Orthogonal test design for optimization of suitable conditions to separate C-phycocyanin from Spirulina platensis by high-speed counter-current chromatography using reverse micelle solvent system.

High-speed counter-current chromatography (HSCCC) was applied to separate C-phycocyanin (C-PC) from Spirulina platensis in the article. The suitable conditions were optimized by an orthogonal test design (L(9)(3)(3)), including the stationary phase of reverse micelle solvent system (0.10 g/mL cetyltrimethylammonium bromide [CTAB]/isooctane-hexylalcohol), mobile phase A (0.05 mol/L sodium phosphate buffer, pH 4.0, containing 0.2 mol/L KCl) and mobile phase B (0.05 mol/L sodium phosphate buffer, pH 8.0, containing 0.4 mol/L KCl). Under the selected conditions, 78.7 mg protein was purified from 200 mg crude extract of S. platensis, and the purity of the product was 4.25 based on the absorbance ratio of A(620)/A(280) , which was increased 6.85 times compared with the crude extract. Then, the protein was identified to be C-PC by MALDI-TOF/TOF-MS and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis compared with the standard. The application of HSCCC used in the separation of C-PC from S. platensis was first reported in the article. Furthermore, three kinds of tumor cell lines including human hepatoma cell line SMMC-7721, human ovarian carcinoma cell line ES-2, and human lung adenocarcinoma cell line SPCA-1 were used to evaluate the anticancer activities of the separated product, and the results showed that the separated C-PC had excellent anti-tumor actions with the IC(50) values at 2.998, 4.854, and 8.423 μg/mL, respectively, for 48 h treatment. The outcome indicates that an effective method for C-PC purification by HSCCC has been established.