Inhibition Of N-nitrosamine Synthesis And Carcinogenesis By Diospyros Lotus L.leaves And Its Mechanism

Cancer has become the second leading cause of death worldwide.N-nitrosamines(NAs)are recognized carcinogens in the world.NAs not only exist in drinking water,soil,tobacco smoke,food,cosmetics and some industrial products,but also can be synthesized in the human body.Nitrite(NIT)and biogenic amines are precursors to the synthesis of NAs.NIT widely exists in a variety of food raw materials and living environments,and is also a commonly used additive in meat processing.Biogenic amines are breakdown products of amino acids.Due to the ubiquity of NAs and its precursors(NIT and biogenic amines),the human body cannot avoid exposure to NAs.How to prevent the harm of NAs to human body has become a concern problem.Inhibition of NAs formation or blocking of NAs induced carcinogenesis is an effective way to prevent NAs hazard.Therefore,it is of great significance to separate and prepare effective ingredients from natural products to inhibit the formation of NAs or block NAs induced carcinogenesis,and further explore their mechanism of action,and then study their application in drug and food production.In this paper,the leaves of Diospyros Lotus L.(D.Lotus)is studied.D.Lotus is a persimmon plant that widely distributed in China.The main active fraction inhibiting the formation of NAs were prepared and screened;The active ingredients inhibiting the formation of NAs were analyzed and identified;The main active ingredient myricitrin(Mytr)was isolated and prepared;The inhibitory effect and its mechanism of Mytr on NAs-induced L-02 cell damage were studied;The mechanism of Mytr inhibiting endogenous NAs formation and blocking NAs induced carcinogenesis was explored;The inhibitory effect and its mechanism of Mytr as additive on the formation of NAs in cooked sausage were investigated.The main contents are as follows:1.The main active fraction inhibiting the formation of NAs in D.Lotus leaves was prepared and screened.The water extract of D.Lotus leaves was separated and purified by means of alcohol precipitation and D101 macroporous resin column,and seven sample fractions(A,Z,Fr₁-Fr₅)were obtained.Then,the contents of polyphenols,the scavenging activities against DPPH·and NIT,the inhibitory effects on the formation of NAs in simulated gastric juice,and the inhibitory effects on Staphylococcus aureus and Escherichia coli were compared.The results showed that the polyphenols were mainly concentrated in Fr₂(38.37 g/100g)and Fr₃(49.79 g/100g).The scavenging effect of Fr₃on DPPH·and NIT,the inhibitory effect of Fr₃on the formation of N-dimethylnitrosamine(NDMA)and N-diethylnitrosamine(NDEA),and the inhibitory effect of Fr₃on Staphylococcus aureus and Escherichia coli were obviously stronger than those of other fractions(p<0.05).These results indicated that Fr₃was the main active fraction that inhibited the formation of NAs in D.Lotus leaves.2.The main active ingredient of Fr₃inhibiting the formation of NAs was identified and prepared.Firstly,by comparing the chromatograms of Fr₃inhibit nitrosation reaction before and after,infer the active ingredient peaks that suppression of NAs formation in Fr₃,and four active ingredients in Fr₃,including myricetin(Myt),Mytr,myricetin-3-O-?-D-galactoside(Myt-Gal)and myricetin-3-O-?-D-glucoside(Myt-Glc),were identified by mass spectrometry and other methods.Secondly,a HPLC method was established for the simultaneous determination of four active ingredients,and the contents of four active ingredients in the D.Lotus leaves and Fr₃were determined.The results showed that the content of Mytr in the D.Lotus leaves(2107.49 mg/100g)and Fr₃(28240.69 mg/100g)was significantly higher than that of Myt-Gal,Myt-Glc and Myt,and was 164.39 and 33.61 times higher than that of Myt.Then,the activity of the four active ingredients on antioxidant,scavenging NIT and inhibitory NDMA formation were compared.The activity of Mytr were significantly higher than that of Fr_3,Myt-Gal and Myt-Glc,but slightly weaker than that of Myt.Finally,Fr₃was separated and purified by gel chromatography to obtain Mytr with a purity of 95.18%.On the basis of the above results,it was determined that Mytr was the main active ingredient inhibiting the formation of NAs in the D.Lotus leaves.3.Inhibitor effect and its mechanism of Mytr on L-02 cell damage induced by NAs.The inhibitory effect of Mytr on NDEA-induced L-02 cell damage and its mechanism were studied by observing the cell morphology,and comparing the cell survival rate,intracellular ROS level,cell apoptosis rate and cell cycle of the sample group and model group.The results showed that 50?200?M Mytr had no damage to L-02 cells.NDEA could induce L-02 cell damage and apoptosis.Compared with the normal control group,the survival rate of cell in NDEA model group was significantly decreased(p<0.05),the apoptosis rate and the intracellular ROS level were significantly increased(p<0.05),G0/G1 phase cells increased,S phase cells and G2/M phase cells decreased.The groups of Mytr could effectively reverse the levels of various indicators in the model group.Compared with the model group,the survival rate of cells in the high-dose Mytr group(200?M)increased by 49.80%,the apoptosis rate decreased by 47.40%,the intracellular ROS level decreased to 38.68%,G0/G1 phase cells decreased,S phase cells and G2/M phase cells increased.The results indicate that Mytr can effectively inhibit normal liver L-02 cell damage induced by NAs,the mechanism of which is related to blocking apoptosis pathway,regulating cell cycle,and reducing intracellular ROS level.4.Mytr inhibits endogenous NAs formation and blocks NAs induced carcinogenesis.The rats were given high dose of sodium nitrite(S-NIT)and aminopyrine inducer for 8weeks.The rats in the sample group were given different doses of samples before ingesting the inducer.At the end of the experiment,the liver morphology,nodular formation,histopathological changes,serum and liver biochemical indexes of rats in each experimental group were compared,to explore the mechanism of Mytr inhibiting the formation of NAs in rats and blocking NAs induced carcinogenesis.The results are as follows:(1)Compared with the normal control group,the weight of rats in the model group increased slowly,the relative liver weight increased significantly,the color was dark,the surface was rough,the gloss was poor,and 3 cases showed tumor on the surface;There were a large number of vacuoles in the liver cells,the destruction of liver lobules,the occurrence of multinucleated and cellular atypia,obvious fibrosis,Sirius scarlet and PCNA staining positive areas increased significantly;The level of ALB and A/G in serum were significantly decreased(p<0.05),but the level of AFP,ALP,ALT,TP,GLB,TBIL and AST were significantly increased(p<0.05),especially the level of AFP was 7.61times higher than that in normal control group;The levels of MDA and 8-OHd G in liver tissues were significantly increased(p<0.05).The GSH content,CAT and SOD activity were significantly decreased(p<0.05);The gene and protein expression levels of CYP1A1(liver?phase metabolic enzymes)and GST-pi(liver?phase metabolic enzymes)were significantly increased(p<0.05),but CYP1A2,CYP2E1(?phase metabolic enzymes)and GST-mu,GST-alpha,UGT1A1,UGT1A6(?phase metabolic enzymes)were significantly decreased(p<0.05).(2)Mytr medium-dose and high-dose groups significantly reversed all the indicators in the model group.Compared with the model group,the weight of rats in the Mytr high-dose group increased significantly,the relative liver weight decreased significantly,the liver color,smoothness and gloss improved significantly,and no tumor was found;The structure of liver lobules was better,fibrosis,vacuoles,cellular atypia,positive areas of Sirius scarlet and PCNA staining were significantly reduced;The level of ALB and A/G in serum were significantly increased(p<0.05),and the level of AFP,ALP,ALT,TP,GLB,TBIL and AST were significantly decreased(p<0.05),especially the AFP level was only29.41%of model group;The contents of MDA and 8-OHd G in liver decreased to 79.64%and 73.85%of model group,while the contents of GSH and the activities of CAT and SOD increased to 1.80,1.42 and 1.80 times of model group;The gene and protein expression levels of CYP1A1 and GST-pi were decreased significantly(p<0.05),and CYP1A2,CYP2E1,GST-mu,GST-alpha,UGT1A1 and UGT1A6 were increased significantly(p<0.05).Conclusion:long-term high dose of S-NIT and aminopyrine can induce liver carcinogenesis due to endogenous NAs formation in experimental rats.Mytr can effectively inhibit liver cancer induced by NAs that formation from S-NIT and aminopyrine.The anticancer mechanism is about related to reduce rat liver lipid oxidation and DNA damage,improve antioxidant activity of enzyme in the liver and regulating the expression level of liver?versus?metabolic enzyme.5.Inhibitory effect of Mytr as additive on the formation of NAs in cooked sausage and its mechanism.By comparing the p H value,total number of bacteria,lipid peroxide(POV),thiobarbituric acid(TBARS),volatile base nitrogen(TVB-N),the contents of NIT and nine kinds of NAs in the cooked sausage of each experimental group during storage(0?28 d),the inhibitory effect of Mytr on the formation of NAs and its mechanism were investigated.The results showed that Mytr could effectively inhibit the formation of NAs in cooked sausage during storage.The mechanism of action was related to the inhibition of bacteria,the delay of protein and fat decay and the elimination of NIT.