Evaluation Of Patchoulene Epoxide On The Activity Of Anti-inflammation And Gastroprotection

ObjectivesIn our previous study,Samples of patchouli oil were assayed to analysis the effect of storage time on compositional variation,of which an unknown compound was found in the long-stored sample,but not exixted in the oil with one-year storage time.The unknown compound was then separated from stale patchouli oil using fractional distillation and underwent structural analysis.The unknown compound was identified as patchoulene epoxide(PAO).Since not so many research on PAO can be searched and these are mainly related to the domain of chemical synthesis,its biological activity still remains unknown,which definitely limites the application of PAO.Considering that PAO belongs to the family of Azulene which are usually deemed the noticeable capability of anti-inflammation and antioxidation.Therefore,to develop the pharmacological application of PAO,this dissertation first conducted a preliminary acute toxicity study to confirm whether PAO was essentially non-toxic and then try to proceed on PAO from the two aspects mentioned above to explore its potential pharmacological effects and mechanisms.Methods 1.The anti-inflammatory effect of PAO in vitroGC-MS technology was adopted to explore the effect of storage time on the composition of different patchouli oil samples.According to the result,there was an unknown component that can only be found in the long-stored oil,which later was identified as patchoulene epoxide(PAO).LPS-stimulated RAW264.7 macrophages was applied to explore the antiinflammatory effect of PAO in vitro.The underlying mechanism was also discussed.Specifically,the level and mRNA expression of pro-inflammatory cytokines such as TNF-α,IL-1β,IL-12 and MCP-1 was measured,accompanied by the detection of COX-2 and iNOS signaling pathway.2.The anti-inflammatory effect of PAO in vivoThis study aims to evaluate the potential anti-inflammatory activity of PAO in vivo by using three animal models: xylene-induced ear edema,acetic acid-induced vascular permeability,and carrageenan-induced paw edema.Further investigation into its underlying mechanism on carrageenan-induced paw edema was conducted.Specifically,TNF-α,IL-1β,IL-6,IL-4,IL-10,PGE2 and NO was measured,while the protein and mRNA expressions of COX-2 and iNOS was also detected.In addition,western blot analysis was used to determinate the protein expression of NF-κB signaling pathways.3.Prophylactic efficacy of PAO against gastric ulcerThis study is committed to evaluate the prophylactic efficacy of PAO against gastric ulcer.Ethanol-induced gastric ulcer model was conducted.Macroscopic examination and histological evaluation are first combined to weigh the anti-ulcerogenic activity of PAO.Four dimensions,i.e.inflammatory response,oxidative stress,apoptosis and gastric mucosal defense are the points that I used to elucidate the potential mechanisms.Specifically,TNF-α,IL-1β,IL-10 and NF-κB pathway-related proteins are the indicators detected to evaluate the anti-inflammatory activity of PAO.GSH,SOD,CAT and MDA are used to measure the antioxidant activity of PAO.Caspase-3,Fas and Fasl belongs to apoptosis pathway and the level of PGE2 and NO represent the capacity of gastric mucosal defense.Results 1.The anti-inflammatory effect of PAO in vitroAccording to the results,pre-treatment with PAO significantly decreased the protein and mRNA levels of pro-inflammatory cytokines including TNF-α,IL-1β,IL-12 and MCP-1 in a dose-dependent manner.In addition,real-time PCR also revealed that PAO could interrupt the mRNA expressions of iNOS and COX-2 and thus,decreased the levels of NO)and PGE2 in LPS-stimulated RAW264.7 macrophages.2.The anti-inflammatory effect of PAO in vivoIn this study,the LD50 value of PAO was greater than 10 g/kg,indicating that there was a relatively wide margin of safety for PAO.Meanwhile,PAO could significantly inhibite the ear edema induced by xylene,lower the vascular permeability induced by acetic acid and decrease the paw edema induced by carrageenan.Moreover,PAO markedly decreased the levels of TNF-α,IL-1β and IL-6,but increased the levels of IL-4 and IL-10.PAO was also shown to significantly downregulate the expression of COX-2 and iNOS signaling pathway.Western blot analysis revealed that PAO remarkably inhibited p50 and p65 translocation from the cytosol to the nucleus by suppressing IKKβ and IκBα phosphorylation.3.Prophylactic efficacy of PAO against gastric ulcerAccording to the results,macroscopic examination revealed that PAO could significantly reduce ethanol-induced gastric ulcer areas as compared with the vehicle group,which was also supported by the histological evaluation result.As for its potential mechanism,the anti-inflammatory activity of PAO contributed to gastric protection through reversing the imbalance between pro-and anti-inflammatory cytokines and modulating the expressions of NF-κB pathway-related proteins including p-IκBα,IκBα,p-p65 and p65.Besides,PAO was able to enhance the expressions of antioxidant enzymes including GSH,SOD and CAT,and down-regulate MDA,an indicator of lipid peroxidation.Furthermore,immunohistochemistry analysis exhibited potent anti-apoptosis effect of PAO,as evidence by down-regulating the protein expression of caspase-3,Fas and Fasl.Additionally,we also demonstrated that PAO could replenish PGE2 and NO mucosal defense.ConclusionAbove all,this dissertation is the first report that discovered and succeed in isolating PAO from the long-stored patchouli oil,which has also been proven non-toxic.Furthermore,the results confirm that PAO possesses anti-inflammatory activity in vitro and in vivo.Also,PAO has significant gastric protective effect,able to resist ethanol-induced gastric injury.In addition,the anti-ulcerogenic mechanism of PAO is associated with its capability of antiinflammation,anti-oxidation,anti-apoptotic and gastric mucosal protection.