Chemical Separation Of Essential Oil From Eupatorium Adenophorum And Its Anti-inflammatory Effect In Vitro

Inflammation is a common defense response of the body in the face of external damage or pathogenic factors.At present,the drugs for treating inflammatory reactions in the clinic are mainly western medicines,and western medicines can cause side effects and drug residues in the treatment of inflammation.Although Ageratina adenophora is a toxic invasive plant,which essential oil(AAEO)contains various anti-inflammatory compounds such as α-pinene,caryophyllene and borneol acetate,and the anti-inflammatory activity hasn,t been reported ever.Therefore,in this study,RAW264.7 cells line was treated with LPS and then disposed with AAEO.Finally,the transcription level of inflammatory factors were determined by elisa and RT-PCR.And the expression level of TLR4 protein and key protein in NF-kappa B signaling pathway were tested by Westen Blot.Thus,it provided a theoretical basis and reference value for the study of anti-inflammatory activity of AAEO in vitro.The research contents are as follows:1.AAEO chemical separation and its component analysis.The results showed that 26 compounds of the AAEO were detected out,and 9substances of them had anti-inflammatory activity.It indicated that the anti-inflammatory substances of AAEO were determined by the GC-MS.2.Effects of AAEO isolates on LPS-induced secretion of inflammatory factors in RAW264.7 cellsThe results showed that there was no cytotoxicity to RAW264.7 cells at drug concentration <20 mg/mL,and the drug concentrations selected in the subsequent experiments were: 1 mg/mL,5 mg/mL,10 mg/mL.The AAEO isolated product at 1mg/mL was able to significantly inhibit the NO content secreted by RAW264.7 cells after LPS treatment,and showed a dose-effect relationship.Compared with LPS group,the drug group could significantly inhibit the secretion of IL-1β,IL-6,IL-8 and TNF-α protein,and significantly up-regulate the secretion of IL-10 protein.Compared with the LPS group,the drug group significantly inhibited the expression of IL-1β,IL-6,IL-8 and TNF-α mRNA at1 mg/mL,and significantly up-regulated the expression of IL-10 mRNA.It was indicated that the extract of AAEO can up-regulate the expression and protein secretion of IL-10 mRNA and inhibit the expression and protein secretion of IL-1β,IL-6,IL-8,TNF-α mRNA in inflammatory response to exert anti-inflammatory activity.3.Western blot was used to detect the effect of the extract of AAEO on TLR4 protein and NF-κB signal transduction pathway with LPS in RAW264.7 cells.The results indicated that the TLR4 protein was inhibited by the extract of AAEO in RAW264.7 cells line with LPS.The expression of inhibitory IκBα protein with LPS was up-regulated.It can up-regulate the expression of IκBα protein inhibited by LPS,and increase the content of p65 and p50 cytoplasmic protein in RAW264.7 cells by promoting LPS,which can inhibit the activation of NF-κB signaling pathway and achieve anti-inflammatory effects.Conclusion:Among the 26 compounds detected by AAEO chemical separation products,9 anti-inflammatory substances were found.They had no cytotoxicity to RAW264.7 cells at concentrations less than 20 mg/mL,and could regulate the secretion of inflammatory factors by regulating the expression of TLR4 protein and key proteins in NF-κB signaling pathway in LPS-induced RAW264.7 cells,thus achieving anti-inflammatory effect.