Analysis Of The Composition Of Ginseng Fermentation Solution And Its Effect On The Liver

Objective: Ginsenosides converted into rare ginsenosides after fermentation are more active and have antioxidant,anti-inflammatory,anti-apoptotic and immune enhancing effects.In this study,Ginseng Fermentation Solution(GFS),which was prepared based on ginseng and millet,was a new ginseng fermentation product.In order to further evaluate the hepatoprotective effect of Ginseng Fermentation Solution on alcoholic liver disease(ALD),this study used a single factor combined with response surface methodology to develop a stable GFS;gas chromatography and liquid chromatography to identify its volatile and non-volatile components;in vitro and in vivo experiments to evaluate its hepatoprotective effect;The mechanism of action of GFS against ALD was elucidated using non-targeted metabolomics and high-throughput sequencing techniques.Methods:1.The Preparation process and quality analysis and evaluation of GFS: single-factor and response surface tests were used to optimize the preparation process of GFS with fermentation time,fermentation temperature and yeast addition as independent variables and mash alcohol as response values for process optimization,followed by three batch experiments to verify the process stability and finally quality analysis and evaluation.2.Composition analysis of GFS based on GC-MS and LC-MS: Gas chromatography-mass spectrometry coupled with Ultra-performance liquid chromatography with quadrupole orbitrap tandem mass spectrometry techniques to establish the qualitative analysis of volatile and non-volatile components of GFS,respectively;to compare the differences of major ginsenosides in GFS and Ginseng Soaked Wine(GSW)by metabolomics methods to screen the signature ginsenosides.3.In vitro evaluation of GFS on alcoholic liver diseases(ALD)hepatocytes: In vitro cell experiments were performed by selecting LO2 cells for modeling;Staining,kits,and flow cytometry were used to observe the level of apoptosis in each group;Western blot was used to observe the changes in the expression of PI3 K,Akt,Bcl-2 proteins and to explore the possible targets of GFS for liver protection.4.Pharmacodynamic evaluation of GFS on zebrafish larvae with alcoholic liver diseases: In vivo experiments were conducted on wild-type AB strain zebrafish to investigate the effects on morphological changes,survival curve,biochemical indexes,and protein expression of zebrafish after the intervention of GFS.5.Regulation of metabolites and intestinal flora in zebrafish with ALD by GFS:Metabolomics techniques were applied to detect liver metabolites in zebrafish larvae and screen biomarkers associated with GFS regulation of ALD;combined with high-throughput sequencing technology to analyze the intestinal contents samples of each group of zebrafish,observe the key strains in the zebrafish intestine that are closely related to ALD,and correlate the metabolites with the strains,and then clarify the metabolic pathways and the mechanism of action of GFS on the intestinal flora of zebrafish with ALD.Results:1.Preparation process and quality analysis and evaluation of GFS: The optimal process determined by response surface method optimization is a fermentation time of 8d,fermentation temperature of 29℃,and yeast addition of 0.5‰.The GFS was obtained with11.56 g/L total sugar,7.21 g/L total acid,5.40 p H,and 8.4% soluble solids.2.The compositional analysis of GFS based on GC-MS and LC-MS: a total of 303 volatile components and 96 ginsenosides were identified in the fermentation solution of ginseng.The metabolomic approach screened 21 rare ginsenosides with significant differences between GFS and GSW,including CK,F2,Rh2,etc..3.In vitro evaluation of GFS on ALD hepatocytes: In vitro cell experiments showed that GFS had a repairing effect on LO2 cell apoptosis;after detection by flow cytometry,it was found that all GFS groups could effectively reduce the level of apoptosis;compared with the model(Alcohol,AL)group,GSH-PX,and SOD activities were increased in all groups of GFS(p<0.05),and TC,TG,and MDA levels were reduced(p<0.05);meanwhile,GFS up-regulated the expression levels of PI3 K,Akt,and Bcl-2 proteins and down-regulated the expression levels of Bax protein,and the effect of GFS high-dose group(GH)was more significant(p<0.05).4.Pharmacodynamic evaluation of GFS on ALD: In terms of morphological changes in zebrafish larvae,GFS significantly improved spinal curvature and oocyst yolk edema in zebrafish after the intervention;meanwhile,compared with the AL group,GFS significantly increased GSH-PX and SOD levels(p<0.05)and significantly decreased TC,TG and MDA levels(p<0.01).At the protein level,the low,medium and high dose groups of ginseng fermentation solution(GL,GM and GH groups)significantly increased the ratio of Bcl-2 to Bax through PI3K-Akt signaling pathway and thus inhibited apoptosis,further demonstrating the hepatoprotective effect of ginseng fermentation solution.5.Regulation of metabolites and intestinal flora in zebrafish with ALD by GFS: 22 and24 metabolic markers potentially related to ALD were identified in positive and negative ion mode,respectively,mainly involving arachidonic acid metabolism,unsaturated fatty acid biosynthesis,and taurine and hypotaurine metabolism.Compared with the AL group,the relative abundance of the genera Proteobacterium,Achromobacter,and Archaea at the genus level increased in the GH group.In contrast,the relative abundance of the genera Flectobacillus and Elizabethkingia decreased.Among the metabolites,Prostaglandin F2 alpha,11,14,15-THETA and Taurocholic acid were negatively correlated with Curvibacter,while 11-epi-Prostaglandin F2 alpha was negatively correlated with mitsuaria and Chryseobacterium,respectively.Conclusion:In this study,we determined the preparation process of GFS,identified 303 volatile components and 96 ginsenosides,and showed that GFS could inhibit apoptosis and regulate ALD through in vivo and in vitro experiments.And we found that the GH group could regulate the microenvironment in the intestine of zebrafish with ALD by increasing the abundance of beneficial genera and decreasing the abundance of harmful flora,thus improving the mechanical hepatoprotective effect.This paper provides data to support the development and clinical application of GFS.