Separation Of Conjugated Linolenic Acid From Lactic Acid Bacteria And Its Death-inducing Effect On Colon Cell

Conjugated linolenic acid?CLNA?is a type of derivants of linolenic acid?LNA?,which is a term for positional and geometrical isomers of conjugated diene and conjugated triene type octadecatriene fatty acids.It has been identified to possess many health-associated functions,such as anti-cancer,anti-inflammatory,anti-oxidation,anti-cardiovascular diseases,and anti-obesity.The structures of CLNA isomers produced by lactic acid bacteria differ from those derived from plant seeds and chemical synthesis,which lead to different activities.However,the function of each CLNA isomer from lactic acid bacteria,as well as the corresponding separation method,are still unclear.Thus,a method for the separation and purification of CLNA isomers derived from lactic acid bacteria was established in this study.Besides,the death-promoting effect of the CLNA isomer on colon cancer cells was also investigated.The main results are as follows:?1?Comparing thin layer chromatography?TLC?and high performance liquid chromatography?HPLC?techniques,optimized HPLC techniques were chosen to separate and purify CLNA from lactic acid bacteria.By optimizing the sample size,the proportion and pH of developing agent in TLC conditions,and chromatographic column type,the composition and proportion of mobile phase in the HPLC conditions,the optimal condition was as follows:HPLC column Ultimate^??5XB-C30;10×250 mm?,methanol-water-formic acid?80?20?0.01,v/v?as mobile phase with the flow rate of 5 mL/min,as well as detection wavelength of 205nm and 233 nm.The pure CLNA monomers purified via the above method was determined.The purities of CLNA1?cis9,trans11,cis15-CLNA?,CLNA2?trans9,trans11,cis15-CLNA?and CLNA3 were 97.48%,99.99%,65.30%,respectively.The production and purity of CLNA3did not meet the requirements of the experiment,in hence,CLNA1 and CLNA2 were selected for follow-up experiments.?2?The effect of CLNA monomers from lactic acid bacteria on colon cancer cells death was studied and compared.Cell viabilities were measured by MTT assay.The results showed that the deaths of three colon cancer cells,Caco-2,SW480 and HT-29,induced by CLNA1 and CLNA2 were dose-and time-dependent.And the IC₅₀ values of CLNA1 and CLNA2 for Caco-2 cells were minimum of about 20?M.So Caco-2 cells were selected as the tested cell.By adding different death mode inhibitors,it was initially implied that CLNA1 might cause Caco-2 cell death through oxidation or apoptosis,and CLNA2 might function through oxidation,apoptosis or necrosis.?3?The mechanism of Caco-2 cell death induced by CLNA from lactic acid bacteria was studied.Further study using flow cytometry showed that CLNA1 and CLNA2 could trigger Caco-2 cells death through apoptosis or necrosis without active oxygen and cell cycle changed,and the cell-death-promoting effect of CLNA2 was better comparatively.The cleavages of proteins associated with apoptosis,such as PARP-1,Caspase-3,Caspase-7,Caspase-9,could not be detected by Western Blot.Moreover,the transcription levels of PPAR?,TNF-?,and TGF-?by RT-qPCR could not be affected by these two isomers.The study indicated that the Caco-2cells apoptosis induced by CLNA1 might be the pathway that was not dependent on Caspase-3,Caspase-7,Caspase-9,the death of Caco-2 cells induced by CLNA2 might be necrosis or apoptosis that is not dependent on Caspase-3,Caspase-7,Caspase-9 pathway,and CLNA2possessed better cell-death-promoting effect.