球等鞭金藻细胞生长抑制物的分离纯化

Purification of Growth-Inhibitor Formed by Isochrysis Galbana

以球等鞭金藻老化培养液的无细胞上清处理液为研究对象,对存在于球等鞭金藻老化培养液中的抑制细胞生长的活性物质进行了分离纯化与抑制活性研究。运用乙酸乙酯萃取,紫外光谱分析,SephadexG-15凝胶过滤,制备薄层层析和C18反相高效液相等光谱分析与纯化方法对其进行活性追踪的分离与纯化。结果表明,采用乙酸乙酯萃取球等鞭金藻老化培养液的无细胞上清处理液可以获得具有明显细胞抑制活性的细胞生长抑制物的粗提物,经200～400nm的紫外光谱扫描,确定了细胞生长抑制物的特征吸收波长为235nm。将粗提物用0.2mol.L-1NaOH溶解,经蒸馏水适当稀释后,以5%床体积的量加载于SephadexG-15凝胶层析柱并用蒸馏水进行洗脱。在紫外235nm下,粗提物经SephadexG-15凝胶过滤,获得3个具有细胞抑制活性的组分。用1mol.L-1HCl将具有细胞抑制活性的组分pH调至2～3之间,用乙酸乙酯提取。于40℃下减压浓缩,将样品点在硅胶G板上,以氯仿为展开剂,进行薄层层析分离与制备,获得4个组分。经检测,仅Rf值为0.63的组分具有明显的细胞抑制活性。此活性组分采用硅烷化键合相C18反相色谱柱,乙腈-水(体积比为63:37)流动相,0.3mL.min-1恒流洗脱,检测波长UV-235nm,获得较好的分离效果。

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 bioaetive 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 fraetionated 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.