| Docosapentaenoic acid(DPA) is the metabolism intermediate of polyunsaturated fatty acid(PUFA), which involves 4, 7,10, 13,16- 22:5(DPAn-6) and 7, 10, 13, 16, 19- 22:5(DPAn-3). The two isomer possess different biological properties. Research on these two fatty acids are far from adequate due to the limited content in natural sources or limited sources. In this study, the fatty acid composition of six type of marine oils were analyzed aiming at selecting the appropriate oils for DPAn-6 and DPAn-3 enrichment and separation, thereafter the optimum parameters of urea complexation, low temperature solvent crystallization and preparative liquid high performance chromatography were systematically studied. Afterwards, the antiinflammatory potential of DPAn-6 and DPAn-3 were investigated by induction with LPS stimulated murine monocytic RAW 264.7 cells. The research would provide theoretical basis for their further investigation on functionality and applications.1. Based on the determination of marine oils resource for enrichment of DPAn-6 and DPAn-3, the fatty acid compositon of six type of marine oils, namely Schizochtrium sp. oil, seal oil, salmon oil, anchovy oil, tuna oil and puffer fish oil were analyzed by GC/MS. DPAn-6 and DHA in Schizochtrium sp. oil are preferentially located in sn-1,3, while MUFA are located in sn-2, SFA were equally distributed. SFA and MUFA in seal oil are preferentially distributed in sn-2 rather than in sn-1,3, EPA, DPAn-3 and DHA are mostly located in sn-1,3; DPAn-3 and DHA in the four type of fish oils showed a preferential distribution in sn-2, while EPA more selectively located in sn-1,3. Schizochtrium sp. oil contribute an appropriate source for DPAn-6 with the content of 15.63% with a comparatively simple PUFA composition. The sources of DPAn-3 were determented by applying urea complexation for the enrichment process, result indicated that different PUFA composition plays a major role on the enrichment of DPAn-3, seal oil provides an appropriate source for DPAn-3 enrichment.2. Based on the enrichment of DPAn-3, the process technologies for improvement of PUFA concentration by urea inclusion was optimized by response surface methodology, the PUFA cocentrate enriched with 12.75% DPAn-3 was achieved by crystallization at 1.3 °C for 17.5 h with 3.7(w/w) urea/ FFA ratio. The final product with 93.12% purity as well as 78.05% recovery of DPAn-3 and 37.87% yield was achieved by preparative HPLC method.3. The low temperature solvent crystallization processing technologies for improving the concentration of PUFA from Schizochtrium sp. oil or its FFA derivatives were investigated. A PUFA concentrate enriched with 26.26± 0.06% and 64.81± 0.69% for DPAn-6 and DHA, respectively was obtained by crystallization at-60 °C for 80 min with 1:5(w/v) FFA/ acetonitril ratio. In terms of Schizochtrium sp. oil, 20.71± 0.06% DPAn-6 and 51.85± 0.67% DHA was achieved by crystallization at-80 °C for 10 h with 10% oil in acetone(w/v). The final product with 95.16% purity as well as 93.7% recovery of DPAn-6 and 50.21% yield was achieved by preparative HPLC method.4. The antiinflammatory property of DPAn-6, DPAn-3 as compared with those of EPA and DHA was investigated by cytokine production. There is no significant variation of cell viability within the concentration of four type of PUFAs. Four type of PUFAs could significantly inhibit the excretion of NO. The ELISA assay indicated that DPAn-6 and DPAn-3 significantyly inhibit the production of inflammatory IL-1β、IL-6 and TNF-α, while help improving the production of IL-10. Real time PCR analysis indicated DPAn-3 is the most potent inhibitor for the expression of inflammatory IL-1β、IL-6 and TNF-α, while DPAn-6 is the most effective in increasing the expression of IL-10. All the trials indicate the antiinflammatory potential of DPAn-6 and DPAn-3. The fatty acid composition of RAW264.7 after incubated with DPAn-6, DPAn-3, EPA and DHA were analyzed by GC, the EPA group showed an improved level of DPAn-3, suggesting a conversion of EPA to DPAn-3. |
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