Researches On The Separation And Purification And Antitumor Activity Of The Alkaloids From Styela Clava

Nowadays, researches on the physiological active substances fromthe marine tunicates are in a high speed progress. Howerver, it has notbeen reported that some tunicate extracts obtained from Styela clava'stunica or body have high effective anticancer potential .And the researchwork carried on Styela clava in homeland is rarely reported. Our countryis rich in tunicates especially coastal areas in Shandong province.Comparing with the overseas, the resources greatly disturbed thecultivation and sailing, not to mention exploiture. Therefore, we shouldpay attention to them. In this paper, we made a basic research on thealkaloids from Styela clava stated below.First, Styela clava's tunica and its body were separated carefully sothat interaction among some substances was prevented. According to thefrequently used methods of extraction and isolation of natural productsfrom organic body, the freeze-dried ascidians' bodies were wet withammonia, then exhaustively extracted with chloroform. The combinedextracts solvent was concentrated under vacuum. The concentratedsubstance was acidified by HCl, then the lipids were extracted by aether.The acid solution was basified by NaOH, then exhaustively extractedagain by chloroform. Finally, the extraction was concentrated undervacuum to yield lipid total alkaloids. The tunicate residues wereextracted by 10% ammonia. The ammonia solution was acidified by HCl,then centrifuge and abandon deposition. The saturated Rain's saltsolution was added into the acid solution, then form many purpledepositions. Depositions were taken out and dissolved by acetone , thenbathed with water and formed again. The depositions were dissolved byacetone again and passed 732 positive ion exchange resin, eluted with10% ammonia. The solutions were concentrated till to dryness undervacuum. The extracts were acidified with HCl, then purified bySephadex LH-20 gel column, semipreparative HPLC, crystal and crystalagain. Finally we obtained three natural products named B-a, C-a, E-a .Second,the method of MS is used to analyze the component of B-a.Results show that B-a is an uncertain structure because of impurity.The methods of MS,IR and X-crystal diffraction are used to analyze thecomponent of C-a. Results indicate that C-a is Betaine chloride. Themethods of HPLC,wavelengh scan and fluorescence scan are used toanalyze the component of E-a because of his strong blue fluorescence.Results show that E-a is probably 1,2-bis(5-methyl-2-benzoxazol)–ethene. The methods of TLC and HPLC are used to anlalyze thecomponent of lipid total alkaloids. Results indicate that lipid totalalkaloids are complicated mixture.At last, we investigated the anticancer activity of B-a,C-a,E-a andlipid total alkaloids by culturing cancer cells such as HepG2 liver cancercell and MCF7 breast cancer cells, and anti-proliferation effects weremeasured by MTT assay .From the data of experiment,we found thatB-a,C-a and E-a were almost ineffective in HepG2 liver cancer cell andMCF7 breast cancer cells;The Lipid total alkaloids can inhibit theactivity of HepG2 liver cancer cell and MCF7 breast cancer cells at 24hand 48h and different dosage. Especially, under the concentration of80μg/mL,it showed remarkable effect at 48h.We believe that through further research, the work in this field inour country will make a rapid progress and a branded-new prospect willcome out.