Optimization Of Ultrasonic Wave-assisted Extraction Process Of Tea-polyphenols And The Antagonistic Effect And Mechanism Of Toxicity Induced By Benz [A] Pyrene

Benzo[a]pyrene(BaP), as a representative polycyclic aromatic hydrocarbons (PAHs), is formed and released into the environment largely as the result of human activities, such as incomplete combustion and thermal decomposition process, resulting in the ubiquity of these toxic chemicals in the marine and air environment. After being activated through a variety of phase I metabolic enzymes, benzo[a]pyrene was demonstrated to bring into play toxic effects as a carcinogenic, teratogenic, mutagens, as well as reproductive toxicity due to its significant antiestrogenic effect through an Ah-receptor mediated mechanism. Tea polyphenols was reported can effectively remove active oxygen and lipid free radicals, with anti-cancer, anti-mutation, anti-genetic toxicity, not been reported about the anti-distortion effect in vivo level, as well as the antagonistic effect on benzo [a] pyrene-induced reproductive toxicity.Based on the optimized extraction of tea polyphenols, the effects of green tea polyphenols (GTP)on the malformations of the fertilized eggs of Japanese Medaka (Oryzias latipes) and the reproductive toxicity induced by benz[a]pyrene was researched using the biological model of Japanese medaka (Oryzias latipes) by the method of intraperitoneal injection, and the real-time PCR was used to detection the mRNA expression of CYP enzymes (CYP1A1, CYP1B, CYP2A, CYP2C, CYP2D, CYP3A) and the GST gene (mGST, GST-A) , hormone synthesis enzymes (CYP17A, CYP19, CYP11A, VTG2, CHG-H), and apoptosis related genes (Bcl-2, Bax, GSH-Px) etc. in liver and gonad organization of Medaka, this was the first application of a d-rR strains Japanese medaka model to reproductive protection studies. The brief conclusions are as follows:1. With ziyang population variety as test material, quadratic orthogonal rotation combination design was used to optimize ultrasonic wave-assisted extraction process of tea- polyphenols substance. A quadratic regression orthogonal model was established to express the relationship between total polyphenol yield and five factors including ratio of solid to liquid, ethanol concentration, ultrasonic time, ultrasonic temperature and ultrasonic power.The factor order on extraction yield from strong to weak was ratio of solid to liquid>ethanol concentration > ultrasonic temperature > ultrasonic time > ultrasonic Power. According to this model,ultrasonic extraction at 72℃, 248 W for 38 min using 77.6%ethanol and 1:35.8(g/mL) of green tea and liquid. The optimal ultrasonic wave-assisted extraction conditions were determined. On the conditions the highest yield of tea-polyphenols,which was21.78%. Compared to the traditional organic solvent extraction method at the same condition,the yield of TP was enhanced by 8.56 % and the extraction time was reduced .2. In this first application of a fish model to the effects of green tea polyphenols on the malformations of the fertilized eggs of Japanese Medaka (Oryzias latipes) induced by benz[a]pyrene. The results showed that the malformation rate of the Japanese medaka fertilized eggs of in the treatment of single injection of benzo [a] pyrene was 15.13%, which was significantly higher by contrast with the DMSO control; The malformation rate in the treatments of tea polyphenols was decreased, the treatments except the lowest dose of tea polyphenols (20μg/gB[a]P+2μg /g TP) were significantly different compared to the B[a]P treatment. Real-time quantitative PCR results showed that the benzo[a]pyrene model group slightly induces the CYP1A1,CYP1B,CYP2A,CYP2C,CYP2D mRNA expression and significantly inhibits the mGST and GST-A mRNA expression of the female Medaka, while tea polyphenols can significantly inhibit the expression CYP1A1 mRNA gene and glutathione-S transferase (GST) gene mGST and GST-A. The antagonism mechanism of green tea polyphenols on the malformations of the fertilized eggs of Japanese Medaka induced by benz[a]pyrene might be correlated with inhibit the of CYP1A1 mRNA gene expression and accelerate the excretion of benzopyrene metabolic product.3. The antagonism effect of green tea polyphenols on B[a]P-induced reproductive toxicity in Japanese Medaka (Oryzias latipes) was studied at the fist time, results from this study demonstrated that exposure to 20ug/g of wet weight B[a]P caused decrease of fecundity in male and female Japanese Medaka, including the reduced egg number, the lower spawning frequency,fertilization success and prolonged the hatch times as well as the poor quality of eggs as exemplified by the egg diameter and the increase of Abnormalities frequencies. The mRNA expression of CYP17, CYP19, CYP11A, Bcl-2, Bax, GSH-Px et al have no significant impact, but the expression of VTG2, CHG-H gene were inhibit significantly, whereas tea polyphenols antagonistic groups and the control did not decrease the expression of VTG2, 50μg/gTP group also increased significantly, illustrating that tea can effectively promote the VTG2 expression. And also we can see that the mRNA expression of CYP1A1 was significantly induced and the GST-A and GST-M were inhibited in liver of female Japanese Medaka. The antagonism effect of green tea polyphenols on B[a]P-induced reproductive toxicity in Japanese Medaka (Oryzias latipes) may by regulation mRNA expression of VTG2, CHG-H gene.