Studies On Effective Substances And Quality Evaluation Of Kumu Injection

Objective:Kumu injection,prepared by the dry sticks or stems of Picrasma quassioides（D.Don）Benn.,is included in Volume 20 of"Drug Standard of Ministry of Public Health of the Peoples Republic of China"（WS3-B-3887-98）.As the result of the repeated acid-base adjustments during the production process of Kumu injection,its chemical compositions are significantly different from P.quassioides.Currently,no literature has reported the chemical constitutes of Kumu injection.The methods of qualitative identification and quantitative control of index components are still absent.In addition,the effective substances of Kumu injection need to be clarified.In our study,the chemical compositions of Kumu injection were systematically separated,and then their anti-inflammatory activity in vitro were evaluated.Finally,based on the effective substances of injection,the improved quality evaluation standard of Kumu injection was established.Methods:1.The Kumu purified extract was separated and purified by silica gel column chromatography,ODS column chromatography,sephadex column chromatography and liquid phase preparation;the structures of the compounds were identified by NMR,mass spectrum,IR and their physicochemical properties.2.Inflammatory cell model was constructed by LPS acting RAW264.7 cell line.It was used to evaluate the inhibitory effects on NO production of the isolated compounds,and the effective substances of Kumu injection was clarified.To investigate the effects of the anti-inflammatory activity compounds on cellular metabolites before and after injury of LPS acting RAW264.7 cell line,and to find potential biomarkers based on cell metabolomics techniques.3.UPLC-Q-TOF-MS/MS was applied to establish the total ion chromatogram of alkaloids in P.quassioides-intermediate-Kumu injection.Combined with the production process of Kumu injection,the chemical composition change rule between P.quassioides-intermediate-Kumu injection were studied.4.The TLC method was established of Kumu injection;The HPLC fingerprint and HPLC quantitative methods of index components of P.quassioides-intermediate-Kumu injection were established.A revised draft of quality standard was established.Results:1.Study on the chemical constituents of Kumu injection:Using HR-ESI-MS,IR,¹H-NMR,¹³C-NMR,HSQC,HMBC,DEPT,¹H-¹H-COSY,ROESY methods combined with their physical and chemical properties to identify the structure of the isolated compounds,finally 43 compounds were isolated from the Kumu injection extract,including 4-methylharman（1）,N-methyl-β-carbolinium（2）,β-carboline-1-propanoic acid（3）,β-Carboline-1-carboxylic acid（4）,1-hydroxy-β-carboline（5）,1-methoxycarbony-β-carboline（6）,1,6-dihydroxy-β-carboline（7）,β-carboline glucopyranoside（8）,1,2,3,4-Tetrahydro-1,3,4-trioxo-B-carbo1ine（9）,3-methylcanthin-5,6-dione（10）,4-methoxy-5-hydroxyeanthin-6-one（11）,picraquanine D（12）,picraquanine E（13）,picrasin B（14）,nigakilactone N（15）,2,5-dihydroxybenzoic acid（16）,3,4-dihydroxybenzoic acid（17）,vanillic acid（18）,syringic acid（19）,（2S）-6-methoxyl-7-hydroxytropic acid（20）,picraquanine A（21）,glycerol-2-（3-methoxy-4-hydroxybenzoicacid）ether（22）,picraquanine B（23）,picraquanine C（24）,picraquanine F（25）,4-hydroxy-3,5-dimethoxybenzamide（26）,3-（4-hydroxy-3-methoxyphenyl）propane-1,2-diol（27）,pedatisectine F（28）,2-methyl-6-（2,3,4-trihydroxybutyl）pyrazine（29）,（E）-4-（6-methylpyrazin-2-yl）but-3-ene-1,2-diol（30）,（2S）-1-[2-（furan-2-yl）-2-oxoethyl]-5-oxopyrrolidine-2-carboxylic acid（31）,nicotinic acid（32）,（E）-3-[5-（hydroxymethyl）furan-2-yl]methylene-4-oxo-pentanoic acid（33）,5-hydroxymethyl-furoic acid（34）,2-Furan acid（35）,polybotrin（36）,glansreginic acid（37）,dihydrophaseic acid（38）,（2E,4E,1’S,2’R,4’S,6’R）-dihydrophaseic acid（39）,2-（3’-hydroxy-5-methoxyphenyl）-3-hydroxylmethyl-7-methoxy-2,3-dihydrobenzofuran-5-carboxylicacid（40）,（+）-isolariciresinol（41）,5-hydroxy-3-methyl-pent-2c-enoic acid（42）,muconic acid（43）.These compounds include 19 alkaloids（4 new compounds）,4 quassinoids（2 new compounds）,9 phenolic derivatives,3 furan compounds,and 8 other compounds.The study on the chemical constituents of Kumu injection provides a material basis for the pharmacological activity and LC-MS identification of Kumu injection.2.In vitro anti-inflammatory activity evaluation of the chemical constituents in Kumuinjection:1-carboxy-β-carboline,1-hydroxy-β-carboline,1-carbomethoxy-β-carboline and 1,6-dihydroxy-β-carboline have anti-inflammatory activity.It explains thatβ-carboline alkaloid is the main anti-inflammatory ingredient in Kumu injection,which provides a theory basis for the discovery of its quality markers.Furthermore,inflammatory cell model was used to compare the metabolic differences before and after the treatment of 1-carboxy-β-carboline,an anti-inflammatory compounds of Kumu injection.15 biomarkers were screened by metabolomics technology and related database analysis,mainly related to glycerophospholipid metabolism,ether lipid metabolism and glutathione metabolism pathway.This study provides a reference for the study of anti-inflammatory activity of Kumu injection.3.UPLC-Q-TOF-MS/MS identification analysis of P.quassioides-intermediate-Kumu injection:In this study,UPLC-Q-TOF-MS/MS was used to establish a qualitative analysis method for P.quassioides-intermediate-Kumu injection.The main contents of P.quassioides areβ-carboline,canthin-6-one and dimer alkaloids.The main contents of intermediate and Kumu injection areβ-carboline,canthin-6-one alkaloids.Combined with the production process of Kumu injection,the chemical composition change rule between P.quassioides-intermediate-Kumu injection were studied.The results deduced that the dimer alkaloids of poorly water-soluble are removed from P.quassioides to the intermediates.There are no significant differences in the chemical constituents between the intermediate and Kumu injection.They mainly containβ-carboline and canthin-6-one alkaloids.The main difference was that the content of canthin-6-one alkaloids in Kumu injection was significantly reduced.β-carboline alkaloids are the highest alkaloid in Kumu injection.This study provides material basis theory for the quality control of Kumu injection.4.The quality improvement of Kumu injection:The TLC method was established of Kumu injection by usingβ-carboline-1-propanoic acid as the indicator component,sticks or leaves of P.quassioides and stems of P.quassioides as control.The HPLC fingerprint of P.quassioides-intermediate-Kumu injection was established.It was found that there were obvious differences among different batches except the intermediate Kumu extract.The common peaks of P.quassioides-intermediate-Kumu injection wereβ-carboline-1-carboxylic acid andβ-carboline-1-propanoic acid.Therefore,the HPLC content determination method was established by assigningβ-carboline-1-carboxylic acid andβ-carboline-1-propanoic acid as index component in P.quassioides-intermediate-kumu injection.This study established a revised draft of quality standard for Kumu injection.Conclusion:This study determined that the effective substances of Kumu injection wereβ-carboline alkaloids.The quality standard of Kumu injection was improved.It is of great significance for the stability of quality and safety of clinical use of Kumu injection.