人参不同炮制品蛋白酶解肽对LPS诱导RAW264.7细胞的抗炎作用研究

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

研究从人参炮制品生晒参、红参、黑参中筛选出具有抗炎作用的蛋白酶解肽并进行对比分析。采用低温浸提法从三种炮制品中提取人参不同炮制品蛋白,采用分步酶解法,利用碱性蛋白酶、中性蛋白酶和胃蛋白酶对三种人参蛋白进行酶解,得到BGP(黑参蛋白酶解肽)、RGP(红参蛋白酶解肽)、SGP(生晒参蛋白酶解肽)三种酶解产物;使用超滤膜进行分离,分别得到不同分子量的超滤组分。通过建立脂多糖(LPS)诱导的RAW264.7炎症模型确定了具有最强抗炎活性的组分。酶联免疫法测定活性组分对RAW264.7细胞分泌一氧化氮(NO)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)的影响。分析三种蛋白酶解肽的氨基酸组成及含量。采用多元统计分析筛选出人参三种炮制品的差异性氨基酸并探究其与抑制RAW264.7细胞分泌细胞因子之间的关系。结果表明,人参三种炮制品蛋白酶解肽小于1 kDa组分较其他组分相比,对RAW264.7细胞增殖的作用最强且在质量浓度为50~200μg/mL时可显著抑制NO、TNF-α、IL-6、IL-1β的分泌(P<0.05)。质量浓度为200μg/mL时,三种蛋白酶解肽给药组对细胞因子释放的抑制作用最强,且BGP-4对细胞因子释放的抑制作用高于RGP-4和SGP-4,具有显著差异(P<0.05)。三种蛋白酶解肽均含有17种氨基酸,但含量存在明显差异。其中,苯丙氨酸含量最高且为三种人参炮制品差异氨基酸与抑制炎症因子的分泌密切相关。本研究初步探讨了炮制对人参抗炎活性的影响,并筛选出不同炮制品的差异性氨基酸,为人参炮制品的加工提供了参考依据。

The purpose of this study was to conduct a screening and comparative analysis of proteolytic peptides with antiinflammatory 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 antiinflammatory 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 50~200μg/mL(P<0.05).Notably,at a concentration of 200μg/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.