Study On The Anti-Inflammatory Mechanism And Anti-Inflammatory Active Components Of Embelia Parviflora

Objective:1.In this research,LPS induced RAW264.7 cells were used as the inflammatory model to screen the fraction with the strongest inhibitory effect on inflammatory reaction by detecting the effects of n-butanol fraction of Embelia parviflora and its different concentrations of polyamide ethanol elution fraction on LPS induced RAW264.7 cells.2.The effects of n-butanol fraction of Embelia parviflora and its anti-inflammatory components on NF-κB and MAPK signaling pathway in LPS induced RAW264.7 cells inflammation model were detected.3.Chemical composition analysis of the anti-inflammatory effective components of Embelia parviflora.4.Establish the fingerprint of anti-inflammatory effective parts of Embelia parviflora.Method:1.In this research,RAW264.7 cells were induced by LPS.MTT was used to detect the safe concentration range of n-butanol fraction of Embelia parviflora and its different concentrations of ethanol eluting fraction on RAW264.7 cells.ELISA was used to detect the secretion of NO、TNF-α、IL-12、TNF-α and IL-12 by RAW264.7 cells induced by LPS In order to screen out the strongest inhibitory effect of n-butanol fraction of Embelia parviflora on inflammatory reaction,the effects of IL-6、IL-1βand other inflammatory factors were studied.2.The effects of n-butanol Embelia parviflora,(Z)and its effective anti-inflammatory components,30%ethanol eluting fraction(ZJ3)and 50%ethanol eluting fraction(ZJ5),on the expression of p-P38,p-ERK,p-JNK and TNF-α in RAW264.7 cells induced by LPS at the same concentration were detected Objective to investigate the effects of anti-inflammatory components of Embelia parviflora,on NF-kB and MAPK signaling pathway.3.Using UPLC-Q-Exactive The chemical constituents of ZJ3 and ZJ5,the effective anti-inflammatory components of Embelia parviflora,were studied by HRMS.The positive and negative ions were collected at the same time,and the two-stage mass spectrometry scanning mode of full scan and automatic trigger was used.The relative molecular weight and multi-stage ion fragment information of the compounds were matched with the database.Combined with the related literature,the mass spectra of the compounds were deduced,and it was speculated that the effective anti-inflammatory components could be used Chemical composition that can be contained.4.Fingerprint research:HPLC was used to establish the fingerprint of n-butanol part of Embelia parviflora from different areas,and the "similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine(2012 Edition)" was used to calibrate the common peaks and evaluate the similarity,and the corresponding mathematical model was established to classify and analyze them.Result:1.When RAW264.7 cells were stimulated with 1～25 ug/mL LPS for 24 hours,the cell growth rate was not inhibited,but increased.With the increase of LPS concentration,the inflammatory response of RAW264.7 cells was more obvious,and the secretion of pro-inflammatory factor no showed a dose-dependent manner.2.RAW264.7 cells were treated with 2～0.06mg/mL n-butanol fraction and fraction of Embelia parviflora stem for 24 and 48 hours respectively.The results showed that with the increase of concentration and the longer the culture time,the inhibition effect of n-butanol fraction and fraction of Embelia parviflora stem on cell viability was stronger.The concentration above 0.5mg/mL had obvious cytotoxicity,while the concentration below 0.5mg/mL had enhanced cell viability.3.Compared with the blank group,the contents of NO、TNF-α、IL-12、IL-6 and IL-1β in raw 264.7 cells stimulated by lug/mL LPS were significantly increased,and the inflammatory model was successful.The results showed that 30%ethanol eluting fraction and 50%ethanol eluting fraction could significantly inhibit the release of inflammatory factors,70%ethanol eluting fraction could only inhibit the release of inflammatory factors at high and medium dose,90%ethanol eluting fraction could only inhibit the release of inflammatory factors IL-12、IL-6 and TNF-α at high dose,but had no inhibitory effect on the secretion of NO.4.LPS induced inflammation in RAW264.7 macrophages,phosphorylated ERK,P38 and JNK,and increased the relative protein expression of p-ERK,p-P38 and p-JNK.Compared with LPS group,the relative protein expression of p-ERK,p-P38 and p-JNK in n-butanol fraction,30%ethanol elution fraction and 50%ethanol elution fraction of Embelia parviflora decreased significantly,which indicated that the relative protein expression of p-ERK,p-P38 and p-JNK in n-butanol fraction and 30%ethanol elution fraction of Embelia parviflora vine decreased significantly 50%ethanol eluted fraction can inhibit the phosphorylation of ERK、P38 and JNK,thus inhibiting the activation of MAPK signaling pathway,thus playing an anti-inflammatory role.5.In this study,the anti-inflammatory active components of 30%ethanol fraction and 50%ethanol elution fraction of Embelia parviflora were detected by high-resolution mass spectrometry in positive and negative ion mode.According to database matching and secondary mass spectrometry,7 and 5 compounds were identified,mainly including organic acids,flavonoids and their glycosides.The 30%ethanol elution fraction may contain mannitol,5-hydroxymethylfurfural,gallic acid and protocatechuic acid Tea acid,catechin,protocatechuic aldehyde,Sophoricoside,50%ethanol elution site may contain protocatechuic acid,catechin,proanthocyanidin B1,1-caffeoylquinic acid,naringenin.6.HPLC fingerprint of n-butanol fraction of Embelia parviflora was established.Four common peaks were identified by HPLC,which were catechin,proanthocyanidin B1,epicatechin and proanthocyanidin B2.Procyanidin B1 with good resolution and stability was selected as the reference peak.The similarity results showed that the similarity of n-butanol parts of 10 batches of Embelia parviflora was greater than 0.9,indicating that the similarity of n-butanol parts of Embelia parviflora from different regions was high.In the cluster analysis,when d<5,the n-butanol fraction of Embelia parviflora mainly clustered into three groups.Principal component analysis extracted two components as principal components,the cumulative contribution rate reached 89.7453%.According to the principal component spatial score map,the classification results were consistent with the cluster analysis results.Conclusion:1.The anti-inflammatory mechanism of the n-butanol and its different polar elution sites of Embelia parviflora is through inhibiting the production of NO、NF-a、IL-12、IL-6 and IL-1β,and the anti-inflammatory ability of 30%ethanol eluting site and 50%ethanol eluting part of Embelia parviflora is stronger.2.Both the n-butanol fraction and its 30%and 50%ethanol eluting fractions from the stem of Embelia parviflora can inhibit the activation of NF-κB/MAPK signaling pathway to slow down the spread of inflammation and thus play an anti-inflammatory role.3.The main anti-inflammatory active components of Embelia parviflora are phenolic acids and flavonoids.The anti-inflammatory components may be gallic acid,protocatechuic acid,1-caffeoylquinic acid,catechin,naringenin,Sophoricoside,proanthocyanidin B1,protocatechuic aldehyde,etc.4.The established HPLC fingerprint of n-butanol fraction of Embelia parviflora has the characteristics of simple operation,stable and efficient method,strong specificity and so on,which can provide reference for the follow-up control of the quality standard of n-butanol fraction of Embelia parviflora and other pharmacodynamic studies.