Inhibition of α-glucosidase by new prenylated flavonoids from euphorbia hirta L. herb

Euphorbia hirta L. (Euphorbiaceae) is a pantropical medicinal rhizomatous herb, traditionally used in the treatment of diabetes, respiratory and gastro-intestinal disorders. To isolate and characterize the constituents of Euphorbia hirta and evaluate their in-vitro α-glucosidase inhibitory activity. The study is also aimed at describing structural activity relationship, type of α-glucosidase inhibition and in-vivo potential to regulate post prandial hyperglycemia in Wistar rats. Methanolic extract of whole plant was suspended in water, and sequentially fractionated with n-hexane and ethyl acetate. Further ethyl acetate fraction was subjected to medium pressure liquid chromatography (MPLC) to isolate the active molecules under the following experimental conditions, pressure (up to 5 kg/cm2) and flow rate (2 in./min). The structural elucidation of isolated compounds was done on the basis of detailed spectral analysis. The α-glucosidase inhibitory potential of isolated compounds was evaluated and compared with standard drug acarbose. In addition, type of inhibition was dwelled by Lineweaver-Burk plot analysis. Further, sucrose tolerance test was performed in Wistar rats pre-treated with the isolated compounds and acarbose (0.015 mM) followed by a sucrose load (2 g/kg, p.o.) and blood glucose level was measured up to 120 min by the glucose oxidase method. The ethyl acetate fraction afforded quercetrin (1), dimethoxy quercetrin (2), along with two new prenylated flavonosides designated as hirtacoumaroflavonoside (3) and hirtaflavonoside-B (4) characterized as 7-O-(p-coumaroyl)-5,7,4′-trihydroxy-6-(3,3-dimethyl allyl)-flavonol-3-O-β-d-glucopyranosyl-(2″→1″′)-O-α-l-rhamnopyranoside and 5, 7, 3′, 4′-trihydroxy-6-(3, 3-dimethyl allyl)-8-(iso-butenyl)-flavonol-3-C-β-d-glucopyranoside, respectively. All the isolated compounds showed dose dependent inhibition of α-glucosidase which was found to be comparable to acarbose. Maximum α-glucosidase inhibition was achieved with compound 3 (IC50 0.022 mM) followed by 4 (IC50 0.071 mM) in comparison to acarbose (IC50 0.092 mM). The results revealed that 5,7,4′- trihydroxyflavone structure is imperative for the inhibitory activity. The prenylation in the flavonoids increase the potency and p-coumaroyl substitution at C-7 further enriched the α-glucosidase inhibition. Compound 3 exhibited non-competitive inhibition while compounds 1, 2 and 4 showed mixed non-competitive inhibitory pattern. The results of sucrose tolerance test corresponded well with the in vitro studies. α-Glucosidase inhibitory activity and sucrose tolerance test demonstrated by the prenylated flavonoids present in E. hirta provide credence to the ethnomedicinal use of the plant in the management of diabetes in folk medicine.