| Daphne giraldii Nitsche is a Chinese native erect shrub,belonging to family Thymelaeaceae and genus Daphne.Its rhizome bark,commonly known as ’ZuShima’,is employed as a folk medicine for the treatment of rheumatoid arthritis and various pains.Now the researches on chemical constituents and pharmacological activities of D.giraldii focus on coumarins and diterpenoids,while other compounds have few reports.On the basis of our previous study,a more systematic study about the chemical composition and biological activities was carried out.By means of various chromatographic methods,68 compounds were isolated from the ethanol extract of the dried stem and root bark of D.giraldii.Among them,68 compounds were identified by extensive spectroscopic and spectrometric methods,including 39 flavans,10 flavones,7 coumarins and 12 other compounds as below:(2S)-daphnegiranol A(A1),(2R)-daphnegiranol A(A2),(2S)-daphnegiranol B(A3),(2R)-daphnegiranol B(A4),(2S)-kazinol U(A5),(2R)-kazinol U(A6),(2S)-kazinol B(A7),(2R)-kazinol B(A8),daphnegiranols C1-C2(A9-A10),daphnegiranols D1-D2(A11-A12),daphnegiralins A1-A4(A13-A16),daphnegiralins B1-B4(A17-A20),daphnegiralins C1-C2(A21-A22),daphnegiralins E-G(A23-A25),daphnegiravans A-F(A26-A31),daphnegiravan H(A32),daphnegiravan K(A33),daphnegiravan M(A34),(2S)-7-hydroxy-3’,4’-dimethoxyflavan(A35),(2S)-7,4’-dihydroxy-3’-prenylflavan(A36),(2S)-kazinol I(A37),(2S)-4’-hydroxy-7methoxyflavan(A38),(2S)-7,3’-dimethoxy-4’-hydroxyflavan(A39),daphnegiravonols A-B(A40-A41),daphnegiravones A-C(A42-A44),broussoflavonol B(A45),morusin(A46),5,4’dihydroxy-7-methoxyflavone(A47),eupatilin(A48),2’,4’-dihydroxy-3-(4-methoxyphenyl)propiophenone(A49),daphnegiratins A-B(A50-A51),umbelliferone(A52),7-dimethylsuberosin(A53),8-hydroxyxanthyletin(A54),luvangetin(A55),nodakenetin(A56),daphnegiranin A(A57),broussonins A-B(A58-A59),5-methoxy-2-(3-(4-methoxyphenyl)propyl)phenol(A60),broussonin E(A61),1-(2’-hydroxy-4’-methoxyphenyl)-3-(4"-hydroxy3"-methoxyphenyl)propane(A62),1-(4’-hydroxy-2’-methoxyphenyl)-3-(3"-hydroxy-4"methoxy phenyl)propane(A63),(-)-pinoresinol(A64),salicifoliol(A65),(4S)-isosclerone(A66),(4S)-4-hydroxy-α-tetralone(A67),3,4,5-trimethoxyphenol(A68).Of all these isolates,36 compounds(A1-A4,A8-A20,A23-A31,A33-A34,A40-A44,A50-A51 and A57)were new and 22 compounds were isolated from genus Daphne for the first time.Previously,12 flavans(P1-P12)were isolated from D.giraldii and were not evaluated for their biological activities.Therefore,these compounds were tested for in vitro cytotoxic experiments together with A1-A63.The results showed that flavans had a selective cytotoxicity towards Hep3B cells.Among them,compounds A11-A12,A16,A17/A18,A19/A20,A28,A33,P2-P4 and P9 displayed potent inhibition on Hep3B proliferation with IC50 values less than 10 μM.Flavone compound A43 exhibited marked cytotoxic activity against all six cancer cells with IC50 values ranging from 2.53 to 20.82 μM.1,3-diphenylpropanes were found to moderately inhibit the proliferation of tested cell lines.In addition,the preliminary structure-activity relationships were also discussed based on MTT results.Further pharmacological research demonstrated that A8 not only induced apoptotic cell death through intrinsic and extrinsic pathways but also triggered autophagy with AMPK activation taking part in this process.Besides,A8-induced autophagy played a protective role in apoptotic cell death.Flavans A28,A33,P2 and P3 inhibited Hep3B cell proliferation by arresting the G2/M phase,while P4 had an inhibitory effect by inducing apoptosis.P2 and P4 could also stimulate ROS generation which probably disrupted normal transition of G2 to M phase and led to cancer cell apoptotic death.A43,a flavone compound,exhibited antitumor activity through the induction of apoptosis.Crotalaria pallida Ait belongs to family Leguminosae and genus Crotalaria.Its herb and roots are employed as a folk medicine for the treatment of dysentery,diarrhea,acute mastitis,cancer and tinea of feet and hands.In addition,C.pallida can also be used for greening and ecological restoration.However,the prolizidine alkaloids in this plant may cause serious hepatotoxicity,which limits its application.Now there are few researches on chemical constituents and pharmacological activities of C.pallida,except for alkaloids.In order to clarify its medical composition,a comprehensive study about the chemical components and biological activities was carried out.By means of various chromatographic methods,15 compounds were isolated from the ethanol extract of the dried seeds of C.pallida.All compounds were identified by extensive spectroscopic and spectrometric methods,including 8 aromatic compounds,2 alkaloids and 5 other compounds as below:crotalariapallin A(B1),crotalariapallin B(B2),(E)-methyl p-hydroxycinnamate(B3),methyl caffeate(B4),trans-ferulic acid methyl ester(B5),methyl isoferulate(B6),methyl rhamnosyl p-coumarate(B7),cis-ferulic acid methyl ester(B8),crotalariapallin C(B9),usaramine(B10),(-)-(9E)-methyl dihydrophaseate(B11),(-)-methyl dihydrophaseate(B12),(S)-5-hydroxy-3,4-dimethyl-5-pentylfuran-2(5H)-one(B13),(-)dimethyl retronecate(B14),1H-indole-3-carboxaldehyde(B15).Of all these isolates,3 compounds(B1-B2,B9)were new and 11 compounds were isolated from genus Crotalaria for the first time.The in vitro cytotoxic activity experiments showed that compounds B1-B15 were inactive towards HepG2 and Hep3B cells,suggesting that they may not be the antitumor constituents of C.pallida.The antioxidant activities of B1-B8 were evaluated by DPPH,ABTS and FRAP assays.The results showed that B4 was the most active antioxidative compound,especially its ABTS’+scavenging and Fe2+reducing capacities,both stronger than that of potive control Trolox. |
