Studies On The Anticancer Constituents Of Vitex Negundo L. And Their Biotransformation

Vitex negundo L.(Verbenaceae)grows widely throughout more than 20 provinces in China.The seeds,leaves,twigs and roots of this plant were used as a folk medicine for catarrhal rhinitis,cough,asthma,malaria, calenture,rheumatism and arthritis.Firstly,bioactive-targeted extraction technique was used to screen anti-tumor fraction from Vitex negundo L..The 40%EtOH extract of the seeds of V.negundo L.was subjected to polyamide column chromatography to afford 6 fractions,anti-tumor effect of the 6 fractions were investigated by MTT assay.These data suggested that the EtOAc fraction could significantly inhibit the growth of MCF-7 cells and the IC₅₀was 66.4μg/mL.Human ovarian cancer,human breast cancer, human uterine cervix cancer,human hepatocelluar cancer and human stomach cancer were established in nude mouse model.This helps to investigate the anti-tumor effect of EtOAc fraction in vivo.The results indicated that EtOAc fraction could inhibit the growth of the above cancers in nude mouse.The inhibitory activity grew stronger along with increasing dosage.Consequently,it was discovered first time that the extract of the seeds of V.negundo L.possessed a broad spectrum anti-tumor activity and EtOAc fraction was anti-tumor effective group.To investigate the anti-tumor constituents of this plant,several methods have been used in the process of separation and purification including polyamide chromatography column(C.C.),sephadex LH-20 C.C.,HPLC and so on.As a result,ten compounds have been isolated, including four organic acids,two phenylpropanoids and four lignans.On the basis of physical-chemical properties and spectral evidences,their structures were elucidated as:p-hydroxybenzci c acid(1), 3,4-dihydroxybenzoic acid(2),sinapaldehyde(3),ferualdehyde(4), caryophyllic acid(5),6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)3-hydro-methyl-7-methoxy-3,4-di-hydro-2-naphthaldehyde(6),6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-3-glucosyloxymethyl-7-methoxy-3,4dihydro-2-naphthaldehyde(7),6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-3- hydroxymethyl-5-methoxy-3,4dihydro-2-naphthaldehyde(8),3-methoxyl-4- hydroxybenzcic acid(9),6-hydroxy-4-(3,4-dimethoxyphenyl)-3-hydroxymethyl-5-methoxy-3,4dihydro-2-naphthaldehyde(10).Among them,compounds 7,10 were new compounds and compounds 3,4 were firstly reported in this plant.Antitumor activity of compound 6,compound 7 and compound 8 were investigated suppressing the proliferation of varied tumor cells in vitro by MTT assay.These studies established that compound 6 exhibited moderate activity against the proliferation of MCF-7,SMMC-7721 and HT-29 cells in vitro,and the activity against COC1 cells was strongest and IC₅₀was 1.79μM;compound 7 showed moderate activity against the proliferation of MCF-7 cells in vitro;compound 8 showed moderate activity against the proliferation of MCF-7 and COC1 cells in vitro.Human ovarian cancer were established in nude mouse model.This helps to investigate the anti-ovarian cancer effect of compound 6,8 in vivo.The results indicated that compound 6 didn't show efficacy in activity assays in nude mice.Compound 8 markedly inhibited the growth of ovarian cancer in nude mice.And compound 8 at a dose(5mg/kg) could effectively inhibit the growth of tumor(control 13874±5652mm³, compound 8 5mg/kg,4495±2087mm³),and the tumor weight inhibitory rate was 53.1%(P＜0.05).At last,the metabolites of compound 6 in SD rats were 6-hydroxy-4-(3-methoxyphenyl)-3-hydroxymethyl-7methoxy-3,4dihydro -2-naphthaldehyde-4'-glucuronic acid,6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxy-3,4dihydro-2-hydromethyl naphthalene and 6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-2,3-dihydroxymethyl-7-methoxy-3,4dihydro-4'-glucuronic acid.The metabolites of compound 6 in SD rats didn't show anti-tumor activity in vitro.