| Objective: As a genuine medicinal material in Gansu Province,Radix Hedysari has the functions of fixing the surface,relieving sweat,invigorating qi and diuresis,supporting poison and restraining sores.Clinically,it is often used to treat diseases such as Qi deficiency,hyperhidrosis,chronic nephritis and heart disease,with remarkable effects.Its active ingredients mainly contain carbohydrates,flavonoids and phenylpropanoids,among which astragalus polysaccharide is one of the main active ingredients,and its pharmacological effects are particularly extensive.With the aging of the population,osteoarthritis is one of the most common diseases in the history of human diseases.The incidence of osteoarthritis accounts for about 20% of the total population.It has seriously affected the quality of people’s life.At present,sodium hyaluronate is commonly used to alleviate osteoarthritis in clinic.In the early stage of this study,it has been found that the total polysaccharides of Radix Hedysari have obvious therapeutic effects on osteoarthritis,and it is clinically proved that sodium hyaluronate is effective in treating osteoarthritis.It is predicted that the combination of the polysaccharides obtained from Sephadex gel chromatography and hydrogel based on sodium hyaluronate will also have a good therapeutic effect on osteoarthritis.Therefore,starting from the purification,separation and pharmacological effects of Radix Hedysari polysaccharide,combined with hyaluronic acid hydrogel to explore the therapeutic effect of Radix Hedysari polysaccharide on osteoarthritis in rats,to study the therapeutic mechanism of Radix Hedysari polysaccharide in bone basis,and to further expand the development and utilization of polysaccharides.Methods: 1.In the previous experiment,the total polysaccharide solution of Radix Hedysari was extracted by decoction,and purified by alcohol precipitation and acetone decolorization.After that,the protein was removed by Sevage method in order to obtain the protein-free polysaccharide of Radix Hedysari polysaccharide.2.Sephadex G-25 chromatographic method was used to separate polysaccharides from Radix Hedysari after protein removal.The polysaccharides were eluted with distilled water at a flow rate of 0.8 mL/min,and the eluents were collected per 3 mL.The phenol-sulfuric acid method was used to trace and detect the value of A and draw the elution curve.To get Radix Hedysari polysaccharide HG-2,the effluent of the main peak was concentrated,and then extracted and purified by alcohol precipitation,static filtration and acetone decolorization.The purity of Hedysarum polyhepagus HG-2 was identified by the Ninhydrin reaction,iodine potassium iodide reaction and polyacrylamide gel electrophoresis.3.TLC and IR spectra were used for the preliminary qualitative analysis.HPLC and GC-MS were used to analyze the composition of the polysaccharide HG-2,and to calculate the molar ratio of each monosaccharide.4.The linear changes of Radix Hedysari polysaccharide HG-2 reference substances and samples were investigated at different temperatures.The contents of total polysaccharides and polysaccharides HG-2 of Radix Hedysari were determined using phenol-concentrated sulfuric acid method.5.Sodium hyaluronate(HA)was reacted with methacrylic acid with pH value maintaining at 8-8.5 for 12 hours.The methacrylated HA(MeHA)was obtained after dialysis and freez-drying.The obtained MeHA macromer was dissolved in distilled water solution,and the hydrogels were formed after the addition of appropriate concentration of ammonium persulfate and tetramethylethylenediamine.For the fabrication of HA hydrogel containing Radix Hedysari polysaccharide HG-2,MeHA macromer was dissolved in the Hedysarum polysaccharide HG-2 solution,followed by the addition of ammonium persulfate and tetramethylenediamine.The gelation time,swelling ratio,release rate and in vitro degradation rate of HG-2 were determined at the same time.6.The rat osteoarthritis model was reproduced by injecting papain into the knee joint.The animals were divided into several groups,and the corresponding drug interference was administered to observe the therapeutic effects of Radix Hedysari polysaccharide HG-2,HA hydrogels,Hedysarum polysaccharide HG-2HA hydrogels on osteoarthritis in rats.Results: 1.The powder of total polysaccharides of Radix Hedysari membranaceus obtained by the method of water extraction and ethanol precipitation was grayish white and loose in texture.The protein-free polysaccharides of Radix Hedysari membranaceus were white and loose in texture,and the deproteinization rate was 50.8%.2.Total polysaccharides of Radix Hedysari membranaceus were separated by Sephadex G-25 chromatography.The elution curve showed that there was only one gradient elution peak.It was preliminarily indicated that only one molecular weight segment of Radix Hedysari polysaccharide HG-2 was obtained,and the yield was 20.05%.The results of the Ninhydrin reaction,iodine potassium iodide reaction and polyacrylamide gel electrophoresis showed that obtained Hedysarum polyhepagus polysaccharide was homogeneous polysaccharide.3.TLC preliminary revealed that HG-2 was composed of different monosaccharides.The Radix Hedysari polysaccharide HG-2 showed three spots of glucose,arabinose and galactose after hydrolysised.IR spectra showed that HG-2 of Radix Hedysari polysaccharide met the general characteristics of polysaccharides.In composition analysis,the HPLC spectra only showed the peaks of glucose and another unknown compound.So further analysis by GC-MS demonstrated that HG-2 of Radix Hedysari polysaccharide was composed of arabinose,rhamnose,xylose and Galact,the molar ratios of which were 3:12:0.1:0.6:17 in order.Glucose accounting for the highest proportion among these compositions.4.By comparing the k value of each reference substance with the samples,glucose was finally selected as the reference substance.The content of total polysaccharide of Radix Hedysari polysaccharide HG-2 was 95.97%,respectively,by phenol-concentrated sulfuric acid method.5.Sodium hyaluronate was used to prepare MeHA,and the yield was 98.89%.The gelation time of Hedysarum polysaccharide HG-2HA hydrogel was 3min±10s,and the swelling rate was 46.43%.The release amount of HG-2 from Radix Hedysari polysaccharide HG-2 reached 86.15% on the fifth day,and the hydrogels were almost degraded on the fifth day.6.Pharmacological experiments showed that HHG-2 could significantly expand the extension angle of knee joint in OA rats,improve joint pathological changes,reduce the thickness of synovium,and increase the content of glycosaminoglycan in synovium.At the same time,HHG-2 could reduce the levels of inflammatory cells and IL-1beta,IL-6 and TNF-alpha in the joint lavage fluid.In addition,two doses of HHG-2 were placed in HA hydrogel,and two doses of HG-2 were given twice a week instead of once a week to investigate the therapeutic effects of different doses of HG-2 on osteoarthritis in rats.The results showed that the effect of HHG-2 in HA hydrogel once a week was similar to that of HHG-2 in HA hydrogel twice a week,suggesting that HHG-2 hyaluronic acid hydrogel can reduce the number of medications used in the treatment of OA,and it is more convenient for clinical use.Conclusion:The content of HG-2 with high purity,which was separated and purified by Sephadex G-25 chromatography.It was found that HG-2 was composed of five different monosaccharides by TLC,IR,HPLC and GC-MS,among which glucose accounted for the largest proportion.Pharmacological experiments showed that the polysaccharide HG-2 of Radix Hedysari had therapeutic effect on osteoarthritis.After combined application of HA hydrogel,the effect of HA hydrogel was significantly increased compared with that of HHG-2,and the times of administration were reduced,which provided a theoretical basis for the study of Radix Hedysari polysaccharide in bone basis. |
PDF Full Text Request