HPLC/MS/MS Study of Phenolic Compounds of Leucaena leucocephala Legumes Monitored with Their in vitro Antihyperglycemic Activity

Abstract

High pressure liquid chromatography coupled with mass spectrometry (HPLC/MS/MS) is considered as one of most the sensitive modern techniques used for determination of phenolic compounds, hence in this research it was utilized for characterization of a variety of phenolic compounds in Leucaena leucocephala legumes growing in Egypt. HPLC gradient elution analysis using water and acetonitrile, both containing 0.1% formic acid was carried out for standard solutions of the available phenolic and flavonoid compounds as well as the investigated fraction in the negative ion mode using MS/MS product ion scans where the deprotonated molecules [M‒H]- were observed for all the studied compounds; for flavonol and flavone glycosides, the spectra exhibit both the deprotonated molecule [M‒H]‒ of the glycoside and the ion corresponding to the deprotonated aglycone [A‒H]–, the latter ion is formed by loss of the sugar residue from the glycosides; different fragmentation pattern was observed for flavone-C-glycosides which involved additional fragmentation in the sugar part while for methyl ether aglycones, formation of [M‒CH3]‒ • was observed. The gained results revealed the elution of eleven phenolic compounds from the ethyl acetate fraction, namely; isovanillin, gallic acid, caffeic acid, apigenin-8-C-glucoside (vitexin), quercetin 3-O-galactoside (hyperoside), luteolin-7-O-glucoside, quercetin, isorhamnetin, luteolin 6-methyl ether (neptein), kaempferol 3,7-dimethyl ether and  5,7,3',4'-dihydroquercetin tetramethyl ether (taxifolin 5,7,3',4'- tetramethyl ether) successively. The methanol extract and different fractions of Leucaena leucocephala legumes were subjected to in vitro evaluation of their α-glucosidase inhibitory activity at different dose levels compared to the reference standard drug “Acarbose”, the gained results revealed that methanol extract and ethyl acetate fraction exhibited significant percent inhibitory activity at dose level of 2.5 mgml-1 (71.55±0.009%, IC50 0.210±0.003 mg ml-1 & 65.44±0.005%, IC50 0.270±0.042 mg ml-1) respectively compared to Acarbose (55.45±0.051%, IC50 0.650±0.051 mg ml-1) which affords both as effective natural antihyperglycemic drugs.