Study On The Chemical Constituents In Ranunculus Sceleratus L. And Their Anti-inflammatory Activity In Vitro

Objective:Ranunculus sceleratus L.has been recorded by ancient books and folk medicine in the treatment of inflammatory diseases,and has also been widely reported in modern pharmacological research.However,there are few studies on relevant chemical components,and the reports on the treatment of inflammation related diseases of R.sceleratus L.also basically remain at the level of extract,and the material basis of its efficacy is still unclear.Although our previous research team also analyzed its chemical composition through liquid chromatography-mass spectrometry technology,this method has some limitations,and it is impossible to substantially obtain the chemical composition information of R.sceleratus L.Therefore,the purpose of this study is to use a variety of separation methods and structural characterization techniques to study the chemical components of different extraction sites of the alcohol extract of R.sceleratus L.At the same time,the anti-inflammatory activity of the isolated monomer compounds in vitro was studied,and the monomer components with better activity were screened out,which provided inspiration for further pharmacological research and laid a foundation for the quality evaluation of R.sceleratus L.Methods:It is planned to systematically separate and purify the ethyl acetate part and n-butanol part of R.sceleratus L.by using preparative high performance liquid chromatography,MCI column chromatography,Sephadex LH-20 gel chromatography,silica gel chromatography,thin layer chromatography,preparative thin layer chromatography and recrystallization.The structure of the isolated compounds was identified by the determination of physical and chemical constants,one-dimensional and two-dimensional spectrum technology(¹H-NMR,¹³C-NMR,DEPT,HMBC,HSQC,¹H-¹HCOSY and NOESY),and searching the micro-spectrum database and comparing the references.CCK-8 method was used to determine the cytotoxicity of isolated monomer compound RAW264.7 on mouse macrophages,and Griess reagent method was used to determine the effect of monomer compound on LPS-stimulated RAW264.7release of NO,and detection of TNF-α,IL-1βand IL-6 by ELISA,for further research on compounds with strong activity,so as to preliminarily evaluate the anti-inflammatory activity of monomer compounds.Results:Thirty-nine monomer chemical components were isolated and identified from the ethyl acetate and n-butanol parts of Euonymus japonicus,are respectively megastigmane（1）,isoscopoletin-β-D-glucopyranoside（2）,salidroside（3）,sinapateglucose-1-ester（4）,quercetin-3-O-β-D-glucopyranoside（3′→O-3′′′）quercetin-3′′-O-β-D-galactopyranoside（5）,quercetin-7-O-β-D-glucopyranoside（6）,camsibriside A（7）,Tangutoside（8）,oresbiusin A（9）,4-[2-formyl-5-（methoxymethyl）-1H-pyrrol-1-yl]butanoic acid（10）,5-（hydroxymethyl）furfural dimer（11）,4-hydroxy-benzal-dehyde（12）,（+）-dehydrovomifoliol（13）,isoscopoletin（14）,ethyl3-（3,4-dihydroxyphenyl）lactate（15）,3-Hydroxy-5α,6β-epoxy-β-ionone（16）,indole-3-acetic acid（17）,ethyl 3,4-dihydroxybenzoate（18）,indazole（19）,5-Hydroxymethyl-furfural（20）,3,4-dihydroxy-β-methoxyphen-ethylalcohol（21）,3,4-dihydroxyphenacyl alcohol（22）,caffeic acid（23）,protocatechuic acid（24）,（E）-4-hydroxyhex-2-enoicacid（25）,nicotineacid（26）,1-O-p-coumaroyl-β-D-glucopyranose（27）,bis（2-ethylhexyl）phthalate（28）,brainoside B（29）,ranunsceleroside A（30）,dihydrodehydrodiconiferyl alcohol-4-O-β-D-glucopyranoside（31）,4-carbonyl-5-hydroxy methyl valerate（32）,γ-keto-δ-valerolactone（33）,ranunscelerosideB（34）,3-（3’,4’-dihydroxyphenyl）-（2R）-lactic acid（35）,cinnacasolide C（36）,coniferin（37）,mongophenoside B（38）,7S,8R-urolignoside-9′-O-β-D-glucoside（39）.Among them,compounds 30 and 34 are new compounds.Except for compounds 6,9,14,23 and 24,other compounds are isolated from Ranunculus for the first time.Further screening the anti-NO activity of monomer compounds in vitro,the results showed that compounds 5-6,9,13,15,18,22-23,27-28,30 and 34-38 had various degrees of anti-inflammatory activity IC₅₀ ranging from 4.29 to 101.08μM;Compounds 6,18,36,37 and 38 have obvious inhibitory effects on NO production,of which compounds 37 and 38 have the most significant effects,with IC₅₀ values of 4.29and 9.29μM respectively,compound 37 and 38 have a certain inhibitory effect on the release of TNF-α,IL-1βand IL-6.Conclusion:In this study,the systematic separation of R.sceleratus L was carried out,and the chemical composition of the n-butanol part of R.sceleratus L was studied for the first time,and the anti-inflammatory activity of the monomer components isolated from R.sceleratus L was preliminarily evaluated.The research results can enrich the chemical composition of R.sceleratus L,provide scientific basis for further research on the material basis of its efficacy and further rational development and utilization.