Study On The Toxicity Intervention Of Anthocyanins From Black Soybean Coats To Aflatoxin B₁

Grain crops,such as soybeans,corn,peanuts,etc.,which are easily contaminated by toxigenic fungi during planting and harvesting period.It is not completely dry during storage or wet weather provides suitable conditions for the growth of molds,which would produce a large amount of toxins and contaminate crops.Aflatoxin B₁(AFB₁)is one of the B group aflatoxins with the largest pollution range,the most serious pollution degree and the strongest toxicity.The natural toxin can be absorbed by the body through the respiratory tract and oral cavity,which can easily cause liver disease.Anthocyanin is a kind of functionally active factor from a wide range of plants,which has anti-inflammatory,anti-oxidant,anti-cancer and other functions to prevent many diseases.However,anthocyanins are unstable in the natural state,it is unstable in their natural state and undergo metabolic degradation under the action of the acid-base environment of the gastrointestinal tract and digestive enzymes in the human body.The vast majority of the physiological activities that enter the blood circulation are its metabolites.In this study,anthocyanins were extracted from black soybean coats[Glycine max(L.)Merr.],then the metabolites of anthocyanins were detected by simulating human gastrointestinal metabolism,so as to study the toxic intervention of anthocyanins and their metabolites on AFB₁.The aim is to reduce the damage of AFB₁ to the human body by increasing the intake of food-borne nutrients—anthocyanins,and provide theoretical support for whether anthocyanins can be used as dietary supplements and prevent liver damage caused by AFB₁.The main research conclusions are as follows:1.Study on preparation of anthocyanins from black soybean coats and gastrointestinal metabolismFirstly,the effects of mobile phase type,gradient elution procedure(?,?,?),elution flow rate(5,8,10,12 mL/min)and injection amount(0.8,1.0,1.2,1.5,1.8 mL)on the purification and preparation of anthocyanins from black soybean coats were investigated.Then,verify the gastrointestinal metabolism of anthocyanins on the basis of previous studies in the simulated in vitro digestion environment.The results show that 5%acetic acid aqueous solution and 100%acetonitrile had the best elution capacity for anthocyanin;the best elution flow rate and injection volume were 8 mL/min and 1.2 mL,respectively.The main component of anthocyanin in black bean coats is cyanidin-3-O-glucoside(C3G).After preparative liquid phase purification,the color value of C3G(1 mg/mL)increased from10.64(U/g)to 70.26(U/g).Anthocyanins can exist stably in the gastric environment,but unstable in the intestinal environment,the cleavage of the C ring of its molecular structure produced two main products:3,4-dihydroxybenzoic acid(Protocatechuic acid,PCA)and 2,4,6-trihydroxy benzaldehyde(Phloroglucinol aldehyde,PGA).2.Study on the interference effect of anthocyanins on the binding of aflatoxin B₁ and human serum albuminThe effects of anthocyanin and its two metabolites(C3G,PCA,PGA)on the interaction between AFB₁ and HSA were studied by fluorescence spectrometry under simulated human physiological conditions(p H 7.4,ion concentration 0.1 mol/L).The results show that C3G and its two metabolites(PCA,PGA)can inhibit the combination of AFB₁ and HSA,the order of inhibition was:PGA>C3G>PCA.AFB₁ quenches the fluorescence of HSA through non-covalent interaction and proceeds spontaneously.In the presence of PGA,AFB₁had a dynamic quenching effect on the quenching of HSA fluorescence.In the presence of PCA,the type of interaction force between AFB₁ and HSA does not change,which was electrostatic;in the presence of C3G/PGA,the type of interaction force between AFB₁ and HSA was van der Waals force and hydrogen bond.Fourier transform infrared(FT-IR)spectroscopy shows that the conformation and main chain structure of HSA changed under the action of exogenous quencher,the content of?-helix decreases and the overall disorder increases.3.Study on the protective effect of anthocyanin on aflatoxin B₁-induced zebrafish toxicityThe young zebrafish were selected as experimental animals,and the oxidative stress model was established by using AFB₁ to induce 3 dpf(days post fertilization,dpf)peeled zebrafish larvae.Then,under the selected induction concentration,the protective effect of C3G/PCA on AFB₁-induced zebrafish toxicity was evaluated.The results showed that AFB₁up to 60 ng/mL had lethal effects on zebrafish,and a large number of deaths occurred at 120ng/mL.At 80 ng/mL,the rate of ROS generation in zebrafish was the highest,and at 100ng/mL,the cell death rate in zebrafish was the highest.When C3G/PCA(5,10,20?g/mL)was incubated with AFB₁,the amount of ROS production and cell death in zebrafish decreased with the advance of the administration time.After AFB₁ treatment for 48 h,ALT and AST in fish homogenate supernatant increased,SOD,CAT and GSH decreased,and Caspase 9 and Caspase 3 activities increased.Compared with the model group,C3G/PCA can inhibit the increase of ALT,AST,SOD,CAT,GSH,Caspase 9 and Caspase 3,which indicating that C3G/PCA may protect zebrafish from oxidative stress and apoptosis caused by AFB₁.