Study On The Difference Of Chemical Composition And Pharmacodynamics Of Gentian Wine Before And After Preparation

Purpose:The polysaccharide content and monosaccharide composition of Gentian and wine-processed one were studied by water extraction and alcohol precipitation method and ELSD-HPLC method.The pharmacodynamics difference in rats by Elisa method of Gentian and processed one were studied,including medicinal properties,protecting liver and anti-jaundice.The mechanism of wine production was preliminarily discussed from the differences in chemical composition and pharmacodynamics.Material and method:Materials:The Gentian medicinal material were collected from the Gentian planting base of Qingyuan,Liaoning Province,which were identified as the rhizome of Gentiana scabra Bge.by Professor Li Feng of the Department of Chinese Medicine Identification of LiaoningUniversity of Traditional Chinese Medicine.The Gentiana pieces were sold in 23 regions,and the wine-processed one were obtained according to the method included in the 2015 edition of the Chinese Pharmacopoeia.Methods:1.The Gentiana pieces and corresponding processed products collected from 23 different areas were treated by water extraction and alcohol precipitation method to obtain crude polysaccharide of Gentiana,coloring of crude polysaccharide by phenol-sulfuric acid,and different sources by UV-spectrophotometry.The content of polysaccharides in Gentiana and wine-processed one were determined.2.The Sevage method was used to treat the crude polysaccharide obtained by water extraction and alcohol precipitation,and the essence polysaccharide of Gentiana was obtained.After hydrolysis of TFA,the monosaccharide composition was detected by HPLC-ELSD method,and the change of the monosaccharide composition in polysaccharide in Gentiana and processed one was compared..3.SD rats were perfused with high and low doses of Gentiana aqueous extract before and after processing,macroscopic signs（body mass and rectal temperature）were recorded,and Elisa kit was used to detect plasma substance metabolism（LA,PA,TG）,liver tissue energy metabolism(Na⁺-K⁺-ATPase,Ca²⁺-Mg²⁺-ATPase),gastric mucosal hormone levels（MTL,GAS,PEG2）similarities and differences,clear the change of bitter cold of Gentiana before and after wine-processing.4.To investigate the protective effects of acute liver injury in rats by using Gentiana aqueous extracts before and after wine-processing.High,medium and low dose Gentiana extracts before and after processed were used to replicate rat acute liver injury model by intraperitoneal injection of CCl4.ALT,AST,TNF-α,MCP-1 and IL-6 in plasma were detected by Elisa kit,as same as SOD,MDA,GSH and GSH-Px in liver tissue.Liver tissue section were observed by HE staining.5.The model of rat damp-heat jaundice was replicated by administrat administrating ANIT combined with high-fat and high-glucose diet and hot and humid environment.The high,medium and low dose Gentiana aqueous extract before and after processing were administrated to rats.ALT,AST,ALP,TBIL,D-BiL and TBA in plasma were detected by Elisa kit,as same as MDA and GSTs in liver tissue.Gene expression of NTCP,BSEP,MRP2 and MRP3 by real-time PCR Level.HE staining was used to observe the liver tissue sections of rats in each group,and the effects of Gentiana and wine-processed one on the elimination of jaundice were studied.Results:1.The content of gentian polysaccharides from 23 different sources ranged from 1.50%to3.31%,and the content of wine-processed Gentiana polysaccharides was between 2.29%and 4.88%.2.The Gentiana polysaccharide was hydrolyzed by TFA and determined by HPLC-ELSD.The monosaccharide composition was determined to be Glc,Fru and Gal.The molar ratio was Glc:Fru:Gal=4.04:0.43:1;the content of each monosaccharide in the polysaccharide after processing is changed,and the molar ratio is Glc:Fru:Gal=4.32:1.19:1.3.After oral administration of Gentiana,the macroscopic signs of rats decreased,and the metabolism of material and energy was slowed down,and the gastric mucosa was stimulated.Among them,body weight,anal temperature,LA and PA content in plasma,Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase in liver tissue,PEG2 and MTL in gastric mucosa decreased.It can also increase the TG content in the plasma and the GAS content in the gastric mucosa.Wine-processed Gentiana has a significant callback effect on the changes of these 10 indicators.4.Gentiana and processed one can significantly decrease the contents of ALT,AST,TNF-α,MCP-1,IL-6 and MDA in rats with liver injury,and TNF-α,MCP-1and IL-6 content in rats in the wine-processed Gentiana group was more significant than that of the raw Gentiana group.Both raw Gentiana and processed-Gentiana can significantly increase the contents of SOD,GSH and GSH-Px.The contents of GSH and GSH-Px in the Gentiana group were more significant than that in the processed group.HE staining results showed that Gentiana aqueous extract and wine-processed one can alleviate acute liver injury induced in rats by CCl4.5.Raw Gentiana and wine-processed one can significantly decrease the contents of ALT,AST,MDA,ALP,TBIL,D-BiL,TBA and GSTs in rats with jaundice hepatitis,and in the regulation of MDA and ALP,the effect of processed Gentiana is significantly better than that of raw Gentiana.The gene expression levels of NTCP,BSEP and MRP2 in rats with jaundice were significantly decreased,and the gene expression level of MRP3 was significantly increased.The Gentiana and wine-processed one have significant reversal effects,and the effect of wine-processed Gentiana is better.Conclusion:1.After processing with wine,the polysaccharide content in Gentiana has different degrees of increase.After hydrolysis of Gentiana polysaccharide,its monosaccharide composition was glucose,fructose and galactose.There was no change in the composition after processing,but the content changed,and the fructose content increased significantly.2.The wine processing can reduce the bitter coldness of Gentiana,improving its inhibitory effect on rat growth,material metabolism and energy metabolism,and reducing its stimulating effect on rat gastric mucosa.Gentiana and processed one have certain protective effects on acute liver injury induced by CCl4 in rats.Among them,Gentiana enhances the anti-oxidation effect of liver tissue,while wine-processed Gentiana has a stronger effect on eliminating inflammatory factors and reducing inflammation.Gentiana and processed one have certain elimination effect on rat jaundice-induced hepatitis caused by ANIT,and it has obvious dose-effect relationship.The effect of wine-processed Gentiana is better than that of raw Gentiana.3.There may be a certain correlation between the change of chemical composition and the change of drug effect,that is,the change of polysaccharide content and monosaccharide composition after wine processing may be the one of material basis for the decline of the bitter cold of the wine-processed Gentiana,the protection of liver and the enhancement of the action of eliminating jaundice.