发酵灵芝主要活性物质变化及对HepG_(2)细胞的抑制作用

The Changes of Main Active Substances of Ganoderma lucidum Fermented by Lactobacillus rhamnosus and the Inhibitory Effect of Alcohol Extracts on HepG_(2) Cells

为探究鼠李糖乳杆菌发酵灵芝子实体粉的主要活性物质变化及对肝癌细胞HepG_(2)的抑制作用,采用固体发酵的方式,以鼠李糖乳杆菌为发酵菌种,灵芝子实体粉为发酵基质,制备发酵灵芝粉,研究其主要活性成分及抗氧化能力。对灵芝子实体粉进行植物代谢组学测定,通过扫描电镜观察其表面结构的变化。采用CCK-8法检测灵芝醇提物对肝癌细胞的抑制作用;细胞划痕的方法检测灵芝醇提物对肝癌细胞迁移的影响;Hoechst 33258检测灵芝醇提物对细胞核的影响,验证凋亡效果;PCR法分析HepG_(2)细胞凋亡相关基因的表达。结果表明,鼠李糖乳杆菌发酵灵芝粉的三萜类物质含量为4.075mg/g、多糖含量为2.347 g/100 g、黄酮含量为4.604 mg/g与发酵前相比均提高;鼠李糖乳杆菌发酵灵芝粉抗氧化能力较原灵芝粉和发酵对照灵芝粉显著提高;植物代谢组学的结果显示,3种样品的物质成分和峰面积均有显著差异。细胞试验结果显示,发酵灵芝醇提物抑制HepG_(2)细胞增长和阻滞细胞迁移能力明显增加,且Hoechst 33258结果凋亡现象更加显著。通过PCR试验验证发酵灵芝醇提物干预细胞使得促进细胞凋亡相关基因Bax、Cyclin-A、CDK-2、P21、P53显著上调,抑制细胞凋亡相关基因Bcl-2显著下调。结论:鼠李糖乳杆菌发酵灵芝可提高灵芝主要活性物质,如灵芝三萜类化合物、灵芝多糖、黄酮的含量,发酵灵芝醇提物对HepG_(2)细胞的抑制作用更强。

In order to explore the changes of main active substances in Lactobacillus rhamnosus fermentation of Ganoderma lucidum fruitum powder and its inhibitory effect on hepatoma cell HepG_(2),a solid fermentation method was used to prepare the fermented Ganoderma lucidum fruitum powder with Lactobacillus rhamnosus as fermentation strain and Ganoderma lucidum fruitum powder as fermentation substrate,and its main active ingredients and antioxidant capacity were studied.Plant metabolomics of Ganoderma lucidum fruitum powder was determined and its surface structure was observed by scanning electron microscopy.The inhibitory effect of Ganoderma lucidum alcohol extract on hepatocellular carcinoma cells was detected by CCK-8 method.The effect of Ganoderma lucidum alcohol extract on hepatocellular carcinoma cell migration was detected by cell scratch method.Hoechst 33258 detected the effect of Ganoderma lucidum alcohol extract on cell nucleus and verified the apoptosis effect.The expression of apoptosis-related genes in HepG_(2) cells was analyzed by PCR.The results showed that the contents of triterpenoids and polysaccharides were 4.075 mg/g,2.347 g/100g and 4.604 mg/g respectively compared with those before fermentation.The antioxidant capacity of Lactobacillus rhamnosus fermented Ganoderma lucidum powder was significantly improved compared with the original Ganoderma lucidum powder and fermented control Ganoderma lucidum powder.The results of plant metabolomics showed that the material composition and peak area of the three samples were significantly different.Cell test results showed that the ability of fermented Ganoderma lucidum alcohol extract to inhibit HepG_(2) cell growth and block cell migration was significantly increased,and the apoptosis was more significant in Hoechst 33258 results.PCR test confirmed that the intervention of fermented Ganoderma lucidum alcohol extract significantly up-regulated the apoptosis-related genes Bax,Cyclin-A,CDK-2,P21 and P53,and significantly down-regulated the apoptosis-related genes Bcl-2.Conclusions:Lactobacillus rhamnosus fermentation of Ganoderma lucidum can increase the contents of main active substances,such as triterpenoids,polysaccharides and flavonoids of Ganoderma lucidum,and the ethanol extract of Ganoderma lucidum fermentation has stronger inhibitory effect on HepG_(2) cells.