both of these cultivars (60.61 μmol TE/g and 59.69 μmol TE/g). Similar pattern was observed in other cultivars with higher values in water extract (67.94 μmol TE/g, 64.39 μmol TE/g, 61.57 μmol TE/g and 59.78 μmol TE/g for BG 20, KHBG-1, GHBG-1 and Noor, respectively) compared to their respective methanolic extract (61.20 μmol TE/g, 57.80 μmol TE/g, 58.41μmol TE/g and 56.54 μmol TE/g for BG 20, KHBG-1, GHBG-1 and Noor, respectively). Moreover, flesh part exhibited maximum ABTS value (68.95 μmol TE/g) and minimum value was observed in seed (51.24 μmol TE/g). The ABTS value for skin and whole fruit was 66.97 μmol TE/g and 60.77 μmol TE/g, respectively.
These results regarding ABTS assay is in agreement with the findings of Tan et al. (2014) showing similar values for ABTS for water and methanol. They further demonstrated that optimization of condition may helpful in increasing the antioxidant activity. Li et al. (2015) utilized different extracts and found that these extracts possessed higher ABTS radical scavenging abilities in that their phytochemical components and contents might have some equivalence regarding to ABTS radical scavenging ability. The IC50 values of methanol was 9.30 mg/mL, exhibiting the highest ABTS radical scavenging ability, followed by acetone, ethanol and ethyl acetate extracts, and the lowest for hexane extracts.
The variations in the values of TPC, TFC, DPPH, FRAP, ABTS, and beta carotene bleaching assay in some studies might be due to variations in the procedures used and also changes in the reference standards however, in all these studies, it is evident that bitter gourd has strong antioxidant ability. It is also ascertain from current study that antioxidants are present abundantly in bitter gourd, hence is the reason to give protection aginst many ailments when regularly intake in diet.
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Table 4.21. Mean values for β-carotene bleaching assay (%) of water and methanolic extract of BG cultivars and parts
Parts Treatments Means Skin Flesh Seed Whole fruit
fg ef stu hijk c T1 64.16±3.06 66.18±1.53 48.00±1.00 61.01±1.00 59.84 jklm fg vwx klmn ef T2 59.90±3.79 64.16±2.52 43.21±0.58 57.27±2.08 56.13 a b stu ghi a T3 81.01±1.15 77.73±1.15 48.56±0.58 61.23±1.53 67.13 de cd uvwx hijk bc T4 69.08±2.08 71.89±2.52 45.20±1.15 60.78±1.53 61.74 de bc stu ijkl b T5 69.13±3.61 75.96±2.65 47.49±0.58 59.12±2.08 62.92 ghij d vwx lmno d T6 61.72±3.79 70.61±1.00 44.61±1.73 57.09±0.58 58.51 ghi fgh tuv lmno de T7 61.28±4.04 63.17±4.51 47.90±2.65 57.21±1.15 57.39 opqr nop wx nopq g T8 54.00±1.00 56.39±2.31 43.12±0.58 54.42±0.76 51.98 jklm ghi uvwx opqr f T9 58.00±3.61 62.43±1.53 45.19±0.58 54.78±1.00 55.10 qr nopq y rs h T10 53.25±5.20 55.67±2.52 37.52±1.15 50.19±0.58 49.16 nopq nopq x pqr g T11 54.29±0.58 55.89±2.65 43.80±1.00 53.21±1.15 51.80 rs st y tuvw i T12 51.90±3.46 49.12±1.53 38.18±1.73 46.03±0.29 46.31 Means 61.48b 64.10a 44.40d 56.03c
T1 = Water extract of BG 20 T7 = Water extract of KHBG-1 T2 = Methanolic extract of BG 20 T8 = Methanolic extract of KHBG-1 T3 = Water extract of Black King T9 = Water extract of GHBG-1 T4 = Methanolic extract of Black King T10 = Methanolic extract of GHBG-1 T5 = Water extract of FSD Long T11 = Water extract of Noor T6 = Methanolic extract of FSD Long T12= Methanolic extract of Noor
78
Table 4.22. Mean values for ABTS (μmol TE/g) assay of water and methanolic extract of BG cultivars and parts
Parts Treatments Means Skin Flesh Seed Whole fruit
ab ab mno cdef ab T1 75.13±1.15 75.21±2.65 55.16±2.89 66.27±1.53 67.94 cde cde opq jkl de T2 66.27±2.08 66.07±0.58 52.45±0.58 60.00±2.65 61.20 a ab mn cd a T3 77.00±2.65 76.55±2.00 55.21±4.16 67.33±2.08 69.02 ijk defg nop hijk def T4 61.89±1.00 65.34±2.08 53.91±2.00 61.32±2.31 60.61 ab b nop defg b T5 75.61±1.53 73.00±2.65 53.56±2.08 65.19±1.53 66.84 ghij defg pqr kl fg T6 63.12±1.00 65.89±2.52 50.63±1.53 59.14±1.15 59.69 cd b nop fghi c T7 67.13±3.79 73.33±2.52 53.92±0.58 63.17±2.52 64.39 hijk defg rst mno hi T8 61.87±0.58 65.08±2.65 48.41±0.58 55.83±1.53 57.80 defg c qrs hijk d T9 65.90±2.65 69.17±2.00 49.83±1.53 61.39±2.08 61.57 fghi efgh st kl gh T10 63.63±2.08 64.23±1.53 46.65±0.58 59.12±2.65 58.41 defg cd qrs lm efg T11 65.02±1.00 67.72±2.00 49.13±0.58 57.26±1.53 59.78 hijk defg t nop i T12 61.09±3.21 65.81±0.76 46.00±1.00 53.26±0.58 56.54 Means 66.97b 68.95a 51.24d 60.77c
T1 = Water extract of BG 20 T7 = Water extract of KHBG-1 T2 = Methanolic extract of BG 20 T8 = Methanolic extract of KHBG-1 T3 = Water extract of Black King T9 = Water extract of GHBG-1 T4 = Methanolic extract of Black King T10 = Methanolic extract of GHBG-1 T5 = Water extract of FSD Long T11 = Water extract of Noor T6 = Methanolic extract of FSD Long T12= Methanolic extract of Noor
79
4.4. Phytochemical or Bioactive Molecules in Bitter Gourd
Analysis of variance (Table 4.23) for ascorbic acid contents in different cultivars and parts illustrated highly significant results. Mean values (Table 4.24) showed that Black King possessed the highest ascorbic acid contents (47.00 mg/100g) followed by FSD Long (44.83 mg/100g), KHBG-1 (36.58 mg/100g), BG 20 (29.00 mg/100g), Noor (19.50 mg/100g) and GHBG -1 (19.08 mg/100g). Mean values also revealed that flesh part is preponderated regarding ascorbic acid contents (42.78 mg/100g) followed by whole fruit (35.33 mg/100g), skin (34.17 mg/100g) and seed (18.39 mg/100g). The maximum ascorbic acid contents (62.33±5.51 mg/100g) were observed in the flesh part of Black King and minimum (15.33±3.06 mg/100g) in the seed portion of GHBG-1.
Ascorbic acid or vitamin C is required in high amount in the body as it is a water-soluble vitamin and lost frequently from the body. It plays a pivotal role in many reversible oxidation-reduction reactions. It is involved in prevention of scurvy disease and also helpful in the synthesis of folic acid derivatives, which are required for synthesis of DNA (Chatterjea & Shinde, 1998). In addition, vitamin C possesses antioxidant property and boosts the immunity system against infections, helps in formation of collagen and thyroxin and enhances absorption of iron (Robert et al., 2003)
The present findings for ascorbic acid in different parts of bitter gourd cultivars are in agreement with the results of Gayathri (2013) who determined 23 mg/100g ascorbic acid in bitter gourd. Earlier, Islam et al. (2011) noted that bitter gourd possessed 50 mg/100g of ascorbic acid contents. Bangash et al. (2011) also reported that bitter gourd is a rich source of vitamin C (65 ± 0.07 mg/100g). The results of instant research are also supported by Choo et al. (2014) indicated that flesh of bitter gourd fruit contained more ascorbic acid contents that the seeds and pith. The ascorbic acid contents of bitter gourd fruits in the present study were lower than Iqbal et al. (2006) who reported higher amount 85 mg/100g in bitter gourd fruit. Myojin et al. (2008) also reported that ascorbic acid contents in bitter gourd are higher in amount (79.7 mg/100g). On the other hand, Somsub et al. (2008) found very low amount (3.8-8.8 mg/100g) of ascorbic acid in bitter gourd. Ullah et al. (2011) showed that ascorbic acid contents in their four cultivars were variable and in the range of 9.41-16.40 mg/100g.
80
Table 4.23. Mean square for Ascorbic acid in different cultivars and parts
S.O.V. df Ascorbic acid
Cultivars (C) 5 1776.30**
Parts (P) 3 1892.70**
C × P 15 145.36**
Error 48 6.96
Total 71
** = Highly significant
Table 4.24. Mean values for Ascorbic acid (mg/100g) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 29.33±2.31f 38.33±2.08e 17.67±1.53ijk 30.67±1.53f 29.00d
Black King 49.67±4.51cd 62.33±5.51a 21.33±2.08ghi 54.67±1.53b 47.00a
FSD Long 46.33±1.53d 59.33±3.06a 23.00±2.00g 50.67±2.52bc 44.83b
KHBG-1 37.67±1.53e 53.00±4.58bc 16.67±0.58jk 39.00±2.65e 36.58c
GHBG-1 21.33±3.06ghi 21.33±2.08ghi 15.33±3.06k 18.33±1.53hijk 19.08e
Noor 20.67±2.08ghij 22.33±3.06gh 16.33±1.15k 18.67±1.53hijk 19.50e
Means 34.17b 42.78a 18.39c 35.33b
81
The differences in ascorbic acid contents are might be due to selection of different cultivars as fruit used by these authors is not mentioned. There is strong influence of genetic makeup on the absorption capacity of different components from the soil. Many other factors may also involve in occurrence of ascorbic acid contents including environmental condition, maturity stages of fruit, agronomic practices, harvesting and post-harvesting practices.
The statistical analysis pertaining to total saponin contents are presented in Table 4.25. The ANOVA results highlighted that total saponin contents were significantly varied in different cultivars and in parts.
The mean values pertaining to total saponin contents are presented in Table 4.26. The results revealed that maximum amount of total saponin contents were possessed by Black King with value of 1.95% which is statistically at par with FSD Long with value of 1.91%. The other cultivars including BG 20, KHBG-1, GHBG-1 and Noor possessed lower amount of saponin contents i.e., 1.74%, 1.76%, 1.61% and 1.58%, respectively. The results also revealed that flesh part has maximum amount of saponin (2.75%) followed by whole fruit (1.75%), skin (1.67%) and seed (0.86%). On the whole, flesh part of Black King contained the highest amount of total saponin contents (3.00±0.11%) and the least was determined in seed part of Noor and GHBG-1.
Saponins are good foaming and emulsifying agents due to their amphipathic properties. They are not only used in herbal medicines as active ingredients but also for food and industrial applications (Grover & Yadav, 2004). Notably, they are prescribed as antihyperglycemic herbal medicinal agents for controlling blood glucose levels (Grover & Yadav, 2004; Krawinke and Keding, 2006).
The results regarding total saponin contents in present study are supported by work of Li- Dong et al. (2008) who determined saponin contents in pulp and seeds of bitter gourd in the range of 1.17% to 4.07% and 0.16% to 1.54%, respectively. Earlier work by Li-Dong et al. (2007) quantified saponin contents through advanced optimized spectrophotometry and 2.81% saponin contents was noted in this way. In another study, Wang et al. (2011) used ultrasonic-microwave synergistic extraction for 16 minutes and 2.51% extraction yield of saponins was obtained for bitter gourd. Chen et al. (2010) calculated slightly higher yield
82
(3.2%) in bitter gourd. Oishi et al. (2007) found lower concentration of saponin (1.3%) in bitter gourd. Slightly higher amount of saponin in whole fruit (2.9% to 5.16%) and flesh (3.10% to 5.34%) was observed by Habicht et al. (2011) on dry weight basis. The differences in saponin contents are mainly due to varietal differences and number of environmental factors.
The statistical analysis regarding charantin (Table 4.27) revealed that charantin concentration was significantly affected due to variation in cultivars and in different parts of bitter gourd fruit. However, the interaction of bitter gourd cultivars and their parts showed non- momentous variations. The mean values related to charantin are presented in Table 4.27 which elaborated that Black King possessed maximum amount of charantin (0.10 mg/g) and minimum amount was noted in Noor with value of 0.07 mg/g. Among parts, flesh contained the highest amount (0.11 mg/g) followed by whole fruit (0.09 mg/g), skin (0.08 mg/g) and seed (0.05 mg/g).
Charantin is one of the most important saponin found in bitter gourd and has blood sugar lowering property like insulin (Pitipanapong et al., 2007). The results of present study are comparable with the outcomes of Pitipanapong et al. (2007) quantified 0.126±0.018 mg/g charantin in dried form of bitter gourd fruit. Contrary to this, Lolitkar and Rao (1966) calculated less yield of charantin (0.035%) in dried fruit powder. Later, Chanchai (2003) found that different parts of bitter gourd has ample amount of charantin including fruit (0.0301%), seed aril (0.0417%) and roots (0.0139%). Furthermore, Ham and Wang (2009) purified charantin with the yield of 0.014% in aqueous extract of bitter gourd. The charantin concentration in some studies varied and that is due to effect of different solvent, composition of solvent, solvent flow rate and temperature. These factors influence on the extraction efficiency of charantin.
Mean squares for alkaloids (Table 4.28) indicated highly significant variations in different cultivars and parts. Means regarding alkaloids (Table 4.29) showed maximum value 0.65% in Black King followed by FSD Long (0.62%), BG 20 (0.61%), KHBG-1 (0.56%), GHBG-1 (0.56%) and Noor (0.55%). The highest amount of alkaloid was observed in seeds of cultivar Black King (0.98±0.02%) and the lowest was observed in the skin part of BG 20 (0.43±0.02%). Among parts, seed possessed the highest amount (0.87%) of alkaloids
83
Table 4.25. Mean squares for total saponin and charantin in different cultivars and parts
S.O.V. df Total saponin Charantin
Cultivars (C) 5 0.2699** 0.00127**
Parts (P) 3 10.7825** 0.00948**
C × P 15 0.0405** 0.00014N.S
Error 48 0.0053 0.00009
Total 71
** = Highly significant N.S = Non-significant
Table 4.26. Mean values for total saponin (%) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 1.56±0.05h 2.83±0.08b 0.84±0.06j 1.74±0.06efg 1.74b
Black King 1.79±0.08de 3.09±0.18a 1.06±0.09i 1.86±0.09d 1.95a
FSD Long 1.79±0.01de 3.00±0.11a 0.98±0.07i 1.86±0.06d 1.91a
KHBG-1 1.66±0.06fgh 2.82±0.06b 0.82±0.05j 1.75±0.06def 1.76b
GHBG-1 1.62±0.07gh 2.40±0.06c 0.73±0.05j 1.68±0.05efg 1.61c
Noor 1.63±0.04fgh 2.35±0.05c 0.73±0.06j 1.63±0.05fgh 1.58c
Means 1.67c 2.75a 0.86d 1.75b
84
Table 4.27. Mean values for charantin (mg/g) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 0.09±0.01efg 0.11±0.01bc 0.06±0.01ij 0.09±0.01efg 0.08bc
Black King 0.09±0.01cdef 0.14±0.02a 0.07±0.01hi 0.10±0.02bcd 0.10a
FSD Long 0.09±0.01defg 0.12±0.01b 0.06±0.01ij 0.10±0.02bcd 0.09b
KHBG-1 0.09±0.00defg 0.10±0.02bcd 0.05±0.01ijk 0.09±0.01cdef 0.08c
GHBG-1 0.08±0.01fg 0.10±0.01cde 0.05±0.01jk 0.09±0.01cdef 0.08c
Noor 0.08±0.01gh 0.09±0.02efg 0.04±0.00k 0.08±0.01fg 0.07d
Means 0.08c 0.11a 0.05d 0.09b
85 followed by whole fruit (0.54%), flesh (0.49%) and skin (0.47%).
Alkaloids and their derived compounds are widely accepted as medicinal agents. Owing to this reason, alkaloids in bitter gourd fruit are important and assign bitter gourd as a medicinal plant. The present explorations regarding alkaloids are in agreement with the earlier findings of Krawinkel and Keding (2006) who, studied bitter gourd fruit powder without seeds and found 0.3% alkaloids in their investigation.
Mean squares in Table 4.30 indicated that the amount of momordicin I, momordicin II and vicine were significantly affected by varietal differences and parts of bitter gourd fruit. Means for the amount of momordicin I (Table 4.31) in different cultivars and parts explicated that Black King was predominated (7.12 mg/100g) followed by FSD Long (6.97 mg/100g), BG 20 (6.95 mg/100g), KHBG-1(6.95 mg/100g), GHBG-1 (6.79 mg/100g) and Noor (6.63 mg/100g).
The values for momordicin in different parts revealed that seed part contained the highest amount (9.45mg/100g) of momordicin I followed by whole fruit (6.78 mg/100g), flesh (6.11 mg/100g) and skin (5.27mg/100g).
The results of present study are analogous with the sequels of Beloin et al. (2005) who studied momordicin I in bitter gourd, cultivated in different ecological zones and observed that bitter gourd grown in coastal or humid savannah region contained higher amount of momordicin I (81μg/g) followed by savannah with patchy forests region (56 μg/g). Likewise, Puspawati (2008) fractionated the crude extract of momordicin and found that all fractions were in the range of 5.4 mg/100g to 18.5 mg/100g.
Means concerning momordicin II (Table 4.32) showed that Black King exhibited the highest concentration (8.61 mg/100g) followed by KHBG-1 (8.42 mg/100g), FSD Long (8.39 mg/100g), BG 20 (8.38 mg/100g), GHBG-1 (8.32 mg/100g) and Noor (8.14 mg/100g). Momordicin II also showed variations in different parts with the highest amount 11.51 mg/100g in seed part followed by 7.69 mg/100g in flesh, 7.47 mg/100g in whole fruit and 6.84 mg/100 g in skin. The momordicin II concentration was noted in the range of 6.15±0.14mg/100g for skin portion of Noor to 11.95±0.09 mg/100g for seed of Black King.
86
Table 4.28. Mean squares for alkaloids in different cultivars and parts
S.O.V. df Alkaloids
Cultivars C) 5 0.01928**
Parts (P) 3 0.62074**
C × P 15 0.00357**
Error 48 0.00067
Total 71
** = Highly significant
Table 4.29. Mean values for alkaloids (%) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 0.43±0.02h 0.49±0.04 efg 0.95±0.03a 0.58±0.03d 0.61b
Black King 0.52±0.03e 0.52±0.02e 0.98±0.02a 0.58±0.03d 0.65a
FSD Long 0.52±0.01e 0.51±0.02ef 0.87±0.05b 0.58±0.03d 0.62b
KHBG-1 0.46±0.02gh 0.49±0.02efg 0.80±0.03c 0.51±0.02e 0.56c
GHBG-1 0.46±0.03gh 0.47±0.02efg 0.81±0.03c 0.51±0.02e 0.56c
Noor 0.44±0.02h 0.46±0.04gh 0.80±0.04c 0.49±0.03efg 0.55c
Means 0.47c 0.49c 0.87a 0.54b
87
Table 4.30. Mean squares for momordicin I, II & vicine in different cultivars and parts
S.O.V. df Momordicin I Momordicin II Vicine
Cultivars 5 0.3461** 0.2727** 0.00662** C)
Parts (P) 3 58.8542** 80.9757** 0.61185**
C × P 15 0.1207** 0.1412* 0.00352**
Error 48 0.0453 0.0179 0.00026
Total 71
* = Significant ** = Highly significant
Table 4.31. Mean values for Momordicine I (mg/100g) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 5.27±0.35f 6.23±0.21d 9.60±0.26a 6.70±0.17c 6.95ab
Black King 5.37±0.15f 6.30±0.26d 9.90±0.20a 6.90±0.20c 7.12a
FSD Long 5.30±0.10f 6.17±0.15de 9.70±0.10a 6.73±0.29c 6.97a
KHBG-1 5.33±0.32f 6.06±0.08de 9.70±0.10a 6.73±0.29c 6.95ab
GHBG-1 5.30±0.20f 6.07±0.07de 8.90±0.20b 6.91±0.01c 6.79bc
Noor 5.03±0.15f 5.87±0.05e 8.91±0.45b 6.69±0.07c 6.63c
Means 5.27d 6.11c 9.45a 6.78b
88
The results regarding momordicin II in the present study are in line with the findings of Beloin et al. (2005) who found concentration of momordicin II in bitter gourd range from 11μg/g-197μg/g. According to them, bitter gourd plant in different ecological zones possessed different concentration of momordicin II.
It is obvious from the mean values (Table 4.33) that vicine concentration was found to be higher (0.271 μg/100μg) in Black King, followed by FSD Long (0.250 μg/100μg), KHBG-1 (0.225 μg/100μg), GHBG-1 (0.221 μg/100μg), BG 20 (0.219 μg/100μg) and Noor (0.207 μg/100μg). Among parts, considerable variations were also noted with higher amount of vicine in seed (0.501 μg/100μg) followed by whole fruit (0.204 μg/100μg) and flesh (0.122 μg/100μg) while the lowest concentrations was noted in skin (0.102 μg/100μg). Higher value for vicine concentration was noted in seed portion of Black King (0.630±0.046 μg/100μg) and minimum quantity was noted in skin part of Noor (0.088±0.003 μg/100μg).
Vicine is one of the alkaloids found particularly in the seeds of bitter gourd (Haixia et al., 2004; Tan et al., 2008). It is non-polar protein nitrogenous base, is one of the anti-diabetic components of bitter gourd (Raman & Lau, 1996). The present exploration regarding concentration of vicine is in line with the outcomes of Zhang et al. (2003) who studied different parts of bitter gourd including fruit, seeds and leaves for vicine contents with HPLC. They observed higher amount of vicine in seeds (0.524 μg/100μg) followed by fruit (0.115 μg/100μg) and leaves (0.0456 μg/100μg). Bedi et al. (2014) validated HPTCL method to determine vicine in bitter gourd extract and formulations and recorded 98.01-99.33% recovery in the yield of vicine.
Mean squares in Table 4.34 illuminated that polypeptide P concentration is affected substantially due to variation in cultivars and parts. The results (Table 4.35) explicated that the highest amount of polypeptide P is present in Black King (4.15 mg/g) followed by FSD Long (4.12 mg/g), BG 20 (4.05 mg/g), KHBG-1 (3.96 mg/g), GHBG-1 (3.88 mg/g) and Noor (3.86 mg/g). Different parts also exhibited variations regarding polypeptide P values. The highest concentration was observed in seed (5.44 mg/g) followed by whole fruit (4.34 mg/g), flesh (3.75 mg/g) and skin (2.49 mg/g).
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Table 4.32. Mean values for Momordicine II (mg/100g) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 6.81±0.08j 7.46±0.18f 11.79±0.16a 7.46±0.13f 8.38b
Black King 7.14±0.18gh 7.83±0.07c 11.95±0.09a 7.53±0.13ef 8.61a
FSD Long 7.01±0.10hij 7.77±0.13cd 11.32±0.21b 7.46±0.19f 8.39b
KHBG-1 7.04±0.06hi 7.76±0.12cd 11.35±0.11b 7.51±0.16f 8.42b
GHBG-1 6.88±0.04ij 7.74±0.21cde 11.34±0.13b 7.35±0.13fg 8.32b
Noor 6.15±0.14k 7.56±0.08def 11.33±0.08b 7.54±0.11ef 8.14c
Means 6.84d 7.69b 11.51a 7.47c
Table 4.33. Mean values for Vicine (μg/100μg) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 0.105±0.003ghi 0.128±0.002fgh 0.450±0.044cd 0.193±0.015e 0.219cd
Black King 0.112±0.007fghi 0.130±0.005fg 0.630±0.046a 0.210±0.010e 0.271a
FSD Long 0.109±0.001fghi 0.133±0.001f 0.547±0.025b 0.210±0.000e 0.250b
KHBG-1 0.101±0.004hi 0.123±0.002fgh 0.470±0.010c 0.207±0.021e 0.225c
GHBG-1 0.096±0.002i 0.113±0.005fghi 0.470±0.010c 0.203±0.015e 0.221c
Noor 0.088±0.003i 0.104±0.004ghi 0.437±0.015d 0.200±0.010e 0.207d
Means 0.102d 0.122c 0.501a 0.204b
90
A particular polypeptide, named polypeptide-p or p-insulin, has been isolated from the whole bitter melon fruit, the flesh, and the seeds (Khanna et al., 1981). As the name p-insulin implies, polypeptide-P has been ascribed potential blood glucose lowering properties (Yibchok-Anun et al., 2006). The results of present study are well supported by the work of Gupta et al. (2015) who studied various parts of bitter gourd (seed, fruit, leaf and stem) for the isolation of polypeptide-P. It was found utmost in leaves (9.868 mg/gm), seeds (5.895 mg/gm) and fruits (4.596 mg/gm).
91
Table 4.34. Mean square for Polypeptide P in different cultivars and parts
S.O.V. df Polypeptide P
Cultivars (C) 5 0.1787**
Parts (P) 3 27.1495**
C × P 15 0.0082**
Error 48 0.0289
Total 71
** = Highly significant
Table 4.35. Mean values for Polypeptide P (mg/g) in different cultivars and parts
Parts Cultivars Means Skin Flesh Seed Whole fruit
BG 20 2.50±0.10h 3.85±0.06e 5.51±0.08b 4.36±0.15d 4.05b
Black King 2.58±0.04h 3.89±0.11e 5.73±0.07a 4.39±0.06d 4.15a
FSD Long 2.63±0.06h 3.80±0.06ef 5.69±0.08a 4.37±0.07d 4.12ab
KHBG-1 2.51±0.14h 3.69±0.03fg 5.31±0.09c 4.35±0.11d 3.96c
GHBG-1 2.49±0.05h 3.60±0.04g 5.17±0.06c 4.27±0.07d 3.88d
Noor 2.25±0.08i 3.65±0.09g 5.24±0.21c 4.30±0.07d 3.86d
Means 2.49d 3.75c 5.44a 4.34b
92
4.5. Product Development
4.5.1. Physical Attributes of Bitter Gourd Functional Drink
Mean squares for physical attributes of bitter gourd functional drink are presented in Table 4.36. Mean squares for TSS depicted significant differences due to treatment and storage period however, interaction of treatment and storage days showed non-significant effect on
TSS. Brix for all functional drinks T0, T1, T2 T3, T4 and T5 were 3.28, 3.55, 3.31, 3.93, 4.39 and 5.11, respectively (Table 4.37). Moreover, TSS decreased with the passage of time; maximum value for TSS was recorded at zero day (4.38) whereas minimum at the termination of trial (3.37).
The result of Sheela and Sruthi (2014) well supported the current work; analyzed bitter gourd beverage with different concentration of lemon and mosambi and noted that TSS were in the range of 4.00-4.80o Brix. They observed gradual reduction of TSS for every fifteen day interval and at the end of 60th day, the TSS was found to be in the range of 3.60-4.20o Brix. In another study, Kaur and Aggarwal (2014) studied storage impact on bitter gourd juice with different preservatives and found that TSS value of different samples was in the range of 3.2- 3.3 with no significant changes after storing for six months (3.4-3.6). Singh and Gaikwad (2012) delineated that mean values varied significantly (4.62-4.06%) in different samples prepared with bitter gourd and lemon. Furthermore, a significant decline in TSS (5.033- 4.166%) was also observed during storage. This might be due to hydrolysis of polysaccharides. At zero day of storage, TSS of all drinks was in the range of 4.5-5.6 per cent which gradually reduced to 3.7-4.2%. On the other hand, Satkar et al. (2013) found that the fresh bitter gourd RTS had TSS on an average 13.00º Brix which increased to 14.14º Brix in 90 days storage period.
Mean squares for pH of functional drinks indicated significant effect due to treatment and storage while non-substantial effect was noted for the interaction of treatments with storage period. The highest pH value 4.96 was noted in T5 whilst minimum 4.34 was observed in T0 and T1 (Table 4.38). Likewise, decrease in pH value was noted with passage of time; minimum pH was noted at 0 day (4.07) whereas maximum at the termination of trial (5.03).
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Table 4.36. Mean squares for physical attributes of bitter gourd drink
S.O.V. df TSS pH Acidity L* a* b*
Treatment (T) 5 6.15** 0.63** 0.62** 1289.71** 4.30** 11.44**
Days (D) 3 3.18** 3.00** 3.82** 63.15** 0.20** 0.10**
T × D 15 0.05 N.S 0.02 N.S 0.05** 1.54 N.S 0.003 N.S 0.003**
Error 48 0.03 0.06 0.02 1.20 0.01 0.001
Total 71
** = Highly significant N.S = Non-significant
Table 4.37. Effect of treatments and storage on TSS of bitter gourd functional drink
Storage Days Treatments Means 0 15 30 45
ef hi hi j e T0 3.8±0.64 3.3±0.1 3.3±0.1 2.7±0.1 3.28
ef fg gh i d T1 3.9±0.1 3.7±0.15 3.5±0.1 3.06±0.15 3.55
ef h h j e T2 3.9±0.17 3.4±0.15 3.4±0.15 2.5±0.31 3.31
d e ef h c T3 4.4±0.12 4.03±0.23 3.9±0.1 3.4±0.2 3.93
c d d e b T4 4.77±0.12 4.37±0.21 4.37±0.21 4.07±0.25 4.39
a b b cd a T5 5.57±0.15 5.2±0.1 5.12±0.15 4.5±0.26 5.11
Means 4.38a 4.01b 3.94b 3.37c
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The result of Sheela and Sruthi (2014) supported the current findings regarding pH. According to them, addition of bitter gourd has resulted in increase in pH. In their study, storage period also resulted in gradual rise in pH for every fifteen day until 60th day of storage. Singh and Gaikwad (2012) showed significant effect of storage period and different sample on pH of all drink samples of bitter gourd with decrease in acidity and increase in pH from 2.66 to 3.66 during storage. pH is inversely proportional to the acidity of any medium. The changes in pH are due to enhanced activity of microorganisms with the passage of time and effect of temperature.
Means squares for acidity showed significant differences due to treatments, storage and their interaction. Higher acidity value i.e. 4.49 was estimated in T0 whilst minimum 3.85 was observed in T5. Storage of drinks resulted marked decrease in acidity from 4.82 to 3.72 at 0 and 45th day (Table 4.39).
The result of instant study regarding acidity and pH are comparable with the study of Sheela and Sruthi (2014), narrated variations in acidity with substitution of bitter gourd in lemon from 5.6% to 5.5%. They also observed that over the period of storage there was a gradual reduction in the acidity for every fifteen days until the 60th day of storage. On the final day of the acidity was found to be in the range of 4.0%-4.7% in different samples.
Satkar et al. (2013) also monitored decline in acidity from 0.30 to 0.23 % in 90 days storage studies. They observed that temperature is an important factor in this change. The decrease in acidity was more obvious in the drinks placed in ambient temperature than the refrigerated temperature. This decrease could be attributed to the chemical interaction between organic constituents of the beverage induced by temperature and action of enzymes. Contrary to this, Kaur and Aggarwal (2014) studied bitter gourd beverages and role of different preservatives on the storage stability and noted that there is gradual increase in acidity with the passage of time.
Product color is one of important indicators reflecting the extent of consumer acceptance. The color measurement is mainly performed with CIELAB color system and its attributes are L*, a* and b* where L* is the indicator of lightness-darkness, a* indicates greenish to reddish tonality, whereas b* represents bluish to yellowish tonality.
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Table 4.38. Effect of treatments and storage on pH of bitter gourd functional drink
Storage Days Treatments Means 0 15 30 45
j ghi fghi bc c T0 3.8±0.1 4.23±0.12 4.4±0.26 4.93±0.25 4.34
j ghi fghi bc c T1 3.8±0.1 4.23±0.12 4.4±0.26 4.93±0.25 4.34
ij fghi bcde ab bc T2 4.03±0.21 4.37±0.21 4.8±0.2 5.07±0.21 4.57
hij efgh cdef bcd bc T3 4.1±0.2 4.43±0.15 4.63±0.21 4.9±0.17 4.52
ghi defg bcde abc b T4 4.2±0.15 4.53±0.32 4.8±0.1 5.00±0.17 4.64
efgh bcd ab a a T5 4.43±0.15 4.87±0.25 5.12±0.32 5.37±0.67 4.96
Means 4.07d 4.44c 4.70b 5.03a
Table 4.39. Effect of treatments and storage on acidity of bitter gourd functional drink
Storage Days Treatments Means 0 15 30 45
a cd def hi a T0 5.33±0.12 4.5±0.1 4.27±0.06 3.87±0.06 4.49
a cde fg hi a T1 5.2±0.1 4.47±0.25 4.17±0.06 3.83±0.12 4.42
b def gh hi b T2 4.83±0.12 4.27±0.21 3.97±0.15 3.81±0.04 4.22
bc def fg hi b T3 4.67±0.25 4.3±0.2 4.17±0.15 3.8±0.2 4.23
bc ef gh i b T4 4.63±0.21 4.23±0.12 3.93±0.15 3.67±0.15 4.12
def hi hi j c T5 4.27±0.06 3.9±0.1 3.87±0.12 3.37±0.15 3.85
Means 4.82a 4.28b 4.06c 3.72d
96
Mean squares elucidated that treatments and storage days imparted substantial effect on L* value, however, interaction of treatment and study interval affected non-significantly. Means regarding L* values of treatments are presented in Table 4.40. The L* values for drinks T0,
T1, T2, T3, T4 and T5 were 56.08, 45.75, 39.71, 33.71, 31.17 and 28.50, respectively. Storage days resulted in substantial decrease in L* value from 41.08 to 36.83 from 0 day to 45th day.
Means squares for a* value of bitter gourd functional drinks indicated momentous effect of treatments and storage period on this trait while their interaction showed non-significant differences. Means for a* value (Table 4.41) explicated that the addition of bitter gourd in drink formulations resulted marked increase for this attribute from -0.20 in T0 to -1.34, -1.55,
-1.70, -1.73 and -1.77 in T1, T2 T3, T4 and T5, respectively. With regard to storage days, a* th value at 0, 15, 30 and 45 day were -1.49, -1.43, -1.36 and -1.25, correspondingly.
It is evident from the Table 4.36 that treatment, storage days and their interaction imparted highly significant differences regarding b* values. The mean values for b* (Table 4.42) showed that various formulations possessed different values for this trait. The b* values for
T0, T1, T2 T3, T4 and T5 were 4.27, 1.72, 1.75, 1.80, 2.26 and 2.34, respectively. During storage gradual decrease in b* value was recorded from 2.45 in zero day to 2.27 on 45th day.
The results of instant research are in agreement with the values of colour attributes (L*, a* and b*) observed by Kaur and Aggarwal, (2014). In their experiment, value of colour varied significantly in different treatments and for storage.
4.5.2. Sensory Evaluation of Bitter Gourd Functional Drink
Mean squares for sensory profiling (colour, aroma, flavor, taste and overall acceptability) of bitter gourd functional drink showed highly significant differences due to treatments and storage days. However, the data pertaining to their interaction elucidated non-momentous effect (Table 4.43).
Means for color score in various treatments (Table 4.44) illustrated that scores varied from
4.08 to 7.40 for T0 and T4 whereas, T1, T2, T3 and T5 were assigned 5.73, 7.15, 5.20 and 4.60 scores, respectively. Storage depicted substantial decline in colour scores from 0 to 45th day i.e. 6.05 and 5.25 whilst 5.85 and 5.62 scores was obtained at 15th and 30th day.
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Table 4.40 Effect of treatments and storage on L* value of bitter gourd functional drink
Storage Days Treatments Means 0 15 30 45
a ab bc c a T0 57.67±1.52 57.00±1.00 55.67±0.58 54.00±0.00 56.08
d de ef f b T1 47.33±1.15 46.33±0.58 45.00±1.00 44.33±0.58 45.75
g gh hi i c T2 41.67±1.53 40.00±1.00 39.17±0.29 38.00±1.00 39.71
j k kl lm d T3 35.83±0.29 34.00±2.00 33.33±0.58 31.67±1.53 33.71
k kl m n e T4 33.67±2.08 33.33±0.58 30.00±1.00 27.67±0.15 31.17
m m n o f T5 30.33±1.53 30.33±0.58 28.00±1.00 25.33±0.58 28.50
Means 41.08a 40.17b 38.53c 36.83d
Table 4.41. Effect of treatments and storage on a* value of bitter gourd functional drink
Storage Days Treatments Means 0 15 30 45
b ab a a a T0 -0.37±0.12 -0.23±0.06 -0.13±0.06 -0.08±0.15 -0.20
def de d c b T1 -1.43±0.06 -1.4±0.1 -1.3±0.17c -1.23±0.06 -1.34
ghi gh efg def c T2 -1.63±0.06 -1.6±0.1 -1.53±0.21 -1.43±0.12 -1.55
k ijk ghij efg d T3 -1.83±0.06 -1.77±0.06 -1.67±0.12 -1.53±0.06 -1.70
k jk hijk fg d T4 -1.83±0.06 -1.8±0.1 -1.73±0.21 -1.57±0.06 -1.73
k jk jk ghi d T5 -1.83±0.06 -1.8±0.1 -1.8±0.1 -1.63±0.06 -1.77
Means -1.49c -1.43c -1.36b -1.25a
98
Table 4.42 Effect of treatments and storage on b* value of bitter gourd functional drink
Storage Days Treatments Means 0 15 30 45
a b b b a T0 4.34±0.02 4.26±0.3 4.25±0.04 4.24±0.02 4.27
hi j jk k f T1 1.82±0.07 1.72±0.03 1.68±0.02 1.65±0.01 1.72
gh i jk k e T2 1.85±0.04 1.79±0.04 1.70±0.04 1.66±0.03 1.75
g hi hi jk d T3 1.88±0.04 1.81±0.03 1.81±0.02 1.69±0.02 1.80
c d e f c T4 2.39±0.01 2.32±0.01 2.22±0.06 2.13±0.06 2.26
c cd d e b T5 2.41±0.02 2.37±0.03 2.32±0.04 2.26±0.04 2.34
Means 2.45a 2.38b 2.33c 2.27d
99
The aroma scores in Table 4.45 showed the highest rating for T3 (7.33) which is statistically at par with T2 (7.08) followed by T1 (5. 98), T4 (5.65) and T5 (4.73) whilst, the lowest in T0 (3.83). Storage days showed a declining tendency in aroma scores as the highest score 6.18 was obtained at 0 day which is reduced to 5.37 on 45th day during the present study.
Mean scores for flavour (Table 4.46) were 4.48, 5.40, 6.73, 7.05, 4.70 and 4.18 scores for T0,
T1, T2, T3, T4 and T5, respectively. Besides, score rating for taste at the initiation of study (0 day) was 5.83 which reduced to 5.00 at the termination of study. The recorded scores during 15 and 30 day interval were 5.58 and 5.27.
The taste scores in Table 4.47 elucidated the highest value for T3 (7.15) followed by T2 (7.00), T1 (6.03), T4 (4.48), T1 (3.65) and T5 (3.48). Alongside, the storage imparted waning trend in scores like 5.75, 5.52, 5.20 and 4.72 at 0, 15, 30 and 45th day for this parameter.
Finally, with respect to overall acceptability, the highest rating was earned by T3 (7.00) followed by T2 (6.88), T1 (5.65), T0 (4.50), T4 (3.05) and T5 (2.98) (Table 4.48). Storage days also imparted significant impact on the acceptability of these functional drinks with maximum score at the start of trial (5.33) which gradually reduced to 5.07, 4.93 and 4.70 after 15, 30 and 45th day interval.
The results were in agreement with Satkar et al. (2013) conducted sensory evaluation of fresh bitter gourd beverage and indicated that colour and appearance score of fresh beverage was 7.3 while flavor score was 7.7, taste score was 7.5 and overall acceptability score was 7.6. The appearance of beverage was influenced by the TSS levels. However, addition of citric acid contributed towards better flavour. The taste of these drinks was greatly attributed to the appropriate sugar-acid blend in the product. The sensory score of all parameters decreased continuously during storage. The overall acceptability score decreased from 7.2 to 5.9 after storage. This decrease may be due to degradation of colour and changes in flavour and taste of stored samples.
However, the average score by the taste panelists was low for all the sensory attributes of bitter gourd by Waghray et al. (2012). Bitterness was the characteristic that determined the preference of consumers. Bitter sensation is not by itself appealing to most people (Drewnowski & Carmen, 2000), so it could be the reason for low acceptability of
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Table 4.43. Mean squares showing treatments and storage effect on sensory attributes
Overall S.O.V. df Colour Aroma Flavour Taste acceptability
Treatment (T) 5 72.75** 72.42** 58.57** 108.57** 128.72**
Days (D) 3 7.08** 7.58** 7.95** 11.99** 4.19**
T × D 15 0.04 N.S 0.13 N.S 0.27 N.S 0.07 N.S 0.05 N.S
Error 216 1.11 0.57 0.70 0.57 0.65
Total 239
** = Highly significant N.S = Non-significant
Table 4.44. Effect of treatments and storage on colour scores of drink
Storage Days Treatments Means 0 15 30 45
ghij hij ij j e T0 4.40±0.97 4.20±0.79 4.10±0.88 3.60±0.70 4.08
cde de ef efg b T1 6.00±1.05 5.90±0.88 5.70±0.67 5.30±0.82 5.73
ab ab ab bcd a T2 7.60±0.97 7.30±0.67 7.00±0.82 6.70±0.95 7.15
a ab ab abc a T3 7.80±1.03 7.60±0.70 7.30±0.95 6.90±0.88 7.40
ef efg efgh fghi c T4 5.60±0.97 5.30±0.67 5.10±0.88 4.80±0.79 5.20
fghi fghi ghij hij d T5 4.90±2.02 4.80±1.87 4.50±1.27 4.20±1.62 4.60
Means 6.05a 5.85ab 5.62bc 5.25c
101
Table 4.45. Effect of treatments and storage on Aroma scores of drink
Storage Days Treatments Means 0 15 30 45
jkl jkl kl l d T0 4.10±0.99 3.90±0.57 3.70±0.48 3.60±0.52 3.83
cd de efg fg b T1 6.50±0.97 6.20±0.63 5.70±0.67 5.50±0.53 5.98
ab ab bc cd a T2 7.50±0.97 7.30±0.48 6.90±0.57 6.60±0.52 7.08
a ab ab bc a T3 7.60±0.52 7.50±0.97 7.30±0.95 6.90±0.74 7.33
def efg fg gh b T4 6.10±1.29 5.70±0.67 5.50±0.71 5.30±0.48 5.65
gh hi ij ijk c T5 5.30±0.82 4.80±0.42 4.50±0.71 4.30±1.06 4.73
Means 6.18a 5.90b 5.60c 5.37c
Table 4.46. Effect of treatments and storage on Flavour scores of drink
Storage Days Treatments Means 0 15 30 45
efg fg gh hi cd T0 5.00±1.05 4.70±0.95 4.30±0.67 3.90±0.74 4.48
cd de def efg b T1 5.90±0.99 5.50±0.85 5.20±1.03 5.00±0.94 5.40
ab ab abc bc a T2 7.10±0.88 6.80±0.63 6.60±0.52 6.40±0.52 6.73
a ab ab ab a T3 7.20±1.03 7.10±0.74 7.00±0.82 6.90±1.10 7.05
efg efg fgh gh c T4 5.00±1.15 4.90±0.57 4.60±0.52 4.30±0.95 4.70
efg fgh hi i d T5 4.80±0.79 4.50±0.85 3.90±0.74 3.50±0.53 4.18
Means 5.83a 5.58a 5.27b 5.00b
102
Table 4.47. Effect of treatments and storage on Taste scores of drink
Storage Days Treatments Means 0 15 30 45
ijk jkl lm m d T0 4.20±0.92 3.90±0.57 3.50±0.53 3.00±0.67 3.65
cde de ef fg b T1 6.40±0.52 6.30±0.48 5.90±0.74 5.50±0.71 6.03
ab ab bcd cde a T2 7.50±0.85 7.20±0.63 6.90±0.57 6.40±0.70 7.00
a ab bc cde a T3 7.70±1.06 7.40±0.52 7.00±0.47 6.50±0.97 7.15
gh hi hij jkl c T4 4.90±0.74 4.60±0.84 4.50±0.53 3.90±0.88 4.48
kl kl lm m d T5 3.80±1.03 3.70±0.82 3.40±0.70 3.00±1.05 3.48
Means 5.75a 5.52a 5.20b 4.72c
Table 4.48. Effect of treatments and storage on Overall acceptability scores of drink
Storage Days Treatments Means 0 15 30 45
ef f f f c T0 4.90±0.74 4.50±0.53 4.40±0.70 4.20±0.63 4.50
bc cd cde de b T1 6.10±0.88 5.70±0.67 5.50±0.53 5.30±0.48 5.65
a a ab ab a T2 7.20±0.79 6.90±0.74 6.80±0.63 6.60±0.70 6.88
a a a ab a T3 7.30±0.48 7.10±0.57 7.00±0.47 6.60±0.70 7.00
g g g g d T4 3.30±1.49 3.10±1.20 3.00±0.47 2.80±0.42 3.05
g g g g d T5 3.20±1.55 3.10±1.10 2.90±0.88 2.70±0.67 2.98
Means 5.33a 5.07ab 4.93bc 4.70c
103 bitter gourd juice. However, addition of aspartame in reasonable amount has resulted in increase in the score of overall acceptability of these juices (Waghray et al., 2012).
The present results are further supported by the work of Singh and Gaikwad (2012), showed significant difference for overall acceptability of drinks during the storage of two months. Their study concluded that decreased in level of substitution of lemon juice decreased the overall acceptability for the drinks (Din et al., 2011). There is decrease in overall acceptability of beverages prepared from different ratios of bitter gourd during storage (Singh & Gaikwad, 2012).
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4.6. Bio-evaluation Studies
Bio-evaluation study was carried out through rodent modeling to explore the functional/nutraceutical worth of bitter gourd with special reference to glycemic response and serum lipid profiles. In the present study Sprague-Dawley rats were preferred to human for in vivo evaluation due to ease in handling, giving diet suitable for experimentation, made observation closely and constant environmental settings. Safety concern was also one of the considerable reasons to conduct this study in rodents. Additionally, it is hard to find out volunteers who could restrict themselves on specific diet. Efficacy study comprised of three modules on the basis of different diets i.e. study I (normal diet), study II (high sucrose diet), study III (high cholesterol diet). Furthermore, rats were divided in seven groups in each module based on diet provision including D0 (control), D1 (Diet with 150 mg/kg body weight of skin powder), D2 (Diet with 300 mg/kg body weight of skin powder), D3 (Diet with 150 mg/kg body weight of flesh powder), D4 (Diet with 300 mg/kg body weight of flesh powder),
D5 (Diet with 150 mg/kg body weight of whole fruit powder) and D6 (Diet with 300 mg/kg body weight of whole fruit powder) . To get base-line values, few rats were dissected at the begining. Intake of water and feed was monitored daily whilst body weight was measured after week interval. Half of the rats in each group were anatomized at 28th day whilst remaining were dissected at the end (56th day). The impact of bitter gourd formulations were assessed through determination of glucose & insulin levels and serum cholesterol, LDL, HDL & triglycerides. In addition, liver soundness tests including alkaline phosphatase (ALP), aspartate transferase (AST) and alanine transferase (ALT) and kidney tests i.e., amount of creatinine and urea were also noted. Subsequently, the informations obtained were subject to statistical analysis to come close to conclusion.
4.6.1. Feed Intake
Mean values (Table 4.49) clarified that bitter gourd in diet revealed insignificant effect on intake of food. However, feed intake affected substantially with passage of time (weeks) in all studies. Means in Study I (Normal diet) (Figure 4.1) showed that feed intake at 1st week was 11.29±0.40, 10.89±0.23, 11.07±0.55, 11.34±0.43, 11.17±0.32, 11.11±0.28 and 11.17±0.45 g/rat/day that substantially increased to 19.13±0.70, 18.74±0.84, 18.79±0.70,
105
18.64±0.76, 18.77±0.56, 18.67±0.69 and 18.76±0.53 g/rat/day in D0, D1, D2, D3, D4, D5 and th D6, respectively at 8 week.
Similarly, in Study II (high sucrose diet), overall means (Figure 4.2) showed that increasing time favored feed consumption as it was 11.27±0.48 (D0), 11.04±0.27 (D1), 11.07±0.48 (D2),
11.40±0.37 (D3), 11.26±0.24 (D4), 11.13±0.32 (D5) and 11.20±0.45 (D6) g/rat/day at first week that was lifted up to 19.23±0.48, 18.94±0.66, 18.87±0.70, 18.93±0.96, 18.79±0.58,
18.71±0.74, 18.69±0.47 g/rat/day in D0, D1, D2, D3, D4, D5 and D6, respectively at final week. Likewise, in study III (high cholesterol diet), study days also played a decisive role in enhancing feed intake (Figure 4.3); at the beginning it was 11.37±0.41, 11.23±0.32,
10.87±0.26, 11.33±0.33, 11.07±0.32 , 11.14±0.40 and 11.13±0.36 g/rat/day in D0, D1, D2,
D3, D4, D5 and D6 that increased to 19.31±0.59, 19.01±0.53, 18.91±0.66, 18.91±1.04, 18.90±0.57, 18.84±0.84 and 18.84±0.36, respectively at the end of study.
The non-substantial effect of addition of bitter gourd in diet on feed intake is in harmony with the findings of Klomann et al. (2010). They observed that bitter gourd provision did not impart any significant differences in the feed intake of the rats. In current research slight reduction is noted in groups fed with bitter gourd. The research outcomes of Chen et al. (2003) are in agreement with the current findings that supplementation of bitter gourd has resulted in reduction of feed intake. Similarly Reyes et al. (2006) also depicted that feed intake is decreased in alloxan-induced diabetic rats treated with juice of bitter gourd. The results are also in harmony with the findings of Huang et al. (2008) who reported decrease in feed intake on higher fat bitter gourd powder diet. However, Shetty et al. (2005) found that bitter gourd supplemented diet has resulted in higher consumption of food as compared to control. The difference in the amount of bitter gourd in diet is a key factor in the variation in feed intake in laboratory animals in some investigations.
4.6.2. Water Intake
Mean squares for water intake (Table 4.50) showed that diet containing different amount of bitter gourd and time period imparted significant effect in all the studies. Means for water intake (Figure 4.4) depicted increasing tendency with the passage of time. In Study I, the water intake at 1st week was 19.61±0.18, 19.74±0.18, 19.80±0.22, 19.79±0.21, 19.83±0.23,
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Table 4.49. Mean squares for effect of diet and study weeks on feed intake
Feed intake S.O.V. df Study I Study II Study III
Diets 6 0.310N.S 0.347N.S 0.392N.S
Weeks 7 343.543** 342.913** 349.734**
Error 336 0.164 0.176 0.186
391 Total ** = Highly significant N.S = Non-significant
107
20
D0 17
D1
D2 14 D3
g/rat/day D4 11 D5 D6 8 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
Figure 4.1. Feed intake in Study I (g/rat/day)
20
D0 17
D1
D2 14 D3
g/rat/day D4 11 D5 D6 8 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
Figure 4.2. Feed intake in Study II (g/rat/day)
20
D0 17
D1
D2 14 D3
g/rat/day D4 11 D5 D6 8 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
Figure 4.3. Feed intake in Study III (g/rat/day)
108
19.84±0.14 and 19.86±0.23 mL/rat/day for D0, D1, D2, D3, D4, D5 and D6 that increased to 27.44±0.49, 27.54±0.42, 27.70±0.57, 27.46±0.32, 28.07±0.54, 27.53±0.44 and 27.57±0.37 mL/rat/day, respectively for the same study at 8th week. The water intake in control group was 27.44±0.49 mL/rat/day while in experimental groups; its range was 27.46±0.32 to 28.07±0.54 mL/rat/day at the end of experiment.
Likewise in Study II, the water intake (Figure 4.5) at 1st week was 19.57±0.25, 19.60±0.18,
19.66±0.26, 19.64±0.10, 20.01±0.35, 20.01±0.31 and 19.84±0.24 mL/rat/day for D0, D1, D2, th D3, D4, D5 and D6 that increased with the passage of time and at 8 week noticed as 28.09±1.05, 27.03±0.41, 27.06±0.48, 27.21±0.30, 27.31±0.73, 27.21±0.66 and 27.13±0.52 mL/rat/day, respectively. The maximum water intake (28.09±1.05 mL/rat/day) was noted in control group than the other groups fed with diet containing bitter gourd (27.03±0.41 to 27.31±0.73 mL/rat/day) at the termination of trial.
The rats provided with high cholesterol diet (Study III), showed drink consumption of 19.50±0.18, 19.59±0.21, 19.60±0.28, 19.70±0.16, 19.94±0.30, 19.90±0.22 and 19.83±0.36 mL/rat/day in D0, D1, D2, D3, D4, D5 and D6 at the onset of study that raised to 27.71±0.76, 27.13±0.42, 27.13±0.47, 27.14±0.17, 27.23±0.34, 27.14±0.35 and 27.14±0.56 mL/rat/day, respectively at the termination of efficacy trial (Figure 4.6). The maximum water intake in this study was observed in control group (27.71±0.76 mL/rat/day). The groups fed with high cholesterol diet containing bitter gourd showed water intake in the range of 27.13±0.42 to 27.23±0.34 mL/rat/day at the termination of study.
The results of instant exploration for drink consumption with concomitant intake of bitter gourd based functional diets are in accordance with the work of Shetty et al. (2005) reported that water intake increase in diabetic group but the supplementation of bitter gourd in diet significantly decrease the consumption of water. The excessive water intake is a characteristic sign of diabetes. Parmar et al. (2011) found that there was an increase in intake of water in diabetic rats as compared to rats of control groups. Furthermore, they reported that water intake reduced significantly in diabetic rats after treatment with 50% bitter gourd fruit juice.
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Table 4.50. Mean squares for effect of diet and study weeks on water intake
Water intake S.O.V. df Study I Study II Study III
Diets 6 0.410* 1.461** 0.982**
Weeks 7 365.400** 334.315** 332.037**
Error 336 0.156 0.189 0.177
Total 391
* = Significant ** = Highly significant
110
30
27 D0
D1 24 D2
/rat/day D3 21
mL D4 18 D5 D6 15 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
Figure 4.4. Water intake in Study I (mL/rat/day)
30
27 D0
D1 24 D2
/rat/day D3 21
mL D4 18 D5 D6 15 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
Figure 4.5. Water intake in Study II (mL/rat/day)
30
27 D0
D1 24 D2
/rat/day D3 21
mL D4 18 D5 D6 15 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
Figure 4.6. Water intake in Study III (mL/rat/day)
111
4.6.3. Body Weight Gain
Statistical analysis (mean square) explicated that body weight affected substantially with diets and study intervals in all the studies (Table 4.51). Means in Figure 4.7 illustrated that in Study I, the body weight in different rat groups at week 1 was 128.83±1.51, 131.75±1.17, 129.92±2.54, 132.08±1.80, 133.25±0.88, 132.92±0.80 and 129.60±1.98 g/rat, respectively for groups D0, D1, D2, D3, D4, D5 and D6 that significantly increased with the passage of time to 215.90±2.59, 208.67±2.52, 208.30±2.43, 208.27±2.19, 208.40±0.53, 208.13±1.43 and 208.10±2.17 g/rat, respectively for the same groups at the termination of the trial.
At initiation of efficacy trial, body weight of rats in Study II (Figure 8) for D0, D1, D2, D3, D4,
D5 and D6 were 130.08±0.92, 131.83±0.98, 130.25±1.60, 129.83±2.04, 131.73±1.24, 133.17±0.75 and 129.50±1.87 g/rat, respectively. During 8 weeks, significant weight gain was observed in all groups with values of 229.13±2.50, 233.67±4.73, 234.50±4.27, 234.47±3.44, 235.33±1.15, 236.57±2.38 and 236.60±2.42 g/rat, respectively.
In Study III, the recorded weights (Figure 4.9) at 1st week were 132.42±1.63, 128.83±2.64, 129.67±2.42, 131.58±2.58, 132.25±2.44, 131.50±1.87 and 131.33±2.09 g/rat that substantially elevated at 8th week to 249.50±2.36, 243.27±1.62, 242.30±2.34, 242.10±1.56,
241.10±2.85, 239.67±2.08 and 239.10±1.47 g/rat, respectively in D0, D1, D2, D3, D4, D5 and
D6. It was also noted that weight is reduced in rats by giving bitter gourd fed with normal diet while in sucrose and cholesterol fed rats, weight is increased considerably in experimental groups in comparison to their respective control groups.
The current results regarding body weight are well supported by the earlier findings of Jafri et al. (2009) who pinpointed that there is an increase in body weight in normal control rats but in diabetic control rats that increase was lesser as compared to other diabetic groups fed with bitter gourd. Similarly, in another anti-hyperglycemic study, water extract of bitter gourd seeds involved in increase in body weight in diabetic rats (Sathishsekar & Subramanian, 2005). Likewise, Shetty et al. (2005) also found a marginal increase in body weight of diabetic rats fed with diet containing bitter gourd. Similar findings by Hossain et al. (2012) also strengthen the current results that body weight of diabetic groups treated with bitter gourd was higher than the untreated diabetic group.
112
Table 4.51. Mean squares for effect of diet and study weeks on body weight gain
Body weight gain S.O.V. df Study I Study II Study III
Diets 6 87.9** 59.0** 65.8**
Weeks 7 24390.4** 39623.8** 43650.2**
Error 336 4.0 4.5 4.2
Total 391
** = Highly significant
113
240
210 D0
D1
180 D2 D3
150 /rat/day g D4 120 D5 D6 90 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
Figure 4.7. Body weight gain in Study I (g/rat/day)
270
240 D0 D1 210 D2 180
D3 /rat/day g 150 D4 D5 120 D6 90 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
Figure 4.8. Body weight gain in Study II (g/rat/day)
270
240 D0 D1
210 D2 180
D3 /rat/day g 150 D4
120 D5 D6 90 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
Figure 4.9. Body weight gain in Study III (g/rat/day)
114
The present results of weight gain in hypercholesterolemic condition are in accordance with the findings of Klomann et al. (2010) who reported the highest body weight gain was in control group from onset of experiment till fifth week of study. They observed significant reduction in body weight in mice given whole fruit powder. In another study by Chen et al. (2003), it was observed that addition of bitter gourd in diet has resulted in reduction of body fat and hence, loss in weight. Due to accumulation of visceral fat, there is an increase in body weight and obesity that increases the risk of diabetes and heart diseases (Savini et al., 2013). So, bitter gourd supplementation in diet is helpful in controlling such maladies. It was also noticed that aqueous extract of bitter gourd is helpful in 16% reduction in the body weight. Bitter gourd increases activity of adenosine 5-monophosphate kinase that plays a pivotal role in uptake of cellular glucose and also involved in conversion of glucose into starch and stored it in liver. As a consequence, fatty acids are utilized for production of energy and ultimately lead to the loss in body weight (Bano et al., 2011).
4.6.4. Glucose
Mean squares for serum glucose (Table 4.52) indicated non-significant differences due to treatments whereas significant effect was observed for this trait regarding time interval, however, the interaction of treatment with study interval also imparted non-significant differences in Study I. The results showed highly significant effect of diets, study interval and their interaction on glucose level in Study II and III.
Mean glucose values (Table 4.53) at the termination of trial were 90.37±1.22, 88.60±2.25,
88.03±0.51, 87.47±1.21, 86.73±1.86 and 86.73±1.65 mg/dL for D0, D1, D2, D3, D4, D5 and D6 in study I; 142.93±2.70, 117.83±3.07, 112.23±2.46, 110.80±3.36, 104.53±4.23, 103.57±3.00 and 97.70±2.17 mg/dL in study II, and 106.33±2.52, 100.33±1.53, 99.87±1.86, 98.57±1.03, 97.30±2.56, 95.27±1.99 and 93.50±0.60 mg/dL in Study III, respectively. The highest glucose value of 142.93±2.70 was observed in hyperglycemic control D0 and the lowest in D6 (diet containing 300 mg/kg body weight of whole fruit powder) in all the studies.
The study intervals (0, 28th & 56th day) led to an enhancement in the glucose level of control groups i.e., 88.23±1.14, 89.90±2.55 & 90.37±1.22 in Study I, 88.17±0.99, 113.80±2.03 & 142.93±2.70 in Study II and 87.50±0.80, 98.43±1.21 & 106.33±2.52 mg/dL in Study III,
115 respectively. However, bitter gourd enriched diets substantially suppressed this trait with passage of time and the lowest glucose concentration was observed in D6 with values of 89.93±2.49, 87.80±2.08 & 86.07±1.23 in Study I, 90.40±1.93, 94.47±3.70 & 97.70±2.17 in Study II and 87.83±2.00, 94.63±1.72 & 93.50±0.60 mg/dL in Study III for 0, 28th and 56th day, respectively.
The Figure 4.10 indicated percent decrease in level of glucose by giving diet containing different preparations of bitter gourd fruit powder in rat groups of different studies. The study I indicated non-significant variation among treatment and noted 1.96%, 2.59%, 3.21%,
4.03%, 4.03% and 4.76% decline in D1, D2, D3, D4, D5 and D6, respectively compared to control. Study II showed the highest glucose reduction with values of 17.56%, 21.48%,
22.48%, 26.87%, 27.54% and 31.64% in D1, D2, D3, D4, D5 and D6, respectively. In case of hypercholesterolemic diet (Study III), addition of bitter gourd supplementation has resulted in marked reduction in glucose level as well. In this study 4.20%, 4.52%, 5.43%, 6.32%, 7.74% and 8.98% decrease in glucose was noted in respective groups.
The hypoglycemic ability of bitter gourd was well established by the outcomes of different investigations, highlighting importance of consumption of its different forms. The significant reduction in glucose level after consuming bitter gourd in hyperglycemic rats was reported by Jafri et al. (2009) which is in accordance with the present study. Singh and Gupta (2007) prepared extract of whole bitter gourd fruit in acetone and given in different doses (25, 50 and 75 mg/100 g body weight). They observed that blood glucose level decreased 13.30 to 50 percent in alloxan diabetic albino rats after 8, 15 and 30 days treatment. They confirmed antihyperglycemic property of this plant in diabetic condition in both animals as well as in humans. Later, Yuan et al. (2012) investigated that some protein hydrolysates from bitter gourd resulted in 46.15-52.59% reduction in blood glucose level. Srivastava et al. (1993) also reported that feeding of powdered bitter gourd for 3 to 7 weeks led to fall of 11 to 48% in post prandial blood glucose in diabetic patients. Virdi et al. (2003) also observed antihyperglycemic property of bitter gourd. They used various extracts of fresh and dried whole fruit of bitter gourd and found that water extract of 20 mg/kg of body weight when administered orally in rats has resulted in decline of blood glucose level up to 48%.
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Table 4.52. Mean squares for effect of diet and study intervals on Glucose of rats
Glucose S.O.V. df Study I Study II Study III
Treatments (T) 6 4.30N.S 425.85** 26.42**
Days (D) 2 15.35* 2963.42** 610.94**
T × D 12 3.23N.S 178.57** 15.43**
Error 42 4.63 5.93 2.89
Total 62
* = Significant ** = Highly significant N.S = Non-significant
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Table 4.53. Effect of diet and study intervals on Glucose (mg/dL)
0 Day 28th Day 56th Day Study I (Normal rats) abc ab a D0 88.23±1.14 89.90±2.55 90.37±1.22 ab abc abc D1 89.67±2.18 88.73±1.27 88.60±2.25 abc abc abc D2 88.97±3.29 88.03±1.36 88.03±0.51 a ab abc D3 90.97±2.17 89.63±2.85 87.47±1.21 abc abc bc D4 89.03±1.85 88.10±1.97 86.73±1.86 abc abc bc D5 89.17±2.40 88.00±4.23 86.73±1.65 ab abc c D6 89.93±2.49 87.80±2.08 86.07±1.23 Study II (Hyperglycemic rats) i c a D0 88.17±0.99 113.80±2.03 142.93±2.70 i def b D1 88.80±1.61 102.00±2.26 117.83±3.07 i efg c D2 89.00±3.00 100.33±1.93 112.23±2.46 i fgh c D3 89.10±1.41 98.13±1.91 110.80±3.36 i gh d D4 88.03±0.91 97.00±1.35 104.53±4.23 i gh de D5 89.83±2.27 97.50±1.57 103.57±3.00 i h gh D6 90.40±1.93 94.47±3.70 97.70±2.17 Study III (Hypercholesterolemic rats) h bcde a D0 87.50±0.80 98.43±1.21 106.33±2.52 h def b D1 87.97±0.67 96.77±1.25 100.33±1.53 h def bc D2 88.63±1.38 96.40±1.23 99.87±1.86 h defg bcd D3 89.50±1.57 96.10±2.13 98.57±1.03 h fg cdef D4 88.80±1.31 95.40±2.39 97.30±2.56 h efg fg D5 88.50±2.08 95.70±1.84 95.27±1.99 h fg g D6 87.83±2.00 94.63±1.72 93.50±0.60
D0 = Control D1 = Diet with 150 mg/kg body weight of skin powder D4 = Diet with 300 mg/kg body weight of flesh powder D2 = Diet with 300 mg/kg body weight of skin powder D5 = Diet with 150 mg/kg body weight of WF powder D3 = Diet with 150 mg/kg body weight of flesh powder D6 = Diet with 300 mg/kg body weight of WF powder
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35.00 31.64
30.00 27.54 26.87
25.00 22.48 D1 21.48 D2 20.00 17.56 D3 D4 15.00 D5 D6 10.00 8.98 7.74 6.32 5.43 4.76 4.03 4.03 4.20 4.52 5.00 3.21 2.59 1.96
0.00 Study I Study II Study III
Figure 4.10. Percent decrease in glucose level
119
In another study, Jayasooriya et al. (2000) observed effects of bitter gourd powder in cholesterol free and cholesterol enriched diets fed rats and noted that there was a continuous decline in glucose level in groups of rat fed with cholesterol free diet while no significant influence was noted in rats fed with cholesterol enriched diets. Clouatre et al. (2011) revealed reduction in blood glucose level by giving bitter gourd extracts at 50 mg/kg of body weight in normal rats. They also observed 63-67% reduction in blood glucose level by administering bitter gourd extract (250 mg/kg body weight) in STZ-induced diabetic rats.
Different components and extracts of bitter gourd are believed to possess hypoglycemic properties through different biochemical, physiological and pharmacological ways (Taylor, 2002; Garau et al., 2003; Bhushan et al., 2010). The oral administration of bitter gourd resulted in significant reduction in blood glucose level (Sathishsekar & Subramanian, 2005). A large number of factors may involve in glucose lowering effect of bitter gourd including stimulation of peripheral and skeletal muscles to utilize glucose (Cummings et al., 2007; Akhtar et al., 2011), prevention of glucose uptake from intestine (Uebanso et al., 2007; Jeong et al., 2008; Abdollah et al., 2010), enhancing activity of certain enzymes (Shibib et al., 1993) repairing islet β cells and enhancing their functional efficiency (Gadang et al., 2011). Bitter gourd extracts stimulate pancreatic islets of Langerhans to release insulin (Biyani et al., 2003; Chen et al., 2003). It also improves the function of insulin receptors in liver (Nerukar et al., 2008) and number of beta cells in pancreas to increase the production of insulin (Shetty et al., 2005). A number of phytochemicals are involved in fall of blood glucose level including charantin, polypeptide p, vicine etc.
4.6.5. Insulin
Mean squares in Table 4.54 elucidated non-significant influence of diet in Study I, whereas significant effect was observed for this trait regarding time interval, however, the interaction of treatment with study interval also imparted non-significant differences in this study. The results showed significant effect of diets and study interval on insulin level in Study II and III.
Means values at the end of trial concerning insulin (Table 4.55) for D0, D1, D2, D3, D4, D5 and D6 were 9.03±0.31, 9.10±0.36, 9.33±0.32, 9.07±0.35, 9.37±0.15, 9.13±0.25 and
120
9.87±0.76 in Study I; 12.23±0.74, 14.77±0.81, 14.93±0.75, 14.80±0.79, 15.13±0.49, 15.10±0.30 and 15.33±0.25 in Study II and 11.37±0.96, 12.00±0.62, 12.93±0.38, 12.17±0.32, 12.97±0.67, 12.57±0.47 and 13.10±0.20 µIU/mL, respectively in Study III. Increase in amount of bitter gourd in diet has resulted in production of more insulin.
The study intervals (0, 28th & 56th day) indicated that consumption of bitter gourd supplemented diet increase the production of inulin compared to control and maximum insulin was noted in D6, group fed with 300 mg/kg body weight of whole fruit of bitter gourd.
It is evident from the Figure 4.11 that providing bitter gourd in diets has resulted in elevation of insulin in comparison to control. In this milieu, bitter gourd powder consumption showed
0.78%, 3.32%, 0.44%, 3.77%, 1.11% and 9.30% increase in insulin production in D1, D2, D3,
D4, D5 and D6, respectively in the rats fed on normal diet (Study I). High insulin level was noted in Study II, where addition of bitter gourd in diet has resulted significant increase in the level of insulin 20.77%, 22.08%, 21.01%, 23.71%, 23.47% and 25.35% in respective groups. Increase in insulin was also noted in all the experimental groups (5.15%, 12.76%, 6.54%, 13.08%, 9.81% and 14.15%, respectively) in Study III.
Increase in secretion of insulin in diabetic pateints is highlighted in number of scientific investigation confirming encouraging impact of bitter gourd in diabetic conditions. The results of current study are in accordance with Mohammady et al. (2012), noted effect of bitter gourd on insulin level. In their experiment, significant increase in insulin was observed in diabetic rats treated with bitter gourd as compared to diabetic control group. They reported that insulin level in different groups were 19.6 µIU/mL in control group, 19.8 µIU/mL in bitter gourd control, 6.3 µIU/mL in diabetic control, 7.9 µIU/mL in diabetic with Avandia (synthetic drug against diabetes type 2) and 12.5 µIU/mL in diabetic with bitter gourd. Similarly, Fernandes et al. (2007) observed positive effect of bitter gourd on serum insulin. They reported insulin level in normal control group was 3.5 µIU/mL and in diabetic control was 1.6 µIU/mL. Supplementation of bitter gourd extract in diet resulted in increase in
121
Table 4.54. Mean squares for effect of diet and study intervals on Insulin of rats
Insulin S.O.V. df Study I Study II Study III
Treatments (T) 6 0.22 N.S 6.21** 1.03*
Days (D) 2 0.54* 156.33** 44.23**
T × D 12 0.09 N.S 1.48** 0.37 N.S
Error 42 0.13 0.24 0.25
Total 62
* = Significant ** = Highly significant N.S = Non-significant
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Table 4.55. Effect of diet and study intervals on Insulin (μIU/mL)
0 Day 28th Day 56th Day Study I (Normal rats) abc bcde de D0 9.67±0.32 9.17±0.06 9.03±0.31 abcde bcde cde D1 9.37±0.21 9.17±0.61 9.10±0.36 abcde bcde abcde D2 9.53±0.25 9.20±0.56 9.33±0.32 abcde abcde de D3 9.57±0.23 9.50±0.10 9.07±0.35 ab abcde abcde D4 9.70±0.35 9.30±0.40 9.37±0.15 abcde e bcde D5 9.30±0.10 9.00±0.36 9.13±0.25 abcd abcde a D6 9.60±0.36 9.37±0.31 9.87±0.76 Study II (Hyperglycemic rats) g f e D0 9.40±0.26 10.80±0.46 12.23±0.74 g d abc D1 9.47±0.23 13.83±0.76 14.77±0.81 g cd ab D2 9.60±0.36 14.00±0.44 14.93±0.75 g d abc D3 9.63±0.25 13.90±0.72 14.80±0.79 g bcd ab D4 9.83±0.51 14.50±0.10 15.13±0.49 g bcd ab D5 9.23±0.12 14.40±0.17 15.10±0.30 g ab a D6 9.50±0.10 14.97±0.35 15.33±0.25 Study III (Hypercholesterolemic rats) h g defg D0 9.53±0.15 10.67±0.75 11.37±0.96 h g cde D1 9.57±0.12 10.87±0.74 12.00±0.62 h fg ab D2 9.66±0.23 11.07±0.67 12.93±0.38 h g bcd D3 9.36±0.27 10.77±0.46 12.17±0.32 h efg ab D4 9.68±0.91 11.27±0.32 12.97±0.67 h efg abc D5 9.86±0.19 11.20±0.46 12.57±0.47 h cdef a D6 9.57±0.74 11.83±0.64 13.10±0.20
D0 = Control D1 = Diet with 150 mg/kg body weight of skin powder D4 = Diet with 300 mg/kg body weight of flesh powder D2 = Diet with 300 mg/kg body weight of skin powder D5 = Diet with 150 mg/kg body weight of WF powder D3 = Diet with 150 mg/kg body weight of flesh powder D6 = Diet with 300 mg/kg body weight of WF powder
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30.00
25.35 25.00 23.71 23.47 22.08 20.77 21.01
20.00 D1 D2 D3 15.00 14.15 12.76 13.08 D4 D5 9.81 10.00 9.30 D6
6.54 5.15 5.00 3.32 3.77
1.11 0.78 0.44 0.00 Study I Study II Study III
Figure 4.11. Percent increase in insulin level
124 insulin level of 2.2 µIU/mL by giving oral treatment of 150 mg/kg body weight of bitter gourd extract and 2.6 µIU/mL by giving 300 mg/kg body weight of bitter gourd extract.
The increase in insulin level in the diabetic rats after giving diet supplemented with bitter gourd might be due to recovery of beta cells of Langerhans (Fernandes et al., 2007, Singh and Gupta, 2007). Other studies indicated that addition of bitter gourd in diet enhance the number of beta cells (Mohammady et al., 2012). However, Sundaram and Kumar (2002) reported that bitter gourd did not involve in restoring of these cells rather it enhance the activity of beta cells. Bitter gourd may decrease the oxidative stress by neutralizing release of free radicals and hence death of beta cells. In another study, Xiang et al. (2007) proposed that bitter gourd act as a growth factor for beta cells of pancreas. Among the different bioactive moieties of bitter gourd, hypoglycemic components including charantin, polypeptide p and vicine etc. spontaneously increased the production of insulin.
From the above analysis it is inferred that different bitter gourd preparations are effective to minimize the malfunctioning in the secretion of insulin thus can be helpful in improving the glycemic status of the masses to a reasonable extent.
4.6.6. Cholesterol
The statistical analysis (ANOVA) regarding cholesterol is presented in Table 4.56. The results revealed that diet containing bitter gourd and study interval has highly significant effect on cholesterol among various groups in different studies.
The results pertaining to cholesterol (Table 4.57) in different groups of rats in Study I elaborated that the highest cholesterol was 85.83±0.35 mg/dL in D0 that reduced to
77.13±1.19 (D1), 75.90±2.56 mg/dL (D5), 75.17±2.22mg/dL (D3), 74.67±1.58 mg/dL (D6),
74.60±0.98 mg/dL (D2), 74.00±0.95 mg/dL (D4). The Study II explicated an obvious decrease in cholesterol level by provision of bitter gourd in diet. The cholesterol in diabetic control group D0 was 129.07±1.25 mg/dL that substantially reduced to 103.07±1.83,
100.80±1.30, 99.50±1.13, 97.97±1.77, 98.37±1.52 and 98.00±3.22 mg/dL in D1, D2, D3, D4,
D5 and D6, respectively. In Study III (Hypercholesterolemic rats), enormous increase in cholesterol was observed in control group D0 (160.67±4.16 mg/dL) while experimental groups fed with bitter gourd showed reduction in the level of cholesterol as 129.33±2.52,
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125.30±3.08, 123.37±3.28, 116.70±2.54, 124.70±2.86 and 118.83±1.91 mg/dL in D1, D2, D3,
D4, D5 and D6, respectively. The study interval of 0, 28, 56 day explicated an obvious decrease in cholesterol level from commencement till termination of the study in groups fed with diet containing bitter gourd powder.
The figure 4.12 depicted that diets containing bitter gourd has tendency to lower the level of cholesterol in the body. The highest percent decline in cholesterol for Study I was 13.78%
(D4) followed by 13.08% (D2), 13.00% (D6), 12.42% (D3), 11.57% (D5) and 10.14% (D1).
The observed decrease for this trait in Study II was 20.14% (D1), 21.90% (D2), 22.91% (D3),
24.10% (D4), 23.79% (D5) and 24.07% (D6). Similarly, Study III also elucidated significant cholesterol reduction in respective groups i.e., 24.28%, 27.40%, 28.90%, 34.07%, 27.87% and 32.42% in D1, D2, D3, D4, D5 and D6, respectively.
Many reports revealed that there is high risk of developing dyslipidaemia in diabetic patients (Al-Neaimy, 2008). The reason behind this lipid accumulation is mainly due to non- enzymatic reactions of the lipoproteins that lead to the formation of advanced glycosylation end products in abundance, which increases the hyperlipidaemia in a diabetic condition (Aronson & Rayfield, 2002). The current investigation proved that reducing the cholesterol leven and maintaining its metabolism is because of various bioactive components.
Senanyake et al. (2004) elucidated the role of dietary methanol fraction of bitter gourd at 0.5% and 1.0% in male golden Syrian hamsters in the cholesterol management. They reported that addition of bitter gourd to cholesterol enriched diet has resulted in significant reduction in the level of serum cholesterol i.e., 19.6% to 30.1%. They found an inverse association of serum cholesterol and amount of bitter gourd consumed thus designated it as a therapeutic plant against cholesterol synthesis. In another study, Jayasooria et al. (2000) used freeze-dried powder of bitter gourd to note its effect on lipid parameters in rats fed with diet supplemented with and without cholesterol. They observed 32.0% and 22.4% decline in total cholesterol level in absence/presence of dietary cholesterol. Abas et al. (2015) revealed that cholesterol was significantly increased in diabetic rats compared to control. Consumption of bitter gourd for 28 days showed significant reduction in cholesterol compared to diabetic control group. In an experiment on Wistar rats, decrease in cholesterol was noticed after consuming bitter gourd fruit extract (Fernandes et al., 2007). Similarly,
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Table 4.56. Mean squares for effect of diet and study intervals on Cholesterol of rats
Cholesterol S.O.V. df Study I Study II Study III
Treatments (T) 6 48.46** 198.31** 502.8**
Days (D) 2 29.85** 3103.75** 12837.1**
T × D 12 15.28** 104.30** 159.7**
Error 42 2.98 3.32 9.4
Total 62
** = Highly significant
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Table 4.57. Effect of diet and study intervals on Cholesterol (mg/dL)
0 Day 28th Day 56th Day Study I (Normal rats) bcd a a D0 78.63±1.95 84.13±0.67 85.83±0.35 bc bcd cdef D1 79.70±1.42 78.13±0.70 77.13±1.19 bc defg fg D2 79.53±1.88 76.37±0.87 74.60±0.98 bc cdef efg D3 79.60±3.24 77.27±1.96 75.17±2.22 bcd efg D4 78.47±1.48 75.17±1.98 74.00±0.95g b bcd defg D5 80.27±0.95 78.37±2.12 75.90±2.56 bc defg fg D6 79.60±2.34 75.93±1.70 74.67±1.58 Study II (Hyperglycemic rats) e c a D0 79.50±1.13 98.27±1.12 129.07±1.25 e d b D1 79.55±0.92 91.87±1.34 103.07±1.83 e d bc D2 80.45±1.77 90.50±1.44 100.80±1.30 e d bc D3 79.75±0.64 90.40±2.98 99.50±1.13 e d c D4 79.65±0.78 89.87±1.00 97.97±1.77 e d c D5 78.00±1.84 90.63±2.45 98.37±1.52 e d c D6 79.80±2.12 90.20±3.38 98.00±3.22 Study III (Hypercholesterolemic rats) h bc a D0 80.77±3.15 128.00±4.36 160.67±4.16 h ef b D1 80.47±3.72 114.87±3.82 129.33±2.52 h g bc D2 79.90±2.01 109.33±2.42 125.30±3.08 h fg cd D3 79.47±3.07 110.67±1.31 123.37±3.28 h g e D4 80.47±2.34 107.40±2.26 116.70±2.54 h g bc D5 79.53±3.43 107.33±1.53 124.70±2.86 h g de D6 78.67±3.06 109.33±4.86 118.83±1.91
D0 = Control D1 = Diet with 150 mg/kg body weight of skin powder D4 = Diet with 300 mg/kg body weight of flesh powder D2 = Diet with 300 mg/kg body weight of skin powder D5 = Diet with 150 mg/kg body weight of WF powder D3 = Diet with 150 mg/kg body weight of flesh powder D6 = Diet with 300 mg/kg body weight of WF powder
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40.00
35.00 34.07 32.42
30.00 28.90 27.40 27.87 D1 25.00 24.10 23.79 24.07 24.28 22.91 D2 21.90 20.14 D3 20.00 D4
D5 15.00 13.78 13.08 12.42 13.00 11.57 D6 10.14 10.00
5.00
0.00 Study I Study II Study III
Figure 4.12. Percent decrease in cholesterol level
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Bano et al. (2011) observed significant reduction (21%) in cholesterol after oral administration of aqueous extract of bitter gourd for a period of five weeks. Later, in an antidiabetic study by Wehash et al. (2012) on male Sprague Dawley rats indicated lipid lowering effect of bitter gourd. The ethanolic fraction of bitter gourd and fenugreek at 500 mg/kg body weight was given to STZ induced rats. The extract of bitter gourd resulted in marked reduction in the serum cholesterol level in bitter gourd supplemented diet (93.67 mg/dL) compared to diabetic group fed with control diet (204.67 mg/dL). Hossain et al. (2012) investigated antihyperglycemic and antihyperlipidemic effect of aqueous extract of bitter gourd at daily dose of 250, 500 and 750 mg/kg body weight. They observed 12.88%, 14.44% and 17.21% reduction in serum cholesterol, respectively.
Bitter gourd has the ability to reduce blood cholesterol level but the actual mechanism behind this reduction is still debatable. It was documented earlier that bitter gourd are involved in breaking down of certain enzymes involved in lipid metabolism thus responsible for decline in serum lipid parameters (Bailey & Day, 1989). It was also observed that bitter gourd inhibits the reabsorption of bile acid in the intestine and increases the production of certain enzymes that converts the cholesterol into bile acid (Matsui et al., 2013). In the nutshell, bitter gourd preparations proved beneficial in managing the serum cholesterol level thereby has potential to address lipid related abnormalities.
4.6.7. Low Density Lipoproteins (LDL)
Mean squares in Table 4.58 indicated that serum LDL elucidated non-significant influence of diet and time in Study I. However, highly significant variations in treatments and study intervals were observed during Study II and III.
Means in Table 4.59 showed maximum LDL level in D0 as 29.10±1.51 mg/dL that substantially reduced in D1 (29.00±1.81 mg/dL) trailed by D3 (28.77±2.06 mg/dL), D2
(28.07±3.072 mg/dL), D5 (27.97±1.72 mg/dL), D6 (27.93±1.55 mg/dL) and D4 (27.90±1.92 mg/dL) in Study I. However, the recorded values in Study II showed a diminishing trend with values of 55.80±1.87, 51.07±1.77, 49.67±2.40, 46.33±2.80, 49.57±1.55, 45.63±2.51 mg/dL in D1, D2, D3, D4, D5 and D6, respectively compared to control group D0 (63.85±2.47 mg/dL). Similar trend was observed in Study III with highest value of LDL in control group
130
(74.33±3.31 mg/dL) than D1 (63.20±3.51 mg/dL) D2 (60.23±3.40 mg/dL), D3 (57.00±1.28 mg/dL), D4 (54.67±2.40 mg/dL), D5 (54.57±1.55 mg/dL) and D6 (53.20±2.54 mg/dL).
During Study I, LDL level was varied insignificantly from initiation to termination (0, 28th & 56th day) while in Study II and III, LDL increased with the passage of time but this increase was more obvious in control groups than the groups fed with diet containing bitter gourd.
The Figure 4.13 illustrated the percent reduction in LDL level of rats fed on various bitter gourd preparations compared to control. In Study I, maximum LDL reduction of 4.12 % was noticed in D4, followed by 4.02% in D6, 3.88% in D5, 3.54% D2, 1.13% in D3 and 0.34% in
D1. In study II, 12.61%, 20.02%, 22.21%, 27.44%, 22.36% and 28.54% reduction and in Study III 17.43%, 22.08%, 27.14%, 30.79%, 30.95% and 33.09% decline in LDL was observed in D1, D2, D3, D4, D5 and D6, respectively.
The low density lipoproteins are the major carrier for cholestrerol in blood (Tymoczko et al., 2002). Bitter gourd has the ability to reduce this „bad cholesterol‟ from the blood (Wehash et al., 2012). Temitope et al. (2013) orally administered aqueous extract of bitter gourd at dose of 80, 100, 120, 140 mg/kg body weight for fourteen days. Significant decline in LDL in experimental groups were observed compared to rats fed with normal diet. In another study (Wehash et al., 2012) on Sprague Dawley rats, diabetes was induced by STZ injection. These diabetic rats were fed with diet containing ethanolic fraction of bitter gourd and fenugreek at the rate of 500 mg/kg body weight. The significant reduction was observed regarding LDL level from 141.51 to 31.18 mg/dL in diabetic group fed with control diet and bitter gourd supplemented diet, respectively. Kim et al. (2012) noted increase in LDL in STZ induced diabetic control and decline in all the experimental groups at the end of 28 day study period. Similarly, Chaturvedi et al. (2004) also found reduction in amount of low density lipoproteins in blood by administering bitter gourd extracts. The lowering of LDL by consuming bitter gourd in diet might be due to secretion of Apolipoprotein-B by the liver.
4.6.8. High Density Lipoproteins (HDL)
Mean squares in Table 4.60 elucidated that HDL was significantly affected by treatments and study intervals in all the studies, however, interaction expounded non-significant influence in Study I.
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Table 4.58. Mean squares for effect of diet and study intervals on LDL of rats
LDL S.O.V. df Study I Study II Study III
Treatments (T) 6 5.39 N.S 77.40** 145.87**
Days (D) 2 13.17 N.S 4575.03** 5111.49**
T × D 12 4.26 N.S 18.86** 37.29**
Error 42 4.13 4.69 4.92
Total 62
** = Highly significant N.S = Non-significant
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Table 4.59. Effect of diet and study intervals on LDL (mg/dL)
0 Day 28th Day 56th Day Study I (Normal rats) ab ab ab D0 29.87±2.06 29.97±2.39 29.10±1.51 ab ab ab D1 29.93±2.28 29.50±2.46 29.00±1.81 ab ab ab D2 29.63±3.14 29.77±1.40 28.07±3.07 ab ab ab D3 28.63±1.21 29.33±1.79 28.77±2.06 ab ab abc D4 29.80±2.09 28.60±1.47 27.90±1.92 a bc ab D5 31.03±2.56 27.60±1.15 27.97±1.72 ab bc abc D6 29.07±2.15 27.27±1.30 27.93±1.55 Study II (Hyperglycemic rats) hi f a D0 29.53±3.23 38.93±1.55 63.85±2.47 hi g b D1 28.87±1.37 33.63±1.45 55.80±1.87 hi gh c D2 29.30±2.57 32.10±2.11 51.07±1.77 i gh cd D3 28.17±1.45 31.80±1.95 49.67±2.40 hi hi de D4 28.93±2.66 29.77±2.49 46.33±2.80 hi ghi cd D5 28.70±1.55 31.40±3.02 49.57±1.55 i hi e D6 28.07±1.46 29.17±1.85 45.63±2.51 Study III (Hypercholesterolemic rats) i f a D0 29.73±3.87 49.40±1.35 74.33±3.31 i g b D1 28.63±1.08 40.57±1.63 63.20±3.51 i g bc D2 29.03±2.75 40.17±1.27 60.23±3.40 i gh cd D3 28.67±2.15 39.57±1.53 57.00±1.28 i gh de D4 27.90±0.89 39.00±0.26 54.67±2.40 i gh de D5 28.30±2.10 37.37±2.30 54.57±1.55 i h e D6 28.73±1.50 36.13±1.63 53.20±2.54
D0 = Control D1 = Diet with 150 mg/kg body weight of skin powder D4 = Diet with 300 mg/kg body weight of flesh powder D2 = Diet with 300 mg/kg body weight of skin powder D5 = Diet with 150 mg/kg body weight of WF powder D3 = Diet with 150 mg/kg body weight of flesh powder D6 = Diet with 300 mg/kg body weight of WF powder
133
35.00 33.09
30.79 30.95
30.00 28.54 27.44 27.14
25.00 22.21 22.36 22.08 20.02 D1 20.00 D2 17.43 D3 D4 15.00 12.61 D5 D6 10.00
4.12 5.00 3.54 3.88 4.02
1.13 0.34 0.00 Study I Study II Study III
Figure 4.13. Percent decrease in LDL level
134
Mean HDL values (Table 4.61) at the end of trial were 38.80±3.17, 40.23±1.65, 41.90±2.76,
41.23±2.90, 42.63±1.55, 42.73±1.57 and 42.77±2.70 mg/dL for D0, D1, D2, D3, D4, D5 and D6 in study I; 35.97±2.25, 42.23±1.19, 43.23±1.17, 42.90±1.61, 43.30±0.98, 45.63±2.80 and 47.43±1.12 mg/dL in study II, and 35.97±1.72, 44.90±2.41, 47.03±2.85, 46.00±2.69, 49.30±1.97, 49.20±4.68 and 51.63±4.07 mg/dL in Study III, respectively. The highest increase in HDL level was observed in D6 (diet containing 300 mg/kg body weight of whole fruit powder) in all the studies.
The study interval influenced significantly on the increase in HDL level and the highest increase was observed in D6 with values of 34.20±1.93, 41.13±2.04 & 42.77±2.70 in Study I; 33.53±0.93, 42.00±1.90 & 47.43±1.12 in Study II and 33.40±1.35, 47.70±1.31 & 51.63±4.07 in Study III for 0, 28th and 56th day, respectively.
The Figure 4.14 revealed percent rise in level of HDL by giving bitter gourd in the diet in different studies. The study I indicated significant variations among treatments and noticed
3.69%, 7.99%, 6.26%, 9.87%, 10.13% and 10.23% increase in D1, D2, D3, D4, D5 and D6, respectively compared to control. In the Study II, HDL level also increased in experimental groups with values of 17.40%, 20.18%, 19.27%, 20.38%, 26.86% and 31.86% in D1, D2, D3,
D4, D5 and D6, respectively. In case of hypercholesterolemic diet (Study III), addition of bitter gourd supplementation has resulted in marked increase in HDL level. In this study, 24.83%, 30.75%, 27.88%, 37.06%, 36.78% and 43.54 % increase in HDL was noted in respective groups.
The higher amount of HDL in blood is considered good because it is found good for health and mostly designated as „good cholesterol‟. The decline in HDL concentration in blood leads to wide ranging cardiovascular complications. The lower level of HDL is particularly involved in development of atherosclerosis. Bitter gourd in diet is helpful in increasing the level of HDL in blood. Bano et al. (2011) revealed that increased the serum HDL level rise up to 45 % by administering aqueous extract of bitter gourd for five weeks. Similarly, Wehash et al. (2012) conducted experiment on Sprague Dawley rats which were made diabetic by STZ. These diabetic induced rats were fed with ethanolic fraction of bitter gourd and fenugreek @ 500mg/kg of body weight. It was noted that bitter gourd supplementation
135
Table 4.60. Mean squares for effect of diet and study intervals on HDL of rats
S.O.V. df HDL
Study I Study II Study III
Treatments (T) 6 11.69* 30.65** 75.95**
Days (D) 2 288.17** 392.52** 821.95**
T × D 12 2.97 N.S 14.88** 25.40**
Error 42 4.11 3.28 6.41
Total 62
* = Significant ** = Highly significant N.S = Non-significant
136
Table 4.61. Effect of diet and study intervals on HDL (mg/dL)
0 Day 28th Day 56th Day Study I (Normal rats) g def bcd D0 33.83±1.80 37.30±2.29 38.80±3.17 fg cde abcd D1 34.07±0.71 37.53±1.27 40.23±1.65 efg bcd ab D2 35.23±2.32 39.07±2.05 41.90±2.76 efg abc ab D3 34.77±1.71 40.87±1.56 41.23±2.90 efg ab a D4 35.03±1.64 41.73±1.75 42.63±1.55 g abcd a D5 33.43±1.17 40.53±2.18 42.73±1.57 efg ab a D6 34.20±1.93 41.13±2.04 42.77±2.70 Study II (Hyperglycemic rats) de de de D0 34.93±2.37 35.93±2.27 35.97±2.25 de d c D1 35.20±1.55 37.23±0.95 42.23±1.19 de c bc D2 34.90±2.23 41.73±2.24 43.23±1.17 de c bc D3 35.10±2.66 41.60±1.51 42.90±1.61 de c ab D4 34.50±1.57 41.73±1.75 43.30±0.98 de c ab D5 34.77±0.99 40.87±1.99 45.63±2.80 e c a D6 33.53±0.93 42.00±1.90 47.43±1.12 Study III (Hypercholesterolemic rats) h gh gh D0 34.57±2.12 35.93±1.46 35.97±1.72 h fg cde D1 34.27±1.06 39.13±1.24 44.90±2.41 h ef bcd D2 34.83±2.80 42.40±3.20 47.03±2.85 h def bcde D3 32.23±1.68 43.07±3.82 46.00±2.69 h de ab D4 34.30±1.45 43.33±2.69 49.30±1.97 h bcde ab D5 34.83±1.75 45.73±2.74 49.20±4.68 h abc a D6 33.40±1.35 47.70±1.31 51.63±4.07
D0 = Control D1 = Diet with 150 mg/kg body weight of skin powder D4 = Diet with 300 mg/kg body weight of flesh powder D2 = Diet with 300 mg/kg body weight of skin powder D5 = Diet with 150 mg/kg body weight of WF powder D3 = Diet with 150 mg/kg body weight of flesh powder D6 = Diet with 300 mg/kg body weight of WF powder
137
50.00
45.00 43.54
40.00 37.06 36.78
35.00 31.86 30.75 D1 30.00 27.88 26.86 D2 24.83 25.00 D3 D4 20.18 20.38 19.27 20.00 D5 17.40 D6 15.00
9.87 10.13 10.23 10.00 7.99 6.26 5.00 3.69
0.00 Study I Study II Study III
Figure 4.14. Percent increase in HDL level
138 resulted in increase in HDL level from 14.03 mg/dL to 41.48 mg/dL in diabetic group fed with control diet and bitter gourd supplemented diet, respectively. Temitope et al. (2013) examined different doses of bitter gourd to analyze HDL level in blood and found diets containing100 mg/kg and 140 mg/kg body weight as suitable dietary approaches and resulted in marked increase in the HDL level than the rest of the treatments. Many bioactive moieties in bitter gourd are responsible this rise in HDL.
4.6.9. Triglycerides
Mean squares regarding triglycerides (Table 4.62) represented that triglycerides were non- significantly affected by treatments and study intervals in Study I, whereas significant differences were observed in Study II and III because of diets and study intervals along with their interaction.
Mean values for triglycerides (Table 4.63) indicated non-substantial differences in Study I with values of 68.33±3.26, 68.10±1.37, 67.63±1.59, 67.23±1.71, 66.13±2.64, 66.73±2.84 and
66.03±3.16 mg/dL for D0, D1, D2, D3, D4, D5 and D6, respectively. In Study II, 92.87±3.80, 87.00±2.66, 85.43±1.01, 85.53±1.60, 85.10±1.64, 84.90±2.76 and 82.37±2.44 mg/dL of triglycerides were observed in D0, D1, D2, D3, D4, D5 and D6, respectively. The triglycerides in hypercholesterolemic rats (Study III) indicated 124.67±3.06, 103.57±3.37, 99.70±1.25, 99.27±4.34, 99.23±5.19, 98.67±2.34 and 98.23±4.47 mg/dL in respective groups.
The amounts of triglycerides gradually rise with the passage of time. This rise is more noticeable in control groups and supplementation of bitter gourd in diet has resulted in considerable reduction in triglycerides when noticed in 0, 28 and 56 day.
The Figure 4.15 depicted amount of triglycerides in rat groups of different studies. The percent reduction in D1, D2, D3, D4, D5 and D6 was 0.34%, 1.02%, 1.61%, 3.22%, 2.34% and 3.37%, respectively in Study I; 6.32%, 8.01%, 7.90%, 8.37%, 8.58% and 11.31%, respectively in Study II and 22.72%, 26.89%, 27.35%, 27.39%, 28.00% and 28.47%, respectively in Study III.
Increase in triglycerides is related with cardiovascular diseases hence reduction in its amount may lessen the onset of diseases dependent on cholesterol as well.
139
Table 4.62. Mean squares for effect of diet and study intervals on Triglycerides of rats
Triglycerides S.O.V. df Study I Study II Study III
Treatments (T) 6 1.54 N.S 23.12** 254.57**
Days (D) 2 2.83 N.S 1715.81** 6564.50**
T × D 12 1.07 N.S 9.28* 76.48**
Error 42 4.37 4.42 8.46
Total 62
* = Significant ** = Highly significant N.S = Non-significant
140
Table 4.63. Effect of diet and study intervals on Triglycerides (mg/dL)
0 Day 28th Day 56th Day Study I (Normal rats)
D0 68.43±0.71 68.20±1.40 68.33±3.26
D1 67.77±2.29 67.37±1.43 68.10±1.37
D2 67.60±0.89 66.60±1.07 67.63±1.59
D3 67.80±2.00 67.53±0.87 67.23±1.71
D4 68.37±1.65 67.50±2.12 66.13±2.64
D5 67.87±1.19 67.43±3.31 66.73±2.84
D6 67.37±1.50 67.03±3.25 66.03±3.16 Study II (Hyperglycemic rats) g de a D0 67.43±0.76 81.43±2.70 92.87±3.80 g ef b D1 68.30±1.21 78.37±1.56 87.00±2.66 g f bc D2 68.40±2.11 77.37±2.43 85.43±1.01 g f bc D3 68.03±2.31 77.33±1.53 85.53±1.60 g f bc D4 68.13±1.98 76.53±2.15 85.10±1.64 g f bc D5 68.93±1.32 77.30±1.70 84.90±2.76 g f cd D6 67.53±0.78 76.30±2.80 82.37±2.44 Study III (Hypercholesterolemic rats) e c a D0 67.70±2.12 96.67±4.09 124.67±3.06 e d b D1 68.27±1.06 81.67±2.18 103.57±3.37 e d bc D2 67.63±2.80 80.10±2.46 99.70±1.25 e d bc D3 68.80±1.68 81.03±3.10 99.27±4.34 e d bc D4 69.17±1.45 79.43±1.63 99.23±5.19 e d c D5 68.70±1.75 78.57±3.19 98.67±2.34 e d c D6 67.37±1.35 77.03±2.75 98.23±4.47
D0 = Control D1 = Diet with 150 mg/kg body weight of skin powder D4 = Diet with 300 mg/kg body weight of flesh powder D2 = Diet with 300 mg/kg body weight of skin powder D5 = Diet with 150 mg/kg body weight of WF powder D3 = Diet with 150 mg/kg body weight of flesh powder D6 = Diet with 300 mg/kg body weight of WF powder
141
30.00 28.47 28.00 27.35 27.39 26.89
25.00 22.72
20.00 D1 D2
15.00 D3 D4 11.31 D5
10.00 D6 8.37 8.58 8.01 7.90 6.32
5.00 3.22 3.37 2.34 1.61 1.02 0.34 0.00 Study I Study II Study III
Figure 4.15. Percent decrease in triglycerides
142
In present investigation, bitter gourd provision reduced the synthesis of triglyceride in rats. Consistent with current results, Jayasooria et al. (2000) determined the effect of bitter gourd on lipid metabolism and found significant reduction of 39.2% and 40.5% in hepatic triglycerides in cholesterol rich and cholesterol free diets, respectively. Likewise, Senanayake et al. (2004) investigated hamsters fed with diet supplemented with bitter gourd methanol extract. The rats were given cholesterol enriched and cholesterol free diet. They found that bitter gourd supplementation at 0.5% and 1.0% reduced the amount of triglycerides to an extent of 26.1% and 36.2%, respectively. Later, Sato et al. (2011) reported that some members of genus Momordica at 3% in diet significantly reduced the level of triglyceride in the blood. According to them, the decrease was mediated through enhanced excretion of fecal lipid excretion and their lymphatic transport. The present results also supported by the findings of Bano et al. (2011) determined 20% decline in serum triglyceride level due to dietary intake of bitter gourd in diabetic rats. Hossain et al. (2012) observed significant elevation in triglycerides level (47.02%) in diabetic control rat groups. However, bitter gourd at the rate of 250, 500, 750 mg reduced the triglyceride level to 30.30%, 33.84% and 37.43%, respectively. The findings indicated a decline of triglycerides in a dose dependent manner. They observed effect of diet containing bitter gourd on triglyceride level during study period of 90 days. They obtained blood samples at every 15 days interval and observed continuous reduction in triglycerides compared to control group. Recently, Abas et al. (2015) reported that level of triglyceride reduced from 0.57 ± 0.08 mmol/L due to dietary intake of bitter gourd in rats compared to diabetic control group (0.95 ±0.09 mmol/L) following 4 weeks of treatment. No doubt, all bitter gourd forms were beneficial and effective to address lipid related disorders.
4.6.10. Alkaline Phosphatase (ALP)
Mean squares regarding Alkaline phosphatase (ALP) concentration (Table 4.64) showed non significant differences between diets and study interval in Study I. However, substantial differences owing to treatments and study interval were found in Study II and III.
Means for ALP (Table 4.65) depicted that values were 164.87±3.35, 163.17±4.43, 163.33±2.08, 163.47±3.09, 162.10±2.31, 163.13±4.44 and 162.43±1.46 IU/L for groups feeding D0, D1, D2, D3, D4, D5 and D6 diets, respectively under normal study (Study I). In
143 hyperglycemic study (Study II), level of ALP was decreased from 203.40±2.46 IU/L (D0) to 193.67±3.91, 193.60±3.05, 191.53±3.51, 191.20±2.65, 191.47±4.28 and 187.83±1.89 IU/L with consumption of diets D1, D2, D3, D4, D5 and D6, respectively. ALP value was also reduced due to feeding of diet containing bitter gourd in Hypercholesterolemic study (Study III). Values for ALP in this study was recorded as 210.43±4.04, 194.67±2.28, 194.57±3.44,
192.03±4.24, 191.53±3.21, 191.30±4.55 and 189.03±3.48 IU/L for D0, D1, D2, D3, D4, D5 and D6, respectively. Consumption of diet containing bitter gourd has sound impact on ALP when noted 0, 28 and 56 day of trial.
Current findings are comparable with the earlier research investigations regarding ALP. Hossain et al. (2012) illustrated an incredible increase (174.05 U/I) in ALP in diabetic control in comparison to normal control (62.18 U/I). The aqueous extract of fruits of bitter gourd @ 250, 500 and 700 mg in diet resulted in significant reduction in ALP with values of 140.12, 134.60 and 130.20 U/I, respectively. They observed that fruit extract of bitter gourd minimize the elevated levels of ALP by 19.49%, 22.67%, 25.19% by giving doses of 250, 500, and 750 mg/kg body weight, respectively. Earlier, Tennekoon, et al. (1994) reported that bitter gourd has the tendency to significantly reduce ALP concentration in the liver by oral intake of bitter gourd at the rate of 1 milliliter per 100 gram for 30 days. In certain metabolic disorders, activities of phosphatases influence on the movement of certain metabolites in and out of the membrane due to changes in dephosphorylation reactions. In such conditions, levels of phosphatases increased enormously and cause intracellular inorganic phosphate level to rise, which alter the efficiency of ionic pumps and decreased activities of Na+ K+ ATPases (Sailaja, 2000). The bioactive components in bitter gourd reduced the level of ALP in such abnormal conditions and maintain suitable homeostatic conditions within the body.
4.6.11. Alanine Transferase (ALT)
Data pertaining to mean squares in Table 4.66 indicated non-significant effect due to diet but study interval imparted significant effect in Study I. The momentous variations in the ALT as a function of treatments and study interval were observed in Study II however, the interactive effect was found non-momentous. In Study III, substantial differences owing to treatments, study interval and interaction were found.
144
Table 4.64. Mean squares for effect of diet and study intervals on ALP of rats
ALP S.O.V. df Study I Study II Study III
Treatments (T) 6 1.95N.S 69.45* 113.19**
Days (D) 2 29.99 N.S 4709.30** 5442.46**
T × D 12 1.35N.S 19.44* 43.83*
Error 42 16.86 15.06 13.92
Total 62
* = Significant ** = Highly significant N.S = Non-significant
145
Table 4.65. Effect of diet and study intervals on Alkaline Phosphatase (IU/L)
0 Day 28th Day 56th Day Study I (Normal rats) a a a D0 165.20±3.73 163.83±2.76 164.87±3.35 a a a D1 165.77±6.55 162.73±1.94 163.17±4.43 a a a D2 164.47±3.84 161.67±2.31 163.33±2.08 a a a D3 164.57±7.40 163.10±6.58 163.47±3.09 a a a D4 164.83±3.45 162.30±1.61 162.10±2.31 a a a D5 164.83±2.60 163.27±3.48 163.13±4.44 a a a D6 163.77±7.40 162.10±2.76 162.43±1.46 Study II (Hyperglycemic rats) e b a D0 165.33±4.52 193.43±2.54 203.40±2.46 e bcd b D1 164.10±5.85 189.80±6.12 193.67±3.91 e bcd b D2 164.90±1.51 187.47±4.66 193.60±3.05 e cd bc D3 164.53±2.01 185.63±4.57 191.53±3.51 e d bc D4 165.63±4.87 184.33±2.06 191.20±2.65 e d bc D5 164.20±3.28 184.50±3.68 191.47±4.28 e d bcd D6 164.73±3.54 184.33±5.66 187.83±1.89 Study III (Hypercholesterolemic rats) f b a D0 163.93±3.04 196.13±1.70 210.43±4.04 f bcde bc D1 162.27±4.73 190.37±5.93 194.67±2.28 f de bc D2 165.07±3.21 187.53±5.15 194.57±3.44 f de bcd D3 164.93±4.46 186.43±3.57 192.03±4.24 f de bcd D4 165.37±3.75 187.00±3.65 191.53±3.21 f de bcd D5 163.87±3.21 186.17±2.27 191.30±4.55 f e cde D6 164.40±1.66 185.00±3.60 189.03±3.48
D0 = Control D1 = Diet with 150 mg/kg body weight of skin powder D4 = Diet with 300 mg/kg body weight of flesh powder D2 = Diet with 300 mg/kg body weight of skin powder D5 = Diet with 150 mg/kg body weight of WF powder D3 = Diet with 150 mg/kg body weight of flesh powder D6 = Diet with 300 mg/kg body weight of WF powder
146
Means for ALT (Table 4.67) depicted that values were 42.70±2.23, 41.37±0.99, 41.27±0.91,
40.20±1.06, 39.87±0.38, 39.60±2.16 and 39.37±2.40 IU/L for groups feeding D0, D1, D2, D3,
D4, D5 and D6 diets, respectively in Study I. In Study II, ALT values were decreased from
49.37±0.70 IU/L (D0) to 44.57±0.83, 43.93±1.56, 43.87±1.63, 43.30±1.11, 43.37±1.16 and
43.20±1.42 IU/L with consumption of diets D1, D2, D3, D4, D5 and D6, respectively. In Study III, reduction in ALT value was also recoded after feeding of diet containing bitter gourd from 53.77±1.76 (D0) to 48.77±1.50 (D1), 47.27±0.91 (D2), 46.50±3.18 (D3), 45.87±1.53
(D4), 44.57±3.10 (D5) and 44.17±1.70 IU/L (D6). Study interval (0, 28, 56 day) showed significant influence of bitter gourd on ALT of rats with time.
The present outcomes are consistent with the previous findings of Hossain et al. (2012), investigated the impact of aqueous extract of bitter gourd fruit at different doses of 250, 500 and 750 mg against ALT in rats compared to normal control and diabetic rats. They observed that the ALT level in diabetic rats was significantly reduced to 15.37%, 16.68% and 22.25% respectively by the use of aforesaid amount of bitter gourd extract. Similarly, Kim et al. (2012) studied that ALT level in STZ induced diabetic rats is higher (150.20±13.59 and 320.80±14.18) at 6 and 28 day of study, respectively compared to group fed with diet containing bitter gourd i.e., 150.60±13.20 and 264.60±23.64, respectively.
4.6.12. Aspartate Transferase (AST)
Analysis of variance (ANOVA) for AST revealed non-significant impact because of diet whereas study interval highly momentous results for Study I, whereas, in Study II and III, highly significant results were obtained for different bitter gourd based diets, study interval and their interaction (Table 4.68).
In Study I (normal study), means for AST were observed in control group (D0) as 137.67±1.20 IU/L whilst 136.40±0.56, 135.13±1.24, 135.10±1.54, 135.08±1.03, 135.10±1.13 and 135.07±1.95 IU/L for rats fed on bitter gourd supplementary diets D1, D2, D3, D4, D5 and
D6, respectively. In hyperglycemic study (Study II), level of AST was decreased from
151.93±2.10 IU/L (D0) to 145.40±1.35, 145.13±1.24, 145.20±1.54, 144.83±1.42,
145.00±1.41 and 144.60±1.31 IU/L with consumption of diets D1, D2, D3, D4, D5 and D6, respectively. In hypercholesterolemic study (Study III), values for AST were recorded as
147
Table 4.66. Mean squares for effect of diet and study intervals on ALT of rats
ALT S.O.V. df Study I Study II Study III
Treatments (T) 6 3.09 N.S 12.00** 26.61**
Days (D) 2 18.26* 39.44** 161.05**
T × D 12 1.51N.S 3.19 N.S 7.54*
Error 42 2.13 2.19 2.47
Total 62
* = Significant ** = Highly significant N.S = Non-significant
148
Table 4.67. Effect of diet and study intervals on Alanine Transferase (IU/L)
0 Day 28th Day 56th Day Study I (Normal rats) ab a ab D0 42.53±1.90 42.90±1.37 42.70±2.23 a abcde abcde D1 42.90±1.35 41.47±0.49 41.37±0.99 abcde abcde abcde D2 41.37±0.93 41.40±0.87 41.27±0.91 abcd abcde bcde D3 42.00±1.15 41.37±0.93 40.20±1.06 abc abcde cde D4 42.07±1.23 41.40±2.18 39.87±0.38 a abcde de D5 42.93±2.38 40.87±0.85 39.60±2.16 ab cde e D6 42.60±1.21 39.83±0.81 39.37±2.40 Study II (Hyperglycemic rats) bcd bc a D0 42.63±0.90 44.23±1.88 49.37±0.70 bcd bcd b D1 42.20±1.31 42.47±0.93 44.57±0.83 bcd bcd bc D2 42.83±1.75 42.23±1.70 43.93±1.56 d bcd bc D3 41.17±1.08 42.13±1.10 43.87±1.63 bcd d bcd D4 42.17±0.90 41.30±0.87 43.30±1.11 cd cd bcd D5 42.00±2.39 41.93±0.47 43.37±1.16 d cd bcd D6 41.03±2.18 41.83±2.76 43.20±1.42 Study III (Hypercholesterolemic rats) fg bc a D0 42.27±1.25 47.80±1.23 53.77±1.76 fg efg b D1 42.37±0.80 43.40±0.56 48.77±1.50 g efg bc D2 41.17±0.40 43.30±1.92 47.27±0.91 fg efg bcd D3 42.03±1.46 43.40±0.82 46.50±3.18 g fg cde D4 41.20±1.49 43.03±1.26 45.87±1.53 fg fg def D5 42.00±1.55 43.27±1.04 44.57±3.10 g fg def D6 41.43±1.33 43.07±1.00 44.17±1.70
D0 = Control D1 = Diet with 150 mg/kg body weight of skin powder D4 = Diet with 300 mg/kg body weight of flesh powder D2 = Diet with 300 mg/kg body weight of skin powder D5 = Diet with 150 mg/kg body weight of WF powder D3 = Diet with 150 mg/kg body weight of flesh powder D6 = Diet with 300 mg/kg body weight of WF powder
149
172.67±2.11, 162.07±1.99, 161.67±1.46, 161.87±2.03, 161.50±1.47, 161.13±1.79 and
161.53±1.70 IU/L for D0, D1, D2, D3, D4, D5 and D6, respectively (Table 4.69). AST was markedly decreased when noted in 28 day and further decline was observed in 56 day samples in all the treated groups compared to respective control groups.
The current data is comparable to the earlier investigations of Hossain et al. (2012) confirmed a reduction in serum AST of rats treated with bitter gourd extract. They observed 19.75%, 22.56% and27.95% reduction in AST by provision of bitter gourd supplemented diet at 250, 500 and 700 mg in different rat groups. In another study by Kim et al. (2012) noted that bitter gourd is helpful in reducing the amount of AST to 368.00±20.95 and 572.60±50.17 compared to control group 371.80±38.05 and 778.60±47.71 after 6 and 28 day of analysis. Moreover, Sathishsekar and Subramamian (2005) and Senanyake et al. (2004) also reported hepato-protective role of bitter gourd. Hence, bitter gourd is beneficial in reducing the level of AST and ALT.
4.6.13. Serum Creatinine
Mean squares for serum creatinine (Table 4.70) showed non-momentous differences due to treatments and time intervals during the bioevaluation trial in Study I. On the other hand, highly significant effect of diet and study interval was noted in Study II and III.
In Study I, means for serum creatinine (Table 4.71) at the termination of study were observed in control group (D0) as 0.74±0.03 mg/dL whilst for rats fed on bitter gourd slight reduction was noted for D1, D2, D3, D4, D5 and D6 as 0.69±0.01, 0.69±0.01, 0.69±0.02, 0.68±0.03,
0.69±0.01 and 0.69±0.01 mg/dL, respectively. The level of creatinine was recorded for D0,
D1, D2, D3, D4, D5 and D6 as 0.91±0.03, 0.80±0.02, 0.79±0.03, 0.79±0.03, 0.78±0.02, 0.78±0.02 and 0.77±0.01 mg/dL, respectively in Study II. Serum creatinine concentration was also reduced due to feeding of bitter gourd in diet in Hypercholesterolemic study Study III. The values for creatinine in this study was recorded as 1.17±0.05, 0.84±0.03, 0.83±0.04,
0.84±0.02, 0.82±0.02, 0.82±0.01 and 0.81±0.01 mg/dL for D0, D1, D2, D3, D4, D5 and D6, respectively.
During the course of trial, slight increase was observed in creatinine concentration in 28 and 56 day analysis compared to 0 day study but this increase is more prominent in control groups as compared to rat groups fed with bitter gourd in diet.
150
Table 4.68. Mean squares for effect of diet and study intervals on AST of rats
AST S.O.V. df Study I Study II Study III
Treatments (T) 6 3.01N.S 14.37** 50.74**
Days (D) 2 8.56* 494.24** 3748.86**
T × D 12 0.96N.S 7.95* 16.05**
Error 42 2.27 2.23 2.01
Total 62
* = Significant ** = Highly significant N.S = Non-significant
151
Table 4.69. Effect of diet and study intervals on Aspartate Transferase (IU/L)
0 Day 28th Day 56th Day Study I (Normal rats) abc abc a D0 136.63±2.87 136.40±2.26 137.67±1.20 abc abc abc D1 137.40±0.89 135.43±1.89 136.40±0.56 ab abc bc D2 137.57±1.70 135.37±0.85 135.13±1.24 abc abc bc D3 136.37±2.08 135.20±1.54 135.10±1.54 abc bc c D4 136.30±1.35 135.17±0.40 135.08±1.03 abc abc bc D5 136.30±2.38 135.20±1.00 135.10±1.13 abc bc c D6 135.77±1.42 135.13±1.07 135.07±1.95 Study II (Hyperglycemic rats) g b a D0 135.27±2.55 146.67±2.29 151.93±2.10 g cdef b D1 136.90±0.44 142.57±1.96 145.40±1.35 g ef b D2 136.97±1.67 142.37±2.06 145.13±1.24 g def b D3 136.70±1.00 142.50±1.64 145.20±1.54 g ef bcd D4 137.10±0.62 142.27±0.90 144.83±1.42 g f bc D5 136.13±0.67 142.07±1.12 145.00±1.41 g f bcde D6 136.43±0.93 141.90±0.85 144.60±1.31 Study III (Hypercholesterolemic rats) e c a D0 136.03±1.22 156.00±1.73 172.67±2.11 e d b D1 137.00±0.78 148.07±0.71 162.07±1.99 e d b D2 136.63±0.76 147.70±2.02 161.67±1.46 e d b D3 136.47±0.93 148.07±1.12 161.87±2.03 e d b D4 136.43±0.85 147.27±1.10 161.50±1.47 e d b D5 137.23±0.55 147.47±1.52 161.13±1.79 e d b D6 135.73±1.11 147.83±1.05 161.53±1.70
D0 = Control D1 = Diet with 150 mg/kg body weight of skin powder D4 = Diet with 300 mg/kg body weight of flesh powder D2 = Diet with 300 mg/kg body weight of skin powder D5 = Diet with 150 mg/kg body weight of WF powder D3 = Diet with 150 mg/kg body weight of flesh powder D6 = Diet with 300 mg/kg body weight of WF powder
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The present data confirmed that administration of bitter gourd in diet showed a decline in serum creatinine level. In this context, El-Batran et al. (2006) delineated that bitter gourd extracts showed insignificant effect in creatinine concentration in normal rats. On the other hand, bitter gourd extracts caused a significant decrease in serum creatinine in diabetic rats. In another study by Nagy et al. (2012) reflected that serum creatinine concentration was notably increased in streptozotocin induced diabetic rats (1.21±0.06 mg/dL) compared to normal control group (0.6±0.05 mg/dL) while reduction in blood creatinine was observed in diabetic rats (0.43±0.05 mg/dL) after intake of bitter gourd extract. According to the work of Kim et al. (2012), creatinine level was increased in STZ induced diabetic rats from 1.19±0.27 after 6 day of dosing to 2.02±0.15 at 28 day of dosing. The rat groups fed with diet supplemented with biter gourd showed decrease in the level of creatinine as 1.16±0.23 and 1.58±0.13, respectively. From the above debate, it can be concluded that bitter gourd tonify the kidneys by controlling the elevation of creatinine in blood.
4.6.14. Serum Urea
Mean squares for serum urea explicated that treatments, time interval and their interaction exerted highly significant differences in all the studies (Table 4.72).
Means pertaining to serum urea (Table 4.73) at the end of trial in Study I showed values for
D0, D1, D2, D3, D4, D5 and D6 as 27.57±1.12, 24.67±1.45, 23.04±0.82, 24.08±0.78, 24.04±0.70, 23.24±0.41 and 23.00±0.38 mg/dL, respectively. During the study intervals, an increase was recorded in D0 from 26.47±0.60 mg/dL at the start to 27.50±1.06 mg/dL at the middle of study and 27.57±1.12 at the end of trial while a decrease in amount of urea was observed in groups of rats rely on diet containing bitter gourd. In hyperglycemic study (Study
II), level of urea was decreased from 32.48±1.13 mg/dL (D0) to 27.75±0.90, 27.47±1.14,
27.17±2.05, 27.10±1.51, 26.73±1.15 and 26.21±1.58 mg/dL with consumption of diets D1,
D2, D3, D4, D5 and D6, respectively. Serum urea concentration was also reduced due to consumption of bitter gourd parts in Study III. Mean values for urea (mg/dL) in this study was recorded as 37.47±1.12 (D0), 34.00±0.75 (D1), 33.13±0.29 (D2), 33.07±1.31 (D3),
32.50±0.52 (D4), 33.00±0.61 (D5) and 32.23±1.15 (D6). The study days showed gradual reduction in level of urea at 28 and 56 day in groups fed with bitter gourd in comparison to
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Table 4.70. Mean squares for effect of diet and study intervals on creatinine of rats
Serum creatinine S.O.V. df Study I Study II Study III
Treatments (T) 6 0.001 N.S 0.005** 0.027**
Days (D) 2 0.0009N.S 0.087** 0.182**
T × D 12 0.00046N.S 0.002* 0.015**
Error 42 0.0005 0.001 0.001
Total 62
* = Significant ** = Highly significant N.S = Non-significant
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Table 4.71. Effect of diet and study intervals on Serum creatinine (mg/dL)
0 Day 28th Day 56th Day Study I (Normal rats)
D0 0.70±0.02 0.73±0.05 0.74±0.03
D1 0.72±0.02 0.71±0.02 0.69±0.01
D2 0.70±0.02 0.70±0.03 0.69±0.01
D3 0.71±0.02 0.70±0.02 0.69±0.02
D4 0.70±0.03 0.71±0.02 0.68±0.03
D5 0.72±0.02 0.70±0.04 0.69±0.01
D6 0.71±0.02 0.69±0.02 0.69±0.01 Study II (Hyperglycemic rats) de a a D0 0.74±0.04 0.91±0.03 0.91±0.03 de b c D1 0.74±0.05 0.87±0.02 0.80±0.02 e b c D2 0.73±0.01 0.86±0.02 0.79±0.03 e b c D3 0.72±0.02 0.86±0.02 0.79±0.03 de b c D4 0.74±0.01 0.85±0.01 0.78±0.02 e b c D5 0.73±0.03 0.85±0.01 0.78±0.02 e b cd D6 0.73±0.02 0.84±0.01 0.77±0.01 Study III (Hypercholesterolemic rats) f b a D0 0.73±0.02 0.98±0.98 1.17±0.05 f c de D1 0.75±0.02 0.91±0.91 0.84±0.03 f c e D2 0.74±0.01 0.89±0.89 0.83±0.04 f c de D3 0.73±0.02 0.91±0.91 0.84±0.02 f c e D4 0.75±0.01 0.89±0.89 0.82±0.02 f c e D5 0.72±0.02 0.89±0.89 0.82±0.01 f cd e D6 0.73±0.02 0.88±0.88 0.81±0.01
D0 = Control D1 = Diet with 150 mg/kg body weight of skin powder D4 = Diet with 300 mg/kg body weight of flesh powder D2 = Diet with 300 mg/kg body weight of skin powder D5 = Diet with 150 mg/kg body weight of WF powder D3 = Diet with 150 mg/kg body weight of flesh powder D6 = Diet with 300 mg/kg body weight of WF powder
155 control in Study II and III as well.
The momentous decline in blood urea level was highlighted by some previous studies as Nagy et al. (2012) reported that serum urea concentration was increased in STZ-induced diabetic rats (59.11±1.33 mg/dL) while groups of rats fed with bitter gourd supplemented diet reduced the blood urea level up to considerable extent (37.86±0.55 mg/dL). El-Batran et al. (2006) performed experiments to evaluate the effect of bitter gourd juice and alcoholic extracts on blood urea level and noted no significant effect in normal rats. However, these extracts induced a significant decline in the amount of urea in diabetic rats. The higher amount of urea is correlated with lower physiological activity of kidney followed by lesser urine production. The chemical constituents in bitter gourd has important role in tonifying kidneys and ultimately increase the functioning of kidneys. This leads to production of more urine and as a result waste products including creatinine and urea can be eliminated out via this urine.
4.6.15. Effect on Different Organs
Mean squares for organ weight (Table 4.74 and Table 4.75) exposed non-significant effect of diet on various organs except for kidney and liver. Means for weight of different organs (Table 4.76) depicted non-significant impact of diet on various organs except for kidney and liver weight that were found higher in diabetic control rat as compared to rats fed with bitter gourd supplemented food. Liver weight was also noted significantly higher in control groups of hypercholesterolemic rats.
Platel et al. (1993) had revealed that the various organ weights of the animals receiving bitter gourd were similar to those maintained on control diet, showing that bitter gourd had no adverse effect on these organs.
The current results about all parameters are fully supported by subsequent experimentation as well.
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Table 4.72. Mean squares for effect of diet and study intervals on urea of rats
Serum urea S.O.V. df Study I Study II Study III
Treatments (T) 6 5.99** 13.05** 6.36**
Days (D) 2 37.52** 3.58 N.S 264.89**
T × D 12 3.00** 3.86* 4.27**
Error 42 0.61 1.76 1.13
Total 62
* = Significant ** = Highly significant N.S = Non-significant
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Table 4.73. Effect of diet and study intervals on Serum Urea (mg/dL)
0 Day 28th Day 56th Day Study I (Normal rats) abc a a D0 26.47±0.60 27.50±1.06 27.57±1.12 ab cde efg D1 27.33±1.00 25.71±0.45 24.67±1.45 a cdef i D2 27.63±0.81 25.71±0.45 23.04±0.82 bcd defg ghi D3 26.10±0.62 24.94±0.87 24.08±0.78 abc efg ghi D4 26.57±0.57 24.44±0.55 24.04±0.70 abc defg hi D5 26.53±0.87 25.04±0.22 23.24±0.41 a fghi i D6 27.53±0.49 24.21±0.86 23.00±0.38 Study II (Hyperglycemic rats) bcd a a D0 27.21±1.03 30.90±1.56 32.48±1.13 bcd b bcd D1 27.31±0.75 28.67±0.87 27.75±0.90 bcd bc bcd D2 27.90±2.11 28.30±1.40 27.47±1.14 bcd bcd bcd D3 27.20±0.40 27.60±1.93 27.17±2.05 bcd bcd bcd D4 27.80±1.49 27.43±1.10 27.10±1.51 d bcd bcd D5 25.90±1.44 26.93±0.81 26.73±1.15 bcd bcd cd D6 27.57±1.01 26.67±1.03 26.21±1.58 Study III (Hypercholesterolemic rats) f b a D0 26.23±0.29 35.07±2.65 37.47±1.12 ef cd bc D1 26.73±0.84 32.83±1.63 34.00±0.75 ef cd cd D2 27.07±1.25 32.53±0.83 33.13±0.29 ef cd cd D3 26.67±0.68 32.43±1.20 33.07±1.31 ef cd cd D4 27.16±1.01 32.29±0.52 32.50±0.52 ef d cd D5 27.25±0.27 31.57±0.96 33.00±0.61 e d d D6 28.05±0.68 31.40±0.89 32.23±1.15
D0 = Control D1 = Diet with 150 mg/kg body weight of skin powder D4 = Diet with 300 mg/kg body weight of flesh powder D2 = Diet with 300 mg/kg body weight of skin powder D5 = Diet with 150 mg/kg body weight of WF powder D3 = Diet with 150 mg/kg body weight of flesh powder D6 = Diet with 300 mg/kg body weight of WF powder
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Table 4.74. Mean squares showing effect of bitter gourd on Heart, Lungs and Kidneys
Heart Lungs Kidneys S.O.V. df Study I Study II Study III Study I Study II Study III Study I Study II Study III
Treatments 6 0.0003N.S 0.0003N.S 0.0003N.S 0.0011N.S 0.0014N.S 0.0008N.S 0.0015N.S 0.0068* 0.002N.S
Error 14 0.0005 0.0004 0.0005 0.0062 0.0039 0.0035 0.0022 0.0023 0.0019
Total 20
* = Significant N.S = Non-significant
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Table 4.75. Mean squares showing effect of bitter gourd on Liver, Pancreas and Spleen
Liver Pancreas Spleen S.O.V. df Study I Study II Study III Study I Study II Study III Study I Study II Study III
Treatments 6 0.0152N.S 0.1499* 0.2238* 0.0004N.S 0.0111N.S 0.0028N.S 0.0003N.S 0.0032N.S 0.0003N.S
Error 14 0.0395 0.0458 0.031 0.0125 0.004 0.0063 0.0014 0.001 0.0015
Total 20
* = Significant N.S = Non-significant
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Table 4.76. Means for organ weight in different studies
Group of rats fed with different diets
Organs Studies D0 D1 D2 D3 D4 D5 D6
Study I 0.95±0.03 0.93±0.02 0.93±0.02 0.92±0.02 0.91±0.03 0.92±0.02 0.90±0.03
Heart Study II 0.95±0.02 0.94±0.01 0.93±0.02 0.93±0.01 0.92±0.01 0.93±0.03 0.92±0.02
Study III 0.95±0.03 0.95±0.01 0.93±0.01 0.94±0.02 0.93±0.01 0.93±0.03 0.93±0.03
Study I 1.59±0.06 1.60±0.06 1.62±0.09 1.57±0.11 1.56±0.07 1.60±0.11 1.60±0.02
Lung Study II 1.59±0.03 1.62±0.05 1.62±0.04 1.58±0.09 1.63±0.03 1.58±0.09 1.59±0.06
Study III 1.61±0.08 1.63±0.04 1.62±0.07 1.60±0.08 1.65±0.04 1.61±0.06 1.61±0.03
Study I 1.94±0.02 1.90±0.03 1.88±0.03 1.89±0.07 1.87±0.05 1.89±0.07 1.88±0.04
kidney Study II 2.01±0.07 1.91±0.04 1.87±0.04 1.90±0.06 1.88±0.04 1.89±0.03 1.87±0.05
Study III 1.96±0.04 1.91±0.04 1.89±0.05 1.90±0.06 1.88±0.03 1.90±0.03 1.90±0.06
Study I 6.72±0.27 6.60±0.10 6.55±0.22 6.59±0.23 6.51±0.20 6.56±0.12 6.53±0.19 Liver Study II 7.23±0.37 6.69±0.24 6.66±0.12 6.69±0.10 6.60±0.14 6.63±0.17 6.59±0.24
Study III 7.58±0.21 7.04±0.20 6.85±0.16 6.96±0.17 6.84±0.09 6.93±0.26 6.77±0.07
Study I 2.02±0.11 2.03±0.20 2.05±0.11 2.02±0.03 2.02±0.13 2.04±0.06 2.03±0.07 Pancreas Study II 1.87±0.06 2.01±0.09 2.01±0.07 2.03±0.04 2.04±0.10 2.04±0.05 2.05±0.07
Study III 1.95±0.11 2.04±0.10 2.02±0.03 2.04±0.04 2.01±0.13 2.03±0.04 2.02±0.03
Study I 0.49±0.04 0.51±0.06 0.49±0.02 0.49±0.01 0.48±0.03 0.49±0.05 0.48±0.03
Spleen Study II 0.41±0.04 0.51±0.04 0.50±0.04 0.50±0.05 0.49±0.04 0.50±0.01 0.48±0.03
Study III 0.48±0.03 0.51±0.05 0.51±0.02 0.51±0.06 0.50±0.03 0.49±0.05 0.49±0.02
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Chapter 5
SUMMARY
A strong relationship between intake of functional/nutraceutical foods and promotion of health has been established in many diet based therapies. Scientific explorations regarding bioactive moieties or phytonutrients as a preventive therapy are rapidly growing owing to their safe status. In this perspective, bitter gourd is one such vegetable that curtail different ailments due to its rich phytochemistry. The present research project was an attempt to probe the health claims of bitter gourd cultivars for the management of serum glucose and cholesterol. For this purpose, bitter gourd fruit samples were subjected to physico-chemical characterization, antioxidant potential quantification of bioactive molecules. Alongside, various treatments of functional bitter gourd drinks were formulated. Lastly, the selected cultivar was utilized in bioevaluation trial.
Nutritional composition of bitter gourd cultivars indicated that moisture content ranged from 6.46% (GHBG-1) to 7.82% (BG 20), ash contents 1.85% (Noor) to 3.18% (Black King), protein contents 14.90 (BG 20) to 24.84% (FSD Long), fat contents 7.86% (Noor) to 10.13% (Black King), fibre contents 6.53% (FSD Long) to 8.47% (Black King) and NFE 56.91% (FSD Long) to 65.45% (BG 20). The moisture contents were 6.92%, 7.91%, 5.82% & 7.23%, ash contents 2.71%, 3.16%, 1.45% & 2.66%, protein contents 23.59%, 24.40%, 11.62% & 21.27%, fat contents 7.19%, 7.35%, 12.14% & 9.15%, fiber contents 2.17%, 2.74%, 18.61% & 6.17% and NFE 64.34%, 62.23%, 56.18% & 60.74% in skin, flesh, seed & whole fruit, respectively.
Mineral composition revealed that potassium ranged from 158.83 (GHBG-1) to 258.83 mg/100g (Black King) , phosphorous 52.08 (GHBG-1) to 92.92 mg/100g (Black King), magnesium 33.61 to 46.74 mg/100g (Black King), sodium 51.05 (Noor) to 61.81 mg/100g (KHBG-1), calcium 25.82 (Noor) to 48.42 mg/100g (Black King), iron 3.28 (FSD Long) to 4.00 mg/100g (GHBG-1), zinc 1.35 (Noor) to 1.78 mg/100g. The composition of mineral was also different in parts. The skin, flesh, seeds & whole fruit possessed 261.78, 291.89, 25.61& 227.11 mg/100g of potassium contents, 88.78, 98.22, 20.49 & 80.72 mg/100g phosphorous, 45.89, 56.11, 3.23 & 50.33 mg/100g magnesium, 73.28, 84.11, 2.27 & 68.28
162 mg/100g sodium, 45.56, 53.42, 2.21 & 44.06 mg/100g calcium, 2.94, 3.29, 4.44 & 3.59 mg/100g iron, 0.68, 0.84, 2.80 & 1.58 mg/100g zinc, respectively.
The extraction efficiency of aqueous and methanolic solvents was also determined at constant temperature and time. The methanolic extract of Black King exhibited highest TPC, TFC, DPPH, FRAP, β-carotene bleaching assay, ABTS as 349.14 mg GAE/100g, 208.68 mg RuE/100g, 72.55%, 94.90 μg FE/g, 67.13%, 69.02 μmol TE/g, respectively. Among parts, flesh exhibited highest extraction efficiency than the rest of the parts with recorded values of 364.56 mg GAE/100g (TPC), 208.61 mg RuE/100g (total flavonoids), 94.56 μg FE/g (FRAP), 64.10% (β-carotene bleaching assay) 68.95μmol TE/g (ABTS) except for DPPH where in whole fruit exhibited higher value (84.25%) followed by flesh (60.81%).
The highest value for ascorbic acid was observed in Black King (47.00 mg/100g) followed by FSD Long (44.83 mg/100g), KHBG-1 (36.58 mg/100g), BG 20 (29.00 mg/100g), Noor (19.50 mg/100g) and GHBG-1 (19.08 mg/100g). The variations in ascorbic acid contents were also observed in parts with highest value in flesh (42.78 mg/100g) followed by whole fruit (35.33 mg/100g), skin (34.17 mg/100g) and seeds (18.39 mg/100g). The maximum amount of total saponin contents (1.95%) were possessed by Black King which is statistically at par with FSD Long (1.91%). The other cultivars including BG 20, KHBG-1, GHBG-1 and Noor possessed lower amount of saponin contents i.e., 1.74%, 1.76%, 1.61% and 1.58%, respectively. Flesh part of Black King contained the highest amount of total saponin contents (3.00±0.11%) and the least was observed in seed part of Noor and GHBG-1. Black King possessed maximum amount of charantin (0.10 mg/g) and minimum amount was noted in Noor (0.07 mg/g). Among parts, flesh contained the highest amount (0.11 mg/g) followed by whole fruit (0.09 mg/g), skin (0.08 mg/g) and seed (0.05 mg/g).
The highest amount of alkaloid (0.65%) was observed in Black King, followed by FSD Long, BG 20, KHBG-1, GHBG-1, and Noor with values of 0.62, 0.61, 0.56, 0.56 and 0.55%, respectively. The highest amount of alkaloid was observed in seed parts with range of 0.80- 0.98% in different cultivars. The amounts of momordicin I & II and vicine were significantly affected by varietal differences with highest values of 9.90±0.20 mg/100g, 11.95±0.09 mg/100g & 0.630±0.046 μg/100μg, respectively in seeds of Black King. The concentration of polypeptide P is varied substantially in different cultivars with highest value in Black King
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(4.15 mg/g). The seed parts possessed highest polypeptide P concentration (5.44 mg/g) followed by whole fruit (4.34 mg/g), flesh (3.75 mg/g) and skin (2.49 mg/g).
In the product development stage, the functional drinks were prepared after the addition of different levels of bitter gourd extracts. The treatments and storage exerted significant differences in total soluble solids, pH and acidity of the prepared functional drinks. TSS decreased with the passage of time; maximum value for TSS was recorded at start (4.38) whereas minimum at the termination of trial (3.37). The momentous decrease in pH was noticed with storage accompanied with substantial increase in acidity. The mean L* values for T0, T1, T2, T3, T4 and T5 were 56.08, 45.75, 39.71, 33.71, 31.17 and 28.50, respectively. Storage days resulted in substantial decrease in L* value from 41.08 in 0 day to 36.83 in 45th day. Bitter gourd in drink formulations resulted marked increase for this attribute from -0.20 in T0 to -1.34, -1.55, -1.70, -1.73 and -1.77 in T1, T2, T3, T4 and T5, respectively. With regard to storage days, a* value at 0, 15, 30 and 45th day were -1.49, -1.43, -1.36 and -1.25, correspondingly. Similarly, various formulations possessed different values for b* as 4.27,
1.72, 1.75, 1.80, 2.26 and 2.34 for T0, T1, T2, T3, T4 and T5, respectively. During storage gradual decrease in b* value was recorded from 2.45 in zero day to 2.27 on 45th day.
Mean squares for hedonic response of different functional drinks elucidated significant variations due to treatments and storage. It is depicted from the means of sensory attributes that T3 exhibited highest scores for color, aroma, flavour, taste and overall acceptability as 7.40, 7.33, 7.05, 7.15 and 7.00. The scores for sensory attributes i.e. color, flavor, taste, texture and overall acceptability imparted diminishing tendency during storage in all the treatments.
In addition, efficacy trials were conducted on experimental Sprague Dawley rats to elucidate the hypoglycemic and hypocholesterolemic acclaims of bitter gourd. During the efficacy study, bitter gourd skin, flesh and whole fruit powder at 150 and 300 mg/kg body weight were given along with control (normal diet). In normal and hypercholesterolemic studies, the weight of rats substantially reduced after the intake of bitter gourd supplementation while increase in weight was observed in all treated groups comparison to diabetic control.
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Bitter gourd extracts imparted positive effect in reducing serum glucose level. The highest glucose value of 142.93±2.70 was observed in hyperglycemic control D0 and the lowest in
D6. The study intervals (0, 28th & 56th day) led to an enhancement in the glucose level of control groups i.e., 88.23±1.14, 89.90±2.55 and 90.37±1.22 in Study I, 88.17±0.99, 113.80±2.03 and 142.93±2.70 in Study II and 87.50±0.80, 98.43±1.21 and 106.33±2.52 mg/dL in Study III, respectively. However, bitter gourd enriched diets substantially suppressed this trait with passage of time and the lowest glucose concentration was observed in D6 with values of 89.93±2.49, 87.80±2.08 & 86.07±1.23 in Study I, 90.40±1.93, 94.47±3.70 & 97.70±2.17 in Study II and 87.83±2.00, 94.63±1.72 & 93.50±0.60 mg/dL in Study III for 0, 28th and 56th day, respectively.
Means values concerning insulin at the end of trial for D0, D1, D2, D3, D4, D5 and D6 were 9.03±0.31, 9.10±0.36, 9.33±0.32, 9.07±0.35, 9.37±0.15, 9.13±0.25 and 9.87±0.76 in Study I; 12.23±0.74, 14.77±0.81, 14.93±0.75, 14.80±0.79, 15.13±0.49, 15.10±0.30 and 15.33±0.25 in Study II and 11.37±0.96, 12.00±0.62, 12.93±0.38, 12.17±0.32, 12.97±0.67, 12.57±0.47 and 13.10±0.20 µIU/mL, respectively in Study III. Increase in amount of bitter gourd in diet has resulted in production of more insulin.
The study intervals indicated that consumption of bitter gourd supplemented diet increase the production of inulin with the passage of time compared to control and maximum insulin level was observed in D6 as 9.87±0.76 (Study I), 15.33±0.25 (Study II) and 13.10±0.20 (Study III).
The hypochlestrolemic perspectives of bitter gourd were evident from reduction of cholesterol from 85.83±0.35 mg/dL (D0) to 74.00±0.95 mg/dL (D4) in normal rats,
129.07±1.25 (D0) to 97.97±1.77 mg/dL (D4) in hyperglycemic rats, 160.67±4.16 (D1) to
116.70±2.54 mg/dL (D4) in hypercholesterolemic rats at the termination of trial. For time intervals, the rats fed with bitter gourd supplementation diet showed substantial decline compared to their respective control.
As far as percent reduction of cholesterol was concerned, D4 result highest decline of 13.78%, 24.10% and 34.07%, respectively in Study I, II and III, respectively. The LDL level in control groups were higher 29.10±1.51, 63.85±2.47 and 74.33±3.31 mg/dL and substantially decreased in treated groups with maximum decline in D6 as 27.93±1.55,
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45.63±2.51 and 53.20±2.54 mg/dL, respectively. The HDL level substantially increased from
38.80±3.17, 35.97±2.25 and 35.97±1.72 in control group (D0) to 42.77±2.70, 47.43±1.12 and
51.63±4.07mg/dL in D6. The concentration of serum triglyceride was also raised in control groups and suppressed significantly by feeding of bitter gourd with highest decrease (66.03±3.16, 82.37±2.44 and 98.23±4.4) in rats fed with 300 mg/kg body weight of bitter gourd whole fruit powder.
Moreover, the bitter gourd extracts imparted positive influence on liver functioning tests, validating safety in consumption. Mean squares regarding ALP, ALT and AST concentration showed non-significant differences due to diets in Study I. However, substantial differences owing to treatments were found in Study II and III. Likewise, serum urea levels were reduced in rats fed with bitter gourd as compared to control diet. The results regarding organ weight exposed non-significant effect of diet on various organs except for kidney and liver that were found higher in diabetic control rat as compared to rats fed with bitter gourd supplemented food.
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CONCLUSIONS
In the nut shell, bitter gourd hold multifarious potential against various life style related disorders owing to its rich phytochemistric The functional components are mainly concentrated in its different parts especially whole fruit that played a significant role in the biological system Among extraction methods aqueous extraction is the most suitable method for the extraction of bioactive moieties Finally bitter gourd extract supplementation in food products especiaaly drinks is pheseable and can be employed to achieve the allied health claims Conclusively, bitter gourd hold potential to be extracted and utilizing functional drinks for quality improvement and value addition Correlation matrix indicated significant association among indices of lipid profile and glucose response in relation to bioactive components present in bitter gourd cultivers
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RECOMMENDATIONS
In diet based therapies bitter gourd extract should be recommended for the vulnerable segments Advanced instrumental techniques, isolation methods and their right application should be adopted in the field of nutraceuticals to enhance excellence Community based programs and such studies should be conducted for further meticulousness The trial should be conducted in industrial premises for the optimization of bitter gourd extract addition for processors and consumers In order to observe these aspects nutrition awareness programs should be arranged among the public
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APPENDICES
Appendix I
Performa for sensory evaluation of functional drink
Name of the judge: …………………………………. Date: ……………..
Character T0 T1 T2 T3 T4 T5 Color
Aroma
Flavour
Taste
Overall acceptability
Signature…………………… INSTRUCTIONS
Take a sample of functional drink and score for Color, Aroma, Flavour, Taste and overall 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.
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Appendix II
Composition of experimental diets
Experimental diets:
Ingredients (%) Normal diet High Sucrose diet High cholesterol diet
Corn Oil 10 10 10 Corn Starch 66 26 64 Casein 10 10 10 Cellulose 10 10 10 Salt mixture 3 3 3 Vitamins 1 1 1 Cholesterol - - 1.5 Cholic acid - - 0.5 Sucrose - 40 - Arginine - - -
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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
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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
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