Optimization of the fractional precipitation of paclitaxel from a Taxus chinensis cell culture using response surface methodology and its isolation by consecutive high-speed countercurrent chromatography

摘要

A consecutive preparation method for the isolation and purification of paclitaxel from the Taxus Chinensis cell culture was developed in this study. The process involved alkaline Al_2O_3chromatography, fractional precipitation, and high-speed countercurrent chromatography. The original cell culture materials were first extracted with methanol using ultrasoundassisted extraction, and then the extract (the content of paclitaxel is 1.5%) was separated by alkaline Al_2O_3 column chromatography. Subsequently, fractional precipitation was used to obtain paclitaxel. In particular, response surface methodology was used to optimize the factors of fractional precipitation (methanol concentration, material-to-solvent ratio, and precipitating time were optimized as 48.14%, 8.85 mg/mL, and 48.71 h, respectively) and the yield of fractional precipitation product was 30.64 ± 0.60 mg (the content of paclitaxel is 89.3%, 27.37 ± 0.54 mg) from a 100 mg fraction by Al_2O_3 column separation (the content of paclitaxel is 32.4%). Then, the product was used for further isolation by high-speed countercurrent chromatography. About 1.00 g paclitaxel (200 ± 2 mg in each loading) with a purity up to 99.61% was isolated from 1.25 g of fractional precipitation product with a solvent system of n-hexane/ethyl acetate/methanol/water (1.2:1.8:1.5:1.5, v/v/v/v) in one run of five consecutive sample loadings without exchanging a new solvent system.