Study On Isolation And Identiifcation Of Active Ingredient From Astragali Complanati Semen And Its Pharmacological Activity

Astragali complanati semen comprises the dry mature seeds of Astragalus complanatusR.Br. It has been extensively used for clinical treatment of reinforcing liver and benefitingkidney in most authoritative medical book of Ancient China and It is mainly distributed innortheast, north, northwest in China. Some researches have certified that A. complanati semenhas the functions of anti-aging, lowering lipid and hepatoprotection, inhibition plateletaggregation, anti-inflammatory and antitumor. But the systematic researches about thechemical composition and pharmacological action of A. complanati semen are rarely. Thispaper comprehensive analyzed this two aspects which can provide some theoretical basis formore applications and are beneficial to further development.95%ethanol extract of A. complanati semen was hierarchical extraction by petroleumether, ethyl acetate, butanol and water according to priority for four parts, which wasrespectively evaluated by antioxidant （containing reducing power and DPPH·/O₂-· scavenging activity）, anticomplement, anticancer（Hela cervical cancer cell） andanti-inflammatory model in vitro. The result showed butanol fraction had relatively betteranticomplement. The petroleum ether fraction had good antitumor activity that was similar to5-fluorouracil control and when its concentration was200μg/ml, the inhibition rate was67.61%. The ethyl acetate fraction had good DPPH·scavenging activity, anticancer andanti-inflammatory activities. Furthermore, with proper polar of this fraction, this paper chosethe ethyl acetate fraction to further isolation and purification.Macroporous resin was used for separation of total flavonoids and total saponinswith a gradient of ethanol-water（100%water up to100%ethanol）. At the same time, wealso got total alkaloids and total polysaccharides by all kinds of solvent extractions. Thecontent of total flavonoids, total saponins, total alkaloids and total polysaccharides wasrespectively37.10%,45.68%,2.85%and24.27%. Though activity evaluation of antioxidant（containing reducing power and DPPH·/O₂-· scavenging activity）, anticomplement,anticancer and anti-inflammatory model in vitro, the result showed the total flavonoids hadgood DPPH·scavenging activity, anticomplement and anti-inflammatory activities; when theconcentration was0.6mg/ml, the DPPH·scavenging rate was90.72%; when the concentrationwas1mg/ml, the anticomplement inhibition rate was45.70%and when the concentration was200μg/ml, the NO inhibition rate was66.42%. The total saponins had good O₂-·scavengingactivity and anti-inflammatory activity. The total alkaloids had good reducing power and anticancer activities. The total polysaccharides had no obvious activity.The active fraction of ethyl acetate part was separated and purified by various columnchromatography （including silica gel, Sephdeax LH-20gel, ODS columns and TLC analyses）,and the prepared HPLC. Five compounds （EE-A: rhamnocitrin-3-O-β-D-glucoside, EE-B:calycosin, EE-C: formononetin, EE-D: rhamnocitrin and EE-E: Its structure was not workedout.） were gained and their structures were identified by the interpretation of spectroscopicanalyses including MS,1H-NMR and13C-NMR. EE-A, EE-B, EE-C and EE-D were allflavonoids. Five compounds were assessed anticancer activity by Hela cervical cancer celland anti-inflammatory activity by LPS stimulated inflammation factors to mice macrophagesmodel. The result showed formononetin and rhamnocitrin were two active ingredients in A.complanati semen for inhibition effect to Hela cervical cancer cell proliferation. When theirconcentrations were100μg/ml, the inhibition rate were respectively,65.95%and52.60%. Toanti-inflammatory activity, EE-E had relatively good anti-inflammatory activity and it caninhibit more than80%NO release, when the concentration was100μg/ml.