Separation Active Constituents From Chlorella Vulgarisby Counter-Current Chromatography

Chlorella vulgaris, a unicellular green alga grown in fresh water, contains redundant nutrient substance and microelement. Numerous clinical studies have shown that chlorella ingestion can benefit for health, due to its protective role on bacteria, antiproliferative effects on cancer cells, and anti-inflammatory and antioxidant activity. Counter-current chromatography (CCC) is a support-free liquid-liquid partition chromatography, it has numerous advantages of large sample load, moderate operation condition, high sample recovery and low solvent consuming. Therefore, CCC has exhibited high separation efficiency and great potential for separation of active substances. In this work, the new approachs coupling with CCC for preparative isolation and purification of less polar carotenoid and phytosterol from C. vulgaris were presented. The major results are shown as following: 1. The less polar crude of C. vulgaris was gained using ultrasonic extraction with n-hexane-ethanol (1:1, v/v) two phase solvent. Saponification was carried out with eliminating chlorophylls and unwanted lipids. A UPLC method was developed for analysis and determination of low-polarity chemical constituents from C. vulgaris, such as carotenoid and phytosterol.2. The CCC method was developed for preparative isolation and purification of lutein from C. vulgaris. With the two phase solvent system n-hexane-ethanol-water (4:3:1, v/v),30.7mg lutein with purity of94.1%was gained from100mg saponated crude of C. vulgaris in one step. In contrast, semi-preparation HPLC allowed purification and isolation of lutein with significantly higher solvent consumption and lower productivity. The results indicate that CCC is a more economic and scientific separation method and is suitable for isolating active substance from food.3. The green, environmental and natural limonene-ethanol-water solvent system was proposed and applied. The green and environmental CCC approach for preparative isolation and purification of lutein from C. vulgaris was presented. Comparing properties of limonene with n-hexane and ternary volume phase diagrams of limonene-ethanol-water with n-hexane-ethanol-water, limonene which could become a green alkane substitute could be used in CCC. With the two-phase solvent system limonene-ethanol-water (10:9:1, v/v),20.4mg lutein with purity of94.6%was obtained from300mg crude of C. vu/garis in one step. The results indicate that the CCC is a green and environmental separation method.4. The recycling CCC method was developed for preparative isolation and purification of phytosterol from C. vulgaris. Using conventional CCC with two-phase solvent system n-hexane-dichloromethane-acetonitrile (10:3:7, v/v),40mg phytosterol mixture was gained from300mg saponated crude of C. vulgaris. Using recycling CCC coupled with two-phase solvent system n-hexane-dichloromethane-acetonitrile (10:3:7, v/v),24.3mg (22E)-Ergosta-5,7,22-trien-3beta-ol and13.7mg26-Nor-25-isopropyl-5,7,22-trien-3β-ol were completely separated from40mg of phytosterol mixture after five cycles, with purity of97.2%and96.4%, respectively. The results indicate that recycling CCC could dramatically improve resolution and is suitable to separate and purify analogue phytosterol from C. vulgaris.