Separation, Structure Identification Of Terpenoid And Phenols Compounds From Rubus Corchorifolius L.f. Leaves And Anti-Tumor Activity

Rubus corchorifolius L.f. is a kind of deciduous shrub and a member of the family Rosaceae Rubus.L. The leaves of Rubus corchorifolius L.f. have comprehensive nutritionaland health care functions. Effects of compounds on cell cycle, apoptosis, mitochondrial membrane potential and calcium ion concentration were analyzed by flow cytometry; effects of compounds on proteins of tumor cells by Westernblot analysis. The proteomics of a novel type diterpene DEK on HCT-116 cells by i TRAQ labeled quantitative proteomic technique.The experimental contents and results were as follows:(1)Purification,separation and structure identification of leaves from Rubus corchorif-olius L.f. leaves.Ingredients of ethyl acetate extraction, obtained from crude ethanol extraction of Rubus corchorifolius L.f. leaves were purified through different separation and purification methods, including column chromatography,TLC(thin layer chromatography),MPLC(medium-pressure column chromatography) and crystallization. Then the chemical structures of this ingredients were determined by comprehensive analysis of NMR(nuclear magnetic resonance:1H-NMR、13C-NMR、HMBC and HSQC), mass spectography and XRSD(x-ray single crystal diffraction), along with comparison with data from former research. According to the results of purification and structural analysis, 10 compounds were separated: 16β,17-dialkyl-3-oxygen-kaurane(1),kaurane-3α,16β,17-tri-ol(2), ent-kaurane-2-ketone-16β, 17,-diol-ketol(3), ent-kaurane-3α, 16β, 17-triol(4), ent-kaurane-16α, 17diol-2ketone(5),ent-kaurane-16β, 17, 19-triol-3-ketone(6),ent-kaurane-2α,16β,17-triol(7), asiatic acid(8), ethyl p-hydroxycinnamate(9), ethyl ferulate(10). Two of the compounds of ent-kaurane-2-ketone-16β, 17,-diol-ketol(3) and ent-kaurane-16β,17, 19-triol-3-ketone(6)are novel compounds.We found the 16β, 17-dialkyl-3-oxygen-kaurane(1), kaurane-3α, 16β, 17-triol(2) and ent-kaurane-3α, 16β, 17-triol(4) in Rubus corchorifolius L.f leaves for the first time. Kaurane-3α,16β,17-triol, ent-kaurane-16α, 17-dihydroxy-2ketone and ent-kaurane-2α, 16β,17-triol were analyzed by XRSD, the single crystal structures of which were not previously recorded in the CA and CDCC data bases, So we reported the single crystal structures for the first time.Antitumor activity screening and echanism monomericcompound from Rubus corchorifolius L.f. leaves(2) Screening of antitumor activity of ten compounds from Rubus corchorifolius L.f.leaves and the anti-tumor mechanism of compounds 4 and 8.Evaluation of the anti-tumor activity of the 10 compounds on Hep G2、MDA-MB-231、SCG-7901、OVCAR3 and HCT-116 cell by MTT assay, the results showed that compound 3,4 and 8 have good inhibitory effect on HCT-116 cell, the IC50 of compounds 3、4 and 8 were 40±1.78 μM,29.84±2.35 μM and 26.84±2.12 μM, respectively. Compounds 9 and 10 have effective inhibitory effects to Hep G2 cells with IC50, 28.17±1.20 μM and 26.2±2.1μM. The inhibitory effects of compound 9 and 10 on normal hepatic cells is that compound 10 does not show obvious inhibition to the normal LO2 cells at 6.25~100 μM, and compound 9 inhibits the LO2 cells’ growth around 25% and 20% at the concentration of 50 μM and 100 μM. Compound 3 does not show obvious inhibitory effects on the growth of CCD-18 co cells at the concentration of 6.25~100μM. Compound 4 shows a little inhibitory effects on the growth of CCD-18 co cells at the concentration of 6.25~100 μM. Compound 8 does not show obvious inhibitory effects on the growth of CCD-18 co cells at the concentration of 6.25~25μM, but it has strong inhibitory effects at the concentration of 100 μM. The effects of compounds 4 and 8 on the cell cycle, apoptosis and proteins of HTC-116 have been evaluated by flow cytometry and Western blot. Compounds 4 and 8 have anti-cancer effects through inducing apoptosis by retarding cell cycle at G0/G1, increasing apoptosis protein Cleaved PARP, Cleaved caspase-3, p53 and decreasing the expression of cycle protein cyclin D1, p27, CDK2 and CDK4.(3) Mechanism of apoptosis on Hep G2 cells by ethyl ferulate from Rubus corchorifolius L.f.leaves.Inhibiting the activity of Hep G2 cells and its mechanism of ethyl ferulate(compound 10) were investigated from the cell morphology, cell cycle, apoptosis and other aspects by using microscopy, flow cytometry and Western blot method. The results showed that: after Hep G2 cells treated with 15~35μM ethyl ferulate for 72 h, the cells had been changed sharply in concentration-dependent that cell’s number was decreased, floating, the shape was becoming round. But the normal liver cell LO2 cells treated at the same conditions had no significantly changes so that ethyl ferulate has no significant toxicity on normal liver cells. Compared with the control, Hep G2 cells treated with ethyl ferulate performed a typical apoptosis form that shrink significantly brighter and lysis nucleus in some apoptotic cells depending on increasing concentration through DAPI staining. Analyzed by with flow cytometry after Annexin V-FITC/ PI staining, the apoptosis rate of Hep G2 cells treated with 15μM and 35μM ethyl ferulate are 1.6 times(from 6.27% to 10.04%), 10 times(from 6.27 % to 62.95%), respectively. Therefore ethyl ferulate could induce Hep G2 apoptosis concentration-dependently. In order to further explore mechanism of ethyl ferulate inducing of Hep G2 cells, the cell cycle was demonstrated by PI staining using flow cytometry. Ethyl ferulate have effect on cell cycle arrest. Ethyl ferulate at the concentration of 15 ~20 μM can blocked cells in G0 /G1 phase; while ethyl ferulate at the concentration of 25~35μM blocked cells in S phase. Ethyl ferulate at the concentration of 15μM~35μM could also reduce the mitochondrial membrane potential in Hep G2 cells dropped from 29 a.u to 14.2 a.u, which had a certain dose-effect relationship. Ethyl ferulate at a concentration of 15 and 35μM could increase the concentration of calcium in Hep G2 cells 3.0 and 4.5 times, respectively, compared with control group. Ethyl ferulate was further confirmed that activating expression of pro-apoptotic protein cleaved PARP, Cleaved caspase-3, Cleaved caspase-9, p53, Bax and cycle arrest protein p21, inhibiting expression of cycle progression protein cyclin D1 by Western blot,. Thus, the mechanism of inducing liver cancer cells Hep G2 apoptosis and cycle arrest of ethyl ferulate is acting with DNA, reducing membrane potential, increasing intracellular calcium concentration.(4) Mechanisms of anti-tumor in HCT-116 cell of novel diterpenoid DEK of Rubus corchorifolius L.f. leaves.The mechanisms by which the newly discovered diterpenoid DEK affects a colon cancer cell HCT-116 are inducing cell apoptosis, inhibiting the cell cycle in phase S and lowering the electrical potential of the cell membrane, instead of increasing the concentration of calcium ions. According to the analysis of i TRAQ quantitative proteomics technology, the effect of diterpenoid DEK on a HCT-116 cell is to change the expressed quantity of cell proteins, including 120 different proteins that had a statistically significant change: 53 with decreased quantity and 67 with increased quantity. These proteins mainly participate in the processes of enzyme activity adjustment, electron transfer, and anti-oxidation and protein transcription. They have important cancer inhibitory effects in colorectal cancer pathways, the oxidative phosphorylation pathway, cell cycle pathway and the p53 path way. For example, the proteins of Rac,Rho A,Rac1,Cdc42,SDHB, NDUFS6, COX6B1,ATP6,NDUFA9 and14-3-3σ.Which proteins are related to cancer inhibitory functions, including promotion of cancer cell apoptosis, inhibition of the cell cycle and cell proliferation, alteration of cell morphology, etc. Protein expression tendency of proteins by Western blot results are consistent with i TRAQ results.