| Conjugated linoleic acid(CLA)is an isomer of linoleic acid,which is mainly found in meat and dairy products of ruminants and is the main source of CLA in human diet.Conjugated linoleic acid has anti-tumor,anti-atherosclerotic arteriosclerosis,reduced fat accumulation,enhanced immunity,and growth.With the deepening of research on conjugated linoleic acid,it has gradually been applied to human nutrition and health care.The main research contents of this experiment are as follows:The conjugated linoleic acid in the butter was extracted and purified by freeze separation and urea complexion.Firstly,the frozen crystallization method was used to freeze and crystallize the free fatty acids,and the effects of different organic solvents,free fatty acid and solvent ratio,freezing time and freezing temperature on the purity and yield of CLA were studied.The optimal extraction conditions were obtained by HPLC: the ratio of free fatty acid to absolute ethanol was 1:12,the processing time was 9 h,the processing temperature was-20 °C,and the purity of CLA was 8.49%,and the yield was 85.62%.The CLA was further purified by urea complexion on the basis of frozen crystallization.The effects of reflux time,inclusion time,inclusion temperature,free fatty acid: urea: absolute ethanol on the purity and yield of CLA were studied.The results showed that the reflux time and inclusion time had little effect on the purity and yield of CLA,and the inclusion temperature,urea/anhydrous ethanol and mixed fatty acid/urea had great influence.Then,the inclusion temperature,urea/anhydrous ethanol,and free fatty acid/urea,which have significant influence on the experimental results,are selected to optimize the response surface conditions.The best inclusion process conditions were obtained: the inclusion temperature was 21.70 ° C,the reflux time was 15 min,the inclusion time was 3 h,the urea/anhydrous ethanol was 0.31,and the free fatty acid/urea was 0.37.Urea complexion under these conditions gave an average CLA purity of 22.55% and an average yield of 81.57%.(2)The mouse breast cancer model was successfully established by multiple times of intragastric DMBA.The tumor formation was stable and the specificity was good.Tumors appeared in the model group and the paclitaxel group,and no tumors were found in the CLA group,the LA group,and the blank group.HE staining was used to observe the changes of various organs and the sections of tumor tissues after different treatments.The results showed that DMBA treatment caused damage to liver,kidney and spleen,and the tumor did not see lungs and distant metastasis.The trend of lesions in each tissue was ranked as follows: liver: model group > paclitaxel group > LA group > blank group,CLA group.Lung: paclitaxel group>model group> LA group> CLA group,blank group.Kidney: model group > paclitaxel group,CLA group,LA group > blank group.Spleen: model group > paclitaxel group,LA group > CLA group > blank group.Among them,the paclitaxel group has a greater damage to the lungs,and paclitaxel itself has certain toxicity,and is often used in combination with other drugs in clinical practice.HE staining of tumor tissue showed that the tumor was in a period of vigorous growth.Compared with the model group,the paclitaxel group had a lower degree of infiltration into surrounding tissues.Immunohistochemistry was used to detect the expression of Ki67,P53,PR and VEGF in tumor tissues.The results showed that Ki67 was highly expressed in the model group and significantly different from the paclitaxel group.P53 was highly expressed in both the model group and the paclitaxel group.The expression levels of PR in the model group and the paclitaxel group were both low.VEGF was also expressed in the model group and the paclitaxel group,but there was a significant difference between the two groups.(3)Preliminary study on the anticancer mechanism of conjugated linoleic acid.Peripheral blood of each group of mice was taken for blood routine test.The results showed that albumin was low in each group,but there was no significant difference between them(p < 0.05).The total protein content of each group was basically within the normal range.Among them,the content of alanine aminotransferase in the model group was very high,and there were significant differences with other groups.The high index was also one of the manifestations of liver damage,and the other indicators were within the normal range.The expression levels of bcl-2,bax and p53 mRNA in the blood of mice were detected by quantitative real-time PCR.The results showed that there was no significant difference in the relative expression of bcl-2 and bax mRNA.The relative expression of p53 mRNA was significantly different between the model group and the CLA group(p < 0.05).They were 8.75±0.44 and 4.67±0.33,respectively.There were also significant differences between the CLA group and the other group.This may be because CLA plays an anti-cancer role by inhibiting the expression of p53. |
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