Chemical Constituents From Mussaenda Pubescens And Anti-inflammatory Activity

Objective:To study the chemical constituents from the dried roots and stems of Mussaenda Pubescens Ait.f.The 4 chemical components in the Mussaenda Pubescens were determined and its anti-inflammatory activity was screened in vitro.This topic provides a theoretical basis for further research on the chemical composition and biological activity screening of Mussaenda Pubescens.It lays a foundation for clarifying its anti-inflammatory activity and has important scientific significance for the development and utilization of Mussaenda Pubescens.Methods:The crude powder of Mussaenda Pubescens was extracted by ethanol for 3 times,and the solvent was recovered to obtain the total ethanol extract.The total extract was extracted with petroleum ether and ethyl acetate in turn,and the solvent was recovered.The ethyl acetate fraction and ethanol fraction of Mussaenda Pubescens was selected for further separation.The chemical components were separated and purified by silica gel chromatography,ODS chromatography,MCI chromatography,macroporous resin D101 chromatography and Sephadex LH-20 chromatography.The monomeric compound obtained from Mussaenda Pubescens was verified by HPLC for the purity of the monomer compound,and the structure was identified by Pop methods such as LC-MS,¹H-NMR,¹³C-NMR,DEPT.HPLC was used to determine the content of chlorogenic acid in Mussaenda Pubescens,and the content of isochlorogenic acid A,isochlorogenic acid B and isochlorogenic acid C were determined by using chlorogenic acid of Mussaenda Pubescens as a standard by a method of multi-evaluation.The external standard method and QAMS were used to determine the content of four compound components in different producing areas,and the difference between the two methods was compared.By inducing inflammation of mice macrophages?RAW246.7?with polylipopolysaccharide?LPS?,the cytotoxicity of chlorogenic acid isochlorogenic acid A,isochlorogenic acid B and isochlorogenic acid C in a certain range was determined by MTT,meanwhile,the inflammatory factors of NO,IL-6 and TNF-?were determined.The activity of four compounds was explored by this method.Result:15 compounds were isolated from the ethyl acetate fraction and ethanol extract of Mussaenda Pubescens,and the structure of 14 compounds was identified,namely shanzhiside methyl ester?1?;schimoside?2?,Mussaendiside L?3?,cryptochlorogenic acid methyl ester?4-dicaffeoylquinic acid methyl ester??4?,chlorogenic acid methyl ester?3-dicaffeoylquinic acid methyl ester??5?,mussaenoside?6?,isochlorogenic acid B methyl ester?3,4-dicaffeoylquinic acid methyl ester??7?,isochlorogenic acid A methyl ester?3,5-dicaffeoylquinic acid methyl ester??8?,isochlorogenic acid C methyl ester?4,5-dicaffeoylquinic acid methyl ester??9?,quinovic acid3-O-?-D-glucopyranosyl-28-O-?-L-rhamnopyranosyl ester?10?,chlorogenic acid?3-dicaffeoylquinic acid??11?,isochlorogenic acid B?3,4-dicaffeoylquinic acid??12?,isochlorogenic acid A?3,5-dicaffeoylquinic acid??13?,isochlorogenic acid C?4,5-dicaffeoylquinic acid??14?.The chlorogenic acid in the medicinal materials of the plant has a good linear relationship at 0.2547².5475?g?R²=0.9999?.Using chlorogenic acid as the internal reference material,the relative correction factors of isochlorogenic acid B,isochlorogenic acid A and isochlorogenic acid C were calculated respectively to achieve a multi-assessment.The results showed that there was no significant difference between the results of one test and the external standard.The relative correction factor measured by the experiment was reproducible.Determination of chlorogenic acid,isochlorogenic acid B,isochlorogenic acid A,and isochlorogenic acid C have good linearity in the range of 0.1936⁰.9680?g,0.2016-1.0080?g,0.1956-0.9780?g,and0.2016-1.0080?g,respectively.Relationship,R² is greater than 0.999.The methodological findings in the determination of content all meet the relevant requirements.In the concentration range of 20¹00?M,the cell proliferation of the four components was not statistically significant compared with the blank group,and there was a certain difference in anti-inflammatory activity.The anti-inflammatory effect of chlorogenic acid was not obvious.Isochlorogenic acid B has the best anti-inflammatory effect.Chlorogenic acid has a certain anti-inflammatory effect when administered at 80?M,while isochlorogenic acid B has certain anti-inflammatory activity at 20?M,when the concentration reaches 80?M.It has been able to completely consistent inflammation.When investigating the anti-inflammatory activity of three other compounds except chlorogenic acid,when the concentration is 20⁶0?M,it has a certain inhibitory effect on the release of IL-6,but the release of TNF-?.There is no inhibition effect.Conclusion:Compounds 2,4,5 and 7-14 were isolated from the plant for the first time.The method for the determination of organic acids in Mussaenda Pubescens was simple,feasible and reproducible.The method of one-test and QAMS was applied to the determination of organic acids in Mussaenda Pubescens with low cost and good reproducibility,which was the quality evaluation of Mussaenda Pubescens and provided scientific reference.Compounds of Caffeoylquinic acid from Mussaenda Pubescens have certain anti-inflammatory activities.