桦褐孔菌乙醇粗提物对朊病毒复制的抑制作用

Inhibition on Prion Replication by Ethanol Crude Extracts from Inonotus obliquus

摘要:

优化桦褐孔菌乙醇粗提物提取工艺并初步探究其对朊病毒复制的影响。采用醇提方法，利用正交试验方法，确定最佳提取工艺。使用CCK-8试剂盒对桦褐孔菌乙醇粗提物处理细胞的安全性进行评价，确定其安全质量浓度；根据安全性评价结果，使用蛋白免疫印记（Western blot）方法检测桦褐孔菌乙醇粗提物对朊病毒复制的影响；通过测定过氧化氢及抗氧化因子等对桦褐孔菌乙醇粗提物的抗氧化活性进行研究。桦褐孔菌乙醇粗提物的最佳提取工艺为：料液比（桦褐孔菌生粉:乙醇）1:10（g/mL），乙醇体积分数75%，提取时间2 h，提取温度80 ℃，在此条件下，得率为6.32%。桦褐孔菌乙醇粗提物的最大安全质量浓度为6.25×10-4 mg/mL；在此浓度下，处理细胞24 h后，桦褐孔菌乙醇粗提物对朊病毒的复制有抑制作用（p<0.01），可减少H2O2的含量，增加SOD和抗氧化因子GCLM、GCLC的含量，且具有统计学意义。本试验表明桦褐孔菌乙醇粗提物对朊病毒的复制具有抑制作用，且这种作用可能与其抗氧化活性有关。

Abstract:

To optimize the extraction process of the ethanol crude extracts from Inonotus obliquus and preliminarily study its effect on prion replication. The optimum extraction condition of ethanol crude extracts from Inonotus obliquus were employed by alcohol extraction and orthogonal test. To evaluate the cell safety of the ethanol crude extracts from Inonotus obliquus, CCK-8 kit was used to determine the safe concentration. According to the results of safety evaluation, Western blot was used to detect the effect of the ethanol crude extracts from Inonotus obliquus on prion replication. The antioxidant activity of ethanol crude extracts from Inonotus obliquus was studied by measuring hydrogen peroxide and antioxidant factors. The optimal extraction conditions were as follow: the ratio of solid to liquid was (Inonotus obliquus power: alcohol) 1:10 (g/mL), the volume fraction of ethanol was 75%, the extraction time was 2 h, the extraction temperature was 80 ℃. At this condition, the yield of ethanol crude extracts from Inonotus obliquus was 6.32%. The maximum safe concentration range of ethanol crude extracts from Inonotus obliquus was 6.25×10-4 mg/mL, Under this condition, after 24 h treatment, prion replication were inhibited by the ethanol crude extracts from Inonotus obliquus (p<0.01), which could reduce the content of H2O2 and increase the content of SOD and antioxidant factor GCLM、GCLC，and it was statistically significant. The results showed that ethanol crude extracts from Inonotus obliquus had an inhibitoryeffect on prion replication, which may be related to its antioxidant activity.