Active Compounds Of Rhynchosiae Radix For Female Contraception And Their Mechanism

Rhynchosiae Radix is the dry roots of Rhynchosia volubilis Lour.,which belongs to Leguminous family.It showed diversity activities,such as dispelling wind and dehumidification,promoting blood circulation,detoxification,detumescence and relieving pain.It is the king drug of a folk contraceptive prescription used by native in northwest of Hubei province,China,which has been used for female birth control in clinic for a long time.However,the structures of the active components are still unknown.With the guide of spermicidal activity in vitro,the Rhynchosiae Radix extracts were separated and purified.The results showed that the total extracts of Rhynchosiae Radix could inactivate all sperm in semen at 100 mg/m L.In each extraction layer,the turn of inhibition intensity was:petroleum ether the first,ethyl acetate part and water part the second,and n-butanol part the last.The antifertility components in Rhynchosiae Radix were scattered in all fractions,but the petroleum ether layer was the most active.In order to explore the antifertility substance in Rhynchosiae Radix,forty compounds were isolated from a 70%acetone extract by using a combination of various chromatographic techniques including column chromatography over silica gel,ODS C₁₈,Sephadex LH-20,and semi-preparative HPLC.By using spectroscopic techniques including ¹H,¹³C-NMR,and HR-ESI-MS,thirty-two compounds were identified and their structures were elucidated as Rhynchone A～F（1-6）,（-）-Sigmoidin E（7）,lupinifolin（8）,tonkinensisol（9）,lupinifolinol（10）,cathayanon H（11）,genistein（12）,prunetin（13）,isowighteone（14）,erythrinin B（15）,licoisoflavone A（16）,semilicoisoflavone B（17）,eriosemaone D（18）,formononetin（19）,neobavaisoflavone（20）,erylatissin B（21）,puerarone（22）,3-（4-Hydroxyphenyl）-8,8-dimethyl-8H-pyrano[2,3-f]-chromen-4-one（23）cajanone（24）,5,3’-dihydroxy-4’-methoxy-5’-γ,γ-dimethylallyl-2’’,2’’-dimethylpyrano[5,6:6,7]isoflavanone（25）,precatorin B（26）,sophoraisoflavanone B（27）,3,4-Dihydro-7’-methoxy-2’,2’-dimethyl-3,6’-bi（2H-1-benzopyran）-7-ol（28）,isoneorautenol（29）,lupeol（30）,betulinic aldehyde（31）,Clionasterol（32）.Among them,compounds 1-6 were new compounds,24 compounds include 1-7、9、11、14-18、20-23、25、27-29、31-32 were firstly isolated from this genus,the remaining compounds were isolated from Rhynchosiae Radix for the first time except for cajanone（24）.Sperm is an independent germ cell in vitro with special structure.The spermicidal activity of the isolated constituents was assayed in vitro,and the effect on calcium channel protein Catsper on sperm cells was also evaluated based on the change of intracellular calcium content.The results showed that compounds 2,16,22,26 had a weak effect on inhibiting sperm motility,compared with the positive reference Nonoxnol,compound 2 can inactivate all sperm in normal concentration semen in 300 seconds.Compounds 29 and 30 have affinity for Catsper channel proteins,showed a concentration dependent effect on the intracellular calcium content of sperm compared with the positive control drug progesterone.The more active components and their specific mechanism of antifertility of Rhynchosiae Radix need to be further studied.