Optimized extraction process and identification of antibacterial substances from Rhubarb against aquatic pathogenic Vibrio harveyi

Response surface optimization was applied for the extraction of antibacterial substances from Rhubarb (ASR) against aquatic pathogenic Vibrio harveyi. Based on the experimental results of single factors, the optimal extraction conditions were determined by Box-Behnken design combined with response surface methodology with conditions: 100% ethanol as extraction solvent, liquid/material ratio of 29 mL/g and extraction temperature at 88 degrees C for 148 min. The factual value of inhibition zones can reach 21.31 +/- 0.95 mm and is not different from the predicted value (21.74 mm), which showed that the response surface methodology applied to the extraction optimization of antibacterial substances from Rhubarb against V. harveyi is feasible. Moreover, the yield of ASR was 30.29 +/- 2.27%. Five compounds, namely, aloe-emodin, rhein, emodin, chrysophanol and physcion, were identified in ASR by comparing the HPLC chromatogram of the reference mixtures and the sample. Contents of the five compounds were 0.68 +/- 0.02, 0.24 +/- 0.05, 0.78 +/- 0.07, 6.68 +/- 0.97 and 0.58 +/- 0.15%, respectively. The minimal inhibitory concentration (MIC) values of ASR, aloe-emodin, rhein, emodin, chrysophanol and physcion were 0.625, 0.125, 0.015, >1, > 1, and > 1 mg/mL, respectively, which indicated that aloe-emodin and rhein are the main antibacterial compounds of Rhubarb.