Study On Anti-inflammatory Active Components And Its Mechanism Of Euscaphis Konishii Hayata Peel

Euscaphis konishii Hayata belongs to theaceae and Euscaphis genera,which has strong functions of anti-inflammatory,analgesic,heat-clearing and detoxification,detumescence and dispersal,etc.It has a good medicinal foundation and a long history of medicine in the folk.Some studies have shown that the extract of its pericarp had a good inhibitory effect on the ear swelling inflammation induced by xylene in mice.However,the anti-inflammatory mechanism has not been clarified.The study on the anti-inflammatory activity of natural products from plants has become the need of medical development.In this study,the selection of active parts of the extract from the pericarp of Euscaphis konishii Hayata and the study of its anti-inflammatory effect and mechanism were carried out.The following results were obtained:1.The extract fragment EKH1216 from the pericarp of Euscaphis konishii Hayata with strong anti-inflammatory activity was screened out.We construct an anti-inflammatory cell model of RAW264.7 induced by LPS in vitro,administration of drugs with different extracts from the pericarp of Euscaphis konishii Hayata.According to the content of NO released from the supernatant of cell culture,the inhibitory effects of different extracts from the pericarp of Euscaphis konishii Hayata on the production of NO in the cells were calculated.The extract fragment EKH1216 from the pericarp of Euscaphis konishii Hayata,which had strong anti-inflammatory activity,could inhibit the morphological changes induced by LPS during cell culture.The half inhibitory concentration(IC50)on NO production was 78.47μg·mL-1,and there was no obvious inhibitory effect on the proliferation of RAW264.7 cells at each test concentration,showing low toxicity and strong anti-inflammatory activity.2.Study on the anti-inflammatory molecule mechanism of EKH1216.According to the inhibitory effect of EKH1216 on the production of NO by IC50,the concentration of EKH1216 was 50,100 and 200 μg·mL-1,respectively.The anti-inflammatory molecular mechanism of EKH1216 in RAW264.7 cell was studied by ELISA,qRT-PCR and Western blot.In murine macrophage inflammatory model induced by LPS,EKH1216 inhibits the expression of COX-2 and iNOS proteins by inhibiting the production of COX-2 and iNOS mRNA,thus reducing the synthesis of PGE2 and NO.EKH1216 affects the secretion of TNF-α,IL-1β and IL-6 by regulating the mRNA level of TNF-α,IL-1β and IL-6,and inhibits the expression of the inflammatory factor IL-1β protein.In addition,EKH1216 can block p65 nuclear transport induced by LPS in NF-κB signal transduction pathway,inhibit the expression of p38,P-p38,JNK,P-JNK and P-ERK in MAPK signaling pathway,and down-regulate the expression of STAT1,STAT3 and P-STAT3 protein in JAK-STAT pathway.Thus inhibit the expression of inflammatory related factors and play an antiinflammatory effect.3.EKH1216 has obvious protective effect on immune liver injury in mice.BCG/LPS was used to construct immune liver injury mice model to study the effect of EKH1216 on immune liver injury in mice.The results show that:1)EKH1216 significantly decreased the activity of serum ALT in mice,and the percentage of decreasing enzyme was 42.6%,44.8%and 47.3%,respectively.The activity of serum AST was significantly decreased in the high dose group of EKH1216,and the percentage of decreasing enzyme was 28.8%.2)EKH1216 and bifendate could significantly inhibit the increase of liver index in mice with immune liver injury induced by BCG/LPS.The pathological degree of liver tissue in EKH1216 medium and high dose group and biphenyl diester group was lower than that in model group,the difference was statistically significant.It is suggested thatEKH1216 can promote the regeneration and repair of hepatocytes.The protective effect of EKH1216 on liver injury induced by BCG/LPS in ILI mice was similar to that of biphenyl diester positive control.3)EKH1216 could significantly inhibit the NO content and increase the GSH-Px activity in the liver tissue of mice.The activity of SOD in liver tissue of mice could be significantly increased by EKH1216 high dose group and biphenyl diester group.EKH1216 except for low dose group,and biphenyl diester could significantly inhibit MDA content in liver tissue of mice.4)The low dose group of EKH1216 could significantly inhibit the levels of IL-6 and PGE2 in the liver tissue of mice,and the levels of TNF-α,IL-6 and PGE2 in the liver tissue of mice were significantly inhibited in the biphenyl diester group and in the medium and high dose group of EKH1216.Thus,it can be seen that each dose of EKH1216 has protective effect on the liver of immune liver injury mice and in a dose-dependent manner.In conclusion,the model of macrophage inflammation induced by LPS in RAW264.7 mice was established,and the extract from the pericarp of Euscaphis konishii Hayata was used as the research object.The inflammatory model was used to detect the changes of NO content and cytotoxicity in RAW264.7 cells from different extracts from the pericarp of Euscaphis konishii Hayata.EKH1216,a strong anti-inflammatory site of extracts from the pericarp of Euscaphis konishii Hayata,was screened.The effects of EKH1216 on the morphology of RAW264.7 cells were studied.The anti-inflammatory activity and its molecular mechanism were determined by further determination of the expression of related inflammatory factor genes and proteins and the expression of inflammatory related signaling pathway in the inflammatory model.At the same time,EKH1216 was used to study the animal model of immune liver injury in mice.It was found that EKH1216 had a good anti-liver injury effect.The effect of EKH1216 on inflammatory cytokines in vivo was elucidated,and the anti-inflammatory effect of EKH1216 in vivo and in vitro was further clarified.It can provide a good theoretical and experimental basis for the development and utilization of medicinal resources of Euscaphis konishii Hayata.