Anti-inflammatory Aciticity Mechanisms Of Bioactive Components From Seeds Of Camellia Oleifera Abel

Camellia oleifera Abel. has a long history in China, the seeds are mainly used for the production of Camellia oleifera oil and tea saponin, and there are a large number of studies have shown their biological activities and pharmacological functions. Inflammation which can cause a variety of diseases, such as arthritis, cardiovascular disease, Alzheimer’s, cancer, etc., is a public health problem has been widespread concerned. Through the research of the separation and preparation of the major active ingredients flavonoid glycosides and saponin in C.oleifera seeds, and its anti-inflammatory effects and mechanisms, the study made the following findings:1.Reflux extraction, solvent extraction jointing semi-preparative liquid chromatography, separated and prepared the flavonoid glycosides and saponin in C.oleifera seeds, obtaining four kinds of flavonoid glycosides and five kinds of saponin, namely kaemferol-3-O-[2-O-β-D-glucopyranosyl-6-O-L-rhamnopyranosyl]-β-D-glucopyranoside, kaempferol-3-O-β-D-glucopyranosyl-(1�'4)-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranoside,kaemferol-3-O-[2-O-β-D-xylopyranosyl-6-O-α-L-rhamnopyranosyl]-β-D-glucopyranoside, and kaemferol-3-O-α-L-rhamnopyranosyl(1�'6)-β-D-glucopyranoside; camellia saponin B1/B2or tea saponin H1, tea saponinFl, Assam saponin F, Assam saponinA/E or tea saponin G1and tea saponin Al. The optimal conditions for semi-preparative HPLC separation of flavonoid glycosides and saponin:mobile phase A phase (95%water+5%glacial acetic acid):B phase(acetonitrile)=65%:35%obtained flavonoid glycosides part; A phase (95%water+5%glacial acetic acid):B phase(acetonitrile)=20%:80%obtained saponin part, the flow rate was8mL/min, the maximum sample volume was50mg. The purity of the flavonoid glycosides and tea sopnin were98.58%and75.01%, respectively. Each separation took less than one hour, and the methods of UPLC-MS for identification of flavonoid glycosides and saponin only need10min and15min. 2.Establishing cell model of LPS-induced inflammation and detected the accumulated amount of NO, finding that both of flavonoid glycosides and tea saponin could significantly suppressing the production of NO in cells, showed that both of them had good anti-inflammatory effects. And then, detected the expression levels of the intracellular important pro-inflammatory cytokines TNF-a, iNOS, COX-2and MIP-la in mRNA level and iNOS, COX-2in protein level,finding that both of flavonoid glycosides and saponin could suppressing its expression, explained that the anti-inflammatory effects of flavonoid glycosides and saponin was connected with the suppressing of these cytokines’expression.3. By testing the production of NO in LPS induced inflammation model, we found that crude (oil and refined oil products of camellia seed both have great anti-inflammatory effect. From the analysis results of expression quantities of inflammatory factors including TNF-α、iNOS、COX-2, and MIP-1α, it showed that C.oleifera seeds oil’s anti-inflammatory effects were related to the inhibition of expressions of these factors, which were in accordance with the results from flavonoid glycoside and saponin in it. Moreover, with the same concentration, the inflammatory effect of curde oil was better than refined oil.4. The SPME-GC-MS method to measure the volatile components of C.oleifera seeds were optimized. The best mode of processing for extraction was50℃, for60min. After GC-MS analysis, we knew that there were31volatile componets in crude oil,24kinds in refined oil, and16in both which consisted54.98%of the total quantity in the former,70.23%in the latter. From the perspective of varieties, there was minor difference between these two kinds of oil for the relative amount of hydrocarbon, alcohols, and esters, but with different constitutions. And the relative amount of aldehydes, ketone and acids were different. This indicated that the refine process made a great effect on the varieties and quantities of volatile components.In short, the experiments used active constituents in C.oleifera seeds as study objects, separated flavonoid glycosides and saponins from camellia seed, and also used UPLC-MS to define their compositions and structures. Then we studied the anti-inflammatory effects of general flavonoid glycoside and saponin of C.oleifera seeds and also C.oleifera seeds oil, by building LPS induced RAW264.7macrophages inflammatory models. Furthermore, genes and protein expressions of TNF-a, iNOS, COX-2, MIP-1α involved in inflamatory model were determinated so that the molecular mechanisms of anti-inflammatory were more clear. Besides the method of measuring volatile components of Camellia oleifera seeds was optimized, and the composition and characteristics of the volatile components were determined.