The Separation,Purification,Metabolism And Functional Studies Of Ximenynic Acid

Ximenynic acid is a conjugated octadecenynoic acetylenic acid which was found in some of Santalales families over 70 years. It mostly exists in the seed oils of Opiliaceae, Olacaceae and Santalaceae families, ranged from 1 to 95% in total seed fatty acid content. This unusual conjugated eneyne fatty acid has been demonstrated its beneficial effects of antimicrobial, antifungal, anti-inflammation and larvicidal activities. With the Australian sandalwood (Santalum spicatum R.Br.) seed oil and ximenynic acid as materials, this thesis focuses on the studies of purification of ximenynic acid, in vivo and in vitro metabolism of ximenynic acid, and the anti-cancer activities of ximenynic acid.Part I Separation and purifiation of ximenynic acid and triximenyninXimenynic acid separation. Free fatty acids was extracted from sandalwood seed oil through hydroxylation and acidification, and then dissolved in hexane for low temperature crystallization. Ximenynic acid was obtained from the crystallization. The optimum crystallization conditions were studied.Triacylglycerides separation. Sandalwood seed oil (Santalum spicatum R.Br.) mainly contains three kinds of triacylglyceride. Triximenynin (XXX) contains three ximenynic acids connected with hydroxyls of glycerin; Di-ximenyin, mono-olein (XOX or OXX) is composed of two ximenynic acids and one oleic acid; Di-olein, mono-ximenyin (OOX or OXO) contains one ximenynic acid and two oleic acids. Using reverse phase silica (C18) powder packed column and acetonitrile propan-2-ol (65:35) as the eluent, three kinds of triacylglyceride were easy to be separated.Designing a HPLC method to rapidly separate and identify different triglyceride in sandalwood seed oil. Different kinds of triglyceride were successfully separated with reverse-phase HPLC and UV absorption (236 nm). The eluent system used in HPLC was acetonitrile propan-2-ol (65:35), and the flow rate was set at 2.5mL/min.Part II The metabolism of ximenynic acid in vivo and in vitroIn vivo study. Dietary sandalwood seed oil significantly decreased n-9 MUFA, and increased n-3 PUFA levels in mice liver. In contract, it decreased total PUFA levels in mice brain. Besides, dietary sandalwood seed oil significantly increased blood glucose and low density lipoprotein (LDL) levels of mice serum, and didn’t reduce mice weight.In vitro study. After incubation HepG2 with ximenynic acid, cellular levels of C18:1n-9, C20:1n-9, C24:1n-9, total n-9 MUFA and DHA were significantly decreased. RT-qPCR results indicated that the expression of SCD gene was significantly decreased in ximenynic acid-treated group. Meanwhile, the gene expression of the other enzymesinvolved in lipids metabolism such as FADS1 and FADS2 was also decreased. Western blotting analyses showed that the expression of FADS2 protein was down-regulated after ximenynic acid treatment.Part III The anti-cancer activity of ximenynic acid in vitroXimenynic acid is a unique conjugated enyne fatty acid, and it is interesting for its anti-inflammatory activity. As the close link between inflammation and cancer, this study was designed to investigate the anti-cancer activity of ximenynic acid in human cancer cell line and the underlying mechanisms.Ximenynic acid blocked the G1/S phase transition of HepG2 and HCT116, leading to increasing cell quantity at G0/G1 phase, and decreasing at S phase or G2/M phase. It might due to the inhibition activitieson the expression of cell cycle related protein GCN5L2 and the gene expression of cyclin D3 (CCND3) and cyclin E1 (CCNE1) in HepG2 cells. Meanwhile, it inhibited the gene expression of CCND3, CCNE1 and cyclin K6 (CDK6) in HCT116 cells.Ximenynic acid significantly inhibited proliferation and promoted apoptosis of HepG2 cells. The anti-apoptosis protein Silent Information Regulator T1 (SIRT1) was significantly suppressed by ximenynic acid. Moreover, ximenynic acid repressed the expression of angiogenesis related genes vascular endothelial growth factors VEGF-B and VEGF-C. Lastly, ximenynic acid significantly inhibited the gene and protein expression of cyclooxygenase-1(COX-1) but without altering that of cyclooxygenase-2 (COX-2). It suggested that ximenynic acid inhibits growth of HepG2 cells by selective inhibition on COX-1 expression which leads to cell cycle arrest and variation of the apoptosis pathway and angiogenesis factors.