Separation And Purification Of Antitumor Compounds From Pigeon Pea Leaves And The Anti-breast Cancer Mechanisms

Pigeonpea[Cajanus cajan(L.)Millsp.]belongs to the Cajanus genus of Leguminosae family.It is a valuable perennial or annual woody food crop,and widely distributed throughout the tropical and subtropical regions from 35°N to 35°S.Besides as a popular food,it has high ecological and folk medicinal value.Pigeonpea leaves are traditionally used to treat irritations,sores,coughs,diabetes,the ischemic necrosis of femoral head and cancers et al.However,reports on the chemical constituents and activities of pigeonpea are few.In the present study,the isolation and purification of antitumor active constituents from pigeonpea leaves were guided by MTT assay for the first time.The chemical structures of twenty-two compounds were identified and two new compounds were obtained.The stilbenes in pigeonpea leaves were confirmed as potential antitumor-active constituents following the activity evaluation.Among them,cajaninstilbene acid(CSA)was confirmed as the major active.Furthermore,the preliminary antitumor activity and mechanism of CSA against MCF-7 was investigated in vivo and in vitro.The present research supplied potent references for multiple utilization of pigeonpea,as well as the basis of materials for the development of native agents against cancers.1.Twenty-five compounds were obtained from the active fraction of pigeonpea leaves extracts by using a variety of chromatographic techiques such as macroporous absorption resin,normal or reverse phase silica gel,SephadexLH-20 and semi-prepare HPLC,et al.On the basis of chemical and spectroscopic evidences including UV,NMR and MS,twenty-two compounds were identified as follows:Icosyl palmitate(1),7-hydroxy-5-0-methyl-8-(3-methyl-2-butylene)-4-phenyl-9,10-dihydro-benzopyran-2-one(2*),longistyline C(3),pinostrobin(4)pinostrbin chalcone(5),cajaninstilbene acid(6),3,12-dihydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid(7*),apigenin(8),luteolin(9),naringenin(10)apigenin-8-C-a-L-arabinoside(11),isovitexin(12),vitexin(13),orientin(14),apigenin-6,8-Di-C-a-L-arabinopyranoside(15),apigenin-6-C-?-D-glucopyranosyl-8-C-a-L-arabinopyranoside(16),apigenin-6-C-a-L-arabinopyranosyl-8-C-?-D-glucopyranoside(17),genistin(18),xylose(19),luteolin-6-C-a-L-arabinopyranosyl-8-C-?-D-glucopyranoside(20),luteolin-6-C-?-D-glucopyranosyl-8-C-a-L-arabinopyranoside(21),luteolin-7-O-?-D-glucopyranoside(22).Among of them,both compound 2*and 7*are new chemicals,which are named as cajanuslactone(2*)and 12-hydroxy cajaninstilbene acid(7*).Compounds 11,14-17 and 19-22 were isolated for the first time from pigeonpea.2.The antitumor activity of compounds 2-22 were evaluated by MTT assay.The results showed that three kinds of stilbene compounds 3,6,and 7 were more active than others against MCF-7,A549 and HepG2.IC50 of compound 3 against three cells were 8.53,11.92 and 9.79?g/mL,those of compound 6 were 4.81,5.11 and 5.01 ?M,and for compound 7,they were 10.13,12.01 and 12.78 ?g/mL,respectively.Among them,CSA were considered as the most potential antitumor-active constituent.3.The effect of CSA against MCF-7 xenografts in mice was investigated for the first time.The results proved that CSA could inhibit the proliferation of human cancer cell line MCF-7.The tumor inhibitory ratio was 66.41%and 43.14%when concentration was 30mg/kg and 15 mg/kg,respectively.The effect of CSA against MCF-7cells in mice was active and without inducing body weight loss.Furthermore,the results indicated that CSA could prevent the proliferation of MCF-7 maybe by inhibiting the tumor angiogenesis and inducing apoptosis.4.CSA could remarkably induce apoptosis of human breast cancer MCF-7 cells by inducing the cell cycle arrested at S and G2/M phase.Further experiments revealed that CSA induced MCF-7 apoptosis by caused the mitochondrial membrane potential decreasen,caspase 3 activation,Bcl-2 protein down regulation and Bax up regulation.