Purification of Growth-Inhibitor Formed by Isochrysis Galbana
SUN Ying-ying1,WANG Chang-hai1,2*,CHEN Jing2
1. Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, China 2. School of Ocean, Yantai University, Yantai 264005, China
Abstract：The objective of the present study is to isolate and purify the anti-cell bioactive fraction-cell growth-inhibitor from the old cultured liquid of Isochrysis galbana. By means of extraction with ethyl acetate, ultraviolet scan, Sephadex G-15 column chromatography, preparative thin-layer chromatography and high-performance liquid chromatography, the bioactive component was purified. The growth-inhibitor, a kind of crude ethyl acetate extract isolated from the cell-free supernatant of the old culture liquid of Isochrysis galbana, obviously inhibited the cell growth. It was showed that the growth-inhibitor existing in the old cultured liquid of Isochrysis galbana was efficiently extracted with ethyl acetate. The characteristic absorption of the growth-inhibitor was determined by ultraviolet scan and the wavelength of characteristic absorption for the growth-inhibitor was 235 nm. The residue of crude ethyl acetate extract was dissolved in diluted aqueous NaOH, and then was loaded onto Sephadex G-15 column chromatography and eluted with distilled water. Five-mL fractions were collected and monitored by UV absorption at 235 nm. The crude ethyl acetate extract was fractionated by gel filtration on Sephadex G-15 to obtain three fractions. And the three fractions inhibited the growth of Isochrysis galbana. Those active fractions were adjusted to the pH value 2-3, and extracted with ethyl acetate. And then the extracts were concentrated to the befitting volume under reduced pressure at 40 ℃, and were separated by preparative thin-layer chromatography on silica gel with chloroform. Bands visualized under UV-light (254 and 365 nm) were scraped off and extracted with ethyl acetate, and monitored as well as inhibitory activity against Isochrysis galbana. The extracts were separated into four bands on a preparative thin-layer chromatography plate with chloroform, but only the band at Rf 0.63 was active. This active fraction was finally purified by high-performance liquid chromatography on ODS with aqueous acetonitrile to produce 4 prominent peaks. The selected optimum chromatographic conditions were ZORBAX Eclipse XDB C18 with aqueous acetonitrile at a flow rate of 0.3 mL·min-1,and UV detection at 235 nm. This paper would afford very well condition for further isolation and preparation by HPLC and identification of growth-inhibitor formed by Isochrysis galbana.
[1] Fogg G E. Mar. Biol. Ann. Rev., 1966, 4：195. [2] Hellebust J. A. Limnol. Oceanogr., 1965, 10：192. [3] Robertson P. Amer. J. Bot., 1942,29：142. [4] Liu J G, Zhang C W, Cohen Z, et al. Chinese J. Ocean. Limnol.,2001, 19：21. [5] SUN Yan-ni, YIN Ming-yan, LIU Jian-guo(孙艳妮, 殷明炎, 刘建国). Transactions of Oceanology and Limnology(海洋湖沼通报),2001, (3)：22. [6] Imada N, Kobayashi K, Tahara K, et al. Nippon Suisan Gakkaishi., 1991, 57(12)：2285. [7] WANG Chang-hai, SUN Ying-ying(王长海, 孙颖颖). Marine Science Bulletin(海洋通报),2006,25(3)：29. [8] Proctor V W. Ecolgogy., 1957, 38：457. [9] Legrand C, Rengefors K, Fistarol G O, et al. Phycologia，2003，42(4)：406. [10] Fistarol G O, Legrand C, Selander E, et al. Aquat. Microbial. Ecol.,2004, 35：45. [11] Gross E M. Crit. Rev. Plant. Sci.,2003,22：313. [12] Phatarpekar P V, Sreepada R A, Pednekar C, et al. J. Aquaculture,2000, 181：141. [13] Imada N, Kobayashi K, Isomura K, et al. Nippon Suisan Gakkaishi., 1992, 58(9)：1687. [14] XIA Bing-le, LI Min-li, LIU Qing-liang, et al(夏炳乐, 李敏莉, 刘清亮, 等). Spectroscopy and Spectral Analysis(光谱学与光谱分析)，2004，24(7): 830. [15] LI Yun-xia, SUO Quan-ling, HE Wen-zhi, et al(李云霞, 索全伶, 贺文智, 等). Spectroscopy and Spectral Analysis(光谱学与光谱分析)，2007,27(1)：131. [16] McCrracken M D, Middaugh R D, Middaugh R S. Hydrobiologia., 1980, 70：271. [17] Ikawa M, Hartshorne T, Caron L A, et al. J. Am. Oil. Chemists’ Soc., 1984, 61：1877. [18] Ikawa M, Saner J J, Haney J F. J. Chem. Ecol., 1994,20：2429. [19] Ikawa M, Sasner J J, Haney J. F. Hydrobiologia., 1997, 356：143. [20] Yamada Y, Murakami N N, Morimoto T, et al. Chem. Pharm. Bull., 1993, 41：1863. [21] Craigie J S. Can. J. Bot., 1964, 42：23. [22] Harris D O. Arch. Protistenk., 1971, 113：230.
