黄连解毒汤有效成分的提取及其对体内白色念珠菌的清除作用

Extraction of Effective Fraction of Huanglian Jiedu Decoction and Its Scavenging Effect on Candida albicans in vivo

试验旨在研究黄连解毒汤有效成分的提取及对其体内白色念珠菌的清除作用。分别采用水煎煮和乙醇回流提取法,随后乙醇提取物用3种不同极性的有机溶剂萃取(正丁醇、乙酸乙酯、石油醚),以最小抑菌浓度为指标优化出最佳抑菌效果的提取工艺;观察给药前后系统性感染白色念珠菌小鼠肾脏、肝脏的真菌负荷量及活性氧(ROS)含量水平的变化,并检测血清中细胞因子白细胞介素-6(IL-6)、IL-1β、白细胞介素-1β前体(pro-IL-1β)及肿瘤坏死因子-α(TNF-α)的含量。结果显示,黄连解毒汤抑菌效果最优的提取工艺为:料液比1∶30,乙醇浓度60%,温度80℃,提取时间1 h,提取次数2次,测得乙酸乙酯萃取物MIC 90为0.3125 mg/mL。与模型组相比,黄连解毒汤乙酸乙酯提取物(EAHD)高剂量组能显著降低小鼠肾脏真菌负荷量(P<0.05),完全治愈率达40%,并明显高于氟康唑组;EAHD低、中和高剂量组(50、100及200 mg/(kg·d))均能显著降低肝脏真菌负荷量(P<0.05),其中中剂量组和氟康唑组降低肝脏真菌负荷量效果接近。同时EAHD高剂量组能显著或极显著降低感染后小鼠肾脏和肝脏ROS含量(P<0.05;P<0.01);显著或极显著提高血清中IL-6、IL-1β和TNF-α的含量(P<0.05;P<0.01),同时降低pro-IL-1β含量。综上所述,通过优化后的提取工艺得到的EAHD具有降低模型小鼠肾脏和肝脏ROS含量和调控炎症因子的作用,进而降低炎症反应,起到保护组织和抵抗白色念珠菌的作用。

The aim of the experiment was to study the extraction of effective fractions of Huanglian Jiedu decoction and its scavenging effect on Candida albicans in vivo.The fractions of Huanglian Jiedu decoction were extracted by water and ethanol reflux,and then the ethanol extract was extracted with three different polar organic solvents(n-butanol,ethyl acetate,petroleum ether).The extraction process was optimized with the minimum inhibitory concentration(MIC)as the index.The changes of fungal load in kidney and liver,and reactive oxygen species(ROS)levels in a mouse model with systemic Candida albicans infection(SC)before or after taking medicine were observed.The levels of cytokines IL-6,IL-1β,pro-IL-1βand TNF-αin serum were measured by ELISA.The results showed that the optimal extraction process of Huanglian Jiedu decoction was the following:The material-liquid ratio was 1∶30,the ethanol concentration was 60%,the temperature was 80℃,the extraction time and frequencey were 1 h and 2 times.Under this condition,the MIC 90 of EAHD was 0.3125 mg/mL.Compared with the model group,Huanglian Jiedu decoction ethyl acetate extract(EAHD)high dose group significantly reduced the kidney fungal load of mice(P<0.05),and the complete cure rate was 40%which was obvious higher than that of fluconazole group.The fungal load of liver was significantly decreased in EAHD low,medium and high dose groups(50,100 and 200 mg/(kg·d))(P<0.05),and the effect of EAHD medium dose group and fluconazole group on reducing the hepatic fungal load was similar.The ROS content in kidney and liver of EAHD high dose group were significantly or extremely significantly reduced,respectively(P<0.05;P<0.01),and the levels of IL-6,IL-1βand TNF-αin the serum were significantly or extremely significantly increased,respectively(P<0.05;P<0.01).And the pro-IL-1βcontent in the serum was reduced.In conclusion,the EAHD obtained by the optimal extraction process could reduce ROS content and regulate inflammatory factors in kidney and liver of model mice,thereby alleviate inflammatory reaction to protect tissues and resist Candida albicans.