Study On Anti-inflammatory Mechanism And Pharmacokinetics Of Essential Oil From Artemisiae Argyi Folium

[Background]Artemisia Argyi Folium,the dried leaves of Artemisia argyi Levl.Et Vant.,is mainly used for vomiting blood,metrorrhagia,dysmenorrhea,irregular menstruation,and infertility,etc.It is a commonly used herbal medicine in gynecology of traditional Chinese medicine（TCM）.The essential oil in A.argyi（AAEO）is the major chemical component in the herb and pharmacological studies have shown that it has anti-inflammatory,antibacterial,antitussive,expectorant,and antiallergic activities.Many effects of A.argyi are related to the inhibition of inflammatory response,but the material basis and target of AAEO have not been clarified,which limits its application in clinical practice.[Purpose]In present study,we aimed to reveal the anti-inflammatory mechanism of AAEO by means of network pharmacology and cell metabolomics,and conduct the study on pharmacokinetics and tissue distribution of AAEO components in rats,to provide theoretical basis for clinical guidance.[Methods]1.The anti-inflammatory mechanism of AAEO:The chemical composition of 10 batches of AAEO was analyzed using gas chromatography-mass spectrometry（GC-MS）.Based on the research of chemical composition,network pharmacology was conducted to predict the target of anti-inflammatory activity of AAEO,using path enrichment analysis to find anti-inflammatory related pathways,then cell metabolomics technique was use to observe the lipid oxidation changes in LPS-induced RAW264.7 cells before and after AAEO treatment.Through principal component analysis,cluster analysis and partial least squares discriminant analysis,differential metabolites were screened,and their anti-inflammatory mechanisms were analyzed.Finally,inflammation model of RAW264.7 cells was used to verify the relevant targets screened by network pharmacology and metabolomics.2.Pharmacokinetics and tissue distribution of the main active ingredient of AAEO:The active components in rat plasma after gavage of AAEO were screened by GC-MS analysis in serum pharmacochemistry experiments.A qutitative method of these components by GC-MS/MS was established with the specificity,linearity,precision and accuracy,extraction recovery,matrix effect and stability of the method investigated.After SD rats were given AAEO by injection（25mg/kg）or gavage（125,250,500 mg/kg）,blood was collected at different time points.The plasma samples were processed by liquid-liquid extraction,and the drug concentrations were analyzed by GC-MS/MS to calculate pharmacokinetic parameters.The tissue distribution study was carried out in SD rats gavaged with AAEO（250 mg/kg）,which were sacrificed by decapitation at 0.083,0.5 and 2h after administration.Ten tissues including brain,lung,liver,kidney,heart,spleen,muscle,skin,stomach,small intestine were immediately taken out.After the samples are processed,the content of target components is analyzed by GC-MS/MS.[Results]1.A total of 39 compounds in AAEO were identified by GC-MS,and eight common components were found including eucalyptol,α-thujone,camphor,borneol,eugenol,borneol acetate,β-caryophyllene and caryophyllene oxide,accounting for 61.47%-73.71%of the total volatile oil content.The eight components were imported into the database,and 57 targets of the above components were retrieved with4522 disease targets obtained.By intersecting disease targets and drug targets,a total of 44 potential targets were obtained.Based on these targets,a PPI protein interaction network was established,and then GO and KEGG analysis were used to obtain relevant biological processes and pathways,respectively.It was found calcium signaling pathway,c AMP signaling pathway and arachidonic acid（AA）metabolism are signaling pathways related to inflammation.Finally,the accuracy and reliability of the prediction results were proved by molecular docking.The cellular metabolomics experiment found that the pathway of arachidonic acid metabolism was the main metabolic pathway by KEGG enrichment analysis of the differential metabolites,which is consistent with the results of network pharmacology.The results of cell experiments showed that the three doses of AAEO can significantly reduce the expression of AA in inflammatory reaction.Three doses of AAEO inhibited the expression of PLA2 and had no significant effect on its enzymatic activity,and all doses had a significant inhibitory effect on the enzymatic activity and protein expression of 5-LOX,while only high concentration could decrease the protein expression of COX-2.2.Eight components migrating in blood were chosen for further in vivo disposition study including eucalyptol,α-thujone,camphor,borneol,eugenol,borneol acetate,β-caryophyllene,and caryophyllene oxide.Method validation results show the established GC-MS/MS methods meet the requirements of quantitative analysis of eight components in rat plasma and tissue.Pharmacokinetic results showed that the eight analytes were observed diminished sharply at the first hour after i.v.,and cleared out completely（below LLOQ）from circulation within 8 h,in which camphor displayed the highest elimination rate with t_1/2 of 0.133±0.013h and MRT_0-∞of 0.152±0.007 h,while caryophyllene oxide had the lowest clearance rate with t_1/2of 1.21±0.69 h and MRT_0-∞of 1.059±0.478 h.In p.o.administration,eucalyptol,thujone,camphor,borneol,eugenol and caryophyllene oxide were found instantly appeared in plasma 5 min after dosage,and reached the C_max within 0.5 h,while bornyl acetate and caryophyllene exhibited a relatively slower absorption,which achieved their C_max at 1.5 h.The elimination rates were ranked as eugenol>borneol>camphor>eucalyptol>bornyl acetate>α-thujone>β-caryophyllene>caryophyllene oxide.The tissue distribution results show that these eight components can be rapidly detected in each tissue,then rapidly declined to low levels at 2 h in most tissues.Besides GI tract,the compounds exhibited high exposure mainly in blood-rich tissues including liver,heart,kidney,lung,and spleen.β-Caryophyllene displayed excellent hepatic affinity which made it remain high concentration in liver until 2 h.[Conclusion]1.GC-MS to analyze the chemical composition of AAEO,then combine the analysis of network pharmacology and metabolomics,found that Arachidonic Acid Metabolism pathway may play the main path for AAEO to play an anti-inflammatory effect.Pharmacological experiments further showed that AAEO through inhibiting the expression of PLA2reducing the free AA generation,then inhibit the 5-LOX activity and expression and the expression of COX-2 to inhibit the metabolism of AA,reduce the generation of downstream inflammatory medium.Explain that AAEO plays an anti-inflammatory effect by affecting the generation and metabolism of AA.2.A sensitive,accurate and efficient GC-MS/MS method was established for the quantification of eight components in AAEO in rat plasma and tissue samples,which was successfully applied to evaluate the pharmacokinetics and tissue distribution of AAEO in rats.The pharmacokinetic and tissue distribution characteristics of the main components in AAEO were disclosed,which provided a scientific basis for their quality control and clinical medication.