Study On The Extraction And Separation Of Effective Parts Of Radix Hedysari In Treatment Of UC And Its Mechanism Of Action

Objective:Radix Hedysari is a kind of tonic traditional Chinese medicine,which is one of the authentic medicinal materials in Gansu province.Having the functions including invigorating Qi,promoting Yang,restraining sore,and generating muscle.Radix Hedysari mainly contains polysaccharides,saponins,flavonoids and other components.Ulcerative colitis is a common clinical disease,and the inflammatory reaction caused by colonic ulcer is closely related to the abnormal immunity of the body.Recent studies have shown that Radix Hedysari exhibits pharmacological effects such as anti-inflammation and regulation of immunity.Therefore,this study starts from the extraction and separation of the main components of different extracted parts of Radix Hedysari,to explore the therapeutic effect and material basis of Radix Hedysari on the treatment of ulcerative colitis in mice,and to study its mechanism,and provide theoretical basis for the treatment of ulcerative colitis.Methods:1.Radix Hedysari was extracted with petroleum ether（PE）,dichloromethane（DCM）,ethyl acetate（EA）,ethyl alcohol（EtOH）and water in turn,so that the different extraction parts of Radix Hedysari were obtained by systematic solvent extraction.In addition,Radix Hedysari was slso boiled in water to obtain the water extraction of the total medicinal extract.2.The contents of total polysaccharides,total saponins and total flavonoids in Radix Hedysari and its different extraction parts were determined by phenol-concentrated sulfuric acid colorimetry,vanillin-glacial acetic acid-perchloric acid colorimetry and sodium nitrite-aluminum nitrate-sodium hydroxide colorimetry with anhydrous glucose,astragaloside and rutin as the reference materials.3.Mice model of ulcerative colitis was established with 2,4,6-trinitrobenzenesulfonic acid.We observed the fecal characteristics of mice,evaluated the macroscopic lesions of the colon,observed the pathological changes of the colon,and measured the colonic water content and TNF-α,IL-6 and IL-10 levels in serum through drug intervention to study the therapeutic effect of Radix Hedysari on mice ulcerative colitis and its material basis.4.The total polysaccharides of Radix Hedysari were obtained by traditional water extraction and alcohol precipitation method,decolorized by anhydrous ethanol and acetone in turn,and than deproteinized by Sevage method.5.Sephadex G-100 column chromatography was used to separate the deproteinized Radix Hedysari polysaccharides and elute them with distilled water at a flow rate of 0.5⁰.8mL·min^-1.Eluent was collected every 3 mL,and detected by phenol-concentrated sulfuric acid method,and the elution curve was drawn.The main peak effluents were combined and precipitation by alcohol to get Radix Hedysair polysaccharide HG-3.6.The purity of Radix Hedysair polysaccharide HG-3 was identified by ninhydrin reaction,iodine-potassium iodide reaction and polyacrylamide gel electrophoresis;The composition of Radix Hedysair polysaccharide HG-3 was analyzed by mid-infrared spectroscopy,TLC and GC-MS;The molecular weight and content of Radix Hedysair polysaccharide HG-3 was determined by aqueous GPC and phenol concentrated sulfuric acid method,respectively.Results:1.Radix Hedysari was extracted by systematic solvent extraction so that,the petroleum ether extraction part,dichloromethane extraction part,ethyl acetate extraction part,ethanol extraction part and water extraction part by systematic solvent extraction were obtained in turn,and the extraction rates were 0.34%,0.25%,0.33%,34.20%and 11.50%,respectively.In addition,Radix Hedysari was also boiled in water to obtain the water extraction of the total medicine extract with the extraction rate of 41.73%.2.The contents of the main components in Radix Hedysari and its different extraction parts were determined by UV spectrophotometry.The results showed that the contents of total polysaccharides in the Radix Hedysari,water extraction part by systematic solvent extraction of Radix Hedysari and water extraction of the total medicine extract of Radix Hedysari were10.26%,79.76%and 82.50%,respectively.The contents of total saponins in Radix Hedysari and its petroleum etherextraction part,dichloromethane extraction part,ethyl acetate extraction part,ethanol extraction part,water extraction part by systematic solvent extraction and water extraction of the whole medicine of Radix Hedysari were 4.164%,0.161%,0.124%,0.177%,2.174%,0.454%and 2.560%,respectively;The contents of total flavonoids were0.389%,0.030%,0.032%,0.031%,0.345%,0.035%and 0.474%,respectively.3.The results of pharmacological experiments indicated that compared with the model group,the Radix Hedysari and its extraction parts by systematic solvent extraction（except petroleum ether extraction part）could effectively improve the body weight,fecal traits and occult blood of ulcerative colitis mice（P<0.05）.At the same time,they could not only decrease the CMDI score,colon water content and histopathological score（P<0.05,P<0.01）but also increase the level of serum IL-10 and decrease the level of TNF-αand IL-6 in mice（P<0.01）.The above experimental results revealed that Radix Hedysari exhibited a promising therapeutic effect on ulcerative colitis mice.4.The total polysaccharides of Radix Hedysari obtained by water extraction and alcohol precipitation were off-white powder,and the yield was 4.68%;The deproteinized polysaccharides were white powder,and the deproteinization rate was 43.5%.5.The Radix Hedysari polysaccharide HG-3 isolated by SephadexG-100 column chromatography was white powder with loose texture,and the yield was 22.56%.6.The results of ninhydrin reaction,iodine-potassium iodide reaction and polyacrylamide gel electrophoresis showed that Radix Hedysari polysaccharide HG-3 was a polysaccharide with high purity.The mid-infrared spectra indicated that the HG-3 met the general characteristics of polysaccharides and might beβ-pyran polysaccharides,and TLC and GC-MS results showed that the HG-3 was a polysaccharide composed of glucose.Furthermore,the molecular weight of the HG-3 was determined by aqueous phase GPC,and the results showed that the number-average molecular weight,weight-average molecular weight,mass average molecular weight and the ratio of weight-average molecular weight to number-average molecular weight were 4.25E+03,2.89E+04,3.44E+05 and 6.79,respectively.The content of HG-3 was 96.79%determined by phenol concentrated sulfuric acid method.Conclusion:In this experiment,the contents of total polysaccharides,total saponins and total flavonoids in Radix Hedysari and its different extraction parts were obtained.The results showed that the content of total polysaccharides in the water extraction of the total medicine extract of Radix Hedysari was higher than those in the water extraction part obtained by systematic solvent extraction.Compared to each extraction part,the contents of total saponins and total flavonoids were the highest in the water extraction of the total medicine extract of Radix Hedysari.The contents of total saponins and total flavonoids in each part by systematic solvent extraction of Radix Hedysari were algebraically added,respectively.And the sum was found to be higher than contents of total saponins and total flavonoids in the water extraction of the total medicine extract,respectively.It showed that compared with the single solvent extraction,the systematic solvent extraction could more comprehensively extract the chemical components of Radix Hedysari and distribute them in different extraction parts due to different polarity.Pharmacological experiments showed that compared with the model group,the water extraction of the total medicine extract of Radix Hedysari,ethanol extraction part and water extraction part by systematic solvent extraction demonstrated obvious therapeutic effects on ulcerative colitis in mice,and one of the mechanisms is to reduce TNF-αand IL-6,to increase the level of IL-10.At the same time,those results also revealed that the material basis may be mainly Radix Hedysair polysaccharides,and the total saponins and total flavonoids of Radix Hedysari also play a certain role.The analysis of Radix Hedysari polysaccharide HG-3 isolated by Sephadex G-100 column chromatography showed that it met the general infrared characteristics of polysaccharides and was composed of a component of glucose.This study provided development and utilization value for Radix Hedysari in the treatment of ulcerative colitis and provided reference for the development and utilization of Radix Hedysari resources.