Studies On The Chemical Constituents And Antitumor Activities Of Saponins From The Roots Of Camellia Oleifera C.Abel

As a member of the Theaceae family, Camellia oleifera C. Abel is mainly distributed along the south area of Yangtze River, which covers a huge area of approximate 4 million hectare. The seeds are important oil material used extensively for producing a kind of cooking oil, whose oil is one of the four herbaceous vegetable oil in the world. The oil is rich in unsaturated fatty acids compared to other cooking oil and its content of vitamin E is the double of olive oil.With very high nutritional value. In China the application and medical purposes of Camellia oleifera Abel have long history according to the records. The seeds have the therapeutic effects on skin pruritus, eczema, burns and traumas as well when used externally after pounded. Antioxidant, hepatoprotective and injury-protective activities have been reported for these compounds by pharmacological investigations. Modern chemical studies have led to the isolation of saponins, flavonoids, and tannins from the seeds or seed cake. Recently there are many reports and researches about the seeds of C. oleifera Abel, the roots were rarely studied.In order to illustrate its bioactive components, we conducted a comprehensive phytochemical study on the roots of C. oleifera Abel. After Li Xia isolated 30 chemical compounds from the Et OH extract, we continue to investigate the EtOH extract of the roots of Camellia oleifera, 20 compounds have been obtained including 8 new compounds and successfully enrich known compounds.This research developed the study on Camellia triterpene saponins, provided the reference substance for investigating anti- tumor components of triterpene glycoside in the roots of C. oleifera Abel and laid the material foundation of the study on pharmacology activity about the roots of C. oleifera Abel.In terms of pharmacological activity, to compare antitumor activity of these eight new kinds of sasanquasaponin by MTT assay, YW-8 has been found to perform the best,in which IC50 that can restrain the proliferation of A549 cells has reached 6.73 ± 0.09 μM, at the same time, it presents a time and dose dependent relationship. In result we focus on the mechanism of inhibiting the proliferation of A549 cells in the second half of this project.Cells stained with giemsa showed that YW-8 can make A549 cells present the characteristics of nuclear condensation, chromatin aggregation, cell degeneration, and so on, which indicates that apoptosis may occur; And drug delivery group cells, compared with the control group, that both stained with Hoechst33342, the color becomes bright and the crescent morphology of apoptosis appears, apoptosis was initially identified; After, the rate of apoptosis in A549 cells with different concentrations of YW-8 was evaluated using the Annexin V-FITC/PI assay; The result of analyzing cell cycle by PI staining indicates that the percentage of the G2/M phase on A549 cells, and with the increase of the concentration of drug administration. Sub-G1 peak area also increases.The result of Sub-G1 peak experiment coincides with high concentration of YW-8 in the DNA Ladder experiment; for further investigating the mechanism of YW-8 inducing A549 cell apoptosis. We have carried out statistical analysis on apoptosis related proteins with western blot, such as changes of PARP, caspase-3, Bcl-2/Bax. Cleved-PARP protein which was cut from PARP protein was up-regulated in the experiment, cleved-caspase-3 protein is also the same.However, the rate of Bcl-2/Bax was down-regulated. The result shows that YW-8 acts on A549 cell, which can obviously reduce the membrane potential of cell mitochondria. Meanwhile, the result of the reactive oxygen species(ROS) indicates that A549 cell with YW-8 can significantly increase the intracellular levels of ROS, and has a dose-dependence manner. The results of these two experiments confirmed YW-8 inducing apoptosis of A549 cells is probably associated with mitochondrial and ROS pathways.Autophagy and apoptosis are commonly complementary to each other when organisms protect themselves. We have found that YW-8 can apparently induce A549 cell autophagy and have a dose-dependence manner via Monodansylcadaverine(MDC) staining; western blot was used to carry out statistical analysis on apoptosis related proteins, like Becline-1 and LC-3B. The experiment result expresses protein expression of Becline-1 and LC-3B increase with increasing YW-8 concentration., that YW-8 can induce A549 cell autophagy was proved at the protein level; during the TEM we can not only observe the characteristics of nuclear condensation, chromatin aggregation, double membrane structure of autophagy can also be seen.Finally, in order to demonstrate whether there is a relationship between autophagy, ROS and apoptosis. Before A549 cells were treated with YW-8,we give autophagy inhibitor CQ and antioxidant NAC to stimulate A549 cells two hours early. After that, the apoptosis rate of cells decreases significantly compared with that of the group without.The research results of this project enrich material base exploration of triterpenes chemical composition from the roots of Camellia oleifera Abel. It provides the study on the quality standard of the chemical constituents of saponins in the roots of Camellia oleifera Abel. with a reference. At the same time, it offers a theoretical basis to the development of the research on the anti-tumor drugs.