Extraction methods
A total of 200 g of RG, CG, and MCG were ground to powders. The ginsengs were degreased by petroleum ether for 12 h at room temperature. A concentration of 80% ethanol was used for the extracted ginsengs with a shaking speed of 180 rpm at 50 °C for 48 h. Subsequently, the extracts were liquid partitioned with hexane, chloroform, ethyl acetate, andn -butanol, respectively.
Enzyme-assisted extraction was followed by the previous protocol with slight modification (Lee et al., 2021): the ethanol extract residue was collected and decolorized by pure ethanol. The residue was further extracted by different enzymes (glycosylase, cellulase, ɑ-amylase, β-glucanase, viscozyme®L, papain, compound protease, alkaline protease, neutral protease, chymotrypsin, catalase, trypsin, and pepsin). Extraction was performed at the optimal temperature and pH and at the medicinal powder: enzyme extract ratio of 1: 10. The enzyme-to-power ratio was 1: 100, and the shaking speed was set to 180 rpm. The optimal temperature and pH of enzymes were set in accordance with a previous study (Dai et al., 2019). The optimum temperature and pH of enzyme cellulase/viscozyme® L, compound protease/neutral protease, and chymotrypsin/trypsin are the same. Cellulase+viscozyme® L, compound protease + neutral protease, and chymotrypsin + trypsin were mixed separately and assisted in the extraction of active fractions. After 24 h shaking, the aqueous extracts of ginsengs were obtained. Proteins from the above extracts were removed by Sevag reagent. The active fraction was precipitation by 80% ethanol, and the precipitate was lyophilized and regarded as a crude aqueous extract (CAE).
Step-gradient ethanol precipitation was carried out for purification of the CAE in accordance with a previously described method (Fernando et al., 2020). Briefly, the CAE aqueous solution was adjusted to the ratio of 30% ethanol. The precipitate was harvested after 24 h of equilibrium time at 4 °C. Subsequently, the ratio of ethanol was increased to 60%, 80%, and 90% for precipitation.