Metabolite profiling and in vitro biological activities of two commercial bitter melon (Momordica charantia Linn.) cultivars.

The current study was designed to characterize the metabolite profile and bioactivity of two commercial bitter melon (Momordica charantia Linn.) genotypes. UPLC-high resolution mass spectrometry (HRMS) was used to identify 15 phenolic and 46 triterpenoids in various bitter melon extracts. Total phenolic levels were the highest (57.28 ±â€¯1.02) in methanolic extract of the inner tissue of Indian Green cultivar, which also correlated to the highest DPPH radical scavenging activity (30.48 ±â€¯2.49 ascorbic acid equivalents (mg of AAE)/g of FD). In addition, highest levels of total saponins were observed in chloroform extract of the Chinese bitter melon pericarp (75.73 mg ±â€¯4.67 diosgenin equivalents (DE)/g of FD). Differential inhibition of α-amylase and α-glucosidase activity was observed in response to polarity of extract, cultivar and tissue type. These results suggest that consumption of whole bitter melon may have potential health benefits to manage diabetes.

Magneto-Priming Improved Nutraceutical Potential and Antimicrobial Activity of Momordica charantia L. Without Affecting Nutritive Value.

The need for some economic strategies for increased growth and nutraceuticals of medicinal plants is well acknowledged now. It was hypothesized that external magnetic field treatment (MFT) of seeds affecting internal magnet of cells may affect growth and metabolism. In this study, seeds were subjected to pre-sowing magnetic field (50 mT at 5 mm for 5 s). At vegetative stage, the leaf growth, chlorophyll content, catalase (CAT), peroxidase (POD), amino acids, proteins, flavonoids, soluble sugars, total soluble phenolics, carotenoids, anthocyanins, phenolic profile (HPLC based), and antimicrobial activity of leaves (in terms of the minimum inhibitory concentration against Staphylococcus aureus and Pseudomonas aeruginosa) were studied. Yield was evaluated for nutritive components in fruit (peel+pulp) and peel. MFT improved germination percentage, growth, leaf chlorophyll, antimicrobial activity, peel amino acids, phenolics, and POD with negligible effect on fruit nutritive value. Moreover, photosynthetic pigments and cinnamic acid exhibited direct correlation with antimicrobial potential against both pathogens. However, sinapic acid showed positive correlation against Staphylococcus aureus only. Cinnamic acid, coumaric acid, syringic acid, and quercetin were in direct correlation against Pseudomonas aeruginosa; it was directly correlated with total flavonoids too. In conclusion, magnetic field can be used to manipulate plant cell metabolism promising improvement of growth, antimicrobial activity, and phenolics of interest.

Characterization of a soluble phosphatidic acid phosphatase in bitter melon (Momordica charantia).

Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. The fruit has been widely used as vegetable and herbal medicine. Alpha-eleostearic acid is the major fatty acid in the seeds, but little is known about its biosynthesis. As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, this study focused on a soluble phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) that hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and P(i). PAPs are typically categorized into two subfamilies: Mg(2+)-dependent soluble PAP and Mg(2+)-independent membrane-associated PAP. We report here the partial purification and characterization of an Mg(2+)-independent PAP activity from developing cotyledons of bitter melon. PAP protein was partially purified by successive centrifugation and UNOsphere Q and S columns from the soluble extract. PAP activity was optimized at pH 6.5 and 53-60 °C and unaffected by up to 0.3 mM MgCl2. The K(m) and Vmax values for dioleoyl-phosphatidic acid were 595.4 µM and 104.9 ηkat/mg of protein, respectively. PAP activity was inhibited by NaF, Na(3)VO(4), Triton X-100, FeSO4 and CuSO4, but stimulated by MnSO4, ZnSO4 and Co(NO3)2. In-gel activity assay and mass spectrometry showed that PAP activity was copurified with a number of other proteins. This study suggests that PAP protein is probably associated with other proteins in bitter melon seeds and that a new class of PAP exists as a soluble and Mg(2+)-independent enzyme in plants.

α-Glucosidase inhibitors via green pathway: biotransformation for bicoumarins catalyzed by Momordica charantia peroxidase.

Peroxidase extracted from Momordica charantia catalyzed the H(2)O(2)-dependent oxidative coupling of 7-hydroxy-4-methylcoumarin to form four new dimers (1-4) and two known ones (5, 6). The structures, including the absolute configurations of axially chiral compounds, were unambiguously characterized by NMR spectroscopy, online HPLC-CD, and a variety of computational methods. Bioactive experiments demonstrated that compounds 1 and 2 had significant inhibitory effects on yeast α-glucosidase, much better than the controls. Noncompetitive binding mode was found by the graphical analysis of steady-state inhibition data. The mechanism of enzymatic inhibition confirmed in some depth that the inhibitors altered the secondary structure of α-glucosidase by decreasing the α-helix and increasing the ß-sheet content. In summary, bicoumarins 1 and 2 might be exploited as the lead compounds for further research of antidiabetic agents, and this research provided a "green" method to synthesize compounds with the chiral biaryl axis generally calling for multistep reactions in organic chemistry.

Molecular cloning and characterization of cDNAs encoding carotenoid cleavage dioxygenase in bitter melon (Momordica charantia).

Carotenoid cleavage dioxygenases (CCDs) are a family of enzymes that catalyze the oxidative cleavage of carotenoids at various chain positions to form a broad spectrum of apocarotenoids, including aromatic substances, pigments and phytohormones. Using the rapid amplification of cDNA ends (RACE) PCR method, we isolated three cDNA-encoding CCDs (McCCD1, McCCD4, and McNCED) from Momordica charantia. Amino acid sequence alignments showed that they share high sequence identity with other orthologous genes. Quantitative real-time RT PCR (reverse transcriptase PCR) analysis revealed that the expression of McCCD1 and McCCD4 was highest in flowers, and lowest in roots and old leaves (O-leaves). During fruit maturation, the two genes displayed differential expression, with McCCD1 peaking at mid-stage maturation while McCCD4 showed the lowest expression at that stage. The mRNA expression level of McNCED, a key enzyme involved in abscisic acid (ABA) biosynthesis, was high during fruit maturation and further increased at the beginning of seed germination. When first-leaf stage plants of M. charantia were exposed to dehydration stress, McNCED mRNA expression was induced primarily in the leaves and, to a lesser extend, in roots and stems. McNCED expression was also induced by high temperature and salinity, while treatment with exogenous ABA led to a decrease. These results should be helpful in determining the substrates and cleavage sites catalyzed by CCD genes in M. charantia, and also in defining the roles of CCDs in growth and development, and in the plant's response to environmental stress.

Riboflavin accumulation and characterization of cDNAs encoding lumazine synthase and riboflavin synthase in bitter melon (Momordica charantia).

Riboflavin (vitamin B2) is the universal precursor of the coenzymes flavin mononucleotide and flavin adenine dinucleotide--cofactors that are essential for the activity of a wide variety of metabolic enzymes in animals, plants, and microbes. Using the RACE PCR approach, cDNAs encoding lumazine synthase (McLS) and riboflavin synthase (McRS), which catalyze the last two steps in the riboflavin biosynthetic pathway, were cloned from bitter melon (Momordica charantia), a popular vegetable crop in Asia. Amino acid sequence alignments indicated that McLS and McRS share high sequence identity with other orthologous genes and carry an N-terminal extension, which is reported to be a plastid-targeting sequence. Organ expression analysis using quantitative real-time RT PCR showed that McLS and McRS were constitutively expressed in M. charantia, with the strongest expression levels observed during the last stage of fruit ripening (stage 6). This correlated with the highest level of riboflavin content, which was detected during ripening stage 6 by HPLC analysis. McLS and McRS were highly expressed in the young leaves and flowers, whereas roots exhibited the highest accumulation of riboflavin. The cloning and characterization of McLS and McRS from M. charantia may aid the metabolic engineering of vitamin B2 in crops.

Catalyzed degradation of disperse dyes by calcium alginate-pectin entrapped bitter gourd (Momordica charantia) peroxidase.

Calcium-alginate pectin entrapped bitter gourd peroxidase (BGP) has been employed for the treatment of disperse dyes: Disperse Brown 1 (DB 1) and Disperse Red 17 (DR 17). Peroxidase alone was unable to decolorize DR 17 and DB 1. However, the investigated dyes were decolorized maximally by BGP in the presence of 0.2 mmol/L redox mediator, violuric acid (VA). A slow decrease in percent decolorization was observed when VA concentration was higher than 0.2 mmol/L which could likely be due to the high reactivity of its aminoxyl radical (> N-O*) intermediate, that might undergo chemical reactions with aromatic amino acid side chains of the enzyme thereby inactivating it. Maximum decolorization of the dyes was observed at pH 3.0 and 40 degrees C within 2 hr of incubation. Immobilized peroxidase decolorized 98% DR 17 and 71% DB 1 using 35 U of BGP in batch process in 90 min. Immobilized enzyme decolorized 85% DR 17 and 51% DB 1 whereas soluble enzyme decolorized DR 17 to 48% and DB 1 to 30% at 60 degrees C. UV-visible spectral analysis was used to evaluate the degradation of these dyes and their toxicity was tested by Allium cepa test. The generally observed higher stability of the bioaffinity bound enzymes against various forms of inactivation may be related to the specific and strong binding of enzyme with bioaffinity support which prevents the unfolding/denaturation of enzyme. Thus entrapped peroxidase was found to be effective in the decolorization of the investigated dyes.

Anticlastogenic and anticarcinogenic potential of Thai bitter gourd fruits.

Thai bitter gourd fruits (Momordica charantia Linn., TBG) has been previously demonstrated to possess phase II detoxificating enzymes inducing properties, as well as the ability to reduce phase I carcinogen activating enzyme activity in rat liver. In addition, it was partially inhibited 7,12-dimethylbenz(a)anthracene (DMBA)- induced mammary gland carcinogenesis in female Sprague-Dawley rats. In this study, we therefore examined the anticlastogenic and anticarcinogenic effect of TBG against clastogens, cyclophosphamide (CYP) and DMBA, in mice using the in vivo erythrocyte micronucleus assay and azoxymethane (AOM)-induced colon carcinogenesis in rats, respectively. For anticlastogenicity test, male mice were fed with modified AIN-76 diets containing 6.25% and 12.5% of ground freeze-dried TBG for 2 weeks prior to administration of clastogens till the end of experiment. Blood samples were collected and counted for reticulocytes by using the fluorescent microscope. For anticarcinogeicity test, male Wistar rats were fed with modified AIN-76 diets containing 5% and 10% ground freeze-dried TBG for 2 weeks prior to, during and 1 week after the completion of AOM administration (15 mg/kg once a week for 2 weeks). It was found that TBG at 6.25% resulted in a significant reduction in micronucleated peripheral reticulocytes (MNRETs) induced by only CYP. Study on anticarcinogenic potential demonstrated that rats fed with TBG diets at the concentration tested developed significantly higher incidence as well as the multiplicities of colon tumors than the control group. These results demonstrated that Thai bitter gourd fruits possesses anticlastogenic potential against clastogen in the mouse. Interestingly, it had no preventive potential against AOM-induced colon carcinogenesis in rat, rather increasing the incidence of colonic neoplasm when giving during the initiation stage.

Phenol-mediated decolorization and removal of disperse dyes by bitter gourd (Momordica charantia) peroxidase.

Salt-fractionated bitter gourd (Momordica charantia) proteins were employed for the decolorization of disperse dyes in the presence of H2O2. The effect of various experimental conditions such as concentration of enzyme, H2O2, phenol, reaction time, pH and temperature on the decolorization of dyes was investigated. Dyes were recalcitrant to the decolorization catalysed by bitter gourd peroxidase. However, these dyes were decolorized significantly in the presence of a redox mediator, phenol. Bitter gourd peroxidase (0.215 U/mL) could decolorize about 60% of Disperse Red 17 in the presence of 0.2 mM phenol, whereas Disperse Brown 1 was decolorized by only 40% even in the presence of 0.4 mM phenol. Maximum decolorization of dyes was achieved in the presence of 0.75 mM H2O2 in a buffer ofpH 3.0 and 40 degrees C within 30 min. The K(m) values obtained were 0.625 mg/(L x h) and 2.5 mg/(L x h) for Disperse Red 17 and Disperse Brown 1, respectively. In all the experiments, Disperse Brown 1 was found to be more recalcitrant to decolorization catalysed by bitter gourd peroxidise, as compared to Disperse Red 17.

Conjugated fatty acids accumulate to high levels in phospholipids of metabolically engineered soybean and Arabidopsis seeds.

Expression of Delta(12)-oleic acid desaturase-related fatty acid conjugases from Calendula officinalis, Momordica charantia, and Vernicia fordii in seeds of soybean (Glycine max) or an Arabidopsis thaliana fad3/fae1 mutant was accompanied by the accumulation of the conjugated fatty acids calendic acid or alpha-eleostearic acid to amounts as high as 20% of the total fatty acids. Conjugated fatty acids, which are synthesized from phosphatidylcholine (PC)-linked substrates, accumulated in PC and phosphatidylethanolamine, and relative amounts of these fatty acids were higher in PC than in triacylglycerol (TAG) in the transgenic seeds. The highest relative amounts of conjugated fatty acids were detected in PC from seeds of soybean and A. thaliana that expressed the C. officinalis and M. charantia conjugases, where they accounted for nearly 25% of the fatty acids of this lipid class. In these seeds, >85% of the conjugated fatty acids in PC were detected in the sn-2 position, and these fatty acids were also enriched in the sn-2 position of TAG. In marked contrast to the transgenic seeds, conjugated fatty acids composed <1.5% of the fatty acids in PC from seeds of five unrelated species that naturally synthesize a variety of conjugated fatty acid isomers, including seeds that accumulate conjugated fatty acids to >80% of the total fatty acids. These results suggest that soybean and A. thaliana seeds are deficient in their metabolic capacity to selectively catalyze the flux of conjugated fatty acids from their site of synthesis on PC to storage in TAG.

Partially purified bitter gourd (Momordica charantia) peroxidase catalyzed decolorization of textile and other industrially important dyes.

The aim of this study was to evaluate the enzymatic action of partially purified bitter gourd peroxidase for the degradation/decolorization of complex aromatic structures. Twenty-one dyes, with a wide spectrum of chemical groups, currently being used by the textile and other important industries have been selected for the study. Here, for the first time we have shown peroxidases from Momordica charantia (300 EU/gm of vegetable) to be highly effective in decolorizing industrially important dyes. Dye solutions, containing 50-200 mg dye/l, were used for the treatment with bitter gourd peroxidase (specific activity of 99.0 EU/mg protein). M. charantia peroxidases were able to decolorize most of the textile dyes by forming insoluble precipitate. When the textile dyes were treated with increasing concentration of enzyme, it was observed that greater fraction of the color was removed but four out of eight reactive dyes were recalcitrant to decolorization by bitter gourd peroxidase. Step-wise addition of enzyme to the decolorizing reaction mixture at the interval of 1h further enhanced the dye decolorization. The rate of decolorization was enhanced when the dyes were incubated with fixed quantity of enzyme for increasing times. Decolorization of non-textile dyes resulted in the degradation and removal of dyes from the solution without any precipitate formation. Decolorization rate was drastically increased when the textile and other industrially important non-textile dyes were treated with bitter gourd peroxidase in presence of 1.0 mM 1-hydroxybenzotriazole. Complex mixtures of dyes were prepared by taking three to four reactive textile and non-textile dyes in equal proportions. Each mixture was decolorized by more than 80% when treated with the enzyme in presence of 1.0 mM 1-hydroxybenzotriazole. Our data suggest that the peroxidase/mediator system is an effective biocatalyst for the treatment of effluents containing recalcitrant dyes from textile, dye manufacturing, dyeing and printing industries.