Optimization Of Extraction Process Of Neobractatin From Garcinia Bracteata And Evaluation Of Its Anti-inflammatory Activity In Vitro

Inflammation is an extremely complex process.Mild or rational inflammatory reactions can remove harmful substances or agents and promote tissue repair.Excessive or dysregulated inflammatory reactions may cause tissues to fail to fully recover from health and induce chronic diseases.Although a large number of synthetic anti-inflammatory compounds have been developed over the past few decades,most anti-inflammatory drugs have harmful side effects on the body after long-term use.Therefore,the development of high-efficiency,low-toxic,anti-inflammatory drugs with low side effects from natural medicines has received more and more attention,and the research on its anti-inflammatory mechanism has become a hot spot in the field of new drug research and development.Garcinia bracteata C.Y.Wu is a plant of the genus Garcinia.Cambogia,in which the main active ingredient is caged xanthone has obvious antitumor activity.Clinical studies have shown that the anti-tumor activity of the drug is closely related to the anti-inflammatory activity.Therefore,in this study,neobractatin,a caged xanthone component isolated from G.bracteata,was used as a research object,and mouse mononuclear macrophages were stimulated by LPS（RAW264.7）to establish an inflammatory model to explore the in vitro anti-inflammatory effects of neobractatin.In this study,the response surface methodology,ultrasonic extraction and high performance liquid chromatography（HPLC）were used to establish the method for determining the content of the caged xanthone in the leaves of G.bracteata.Mouse mononuclear macrophages（RAW264.7）were stimulated by LPS for 18 hours to establish an inflammatory model.The levels of TNF-α,IL-6,IL-1β,IL-10 and PGE₂ in the supernatant of RAW264.7 inflammatory cells were determined by ELISA kit after detection of neobractatin（0.4μM、0.8μM and 1.6μM）.The method was established to detect the cytotoxicity of neobractatin on RAW264.7 cells and the content of NO in the supernatant of inflammatory cells after neobractatin treatment by using MTT method and Griess.The effects of neobractatin on the secretion of inflammatory cytokines and inflammatory mediators were further investigated.The effects of neobractatin on the expression of i NOS and COX-2 genes and protein in inflammatory mediators were detected by real-time PCR and Western Blot.The results showed that the optimal extraction process of neobractatin is:liquid-to-liquid ratio 30m L/g,extraction temperature at 30℃,extraction time for 90 min;The actual yield of neobractatin under this process condition is 2.24mg/g,which is basically consistent with prediction result of the model（2.36mg/g）.In the anti-inflammatory activity test,neobractatin significantly reduced the levels of NO,TNF-α,IL-6 and IL-1βsecreted by LPS-induced mouse monocyte-macrophage（RAW264.7）,significantly reducing PGE₂.The content of IL-10 was significantly increased at the same time.At level of the gene and the protein,neobractatin significantly reduced the expression of i NOS and COX-2.The study found that neobractatin could stimulate the levels of inflammatory factors（TNF-α,IL-6 and IL-1β）and inflammatory mediators（NO,PGE₂）in RAW264.7 cells by reducing LPS and exhibit anti-inflammatory effects;Reduce the i NOS,COX-2 gene and protein expression levels to further exert anti-inflammatory effects.In summary,this study preliminarily revealed the content of neobractatin in the plant and its anti-inflammatory effect in vitro,providing a theoretical basis for the development of neobractatin as a novel anti-inflammatory drug and also offer the theoretical basis for the in-depth development and application of the new green veterinary drugs with high-efficiency and low-toxic anti-inflammatory activities from the caged xanthone component.