Bio Active Compounds Of Bitter Melon Genotypes Biology Essay

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Background. Nutritional qualities of Bitter Melons were examined by chemical analyses namely 28 minerals, polyphenolics, protein and amino acids. Analyses for antioxidants and other 29 biochemical attributes as well as cooking qualities are also investigated. Several recipes have 30 been tasted for consumer acceptance. Along with the potential chemo preventative activities, the 31 popular belief of bitter melon improving glucose tolerance in Type II diabetes and lowering 32 blood cholesterol are being investigated. However, it has not been determined if or which 33 alkaloids, polypeptides, or combination of the chemicals, found in bitter melon, are responsible 34 for the beneficial medicinal effect. The functional compounds contained in these foods and their 35 medicinal effects are needed to thoroughly studied and clinically proven. 36

Methods. Four varieties of bitter melon: Indian Green (IG), Indian White (IW), Chinese Green 38 (CG) and Chinese White (CW), were used for phytochemical analyses for protein extraction and 39 determination, protein hydrolysis, determination of amino acid, antioxidant and atimutagenic 40 activity. All the analyses were done as per the standard methods. All values are reported as 41 means of three determinations. Split plot complete randomized design was conducted using JMP 42 5 software package (SAS) and Tukey HSD procedure was performed for the significance of 43 differences at the 5% level. . 44

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Results. Melon flesh contained approximately 93% moisture for all four varieties, whereas the 46 moisture content of melon seeds ranged from 53.3% in Indian Green to 75% in Indian White. 47 Bitter melon flesh contained 8.4% to 9.8% protein, whereas seed contained 27% to 31% protein. 48 Glycine was also higher in bitter melon compared to soy proteins. Other amino acids contents 49

were similar levels as flesh proteins. . Phenolic contents of seed, SCT, and flesh ranged from 50 4.67-8.02, 4.64-8.94, and 5.36-8.90 mg/CAE dry matter, respectively. Phenolic contents of the 51 flesh were significantly higher than those of the SCT and seed and phenolic contents of the seed 52 was the lowest among those of all the tissues. The total phenolic contents of four varieties were 53 significantly different with the highest was Indian white followed by China white, China Green, 54 and Indian green. The antioxidant activities of Indian green, Indian white, China green and China 55 white ranged from 79-88, 79-87, 80-86, and 79-87% inhibition, respectively. The antioxidant 56 activities of the oven-dried samples and the freeze-dried samples were 79-88 and 79-86% 57 inhibition, respectively. Bitter melon varieties IW and CG showed higher antimutagenic effects 58 against benzo(a)pyrene with Salmonella TA98 (92-100% inhibition) and Salmonella TA100 (79-59 86% inhibition) but lower antimutagenic effects against sodium azide. 60

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Conclusion. Bitter melon is a good source of phenolic compounds. The phenolic extracts 62 showed high inhibition effect to Prevent lipid oxidation. These natural plant phenolics can be a 63 good antioxidant which may be applied in many food systems to maintain the food quality. 64

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Background 66

Many exotic vegetables are known for their special nutritional and medicinal properties. Bitter 67 Melon (Momordica charantia L.), an annual vegetable of Cucurbitaceae family, is found to be 68 one of the important vegetables of special nutritional and medicinal qualities in southern United 69 States. Germplasm lines and land races of Bitter Melon were evaluated since 2000 for their 70 adaptability in Southeast Arkansas. Four adaptable lines/varieties were tested in replicated field 71 trials for productivity at the University of Arkansas at Pine Bluff Agricultural Research Center. 72

Melons were harvested at their marketable stages beginning in June and ending in September for 73 yield estimation. Nutritional qualities of Bitter Melons were examined by chemical analyses 74 namely vitamin, minerals, polyphenolics, protein and amino acids. Analyses for antioxidants and 75 other biochemical attributes as well as cooking qualities are also investigated. Several recipes 76 have been tasted for consumer acceptance. Nutrition-related health problems such as 77 hypertension, diabetes, ageing, obesity, arthritis, cardiovascular disease, are prevalent among 78 disadvantaged rural and urban populations, especially minorities in the delta region [1, 2]. Food 79 consumption habits, dietary intakes, and meal preparation methods are believed to contribute to 80 these problems. Human beings are naturally adapted to eating foods which have a balanced ratio 81 of triglycerides and phospholipids. Based on the overall balance of triglyceride/phospholipid 82 ratio, foods from plant sources have less negative effects on human health than foods from 83 animal sources [3]. When these balanced foods are consumed, peroxidation problems of 84 polysaturated fats and lipids, associated with many of the human sicknesses, are remarkably 85 minimized [4]. In addition to the commonly used native vegetable and herbs in the US, many 86 exotic herbs and vegetables are known for their special nutritional and medicinal properties [5]. 87 During the past decade or two, Americans have realized that they could control their own health 88 problems and reduce risks of many chronic illnesses by changing certain dietary behaviors [6]. 89 The popular belief of bitter melon to improve glucose tolerance in Type II diabetes and lower 90 blood cholesterol are being investigated. It is still to be determined if the chemical constituents 91 such as certain alkaloids and polypeptides found in bitter melons are effective individually or in 92 combination. Along with the potential chemo preventative activities, the popular belief of bitter 93 melon improving glucose tolerance in Type II diabetes and lowering blood cholesterol are being 94 investigated. However, it has not been determined if or which alkaloids, polypeptides, or 95

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combination of the chemicals, found in bitter melon, are responsible for the beneficial medicinal 96 effect. 97

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Introduction 99

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Certain vegetables contain an abundance of polyphenolics, terpenoids, isoflavones, 101 anthocyanins, amino acids, minerals, vitamins and other antioxidants that are associated with 102 protection from cancer, aging, cardiovascular diseases, diabetes, hypertension [4,7-12]. The most 103 popular and useful group of the vegetables belong to the family Cucurbitaceae [13]. One of the 104 members of this family is Bitter Melon (Momordica charantia L.), also known as Karela, balsam 105 pear, or foo gwa, an annual fruity vegetable of the cucurbitaceae family. It is a delicacy to the 106 East and Southeast Asian people, especially the people of India, China, Japan, Taiwan, 107 Bangladesh, Pakistan, Thailand, Malaysia, Indonesia, Philippines, Nepal, Bhutan and Sri Lanka. 108 Bitter melon is also a popular vegetable in West Indies, Brazil, Colombia, Cuba, Mexico, 109 Panama, Peru, and some African and European countries. The bitter quality, for which the fruit is 110 named, is due to the alkaloid momordicine. The green warty fruits of this plant are used as a 111 vegetable, rich in vitamin A, vitamin C, and iron [14]. Fruits vary in size (1.0"-9.8" long and 1.0-112 5.9" wide), shape (oval, round, oblong, club, etc.), fruit color (dark green to creamy white), 113 maturity (45-80 days), fruit per plant (6-85) and yield per plant (0.8-12 Ibs). The fruits, leaves, 114 and roots of bitter melon are traditionally believed to have medicinal value in reducing blood 115 sugar levels for diabetic patients [15,16]. The various medicinal properties of bitter melon are 116 well known in eastern Asia [17]. Sofowora [18] reported several uses of M. charantia in 117 traditional medicines in Africa. Pharmacologically, the hypoglycemic properties of the plant 118

organs were established by Lotlikar et al. [19]. The polypeptides from the seeds and fruits of M. 119 charantia were considered as antidiabetic agents [20-24]. Cucurbitacines isolated from several 120 species of the family Cucurbitaceae showed antitumor effects [25-27]7. Lin et al. [28] isolated 121 two lectins from seeds of M. charantia, momordin and agglutinin, the momordin inhibiting 122 protein synthesis by Ehrlich Ascites cells. Licastro et al. [29] announced that two proteins 123 isolated from the seeds of M. charantia inhibited protein synthesis and subsequent DNA 124 synthesis in normal and leukaemic human peripheral blood lymphocytes. Spreafico et al. [30] 125 reported a protein inhibitor from M. charantia showing immunomodulatory activity in mice. 126 Although bitter melon is used by the ethnic people from Asia, Africa and Europe, American 127 consumers have not widely developed a taste and appreciation for it. The crop could be 128 introduced in the US as an additional food item possessing special medicinal properties. 129 Therefore the functional compounds contained in these foods and their medicinal effects are 130 needed to thoroughly studied and clinically proven. Research is required to identify suitable 131 genotypes of this vegetable for their production potential and nutritional and/or nutraceutical 132 values. In these ongoing studies, we have determined that bitter melon can be successfully 133 produced in Arkansas and the southern states. 134

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Methods 136

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Four selected varieties of bitter melon were tested in replicated field trials in 2003-2004 on 138 Calloway silt loam soil at the UAPB Agricultural Research Center in Pine Bluff. Trials were 139 conducted in randomized complete block designs with four replications. Plants were grown on 140 upright trellises with 10 ft. between rows and 12 ft. within rows. Two four-week old seedlings 141

were transplanted in each hole. N, P, and K fertilizers were applied pre-plant using 200 lbs per 142 acre of 13-13-13. Irrigation was provided by drip irrigation system. Weeds were controlled by 143 covering the beds with black plastic. Roundup (Glyphosate) was used in controlling weeds 144 around the beds. Melons were harvested twice a week starting in mid-June and ending mid-145 September. Fruit samples were stored in sealed plastic bags in ice chests for phytochemical 146 analysis. 147

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Bitter Melon Sample Preparation. The bitter melons were washed with deionized water, 149 drained at ambient temperature and cut in half lengthwise. The seeds were removed and flesh 150 was thinly sliced and collected separately from the seeds. Flesh was divided into two halves. One 151 half was freeze dried and the other half was oven dried. Seeds and flesh was oven dried at 80 OC 152 for 3 days. Dried flesh and seeds were then ground to flour and passed through a 60-mesh U.S.A. 153 standard testing sieve (W.S. Tyler, Incorporated, Mentor, OH). 154

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Protein Extraction and Determination. Bitter melon protein isolates were prepared following a 156 standard procedure for soy protein isolation [31]. Ten grams of bitter melon flour were extracted 157 with 100 mL of water at pH 8.0 by stirring at ambient temperature for 2 h. The suspension was 158 then centrifuged at 1500 g for 30 min at 10 OC to separate the supernatant from the solid phase. 159 The supernatant was then adjusted to pH 4.5, held at 4 OC for 2 h and centrifuged again at 1500 g 160 for 30 min at 10 OC. The resulting precipitate was dissolved in 20 mL deionized water, adjusted 161 to pH 8.0 and freeze-dried. Soy proteins were isolated using the same procedure. Protein content 162 of bitter melon flour and its protein isolates were determined using a Kjeldahl Unit (Kjeltec 163 Analyzer Unit, Foss Tecator AB, Hoganas, Sweden). Approximately 50 mg of each sample was 164

digested with concentrated sulfuric acid in the presence of 1 Kjeltab Unit with 0.1 N HCl. Kjet-165 Sorb was used as the receiver during titration. Protein contents were calculated using a factor of 166 6.25. 167

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Protein Hydrolysis. Protein hydrolysis was done using a protocol described by Eveleigh and 169 Winter [32]. Ten mg of proteins were dispersed in 5 mL of 6 N HCl . Two hundred microliters of 170 the suspension were taken into a vacuum hydrolysis tube (Pierce Chemical Co., Rockford, IL). 171 The tube was connected to a vacuum pump for 5 min. The tube was then sealed and placed in a 172 Reacti Heating Module (Pierce Chemical Company) at 150 OC overnight (12 h). After dialysis, 173 the tube was cooled to room temperature before the aliquot was transferred to a 2.5 mL amber 174 vial. The hydrolyzate was then evaporated to dryness under a stream of nitrogen at 60 OC. To the 175 dried amino acids was then added 0.5 mL sample diluting buffer (Beckman Coulter Inc., 176 Fullerton, CA) and the aliquot was filtered through a 0.2 µm filter prior to amino acid analysis. 177

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Amino Acid Composition and Analysis. Amino acid composition was analyzed using an 179 Amino Acid Analyzer 126 Beckman HPLC system with a post column derivatization reactor 180 (Beckman Instruments, Inc. Palo Alto, CA). Bitter melon flour and protein isolates were 181 hydrolyzed with 6 M HCl to produce amino acids and then analyzed with the Amino Acid 182 Analyzer. Chromatographic equipment consisted of a Beckman liquid chromatograph model 126 183 HPLC equipped with a System Gold Nouveau software (Beckman Instruments, Inc, Palo Alto, 184 CA) was used. The absorbance of the effluent was monitored at 570 nm. The mobile phase 185 consisted of sodium buffer Na-E (pH 3.3), Na-F (pH 4.3) and Na-D (pH 6.3) (Beckman Coulter 186 Inc., Fullerton, CA). A Hi-performance regeneration solution (Na-R) (Beckman Coulter Inc., 187

Fullerton, CA) was used to regenerate the column after each run. Flow rate was set at 0.44 188 mL/min and column temperature was set an initial temperature of 50 OC for 7 min and 75 OC for 189 the rest of the test. The initial solvent condition was buffer Na-E. After 19.5 min the buffer was 190 switched to Na-F and at 30 min the buffer was switched to Na-D until 65 min. Then the column 191 was washed with solvent Na-R for 3 min before the solvent was brought to the original 192 condition. The flow rate of the ninhydrin (Nin-RX, Beckman Coulter Inc., Fullerton, CA) was 193 set at 0.23 ml/min. A sample size of 20-100 L was injected during HPLC analysis. The amino 194 acid composition was calculated from standard curves calibrated using the 17 standard amino 195 acids. 196

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Determination of Phenolics. Total phenolics of the fine gourd flesh, SCT, and seed of bitter 198 melons were determined by Folin-Ciocalteu method. One hundred milligrams of each sample 199 was weighted into a screw-cap test tube and vortexed with 10 mL of methanol. The dispersion 200 was heated in a water bath of 65 OC for 2h and allowed to cool at room temperature. To 1 mL of 201 the clear solution in a screw-cap test tube, 1.0 deionized water was added. The tubes were 202 vortexed and allowed to stand for 2h at room temperature. Absorption of the solution at 726 nm 203 was measured using a spectrophotometer. The total phenolic content was expressed as 204 chlorogenic acid equivalents in mg/g dry material. The phenolic acid constituents were analyzed 205 by HPLC [33]. 206

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Antioxidant activity determination. Antioxidant activity was carried out by oxidizing linoleic 208 acid methyl ester (MeLo) in the presence of phenolic extracts as antioxidants [34]. Two mg of 209 the extracts were dissolved in 10 mL of methanol. Five hundred microliters of the extracts 210

solution were added into 0.2 g of MeLo (500 ppm extract in MeLo), and the methanol was 211 evaporated under a stream of nitrogen at ambient temperature. Five hundred microliters of 212 methanol were added into 0.2 g of MeLo for blank as a reference. Oxidation of MeLo in the 213 present of extract was carried out in at 40 C for 72h. Two mg of sample aliquots were taken at 214 the starting point (zero time) and after 72h of oxidation (at 40 C) and dissolved in 10 mL of 215 2,2,4-trimethylpentane (isooctane). The conjugated diene absorption of the aliquots was read 216 using a spectrophotometer (Shimadzu Model UV-1601, Kyoto, Japan) at a wave length of 234 217 nm. The antioxidant activities were expressed as percentage inhibition of conjugated diene 218 hydroperoxides formation of MeLo after 72h of oxidation comparing with blank from MeLo as a 219 reference antioxidant as follows: % inhibition= [(AB(72h) - AB(0h))- (AE(72h) - AE(0h))/ 220 (AB(72h) - AB(0h))] x 100; where A= absorbance, E= extract, and B= blank. 221

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Antimutagenic activity determination. The antimutagenic activity of the methanolic extract 223 from freeze-dried bitter melon flesh and seed from varieties India white and China green was 224 determined by the method of Ames et al. [35]. The histidine requiring stains of Salmonella 225 typhimurium TA98 and TA 100 were used for this test. Benzo(a)pyrene and sodium azide were 226 used as mutagens for mutagenic and antimutagenic tests. The percentage inhibition of 227 mutagenesis was calculated using the following equation: inhibition% = [1-(number of revertants 228 in the presence of fraction/number of revertants in the absence of fraction)] X 100. 229

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Statistical analysis. All values are reported as means of three determinations. Split plot 231 complete randomized design was conducted using JMP 5 software package (SAS 2002) and 232 Tukey HSD procedure was performed for the significance of differences at the 5% level. . 233

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Results and Discussion 235

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Nutritive, Protein and Moisture Content of Bitter Melon. The bitter melon fruit has higher 237 content of vitamin C, potassium, calcium, magnesium, and dietary fiber as compare to some 238 other commercial vegetables [14] (Table 1). It also has niacin, thiamin, riboflavin, vitamin A, 239 protein, organic acids and other nutrients. Physical separation of bitter melons resulted in flesh 240 and seed fractions. The protein and moisture content of flesh and seeds of four varieties of bitter 241 melons are given in Table 2. Melon flesh contained approximately 93% moisture for all four 242 varieties, whereas the moisture content of melon seeds ranged from 53.3% in Indian Green to 243 75% in Indian White. Bitter melon flesh contained 8.4% to 9.8% protein, whereas seed contained 244 27% to 31% protein. 245

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Amino Acid Composition of Bitter Melon. Freeze dried melon flesh was high in lysine (mole 247 percentage) compared to soy protein isolate. Flesh was relative lower in glutamic acid and 248 arginine (Table 3). Essential amino acids, including threonine, valine, methionine, isoleucine, 249 leucine, and phenylalanine are comparable in amount to soy proteins and other legume proteins. 250 On the other hand, oven dried flesh had a much lower percentage of lysine and a lower 251 percentage of arginine while other amino acids were almost similar. Amino acid compositions 252 of seeds are given in Table 4. Seed protein was higher in glutamic acid and arginine but lower in 253 lysine compared to flesh proteins. Glycine was also higher in bitter melon compared to soy 254 proteins. Other amino acids contents were similar levels as flesh proteins. 255

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Quality Criteria. The quality criteria of bitter melon are fresh appearance with uniform 257 coloration and firm without excessive seed development. The bitter melons are harvested, 258 selected for size and uniformity of fruit surface characteristics and commonly packed in carton or 259 wood boxes containing 5, 10, or 20 kg of fruit. It is a chilling sensitive vegetable, and may be 260 air-cooled to 10 OC-12 OC [36, 37]. Bitter melons are intermediate in perishability. The common 261 postharvest defects are seed development, softening and ripening with internal /external color 262 change. The recommended temperature and relative humidity for postharvest handling of bitter 263 melon are 10-12.5 OC and 85-90%, respectively, with estimated shelf-life 7 to 14 days [36, 37]. 264

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Phenolic contents. Total phenolic content of oven and freeze-dried bitter melon tissues from 266 four varieties are given in Table 5. Overall, phenolic content in oven-dried samples were 267 significantly higher than freeze-dried samples. Phenolic contents of the oven dried and freeze-268 dried tissues ranged from 5.39-8.94 mg of chlorogenic acid equivalent (CAE)/g dry matter and 269 4.64-8.90 mg/CAE.g dry matter, respectively. Phenolic contents of seed, SCT, and flesh ranged 270 from 4.67-8.02, 4.64-8.94, and 5.36-8.90 mg/CAE dry matter, respectively. Phenolic contents of 271 the flesh were significantly higher than those of the SCT and seed and phenolic contents of the 272 seed was the lowest among those of all the tissues. The total phenolic contents of four varieties 273 were significantly different with the highest was Indian white followed by China white, China 274 Green, and Indian green. 275

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Antioxidant activities of bitter melon extract. There was no significant difference in the 277 antioxidant activities (% inhibition) of the methanolic extracts from bitter melons among 278 varieties and drying methods (oven and freeze-dried) (Table 6). The antioxidant activities of 279

Indian green, Indian white, China green and China white ranged from 79-88, 79-87, 80-86, and 280 79-87% inhibition, respectively. The antioxidant activities of the oven-dried samples and the 281 freeze-dried samples were 79-88 and 79-86% inhibition, respectively. The antioxidant activities 282 of the methanolic extracts of flesh and SCT were not significantly different, while they were 283 significantly higher than that of seeds. Shu-Jing and Lean-Teik [38] reported that bitter melon 284 extracts possess potent antioxidant and free radical scavenging activities. These antioxidant 285 activities could have contributed, at least partly, to the therapeutic benefits of the certain 286 traditional claims of wild BM. 287

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Antimutagenicity of bitter melon extract. Bitter melon varieties IW and CG showed higher 289 antimutagenic effects against benzo(a)pyrene with Salmonella TA98 (92-100% inhibition) and 290 Salmonella TA100 (79-86% inhibition) (Table 7) but lower antimutagenic effects against sodium 291 azide (data not shown). Similar finding was reported by several authors [39, 40]. Wattenberg 292 [41] discussed chemopreventive agents as blocking agents that prevent carcinogens from 293 reaching or reacting with critical target sites, and as suppressing agents that prevent evolution of 294 the neoplastic process in cells that otherwise would become malignant. Bitter melon extract 295 contains both agents [40]. Although the exact mechanism of the chemopreventive effects of 296 bitter melon is not yet known, these findings suggest that bitter melon is a possible 297 chemopreventive agent against carcinogenesis. 298

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Conclusions 300

Bitter melon is an excellent source of phenolic compounds, antioxidant and antimutagen. This 301 can find application in food products, and dietary supplements. The phenolic extracts showed 302

high inhibition effect to Prevent lipid oxidation. These natural plant phenolics can be a good 303 antioxidant which may be applied in many food systems to maintain the food quality. More 304 Detailed investigations on bitter Melon phenolics, peptides, and proteins are needed to provide 305 information for their nutraceutical values. Bitter melon has high demand in the ethnic market 306 especially in Asian, African and certain South American countries. To the best of our knowledge 307 in normal vegetable usage of bitter melon no toxicity has been reported. However high dose of 308 bitter melon capsule which is now available in the US as well as international market may cause 309 some kind of toxic effect, like over dose of any other polyphenolics it is reported in some non 310 referred sources that higher dose may cause abortion. More Detailed investigations on bitter 311 Melon phenolics, peptides, and proteins are needed to provide information for their nutraceutical 312 values. 313

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