Study On Inhibitory Effects Of Flavones On N-Nitrosodiethylamine Formation In Vitro

Nitrite might interact with amines under the mildly acidic conditions in vitro and in vivo to generate carcinogenic nitrosamines.Thus how to scavenge nitrite or inhibite the formation of nitrosamines in vivo and in vitro has been becoming one of the hot research topics in food safety field. Flavones as secondary metabolite of plants which were widely distributing in the plant kingdom have been applied in developing health food for many biological acitivities,such as antioxidation,antitumor, antiaging,etc.With the further studying of flavone activities,it has revealed that flavones had the ability of scavenging nitrite and inhibiting the formation of nitrosamines.However,the study of flavones on inhibiting nitrosamine formation still focused on crude flavone's extractions,such as bambool flavone,liquorice flavone,buckwheat flavone.In the present paper,we systemically studied the ability of different puri flavones in inhibiting the formation of nitrosamine to provide a preliminary theoretical basis for flavones as nitrosation inhibitor.Contents1.The methods for the determination of NO₂ and N-nitrosodiethyllamine were developed.2.Quercetin,luteolin and chrysin were used to evaluate the relationships between structure,concentration,antioxidant activity and the ability of acting in NDEA formation with EGCG as reference compound.To investigate the relationship between the amount and position of the phenolic hydroxyl and the effect action in NDEA formation, 1,2-dihydroxy benzene,1,3-dihydroxy benzene,1,4-dihydroxy benzene and 1,2,3-trihydroxybenzene were studied.3.The products of quercetin which is the typical representative of flavones after it inhibited NDEA formation in simulated gastric condition were analyzed.The products were separated by preparative high performance liquid chromatography.The products were identified using UV/VIS spectrophotometer,high performance liquid chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy.Then the proportional relationship of products was calculated basing on the character of nuclear magnetic resonance spectroscopy.And the result was validated by analyzing with reversed-phase high performance liquid chromatography/diode array detector.4.Furthermore,the three crude flavone's extractions of Jumi,Ginkgo biloba and Argy wormwood were studied,investigating the influences of extractions' concentration,reaction time,pH and temperature on the scavenging rate and inhibiting rate.Results1.Quercetin,luteolin and chrysin could scavenge nitrite.The scavenging rate was positively related with flavones concentration and appeared to be quercetin＞luteolin＞chrysin.It was discovered that there were twofold influences of flavone in effecting NDEA formation,catalyzing NDEA forming at a low concentration and inhibiting NDEA formation at a high concentration。The scavenging rate and the inhibiting rate were positively related with the antioxidant activity of flavones.By investigating the effects of phenolic compounds with different phenolic hydroxyls in NDEA formation,it was deduced that the twofold influences of flavones in NDEA formation was depending on the concentration and the phenolic hydroxyl position of flavones.There are greatly differences of phenolic hydroxyl activity in flavones,the hydroxyls of B ring＞the hydroxyls of C ring＞the hydroxyls of A ring.Under the high concentration of flavones,NO₂^- completely interacted with the hydroxyls at B ring of flavones to form stable quinine derivatives inhibiting NDEA forming.Under the low concentration of flavones, the hydroxyls at B ring of flavones was not so enough to interact with NO₂^- that the large excess of NO₂^- would interact with the hydroxyls at A ring to generate a more powerful nitrosating agent catalyzing the NDEA formation.In addition,the amount of hydroxyls influenced the extent of flavones in effecting NDEA formation.The inhibiting rates of quercetin,luteolin and chrysin were decreasing successively as well as the amount of hydroxyls decreasing.The definitive mechanism needs to be further studied.2.The products of quercetin reacting with NO₂ ^- at the simulate gastric condition was identified as C₁[2-(3,4-dihydrxybenzoyl)-2,4,6-trihydroxybenzofuran -3(2H)-one],which could decompose to generate C₂[3,4-dihydroxybenzoic acid]and C₃[2,4,6-trihydroxyphenylglyoxylic acid].The proportional relationship of C₁,C₂ and C₃ was 1:0.2:0.8.3.By studying the effect of the crude flavone's extractions of Jumi, Ginkgo biloba and Argy wormwood effecting on nitrosation,it was shown that the two crude flavone's extractions containing flavones at the high concentration(Jumi:7.17 mg/mL,Argy wormwood:5.20 mg/mL) had the effects of scavenging nitrite and inhibiting NDEA formation.The scavenging rate and inhibiting rate were positively related with the reacting time and flavones' concentration and negatively related with the pH value.That the temperature was too high or low was unfavorable to the inhibiting effect of flavones.The crude flavone's extraction containing flavones at the low concentration(Ginkgo biloba:0.85 mg/mL) always catalytic NDEA forming and the scavenging rate was the lowest.ConclusionsIt was suggested that flavones could redox react with nitrite under mildly acidic condition and inhibite the formation of nitrosamine if the concentration of flavones was enough.The blocking rate was positively related with its concentration and antioxidant activity.Flavones could accelerate NDEA forming at a low concentration.The twofold influences of flavones in effecting the NDEA formation might be related with the structure of phenolic hydroxyl of flavones,which need to be further studied.By analyzing the products of quercetin reacting with nitrite,it was concluded that flavones were oxidated on the process of inhibiting the nitrosamine formation and produced relevant oxidation products.