Studies On The Extraction And The Anti-diabetic Effect Of Entagenic Acid

Entada phaseoloides(L.)Merr.,belonging to the Entada Leguminosae plants,is a traditional Chinese folk medicine.Their seeds have been long used by Dai people,one of the ethnic minorities in China for the treatment of haemorrhoids,stomachache,edema,and constipation.In addition,antibacterial,antioxidant,hypoglycemic,antitumor,anti-inflammation and analgesia effect and other biological activity had been reported by researchers through Modern pharmacological research methods.Previous phytochem-ical studies on E.Phaseoloides showed that triterpene saponins are the main active constituents.We have earlier reported that the saponins from Entada phaselolides(Linn.)Merr.have antidiabetic effect in type 2 Diabetes mellitus rats.However,the antihyperglycemic effect of Entagenic acid(EA),as the main aglycon of the total saponins,remains to be elucidated.Here,we extracted EA from the seeds of Entada phaselolides(Linn.)Merr and explored the antidiabetic effect of EA by in vivro and in vivo models.The powdered seeds kernel material was extracted with 70% EtOH at room temperature.The ethanol extract was poured into Diaion HP-20 column and eluted with water to remove sugars and ionic substances,successively eluted with EtOH to afford a crude saponin mixture.The total saponin was heated at 90 ?in a 2N HCl solution(1:10 g/ml)for 3 h.The mixture was adjusted to pH 6 with a dilute NaOH solution and then extracted with EtOAc.The combined EtOAc extracts were evaporated then and fractionated by MCI column using MeOH as eluent to afford Entagenic acid.The purity of EA was confirmed to be 98.0 % by HPLC.We explored the effect of EA on the glucose consumption with the L6 myotubes and IR HepG2 cells as the in vivro models.The results showed that EA dose-dependently increased L6 myotubes and IR HepG2 cells glucose consumption.This indicate that EA may have potential hypoglycemic activity.To compare the hypoglycemic effects of EA and it's similarly derivatives,we conducted the glucose consumption assay on L6 myotubes.The results showed that the entagenic acid(EA),ursolic acid(UA)and oleanolic acid(OA)significantly increased L6 myotubes glucose consumption,of which EA possessed the strongest hypoglycemic activity.In order to reveal the mechanism of the hypoglycemic effect of EA,we conduct the IRAP translocation assay on the L6 myotubes which were transected with pIRAP-mOrange cDNAs.The result showed that EA significantly increased the translocation of IRAP which indicated that EA may have effect on the translocation of GLUT4,as IRAP and GLUT4 showed colocalization relationship.To further confirm this effect,we detect the effect of EA on the expression of GLUT4 by western blotting assay.The results showed that EA dose-dependently increased the expression of GLUT4.In addition,EA significantly increased the phosphorylation of AMPK and ACC without signifficant effect on the phosphorylation of Akt.Our study indicated that EA increase glucose consumption by increase the translocation and expression of GLUT4 through AMPK pathway.We used the db/db mice as the T2 DM model to detect the antidiabetic effect of EA in vivo.After six weeks of treatment,EA lowered postprandial blood glucose levels in db/db mice and suppressed the TC,TG,LDL-C and TNF-? levels and increased HDL-C level in the serum of db/db mice.The histological analysis showed that EA alleviated the fatty degeneration of hepatocyte in db/db mice,and also alleviated the abnormity caused by diabetes in the pancreas of db/db mice.We also fund that EA lowered the MDA level and increased the SOD activity in the liver of db/db mice.These findings suggest that the therapeutic efficacy of EA for type 2 diabetes is attributable to it's improvement for the carbohydrate and lipid metabolism disorder and it's protection for liver and pancreas damage,as well as it's regulation effect on the GLUT4 expression in the muscle of db/db mice.