Purification and Characterization of 3-o-methyl-d-glucose from the Seed Coat of Vigna Mungo (L.) Hepper

Abstract

Vigna mungo. L (Black gram) belonging to the Leguminosae family is a most abundant source of protein for human and animal diets. Over 8.5 million tonnes of black gram are being produced worldwide, and after confiscating the endosperm for the protein source, the seed coat is dumped as agro-waste and used as feed for the cattle. This study aims to investigate and profile the metabolites in three distinct parts of V. mungo seeds: coat, cotyledon, and intact seed. Also, it has been hypothesized that sugar methylation plays a pivotal role in facilitating black gram seed germination by serving as an energy source during metabolic processes. In our study, A total of forty-nine compounds belonging to the class such as fatty acids, saccharides, triterpenes, sterols, and vitamins were detected in various parts of V. mungo seed by gas chromatography-mass spectrometry (GC-MS). Most interestingly, 3-O-methyl-D-glucose (3-OMG) was commonly detected in methanolic extract of coat, intact seed, and cotyledon with peak area percentages of 3.076, 1.157, and 1.100 %, respectively. Based on the highest peak area percentage in the coat, this compound was purified by using a silica gel column with a mesh size of 60–120 followed by Biotage SNAP cartridge KP-C18-HS (12 g). The yield and purity of the purified fractions were found to be 11.78 & 94.19 %, respectively by high-performance liquid chromatography (HPLC) analysis. Further, the mass confirmation of purified fraction containing 3-OMG (m/z - 217.06 g/mol) was done by liquid chromatography-mass spectrometry (LC-MS/MS) and reconfirmed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The MS/MS analysis of the fragment showed m/z 206.91 for 3-OMG which corresponds to the association of the sodium adduct. The concentration of 3-OMG was quantified as 332.65 µg/g of dry weight in the coat of V. mungo by HPLC analysis. Also, among the antioxidant assays conducted, such as DPPH, ABTS, FRAP, and NOS, it was observed that the methanolic extract of coat exhibited the most significant antioxidant activity. The 3-OMG is commonly used as a non-metabolized analogue of glucose by regulating glucose transporter, especially GLUT-4 that regulates the absorption of glucose by insulin into muscle and fat cells. This study supports the black gram seed with coat as an alternative medicine to regulate blood glucose.