Preliminary Study On Chemical Constituents And Hepa-toprotective Effect Of Ethyl Acetate Extract Of The Roots Of Indigofera Stachyoides

Objectives: To Screen hepatoprotective activity of different extracts of the root s of Indigofera stachyoides of the Papilionoideae family, and study the chemical constituents of ethyl acetate fraction which has hepatoprotective activity, and investigate the hepatoprotective effects of some of the isolated compounds from this plant. Methods:1.The roots of Indigofera stachyoides was extracted by ethanol, and was obtained by extraction of pe-troleum ether, ethyl acetate fraction, n-butanol. hepatoprot-ective activity screening perform ed by pharmacological animal experiments to determine hepatoprotective active site. 2.Screening of hepatoprotective effect of mice.Weighing 20 ± 2g healthy and clean Kunming male mice were randomly divided into normal control group, the carbon tetrachloride model group, DDB group(100mg / kg), intragastric irrigation group which given ethyl acetat e(200mg / kg), subcutaneous group which given ethyl acetate(200mg / kg), intragastric irr igation group which given n-butanol(500mg / kg), subcutaneous group which given n-buta-nol(500mg / kg), n = 10. Each group was given medicine for consecutive 4 day, intrape-ritoneal injection with 0.1% CCl4- vegetable oil 10 ml / kg 8 hours after the last adminis-tration, fasted for 16 hours. collecting the blood of mice and separating serum from it and screening activity of ALT. 3.Different chromatographic methods and technologies such as silicagel column chromatography, Sephadex LH-20, RP-18, HPLC were applied to the isolation and purification of acetic ether extraction of the roots of Indigofera stachyoides, and the structures were elucidated by their physical and chemical properties and spectrosco-pic techniques. 3.Screening hepatoprotective activity of chemical compounds(HIL-2, 3, 4, 6, 7, 8, 9, 10, 11) with tetrachloride(CCl4)-induced acute liver injury, MTT assay was used to observe cell survival and calculated cell survival rate. Rusults: 1.CCl4 can increased the level of ALT of mice, the difference was significant compared with the normal group; The level of ALT of subcutaneous group which given ethyl acetate was decreased compar e-d with the model group, the difference was significant. 2.Eleven compounds were isolate d from the roots of Indigofera stachyoides and elucidated as stigmasterol(HIL-1), L-Maa ckiain(HIL-2), Medicarpin(HIL-3), Stigmast-4-en-3-one(HIL-4), Stigmastan-3-one(HIL-5), 3-Hydroxy-8,9-dimethoxypterocarpan(HIL-6),(-)-epicatechin(HIL-7), 2α,3α-epoxyflavan-5,7,3′,4′-tetraol-(4βâ†'8)-flavan-5′′,7′′,-4′′′-triol(HIL-8), 2α,3α-epoxy-5,7,3′,4′-t etrahydroxyflavan-(4βâ†'8)-epicatechin(HIL-9),(+)-epicatechin(HIL-10), schizandriside(HIL-11). Compound HIL-2 is isolated from this plant for first time. Compounds HIL-3, HIL-6 are isolated from Indigofera for first time. 3. HIL-7, HIL-8, HIL-9, HIL-10 and HIL-11 has protective effect on carbon tetrachloride-induced damage on human liver cell HL-7702, the survival rate increased significantly; HIL-2, HIL-3, HIL-4 and HIL-6 had no signif-icant effect. Conclusions: 1.This study enrich and improve the study of chemical constitu-ents of Indigofera stachyoides. 2. Subcutaneous injection of ethyl acetate extract of Indig ofera stachyoides has a protective effect on liver injury of mice caused by CCl4. HIL-7, HIL-8, HIL-9, HIL-10 and HIL-11 demonstrated the protective effect on liver cell damag e in vitro.