| Apostichopus joponicus shlenka belonging to the Echinodermata is rich in glycosides with great biological and pharmaceutical effects. In recent years, numerous studies have shown that glycosides have biological and pharmacological activities in antifungal, cytotoxic, hemolytic, immunomodulatory and inhibition of Na+-K+-ATPase activities. At present, glycosides are normaly extracted by ethanol or methanol, which need large amount of organic solvent, can cause environment pollution and cost a lot, so it is not applicable for the industrial production. And also, the holothurian glycosides obtained by ethanol or methanol have lower water-soluble activity which limits its approach to clinic use. Unfortunately, at present there is no report about extraction and purification research of water-soluble holothurian glycosides and its biological activities. In this study, water-soluble holothurian glycosides from Apostichopus joponicus have been isolated and its biological activities have been studied. Water-soluble holothurian glycosides were isolated from the water-extracts of Apostichopus japonicus by using macroporous resin column chromatography, and its antimicrobial, antitumor, antioxidant and dispel-drosophila activities were examined. The extraction was carried out from the leftover solution of freeze-dry manufacture of Apostichopus japonicus and orthogonal experiments were performed in multifactors and at multilevels to obtain as numerous water-soluble holothurian glycosides as possible. The optimal extraction process was established and the constitiuents obtained from the leftover solution were investigated for the antifungal and antitumor activity. Those high bioactive constitiuents were further purified by various chromatography methods including macroporous resin, silica gel and Sephadex LH-20 respectively. At last, the antitumor effecst of purified water-soluble holothurian glycosides in vitro and in vivo were studied by pharmacological experiments.The results showed that the method of water-extraction and macroporous resin column chromatography were good ways in preparing water-soluble holothurian glycosides. Using these methods, massive production of water-soluble holothurian glycosides was obtained. The key factors in preparation technics of water-soluble holothurian glycosides were optimized as follows, the pH value of the waste holothurian solution was at 10, the loading velocity was twice that of the column volume per hour, the concentration of eluting ethanol was 70 %, and the eluting volume was 6 times that of the column volume.The results of antimicrobial activity research showed that water-soluble holothurian glycosides exerted high antifungal activity against Schizosaccharomyces pombe, Saccharomyes cerevisiae, Candida albicans, Colletotrichum gloeosporioides, Fusarium oxysporum f.sp.cucumerinum and Aspergillus niger. The minimum inhibitory concentration (MIC) was at 0.002,0.016,0.063,0.125,0.125,0.250 mg/mL respectively, but no antibacterial activity against Gram-positive bacterium Bacillus subtilis, Staphylococcus aureus and Gram-negative bacterium Escherichia coli, Pseudomonas aeruginosa. The results of antitumor activity research showed that water-soluble holothurian glycosides exerted obvious antitumor activity against HeLa, A-549, SGC-7901 and Bel-7402, the IC50 was at 16.39,13.98,14.51,15.29μg/mL respectively.The results of antioxidant activity research showed that water-soluble holothurian glycosides had no obvious antioxidant effects, so it could not be treated as antioxidant substance. In this study, it was found that the water-soluble holothurian glycosides extracted from Apostichopus joponicus had dispel-drosophila activities, which was the first time to announce these phenomena, and this effect could not sustain too long. After 24 h, the glycosides lost its dispel-drosophila activity.By using macroporous resin, silica gel and Sephadex LH-20 chromatography, four compounds were purified from water-soluble holothurian glycosides. Among these compounds, the purification rate of SC-2, SC-3 and SC-4, was at 99.6%, 99.8% and 96.2%, respectively tested by HPLC. It was shown that SC-2 and SC-3 had the same MIC value against S. pombe, S. cerevisiae, C. albicans, C. gloeosporioides, F. oxysporum and A. niger, which were at 0.25,4,8,﹤32,32,﹤32μg/mL, respectively. The MTT results showed that the IC50 of SC-2 against HeLa, A-549, SGC-7901 and Bel-7402 was at 3.23,4.13,4.41,3.04μg/mL and the SC-3 at 3.49,4.11,4.26,3.15μg/mL respectively.The results of antitumor experiments in vivo showed that SC-2 could evidently inhibit the neoplasia and effectively enhance the thymus exponent of mouse, at doses 10, 5, 2 mg/kg B.W., and the inhibition rate was 47.84%,38.93% and 33.88% respectively. When the dose was at 10 mg/kg B.W., it could obviously improve the spleen exponent of the tested mouse.The result of antitumor experiments in vitro showed that SC-2 could induce apoptosis of cultured HeLa cells. After being treated with 2μg/mL of SC-2 for about 12 h, 10 percent HeLa cells apoptosised, and 80 percent apoptosised after being treated with 10μg/mL of SC-2 for about 12h. When the concentration was at 50μg/mL, it caused nearly 100 percent of HeLa cells apotosis tested for 12 h. The results of DNA electrophoresis showed that the HeLa cells underwent much remarkable apoptosis after being treated with 10 and 50μg/mL SC-2 for 12h, and obvious DNA ladders were found in electrophoresis.In summary, through this study we established the extraction method of water-soluble holothurian glycosides for the first time and purified two kinds of water-soluble glycosides which showed high antitumor and antifungal activities. It not only lay important foundations for finding new antitumor and antifungal drugs, but also have theoretical and practical significance to the study of other echinoderm water-soluble glycosides.
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