Study On Sepratation Of Bioactive Substances From Marine Microalgae By High-speed Countercurrent Chromatography

Marine microalgae usually contain some bioactive substances with specific structurefor its special growing environment, and have some characteristics such as fast growth,high output, directional training and easy to be controled. So marine microalgae havebecome new potential sources of natural medicine.As a liquid-liquid partition chromatography, High-speed counter currentchromatography （HSCCC） has some excellent characteristics such as simple operation,large separation quantity, avoiding sample adsorption and so on. In this study, thistechnology was applied to separate and purify the active substances from marinemicroalgae. And the experiment purpose was to establish an efficient and rapid extractionmethod of microalgae antimicrobial active substances, to explore a new solution ofseparation and extraction of the active substances from marine microalgae.Two kinds of marine Chrysophyta were used as the experimental materials. Basis onselection of solvent systems, the separation and extraction methods of active substancesfrom two microalgaes by HSCCC were studied. The structure of the separated compoundswas identified.The antibacterial experiments were made in order to observe theantibacterial activity of the compounds.（1） In the experiment of using HSCCC to extract and separate active substance fromcrude extractings of microalgae Ruttnera spectabilis, the method of referencing solventsystems was used to determine the solvent systems. According to the solvent for thesolubility of substance, the time of layering, the stationary phase retention for the selectionof the solvent system, five kinds of relatively ideal three-phase solvent systems wereselected. The five kinds systems were used to separated the crude extract of themicroalgae Ruttnera spectabilis by HSCCC. According to the separation effects, thethree-phase solvent system n-hexane-acetonitrile-methylacetate-water （4:4:3:4, v:v:v:v）was determined as the optimal separation system. The upper phase and middle phase wasstationary phase and lower phase was mobile phase. Three components were obtained named as RS1, RS2,RS3, respectively. Three groups of substanceswere detected by HPLC, and the RS1was a polar single compound. The substance wasidentified as12-hydroxy-（cis）-9-octadecenoic acid by GC-MS, its purity was87%. Theantibacterial results showed that the RS1had a good antibacterial effect on Gram-positivebacteria, Gram-negative bacteria and fungi. RS2had good effects on bacteria antibacterial;but had no inhibition on fungus. RS3had no inhibitory activity.（2） The methods of reference solvent systems and determination of partitioncoefficient by HLPC was used to select the solvent system. The two-phase solvent systemof hexane-ethylacetate-methanol-water （5:2:5:2, v:v:v:v） was determined for best solventsystem. The upper phase was stationary phase and lower phase was mobile phase, and theseparation methods of crude extractions for microalgae Sarcinochrysis marina Geitler wasstudied. The content of formic acid in the solvent system, sample injection volume andflow rate of mobile phase were optimizated in turn. The optimal separation conditionswere as follows: the solvent system was n-hexane-ethylacetat-methanol-water（5:2:5:2, v:v:v:v） with1%formic acid; the weight of the sample was75mg, the flow rateof mobile phase was2mL/min, the detection wave-length was280nm, andthe instrument speed was900r/min. Three components of SG₁, SG₂, SG₃were obtained.After the purifity detection by HPLC, the SG₃was myristic acid with the purity of92%.The anti-bacterial results showed that the SG₁and SG₂had good anti-bacterial activitieson all experimental strains, and SG₃was not obvious effects on E. coli, but it had stronganti-bacterial activity on other bacterias.（3） Further separation for SG₁: the method of determining partition coefficient wasused. the solvent system of n-hexane-ethylacetate-methanol-water （4:5:4:3.5, v:v:v:v） wasdetermined as optimum solent system. Subsequently the quatity of injected sample wasoptimized, and the optimal separation conditions were as follows: the solvent systemwas n-hexane-ethylacetat-methanol-water （4:5:4:3.5, v: v: v: v） with1%formic acid; thequatity of injected sample was50mg, the flow rate of mobile phase was2mL/min, thedetection wavelength was280nm, and the instrument speed was900r/min. Fraction Ⅲand Ⅳ were obtained. The results of the structure identification showed that the twocomponents were cis-15-teracosenic acid and （Z）-9-Octadecenoic acid and the their purity were94%and97%, respectively. The results of anti-bacterial experiment showed that twocompounds had antibacterial activities on the bacteria and fungi, and their anti-bacterialactivity were stronger than the anti-fungal activity. Anti–bacterial activity of fraction IIIwas weaker than fraction IV, but its anti-fungal activity was stronger than that of thefraction Ⅳ. The antibacterial activities of two compounds on Gram-positive bacteria weregreater than the antibacterial activity against gram-negative bacteria.