Anti-inflammatory Effects of Proteolytic Peptides from Different Ginseng Concoctions on LPS-induced RAW264.7 Cells

The purpose of this study was to conduct a screening and comparative analysis of proteolytic peptides with anti-inflammatory properties derived from three different ginseng concoctions: Sundried ginseng, red ginseng and black ginseng. Ginseng proteins were extracted from three different types of ginseng products using a low-temperature leaching method. Subsequently, the extracted proteins underwent enzymatic digestion using alkaline protease, neutral protease, and pepsin through a stepwise enzyme digestion method. This process yielded three distinct enzyme digestion products, namely BGP (black ginseng proteolytic peptide), RGP (red ginseng proteolytic peptide), and SGP (sundried ginseng proteolytic peptide). The samples were subjected to separation using ultrafiltration membranes, resulting in the acquisition of ultrafiltration fractions with distinct molecular weights. Subsequently, the ultrafiltration fractions were further separated utilizing ultrafiltration membranes to obtain fractions with varying molecular weights. The fraction exhibiting the most potent anti-inflammatory activity was determined through the application of a lipopolysaccharide (LPS)-induced RAW264.7 inflammation model. The impact of the active fractions on the secretion of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β ((IL-1β), and interleukin-6 (IL-6) by RAW264.7 cells was assessed using enzyme immunoassay. The amino acid composition and content of the three proteolytic peptides were examined. Multivariate statistical analysis was employed to identify the distinct amino acids in the three ginseng concoctions and investigate their correlation with the inhibition of cytokine secretion by RAW264.7 cells. The findings of the study indicated that the proteolytic peptide fraction with a molecular weight of less than 1 kDa in the three ginseng products exhibited the most pronounced impact on the proliferation of RAW264.7 cells compared to the other fractions. Additionally, this fraction significantly suppressed the secretion of NO, TNF-α, IL-6, and IL-1β at concentrations ranging from 50~200 μg/mL (P<0.05). Notably, at a concentration of 200 μg/mL, the three groups receiving proteolytic peptide administration demonstrated the most potent inhibitory effect on cytokine release. Furthermore, the inhibitory effect of BGP-4 on cytokine release surpassed that of RGP-4 and SGP-4, exhibiting a statistically significant difference (P<0.05). All three peptides consisted of 17 amino acids, however, their compositions exhibited significant variations. Notably, phenylalanine exhibited the highest content, and the differential amino acids present in the three ginseng concoctions were closely associated with the inhibition of inflammatory factor secretion. This study represents an initial exploration into the impact of concoctions on the anti-inflammatory properties of ginseng, identifying distinct amino acids among different concoctions. These findings offer a valuable reference for the formulation of ginseng concoctions.