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.