| Astragalus is one of the commonly used traditional Chinese drugs. The maineffective components-astragalus polysaccharides (APS) of the crud drug has manypotency effects, such as improving immunity, antibiosis, anti-virus, hepatoprotection,protecting kidney and so on. While, the yield rate of astragalus polysaccharides fromastragalus is low, therefore its clinical use was restricted to some extent. Previousstudies have shown that the average polysaccharides yield rate of probiotics M-9fermented astragalus was improved significantly (P<0.01). However, the qualitycontrol of the fermented astragalus, the contents of the active ingredients andpharmacologic effects of them were not clear yet, therefore, this study carried outquantitative determination of astragaloside Ⅳ and calycosin-7-glucoside of fermentedastragalus and crude drug astragalus by high performance liquid chromatography,extraction technology of astragalosides and polysaccharides, effects of the FAPS onexperimental hepatic fibrosis in rats and analysis of digital gene expression profiling(DGE) on the differences of gene expression in every group of rats liver tissue. Inorder to lay the theoretical foundation for the further pharmacological research,development and utilization of fermented astragalus and interpretation the mechanismof biotransformation.(1) Quantitative determination of astragaloside Ⅳ and calycosin-7-glucoside offermented astragalus and crude drug astragalus by high performance liquidchromatographyAstragaloside Ⅳ and calycosin-7-glucoside contents of fermented astragalus andcrude drug astragalus were determined by high performance liquid chromatography(HPLC) according to the pharmacopoeia of the people's republic of China (2010). Theresults were0.049%,0.087%,0.030%and0.040%respectively. Results showed thatcontent of astragaloside Ⅳ of fermented astragalus (with equal quantity of crude drugastragalus) and crud drug astragalus were0.063%and0.087%, the content ofcalycosin-7-glucoside of fermented astragalus(with equal quantity of crude drugastragalus) and crude drug astragalus was0.038%and0.040%. Changes ofCalycosin-7-glucoside content before and after fermentation was not significant (P<0.05) after the quality conversion, while the content of astragaloside Ⅳ infermented astragalus was reduced by27.6%comparing with crude drug astragalus.Therefore, content of calycosin-7-glucoside and astragaloside Ⅳ could be used forquality control of fermented astragalus.(2) Extraction, isolation and quantitative determination of the astragalosides andpolysaccharides of fermented astragalus and crude drug astragalusMethod of comprehensive extraction of astragalus polysaccharides andastragalosides was carried out to screen the extraction process. The results showedthat the optimum extracting conditions of astragalosides and polysaccharides were asfollows: Weighed a certain amount of astragalus powder and soaked in10times of themedicine amount of80%ethanol at room temperature for12h in a closed container,then it was reflux extracted at80℃twice,1h per time. The mixture of twice medicalliquids was used in extracting astragalosides and the herbal residues were dried andused in extracting polysaccharides.(A) Process for extraction of astragalosides:Solution of ethanol extracting was filtrated and concentrated by reducing pressuredistillation into liquid extract. Dissolved a certain amount of liquid extract in50timesof the medicine amount (mL/g) of purified water then were absorbed by AB-8macropore resin, there was eluted with4times in column volume of80%ethanol. Theeluates were concentrated by reducing pressure distillation and derived astragalosidesenrichment site.(B) Through comparision the extraction method of immersionmethod, traditional decoction method, assisted extraction with cellulase and saturatedlimewater method. The immersion method was optimized for polysaccharides'extraction: Herbal residues were extracted by20times water (purified water to herbalresidues amount, mL/g) at80℃for4.5h, got water extracts, then was centrifuged,condensed, deproteinized by Sevag method, dialysed, added ethyl alcohol to4timesof the volume of the resulting dialyzed filtrate, the mixture was stirred and depositedfor12h and then centrifuged, the precipitate was washed with absolute ethyl alcohol,acetone, and diethyl ether,2times separately. Polysaccharides was oven dried at60℃.(C) Vanillin-sulfuric acid method for determing astragalosides content, results showedthat content of crude drug astragalus extract and fermented astragalus extract were16.87%and10.81%, extraction ratio of astragalosides from crude drug astragalus and fermented astragalus were0.0654%,0.0558%. Astragalosides content of AB-8derived astragalosides enrichment site was81.23%. Phenol-sulfuric acid method fordeterming polysaccharides content, results showed that content of fermentedastragalus polysaccharides (FAPS) and yield rate were66.59%and4.35%; crude drugastragalus polysaccharides (APS) and yield rate were36.01%and2.73%. Thecomponent sugars of FAPS-2and FAPS-3were confirmed by thin layerchromatography (TLC). Results showed that FAPS were composed of mannose(D-Man) and glucose (D-Glu), et al. Content of the main FAPS fraction FAPS-2-1was95.41%, its weight average molecular weight (Mw) was1,753,456Da and thenumber average molecular weight (Mn) was1,564,267Da. Results showed thatpolysaccharides content in fermented astragalus was increased by84.92%, the gainratio was increased by59.34%, while the extract ratio of astragalosides was reducedby17.20%.(3) Effects and mechanism of the FAPS on experimental hepatic fibrosis in ratsSixty male SD rats were divided into six groups randomly, liver fibrosis modelwas made by subcutaneous injection of CCl4in5groups of the rats for8weeks.Simultaneously, administered FAPSH200mg·kg-1·d-1, FAPSM100mg·kg-1·d-1, FAPSL50mg·kg-1·d-1, colchicine (Col.)0.2mg·kg-1·d-1to the groups of treatment, and themodel group and control group were administered with the equal volume ofphysiological saline solution. Rat's body weight in groups of FAPS were higher thanthe model group, after treating with FAPSH, ALT, AST, and ALP activity in serumwere significantly reduced compared with model group (P<0.01), content of Hyp inliver tissue (P<0.05) and PⅢNP, CⅣ, LN, HA in serum were also reducedsignificantly. FAPSHcould effectively increase the low level of GSH-Px (P<0.01),GST in serum and T-AOC (P<0.05) in liver tissue. High levels of GSH and MDA inliver tissue were tended to normal. H.E. staining, Masson trichrome staining andelectric microscope were used to observed the histopathological changes of livertissues, pathological examination showed that hepatocyte fatty vacuolation, apoptosis,necrosis and liver fibrosis in CCl4model group were serious, pathological changeswere improved in the treated groups. Functions of anti-liver fibrosis of FAPS were proportionated to its dosage. FAPSHanti-liver fibrosis effect was equivalent to Col.,and the mechanism may be related to antioxidation.(4) Analysis of digital gene expression profiling (DGE) on the differences of geneexpression in every group of rats liver tissueDifferences of gene expression and function in every group of rats liver tissuewere analyzed by DGE. Results showed that, there were162down-regulated genesand2162up-regulated genes between CCl4model group and control group.Differentially expressed genes took part in the formation of liver fibrosis through218signaling pathways of multiple cell biological processes. Multiple genes, particularlyin the PPAR signaling pathway, TGF-β signaling pathway, MAPK signaling pathways,VEGF signaling pathway and the Jak-STAT signaling pathway were down-regulated,which built the basis for screening new anti-liver fibrosis drugs.Comparing with the DGE of CCl4model group, results showed that FAPS couldinhibited the increasing of several key genes in the TGF-βand PPAR signalingpathways significantly, so that inhibited HSCs activity. Especially, expression ofSCD-1decreased significantly which showed that FAPS could inhibit the activity ofSCD. It is indicated that the mechanism of anti-liver fibrosis of FAPS was related withdecreasing the activity of SCD, the generation of monounsaturated fatty acids wasweaken, led to the reduction of hepatic lipid accumulation, so that inhibited theactivation of HSCs.
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