Multistep Separation and Purification of Huangjiu Peptides and its Effects on the Immune Regulation of Macrophage in Mice

In this study, the active peptide components in huangjiu were purified by macroporous resin adsorption, gel filtration chromatography and other methods. The effects of different peptide components on immune regulation of mouse macrophages were investigated.In vitro cell validation model was created by stimulating RAW264.7 cells with lipopolysaccharide (1 μg/mL), different concentrations of purified huangjiu peptide samples were used as intervention to determine their immune regulatory activity, the structure of the peptides in huangjiu with the highest inhibition rate of NO release was analyzed. The results showed that the active peptides could be separated and purified by macroporous resin. The adsorption and desorption capabilities of three different types of macroporous resin were compared, and DA201-C macroporous resin was finally selected to enrich the huangjiu peptide, and the eluent was collected to obtain the huangjiu peptide SJ after rotary evaporation and freeze-drying. The RAW264.7 cells were treated with different concentration of huangjiu peptides, cell viability was determined with MTT methods. The safe range of huangjiu peptide SJ was less than 2.5 mg/mL.The Griess Reagent method was used for the detection of nitric oxide (NO) content in cell culture solution. Compared with no intervention group (LPS model group), the huangjiu peptides SJ in the concentration of 0.3, 0.6, 1.2, 2.5 mg/mL could significantly inhibit the release of NO (the inhibition rates were 20.85%, 36.42%, 68.58% and 95.01%, respectively).The huangjiu peptide SJ were further separated by gel filtration on Sephadex G-15 into 3 peaks, namely G₁, G₂ and G₃.Elution peaks of these three groups were collected.The study found that immune regulatory activity of G₁ component was higher than the other two components, and the semi-inhibited concentration of NO release of G₁, G₂ and G₃ were 0.68, 0.86 and 0.75 mg/mL, respectively. The amino acid composition analysis of G₁ component showed that the proportion of hydrophobic amino acid was 32.63% and the proportion of essential amino acid was 28.46%. The results showed that huangjiu peptide could significantly inhibit LPS-induced RAW264.7 secretion of NO, and had a protective effect on LPS-stimulated RAW264.7 cell inflammation, thus promoting macrophages to play an immune role.