| At present, foreign research on physiological active substances from the marine tunicates has been in a high speed progress. Many of active substances which were isolated from marine tunicates have been under clinical studies. However, domestic research on ascidians, especially the separation and activity study of the natural products is still at an early stage, both breadth and depth of studies should be enhanced. Our country is rich in ascidians resources, especially in Shandong province. Compared with other countries, ours ascidians resources not only has not been effectively exploited, but has troubled fisheries and maritime development. Therefore, this issue should be great concerned. In this paper, we made a preliminary study on the physiological active substances from Styela clava, with the alkaloids as the main objective, in following aspects stated below.First, the fresh animal material Styela clava was deep frozen, then the tunica and the body were separated carefully so that interaction among some substances was prevented. The body tissues were grinded, and the freeze-dried ascidians'bodies were fully wet with 10% ammonia, then exhaustively extracted with chloroform. The extracts solvent was concentrated under vacuum. The concentrated substance was acidified by HCl, then the fat-soluble impurities were removed by aether. The acid solution was basified by NaOH, and then exhaustively re-extracted respectively by aether and chloroform. Finally, the extractions were concentrated under vacuum, respectively named as alkalization ether phase (JY) and alkalization of chloroform (JL).The residues continued to extract with ethyl acetate, and then concentrated to red brown paste. Through the semi-preparative HPLC, we gained the purified sample YSYZ-P. The residue thirdly extracted by methanol, and the concentrations were reddish brown dopes. Through hydrochloride acid treatment and crystallization, we received colorless crystal JC-C.Secondly, we analyzed the structure of the sample. The sample YSYZ-P is the pale yellow liquid at room temperature, dried sample takes on a solidified gum and freeze-dried sample takes on small droplet. HPLC analysis showed that it is a pure sample. According to the UV spectrum, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, we preliminarily inferred it is the thymidine after contrasting compounds database. Samples JC-C is a colorless transparent crystal, a cuboid-shaped, structured and rigid. The X-ray powder diffraction analysis indicated it is an unknown compound. It was unable to identify its exact structure, but we initially estimated it is a alkaloid chloride.At last, we investigated the anti-tumor activity of Styela clava extracts by using cell culture technology and anti-proliferation effects were measured by MTT assay. From the data of experiment, we found that the vitro anti-tumor activities of YSYZ-P and JC-C was instable, no regular concentration and time dependence. The maximum inhibition rate of YSYZ-P was 49.42% (HepG2, 200μg/ml, 24h), and the maximum inhibition rate of JC-C was 48.97%( HepG2, 200μg/ml, 72h).According to the current available information, we purified the thymidine from Styela clava for the first time in this experiment, so this research is a certain innovation in the field of domestic work. Basing on this paper, further extensive screening, chemical and biological activities study should be developed, in order to find the novel bioactive substances to the development of a new drug or drug design as a precursor compounds. It can also provide valuable tool medicine for the basis life science research. We believe that further experimental studies can push forward the domestic work in the field of natural products research and development, and start the new situation of ascidians physiology active material studies. |
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