Studies On Antioxidant Chemicals Of The Fruits Of Cudrania Tricuspidata And Research The Anti-breast Cancer Apoptosis Pathway Of Scandenolone

Objective:To obtain compounds from the the antioxidant component of fruits of Cudrania tricuspidata (Carr.)Bur., several separation equipments were used to separate, extracte and purify the fruits, NMR and MS were used to identify the structure of compounds. We researched the cell apoptosis pathway and analyzed the mechanism that Scandenolone inhibited growth of the breast cancer cell M231. After a series of studies, We could obtain the finger-print, medicinal value and mechanism of the fruits of Cudrania tricuspidata (Carr.) Bur.Methods:(1)First, ethyl alcohol was used to extract the fruits. Secondly, extractum was separated by macroporous resin and ODS. Finally, the antioxidant component of monomer was obtained by preparative liquid phase and the structure of monomer was identified by NMR and MS.(2) MTT was used to detecte the growth situation of M231 cells exposed Scandenolone; cellular surface information was collected by atomic force microscopy (AFM); Flow cytometry (FCM) was applied to observe the influence of cell cycle and the changes in mitochondrial membrane potential (MMP); fluorescence microplate was used to detect the level of ROS; western blot detected the proteins expression of the cell apoptosis pathway, including casepase-3, PARP, Bcl-2, Bax, Bcl-xL, Bad, (p)-p53, (p)-JNK, (p)-ERK, (p)-AKT, (p)-p38, GADPH.Results:(1) Four kinds of crude extract that were the 30%-ethyl alcohol(2.8g),50%-ethyl alcohol(4.0g),75%-ethyl alcohol (3.7g) and 95%-ethyl alcohol(0.3g) were obtain when the fruits were separated by macroporous resin. The oxidation resistance of the four crude extracts were detected, the result showed that oxidation resistance of the 75%-ethyl alcohol was the strongest.The crude extract of 75%-ethyl alcohol was further separated and 13 kinds of monomers was obtained, four monomers amony them had comfirmed the specific chemical structure. They were Arctiin, erythrivarone A, lappaol F, 2-(3,4-dimethoxybenzyl)-3-(3-methoxy-4-hydroxybenzyl)-c-butyrolactone。(2) Scandenolone could obviously inhibit the grow of breast cancer cell M231.The cells nearly stopped grow by culture 72h when concentration of Scandenolone reached 20ug/ml. Three different concentration (10,12.5,15ug/ml) were selected to complete next experiments. AFM showed that after Scandenolone induced M231 cells with 24 h, with the increase of concentration, the rhombuslittle cell changed circular shape gradually, fell off from the wall of culture dish and cell volume also reduced gradually. FCM showed that after Scandenolone induced M231 cells with 24 h, Scandenolone resulted in a sudden increased of the Sub-G1 and the data was inceased from 17.9% of the low-dose group (lOug/ml) to 99.3% of the high-dose group (15ug/ml). There was significant differences above comparison (p<0.05).But there were little or no impact on G0/G1, S and G2/M. With the increase of concentration, MMP was decreaced. ROS assay showed with the increase of concentration, ROS levels was increased. ROS levels reached maximum at 30min, later the ROS levels kept to stabilize. Western blot showed that M231 cells were exposed to different concentrations of the drug for 24h, the expressions of casepese-3 protein in stimulation groups were decreased compared to that of control group (p<0.05). And the expressions of RARP protein in stimulation groups were decreased compared to that of control group (p< 0.001). After Scandenolone induced M231 cells with 24 h, the expressions of p53 protein every groups were no significant differences by statistical analysis (P>0.05), but the expressions of of p-p53 protein increaced in stimulation groups compared to that of control group (p<0.001). The expressions of Bax and Bcl-2 increased, and the ratio of Bax/Bcl-2 increaced in stimulation groups copared to that of control group (p<0.001).Meanwhile the expressions of Bad and Bcl-xl protein had no significant differences. After Scandenolone induced M231 cells with 24 h, the the expressions of p38 protein every groups were no significant differences by statistical analysis (P>0.05), but the expressions of p-p38 protein increaced in stimulation groups compared to that of control group (p<0.01), except the low-dose group(5ug/ml).The level phosphorylation of AKT, ERK, JNK every groups were no significant differences by statistical analysis (P>0.05).Conclusion:(1) We obtained the chemical structure of four compounds. They were2-(3,4-dimethoxybenzyl)-3-(3-methoxy-4-hydroxybenzyl)-c-butyrolactone, Arctiin, Arctiin, erythrivarone A, lappaol F.(2) Scandenolone had the activity of anti-breast cancer and the apoptosis pathway was:after Scandenolone induced M231 cells, the high level of ROS led to oxidative damage in M231 cells. Then, AKT, ERK and JNK were inhibited, p38 and p53 were activated. The decreace of MMP and the expressions of Bcl-2 family led to activate apoptosis perform protein of casepase-3, finally induced apoptosis of M231 cells.