至真方固体双向发酵工艺的建立及其药效评价

Establishment of Bi-directional Solid Fermentation Process for Zhizhen Recipe and Evaluation of Its Efficacy

目的以中药固体双向发酵方法,建立至真方的新型发酵工艺。方法①通过文献研究拟定候选发酵菌种。对候选发酵菌种进行平板混合实验,考察各菌种间生长兼容性。采用CCK-8细胞增殖抑制实验,比较不同发酵条件下所得产物对人结直肠癌细胞(HCT-8)的半数抑制浓度(IC50),评判发酵体系的优劣,从而确定发酵菌种、发酵时长等关键发酵参数,建立发酵工艺。②通过HCT-8细胞增殖抑制率,以及巨噬细胞(RAW264.7)NO释放率和吞噬活性测定,比较至真方发酵前后的体外药效差异。结果黑曲霉和酵母菌为最优发酵菌种组合,4 d为最优发酵时长。在此发酵条件下,发酵组对HCT-8细胞的IC50为67.41μg·mL^(-1),生药组对HCT-8细胞的IC50为1032μg·mL^(-1);与生药组比较,发酵组能显著抑制RAW264.7释放炎症因子NO,并显著提升其吞噬活性。结论该研究建立了至真方固体双向发酵新工艺。在该发酵体系下,至真方的体外抗结直肠癌活性、抗炎活性和促免疫活性得到显著提升。

Objective To establish a new fermentation process for Zhizhen Recipe by using bi-directional solid fermentation method of traditional Chinese medicine.Methods①Candidate strains for fermentation were primarily recruited through literature research.Then plate incorporation assay of candidate strains were conducted to investigate the growth compatibility between the strains.CCK-8 cell proliferation assay was used to compare the half proliferation inhibitory concentration of the products obtained under different fermentation conditions against human colorectal cancer cell line HCT-8.The advantages of different fermentation systems were evaluated and the key fermentation parameters including strains and incubation periods were determined,which gave rise to the establishment of the optimal fermentation process for Zhizhen Recipe.②In vitro pharmacological effects were compared between the fermented and unfermented Zhizhen Recipe by measuring proliferation inhibition of HCT-8 cells,as well as NO release and phagocytic activity of RAW264.7 cells.Results The results showed that Aspergillus niger/Saccharomyces cerevisiae was the optimal combination of strains,and the optimal fermentation time is 4 days.Under optimized conditions,the half inhibitory concentration(IC50)of the fermentation group on HCT-8 cells was 67.41μg·mL-1,while IC50 value of the unfermented Zhizhen Recipe was 1032μg·mL-1.Compared with unfermented preparation,the fermented preparation exhibited significant effects on inhibiting the release of inflammatory factor NO of macrophages RAW264.7 cells and obviously enhanced their phagocytic activity.Conclusion In this present study,we established a new bi-directional solid fermentation process for Zhizhen Recipe.Under this fermentation system,the anti-colorectal cancer activity,anti-inflammatory activity and pro-immune activity of Zhizhen Recipe in vitro were significantly improved.