Study On The Preparation Of Methyl Salicylate Glycoside, An Anti-inflammatory Active Ingredient In Dianbaizh

Dianbaizhu a folk medicine used as an antirheumatic,is made from Gaultheria leucocarpa Blume var.yunnanensis(Franch.)T.Z.Hsu&R.C(Ericaceae).Chinese medicine Curcuma Radix first appeared in "Yao Xing Lun" and has a long medicinal history in China.Curcuma phaeocaulis Val.has a thousand years of cultural history in Sichuan,and is one of geo-authentic crude drugs of Curcuma Radix.However,compared to the homologous medicine Curcuma Rhizoma,researchers Fang(Ericaceae)and have multiple plant origins and officinal parts.In the previous HPLC chromatographic study,we found that the active ant rheumatic fraction(alcohol extract 30%ethanol elution)mainly contains methyl salicylate glycosides.The methyl salicylate glycosides(Gaultheria、MSTG-A、MSTG-B),The components were similar to the chemical structure of aspirin and has a significant anti-inflammatory effect which has attracted great attention of many researchers.Some scholars think that it may become a new type of non-steroidal anti-inflammatory drugs.MSTG-A and MSTG-B belong to the same structure mother nucleus,MSTG-A and MSTG-B belong to methyl salicylate glucoside of triose glycoside.The compound MSTG-A is linked with two β-D-xylopyranose groups in 2 and 6 positions of glucose.MSTG-B isglucose 6-position linked to xylose group,2-position is glucosyl group.Both polarities are similar,and their separation is difficult.According to the literature search,the preparation of methyl salicylate glycoside component(glucoside),MSTG-A and MSTG-B(triglucoside)monomer has not been reported.The research results provide a material foundation for exploring the mechanism of anti-inflammatory and analgesic action of this kind of components and in vivo metabolism,as well as improving the quality control standard and guaranteeing clinical curative effect.The main contents of this paper are as followsThe First Part Literature ReviewIn this paper,57 literatures on methyl salicylate glycoside and Dianbaizhu were reviewed systematically,including the properties,structural,characteristics,pharmacological activities,functions and research prospects of methyl salicylate glycoside.At the same time,the preparation methods and techniques of traditional monomers were reviewed,in order to provide reference for the preparation of active components and the formulation of quality standards of Radix Dianbaizhu.The Second’ Part the main research results of this paper1.Studies on determination of the major components from DianbaizhuHPLC method is used to determine the contents of MSTG-A,MSTG-B and Gaultherin in Dianbaizhu.The Chromatographic conditions are as follows:column YMC-ODS-AC18(4.6 mm×250 mm,5μm);The mobile phase consisted of 0.2%acetic acid aqueous solution and acetonitrile solvent B.The gradient elution conditions were as follows:0-3 min～7-9%B;3-8 min,910%B;8-13 min～10%B;13-18 min,10-13%B;18-30 min～13%B;30-40 min～13-95%B,flow rate=1 mL/min,column temperature=30℃,sample quantity=10 L.Results:The linear range of methyl salicylate glucoside MSTG-A MSTG-B and Gaultherinis is 24.00～1535.00 24.14～1545.00;5.36～343.30(ug/mL),respectively.The RSD of precision test was less than 1.63,the repeatability of RSD was 1.92,and the recovery rate of adding sample was 98.56-101.250.The RSD of precision test was less than 1.65.The monomer contents of MSTG-A、MSTG-B and Gaultherin in Dianbaizhu were 1.54%and 1.64%,0.71%,respectively.2.Study on extraction Technology of Methyl Salicylate glycoside from DianbaizhuIn this paper,the content of each monomer,the total content of three methyl salicylate methyl glucoside(MSTG-A),and the combination of MSTG-A and MSTG-B,Gaultherin were used as evaluation indexes,and four factors affecting the extraction solvent,extraction time,ratio of material to liquid and times of extraction were selected.On the basis of single factor experiment,orthogonal experiment was carried out.The results showed that the optimum extraction process was concentration of ethanol B%,ratio of material to liquid 1:L2 heating reflux extraction for S3 h each time.T3times.The results showed that the contents of methyl salicylate MSTG-A MSTG-B and Gaultherin were 3.67%,3.26%and 0.97%,respectively.3.Study on the enrichment process of Methyl salicylate glycoside in DianbaizhuBy screening the type of macroporous resin,the AB-8 macroporous adsorption resin was confirmed,and the effects of sample concentration,sample quantity,eluent and elution volume on the enrichment process of methyl salicylate glycoside were investigated.According to the results of investigation and comprehensive analysis of various factors,the best enrichment process of macroporous adsorption resin was determined:the concentration range of sample was S1～S3 g/mL,and the adsorption rate was B2 mL/min,D1BV deionized water to remove impurity.The flow rate of elution was A1 mL/min.After the macroporous adsorption resin was enriched,and purified,the content of MSTG-A,MSTG-B and Gaultherin in dry paste increased from 1.54%,1.64%and 0.71%to 6.99%,5.82%and 3.18%,respectively.After being enriched and purified by macroporous adsorption resin,the content of MSTG-A,MSTG-B and Gaultherin were increased by 5 times.This step lays a good foundation for the simultaneous preparation of three monomers of MSTG-A,MSTG-B and Gaultherin by a silica gel column chromatography at a later stage.4 The preparation of methyl salicylate glucoside by silica gel was studied.Under the guidance of TLC information,we tried to optimize the composition and polarity of eluent,the ratio of diameter to height,the filling amount of silica gel,the amount of sample,the flow rate of elution,and so on.Three methyl salicylate glycosides were purified by silica gel column chromatography.The optimum purification process of silica gel column chromatography was determined as follows:diameter to height ratio 1:J3,optimum sample amount 1:S2 mixed solvent(chloroform-methanol L5:J1)gradient elution,The flow rate of elution was A1 mL/min through this process,The contents of MSTG-A,MSTG-B and Gaultherin were increased to 78.3%,80.11%and 83.05%,respectively.5 Study on recrystallization of methyl salicylate glycoside.In this experiment,A%,B%,C%,D%ethanol was used as solvent,the effects of different concentration ethanol solution on the crystal purity.Experiments show that the three Methylis salicylas respectively with D%ethanol pure crystalline glucoside can be prepared with purity of 97%monomers,but the crystal appearance is not ideal.In order to make the crystal form better,the first recrystallization was carried out with A%ethanol as solvent and the second recrystallization with D%ethanol.,after recrystallization of three Methylis salicylas glucoside monomer content can reach more than 97%.This method has the advantages of simple operation,low cost,prepared by MSTG-A,MSTG-B,Gaultherin with high purity.The study of Methylis salicylas glucoside mass preparation provides an effective and eliable method.InnovationThe above three monomers were prepared simultaneously by a single silica gel column chromatography combined with crystallization method.The purity of the monomers was above 97%.