Optimization protocol for the extraction of 6-gingerol and 6-shogaol from Zingiber officinale var. rubrum Theilade and improving antioxidant and anticancer activity using response surface methodology

Background: Analysis and extraction of plant matrices are important processes for the development, modernization, and quality control of herbal formulations. Response surface methodology is a collection of statistical and mathematical techniques that are used to optimize the range of variables in various experimental processes to reduce the number of experimental runs, cost, and time, compared to other methods. Methods: Response surface methodology was applied for optimizing reflux extraction conditions for achieving high 6-gingerol and 6-shogaol contents, and high antioxidant activity in Zingiber officinale var. rubrum Theilade. The two-factor central composite design was employed to determine the effects of two independent variables, namely extraction temperature (X-1:50-80 degrees C) and time (X-2:2-4 h), on the properties of the extracts. The 6-gingerol and 6-shogaol contents were measured using ultra-performance liquid chromatography. The antioxidant activity of the rhizome extracts was determined by means of the 1,1-diphenyl-2-picrylhydrazyl assay. Anticancer activity of optimized extracts against HeLa cancer cell lines was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Results: Increasing the extraction temperature and time induced significant response of the variables. The optimum extraction condition for all responses was at 76.9 degrees C for 3.4 h. Under the optimum condition, the corresponding predicted response values for 6-gingerol, 6-shogaol, and the antioxidant activity were 2.89 mg/g DW, 1.85 mg/g DW, and 84.3 %, respectively. 6-gingerol and 6-shogaol were extracted under optimized condition to check the viability of the models. The values were 2.92 and 1.88 mg/g DW, and 84.0 % for 6-gingerol, 6-shogaol, and the antioxidant activity respectively. The experimental values agreed with those predicted, thus indicating suitability of the models employed and the success of RSM in optimizing the extraction condition. With optimizing of reflux extraction anticancer activity of extracts against HeLa cancer cells enhanced about 16.8 %. The half inhibition concentration (IC50) value of optimized and unoptimized extract was found at concentration of 20.9 and 38.4 mu g/mL respectively. Optimized extract showed more distinct anticancer activities against HeLa cancer cells in a concentration of 40 mu g/mL (P < 0.01) without toxicity to normal cells. Conclusions: The results indicated that the pharmaceutical quality of ginger could be improved significantly by optimizing of extraction process using response surface methodology.