| Conjugated linolenic acid (Conjugated linolenic acid, CLnA) is a group of position and geometric isomers of octadecenoic polyunsaturated fatty acid with three double linkage, CLnA has avariety of biological activities, such as:inhibiting carcinogenesis, enhanceing immunity and altering lipid metabolism activities. Punicic acid (c9, t11, c13-CLnA, PA) and alpha-eleostearic acid (c9, t11, t13-CLnA, a-ESA) is the most common isomers of conjugated linolenic acid. The punicic acid was extracted and purified by fractional crystallization and the apoptosis of punicic acid and alpha-eleostearic acid was studied on human endometrial carcinoma RL95-2, whose possible mechanism is discussed.Firstly, the effect of the crystallization time on the purity of punicic acid under-18℃、4℃、25℃conditions were studied through the single factor test. The results indicated that:-18℃, crystallization for1h,4℃crystallization for2h,25℃crystallization for16h, the ratio of ethanol to fatty acid1:2(v/v), the purity of punicic acid was95.2%.Secondly, the apoptosis of endometrial cancer cell RL95-2by treatment of a-ESA and PA was investigated. MTT analysis showed that the punicic acid and alpha-eleostearic acid could inhibit the viability of endometrial carcinoma RL95-2cell after treatment for48hours, in the methods of time-dependent and dose-dependent. Half of lethal concentration (IC50) of punicic acid and alpha-eleostearic acid were30μM and60μM individually. Hoechst staining analyzed morphological changes of RL95-2cell by fluorescence microscopy, after the treatment of punicic acid and alpha-eleostearic acid for48h. The nucleus was significantly smaller, broken into pieces with granular fluorescence. Flow cytometry analysis showed that punicic acid and alpha-eleostearic acid can induce the apoptosis of endometrial carcinoma RL95-2. The apoptosis rate was68.24%and44.91%at40μM of punicic acid and alpha-eleostearic acid after48h. Western blot analysis showed that pomegranate acid and a-palmitic acid increased the expression of apoptosis-related proteins Bax, active type caspase-3, p53and decreased the expression of Bcl-2apoptosis-related protein. In addition, punicic acid and alpha-eleostearic acid also increased the expression of peroxisome proliferator-activated receptor-γ protein.In summary, the punicic acid was extracted and purified by fractional crystallization. Punicic acid and alpha-eleostearic acid could significantly inhibit the viability of endometrial carcinoma RL95-2cell and induce the apoptosis of endometrial cancer cell RL95-2. These results sugguested that the apoptosis may be related with the regulation of Bax, Bcl-2, caspase-3, p53and peroxisome proliferator-activated receptor-γ pretein expression. |
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