茯苓皮总三萜大孔树脂纯化工艺及体外抗炎活性研究

Macroporous Resin Purification Process and in Vitro Anti-inflammatory Activity of Total Triterpenoids from Cortex Poriae Cocos

目的 为提高茯苓皮的综合利用价值,建立一种简便的茯苓皮总三萜纯化方法,并通过体外实验验证其抗炎作用。方法 采用HPD-100型大孔树脂吸附法纯化茯苓皮总三萜。基于单因素实验,以总三萜含量为指标,经正交试验优化茯苓皮总三萜纯化工艺。建立脂多糖诱导的RAW 264.7细胞炎症模型,考察茯苓皮总三萜对细胞活力的影响,检测给药后RAW 264.7细胞内一氧化氮(nitric oxide, NO)释放量和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)水平。结果 纯化后样品总三萜含量增加至70.36%,明显高于粗提总三萜含量(29.76%),纯化工艺为上柱HPD-100型树脂20 g,上样浓度15 mg/mL,35%乙醇洗脱杂质容积为5倍床体积(bed volume, BV),95%乙醇洗脱三萜容积为4 BV,经实验验证该纯化工艺重复性良好。体外实验结果表明:茯苓皮总三萜在50~250μg/mL范围内对细胞活力无明显影响;与模型组比较,给药后RAW 264.7细胞内NO释放量和TNF-α、IL-1β、IL-6水平明显降低(P<0.05)。结论 HPD-100型大孔树脂吸附法纯化工艺能明显提高样品总三萜含量,茯苓皮总三萜具有减轻炎症的作用。

Objective To establish a simple and convenient method for the purification of total triterpenoids from Cortex Poriae Cocos with the purpose of improving the comprehensive utilization value of Cortex Poriae Cocos,and to investigate its anti-inflammatory effect by in vitro experiments.Methods The method of HPD-100 macroporous resin absorption was used for the purification of total triterpenoids from Cortex Poriae Cocos.Based on the single-factor experiment and the content of total triterpenoids as the index,the orthogonal test was used to optimize the purification process of total triterpenoids from Cortex Poriae Cocos.RAW 264.7 cells were used to establish a model of lipopolysaccharide-induced inflammation, and the effect of total triterpenoids from Cortex Poriae Cocos on cell viability was analyzed.After administration, the RAW 264.7 cells were measured in terms of the release of nitric oxide (NO) and the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β),and interleukin-6 (IL-6). Results After purification,the content of total triterpenoids in sample increased to 70.36%, which was significantly higher than that of the crude extract of total triterpenoids (29.76%).The purification process was 20 g of HPD-100 resin on the upper column,a loading concentration of 15 mg/mL, 5 bed volumes (BVs) of 35% ethanol-eluted impurity volume,and 4 BVs of 95% ethanol-eluted triterpenoid volume,and the experiment showed that this purification process had good repeatability.The in vitro experiment showed that total triterpenoids from Cortex Poriae Cocos had no significant effect on cell viability within the range of 50-250 μg/mL,and compared with the model group,there were significant reductions in the release of NO and the levels of TNF-α,IL-1β,and IL-6 in RAW 264.7 cells after administration (P<0.05). Conclusion The purification process of HPD-100 macroporous resin adsorption can significantly increase the content of total triterpenoids in the sample,and the total triterpenoids from Cortex Poriae Cocos can alleviate inflammation.