Study On The Fermentation Extraction Process And Antiviral Activity Of Scorpio,Atractylodes Macrocephala And Holotrichiae Larva

Objective:For three groups of traditional Chinese medicine combination fermention,such as Scorpio,Atractylodes macrocephala and Pulsatilla chinensis,Scorpio,Atractylodes macrocephala and Holotrichiae larva,Scorpio,Atractylodes macrocephala,Pulsatilla chinensis and Holotrichiae larva,that have better anti-RSV virus effect screened in the previous experiments,a plurality of viruses including RSV,EV71 and COX-B2 were further selected in vitro for antiviral activity to determine the traditional Chinese medicine combination fermentation with the best antiviral effect.The fermentation process and extraction process were further optimized.The antiviral activity and mechanism of the traditional Chinese medicine combination fermention in vivo were studied.The differences of the main components of combination before and after fermentation were explored,so as to provide a new idea for the study of Chinese medicine fermented by medicinal fungi fermentation and its combinations.Methods:1.The three traditional Chinese medicine combination fermentions,such as Scorpio,Atractylodes macrocephala and Pulsatilla chinensis,Scorpio,Atractylodes macrocephala and Holotrichiae larva,Scorpio,Atractylodes macrocephala,Pulsatilla chinensis and Holotrichiae larva,were extracted by water ultrasonic extraction,50%alcohol reflux extraction,50%alcohol ultrasonic extraction and 80%alcohol reflux extraction.And the study of anti-RSV,EV71 and COX-B2 viruses in vitro were studied by MTT method respectively to determine the traditional Chinese medicine combination fermention with the best anti-virus effect and extraction method.2.Taking the comprehensive score of total protein and total polysaccharides as an index,the fermention process of the traditional Chinese medicine combination fermention was optimized by taking fermentation temperature,fermentation thickness and fermentation time as three factors.The effects of light and water content on spore germination rate under the optimal fermentation time were investigated.3.Taking antiviral therapeutic index（TI）in vitro as evaluation index,three factors including extraction time,solid-liquid ratio and ethanol concentration were investigated by response surface methodology to determine the optimal extraction conditions.4.After establishing a mouse model of RSV virus,the traditional Chinese medicine combination fermention was divided into large,medium and small dosage groups to carry out antivirus experiments in vivo.And the ELISA method was used to detect the concentration of cytokine in mice serum,such as tumor necrosis factor（TNF-α）,interferon-γ（IFN-γ）and nitric oxide（NO）,to explore the anti-RSV mechanism in vivo.5.The content of protein,polysaccharide,flavone and nucleic acid before and after fermention were determined by modified Lowry method,phenol-sulfuric acid method,Na NO₂-Al Cl₃-Na OH colorimetric method and phosphorus determination method respectively.The changes of protein molecular weight before and after fermentation under the optimal extraction conditions of traditional Chinese medicine combination fermention were compared by Tricine-SDS-PAGE electrophoresis.Results:1.The 50% alcohol ultrasonic extraction of Scorpio,Atractylodes macrocephala and Holotrichiae larva combination fermention had the best effect in vitro.2.The optimal fermentation process of Scorpio,Atractylodes macrocephala and Holotrichiae larva combination fermention was as follows:fermentation temperature was25℃,fermentation thickness was 2 cm,fermentation time was 216 h,illumination condition was 12 h:12 h and water content of culture medium was 300%.3.The optimal extraction process of Scorpio,Atractylodes macrocephala and Holotrichiae larva combination fermention was as follows:extraction time was 150 min,extraction solid-liquid ratio was 1:40,ethanol concentration was 50%,and TI under this extraction condition was 45.82.4.In the experiment in vivo,the lung index of the high,medium and low dose groups of the traditional Chinese medicine combination fermention were lower than that of the model group,showing good antiviral effect.The combination fermention could obviously improve the levels of TNF-α,IFN-γand NO of virus mice,suggesting that the antiviral mechanism is related to enhancing immunity.5.The content of protein,polysaccharide,flavone and nucleic acid before fermentation were 49.49 mg/g,123.94 mg/g,2.02 mg/g and 15.78 mg/g respectively.After fermentation,the content of protein,polysaccharide,flavone and nucleic acid were 76.30 mg/g,163.62mg/g,2.86 mg/g and 38.38 mg/g respectively.The molecular weight of the protein after fermentation was significantly smaller than that before fermentation.Conclusion:The traditional Chinese medicine combination fermented by Scorpio,Atractylodes macrocephala and Holotrichiae larva with the best antiviral activity is screened through various viruses.The optimal fermentation process and extraction process are determined.The antiviral effect of the traditional Chinese medicine composition fermention in vivo is confirmed,and the mechanism is related to enhancing immunity.Moreover,the content of protein,polysaccharide and nucleic acid in the combination fermented by Scorpio,Atractylodes macrocephala and Holotrichiae larva is obviously increased and the molecular weight of the protein is reduced.