Study On Chemical Constituents Of Clerodendron Cyrtophyllum And Purification Technology Of Acteoside

Clerodendron cyrtophyllum Turcz,a member of the genus Verbena,is one of the Miao medicine commonly used in Guizhou with abundant resources.In order to explore the chemical constituents of C.cyrtophyllum and provide basis for the utilization of this resource,this project investigated the chemical constituents of C.cyrtophyllum and the extraction and purification process of acteoside.The results are as follows:1.Study on chemical constituents and biological activities of C.cyrtophyllumLiquid-mass spectrometry（LC-MS）was used to qualitatively analyze the n-butanol extraction parts of C.cyrtophyllum.Forty-two compounds were identified.There were 15 phenylpropanoids,which were verbascoside,forsythoside E,chlorogenic acid,esculetin,caffeic acid,7-methoxycoumarin,isopsoralen,skimmin,ferulic acid,methyl 4-hydroxycinnamate,cimifugin,secoisolariciresinol diglucoside,isoacteoside,ferulaldehyde,methy 4-hydroxy-3-methoxycinnamate.There were 12flavonoids,including cynaroside,scutellarin,scutellarin methyl ester,hyperoside,oroxin A,diosmetin,homoplantaginin,kaempferol-7-O-β-D-glucopyranoside,apigenin-7-O-β-D-glucoside,jaceosidin,ononin.Four were terpenoids,including curcumol,curdione,rubescensin B and steviol-19-O-glucoside.There were 11 fatty acids,including methyl gallate,isovanillic acid,ligustilide,α-linolenic acid,stearic acid,protocatechuicacid,protocatechualdehyde,vanillin,3,5-dimethoxy-4-hydroxybenzaldehyde,azelaic acid,anisic aldehyde.The n-butanol fractions were separated and purified by macroporous resin column,Sephadex LH-20 dextran gel column,RP-18 reversed phase column and semi-preparative HPLC column.The structures of n-butanol fractions were identified by IR,UV,MS and NMR.As Cyrtophyllumoside A,Acteoside,Luteolin and Acantrifoside E,respectively.Cyrtophyllumoside A was A new compound,and Acantrifoside E was isolated from C.cyrtophyllum for the first time.Cyrtophyllumoside A showed significant DPPH radical scavenging activity with IC₅₀of 26.974±0.442μg/mL.2.Extraction and purification process of acteoside from C.cyrtophyllumThe L₁₆（4⁵）orthogonal test method was designed with the quality of acteoside as the index.The extraction times were as follows:ethanol concentration of 30%,50%,70%,90%,particle size of 20 mesh,40 mesh,60 mesh,80 mesh,solid-liquid ratio（g/mL）1:15,1:20,1:25,1:30,extraction times:1,2,3,4,extraction time 1.0 h,1.5 h,2.0 h,2.5 h.After obtaining the optimal results,influence of temperature on the extraction rate was investigated.The optimal extraction conditions were as follows:the particle size of 60 mesh,50%ethanol,the ratio of solid to liquid（g/mL）1:15,extraction for 4 times,1 h each time,and the boiling temperature of 81.9℃.Under this condition,the average extraction yield was 18.60±0.66%,and the average yield of acteoside was 1.59±0.51%.Static adsorption and desorption experiments were used to select the best resin types for enrichment of acteoside from D-101,LSA-21,AB-8,HPD600,NKA-9 and S-8.Static experiments were conducted to investigate the concentration of sample and ethanol eluent.The dynamic adsorption experiments were carried out to investigate the flow rate of sample solution,washing water,elution flow rate to optimize the purification process of acteoside.The optimal purification conditions were as follows:1.25 BV（7 mg/mL）of the initial extract of C.cyrtophyllum was loaded into the column with HPD600 resin at the flow rate of 5 mL/min,the macroporous resin column was eluted with 4 BV pure water,and then eluted with 5 BV 50%ethanol solution at the flow rate of 3 mL/min.The average recovery and purity of acteoside were 91.65±0.49%and 28.17±0.32%,respectively.The extracts were characterized by NMR.DPPH free radical scavenging assay showed that the extracts of acteoside had significant antioxidant activity,with IC₅₀of 7.341±0.422μg/mL.The research on chemical constituents and extraction and purification technology of acteoside enriched the understanding of chemical constituents of C.cyrtophyllum,and also provided basis for subsequent resource utilization.