The Effects Of Four Polysaccharides On The Intestinal Absorption And Mutagenicity Of Tryptophan-P-1

Tryptophan-P-1,3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole is widely found in meat products,which shows high carcinogenicity and mutagenicity and has been identified as 2B carcinogens by the international agency for research on cancer(IARC).Therefore,the reduction of Tryptophan-P-1 's mutagenicity in meat products is extremely important for the safety of meat products.Three traditional methods to reduce Tryptophan-P-1's mutagenicity are commonly used:to reduce its formation by controlling cooking time/temperature or adding other substance,to inhibit its mutagenicity by using antimutagenic substance.However,these reduction methods are limited in practical applications:it is difficult to precisely control cooking time or temperature in home-prepared meals;the addition of other substances may bring an undesired flavor;the antimutagenic substance itself may have other biological effects.Therefore,a new method to reduce Tryptophan-P-1's mutagenicity is very important for the quality control of meat products.In this study,we systematically verified method to reduce Tryptophan-P-1's mutagenicity,which is to reduces the absorption of Tryptophan-P-1.Four polysaccharides,Arabic gum,carrageenan,xanthan gum and sodium carboxymethyl cellulose(CMC),were selected as potential inhibitors for absorption and mutagenicity of Tryptophan-P-1.We evaluated their effects on the mutagenicity and absorption of Tryptophan-P-1 in vitro and in vivo,and investigated the involved mechanism.The present study mainly included the following four parts:1.The effects of the four polysaccharides on the absorption of Tryptophan-P-1 in vitroThe MDCK-MDR1 cell monolayer model and intestinal mucosal tissue model were used to study the absorption characteristics of Tryptophan-P-1 and to evaluate the effects of the four polysaccharides at low concentrations(<1%,w/v)on the absorption of Tryptophan-P-1 in vitro.Tryptophan-P-1(20 ?M)was transported through the intestinal absorption models alone or together with different concentrations of polysaccharides(10 mM,20 ?M or 2 ?M,in terms of monomer).The concentration of Tryptophan-P-1 in the absorption tank at each time-point was measured in 120 min(MDCK-MDR1 cell monolayer model)or 90 min(intestinal mucosal tissue model),respectively.The absorption amount and absorption parameters(Apparent permeability coefficient,Papp)of Tryptophan-P-1 were calculated and analyzed,as well as the inhibition rate of Tryptophan-P-1 intestinal absorption by the four polysaccharides.The results showed that:(1)Different polysaccharides had different effects on the intestinal absorption of Tryptophan-P-1.Arabic gum had no significant effect on intestinal absorption of Tryptophan-P-1.Xanthan gum,carrageenan and CMC significantly inhibited the intestinal absorption of Tryptophan-P-1.(2)The degree of inhibition of Tryptophan-P-1's intestinal absorption by xanthan gum,carrageenan or CMC was related to the concentration of the polysaccharides.Polysaccharides of 10 mM exhibited the highest inhibition of intestinal absorption.10 mM xanthan gum,carrageenan,and CMC decreased the intestinal absorption of Tryptophan-P-1 through MDCK-MDR1 cell monolayer by 49.5%,72.9%,and 31.5%,respectively;At the same concentration,the three polysaccharides reduced the intestinal absorption of Tryptophan-P-1 through intestinal mucosal by 83.4%,64.1%and 64.6%,respectively.(3)The inhibition rate of Tryptophan-P-1 intestinal absorption by the three polysaccharides was nonlinearly related to their concentration.At some conditions,the inhibition rate was similar at different concentrations of the same polysaccharides.2.The effects of the four polysaccharides on the absorption of Tryptophan-P-1 in vivoHealthy mice were used as an animal model,and the test substance was administered by gavage.Animals were divided into 5 groups.Tryptophan-P-1(10mg/Kg)was intragastric administrated alone or with different polysaccharide(125mg/Kg).Blood samples were taken at 20min,40min,1h,1.5h,2h,3h,5h,8h,18h and 24h after gavage,and the concentration of Tryptophan-P-1 in blood samples was determined by HPLC using internal standard method.The pharmacokinetic software DAS2.0 was adopted to analyze the Tryptophan-P-1 pharmacokinetic parameters of each group using the statistical moment model.The results showed that Arabic gum exhibited no significant difference on the absorption of Tryptophan-P-1 in vivo.The other three polysaccharides reduced absorption degree(Cmax)and absorption amount(AUC(0-t)and AUC(0-?)of Tryptophan-P-1,while having no obvious effects on the absorption rate or the elimination rate of Tryptophan-P-1.Calculated basing on AUC(0-t),the absorption of Tryptophan-P-1 was reduced by 45.5%,63.6%%and 35.6%,respectively.Calculated Basing on AUC(0-?),xanthan gum,carrageenan or CMC in the test groups reduced the absorption of Tryptophan-P-1 by 44.9%,64.8%%and 41.5%,respectively.3.Effects of the four polysaccharides on the mutagenicity of Tryptophan-P-1Ames test was adopted to study the effects of the four polysaccharides(Arabic gum,xanthan gum,carrageenan,xanthan gum and CMC)at different concentration on the mutagenicity of Tryptophan-P-1.TA98 was selected as the experimental strain.The concentration of Tryptophan-P-1 was 20 nmol/plate,and the concentration of polysaccharide was expressed as Tryptophan-P-1:polysaccharide,which was 1:0.1,1:1,1:5,1:25,1:50,1:100,and 1:500,respectively.The results showed that:(1)Four polysaccharides exhibited no mutagenicity to TA98 in Ames test.(2)Different kinds of polysaccharides showed different effects on the mutagenicity of Tryptophan-P-1.Arabic gum had no significant effect on Tryptophan-P-1 mutagenicity.Xanthan gum,carrageenan and CMC had an inhibitory effect on the mutagenicity of Tryptophan-P-1.(3)The inhibition rate of Tryptophan-P-1's mutagenicity by xanthan gum,carrageenan and CMC increased with the concentration of the three polysaccharides.However,at some conditions,the inhibition rate was similar at different concentrations of the same polysaccharides.When the concentration of polysaccharides was 10?mol/plate(Tryptophan-P-1:polysaccharide=1:500),the inhibition rate of Tryptophan-P-1 mutagenicity by xanthan gum,carrageenan and CMC was 94.0%,95.3%and 88.0%,respectively.The mutagenicity of Tryptophan-P-1 was almost completely inhibited.The involved mechanisms were also investigated:(1)to investigate whether the effect of polysaccharides on Tryptophan-P-1's mutagenicity was related to metabolic system activation by setting parallel experiments of metabolic system activation conditions(S9+)and non-metabolic system activation conditions(S9-);(2)to investigate whether there was an interaction among the polysaccharides,Tryptophan-P-1 and TA98 Strain by setting different experimental method.The results demonstrated that the involved mechanism in the inhibition of Tryptophan-P-1 's mutagenicity by polysaccharides may be related with the interaction between Tryptophan-P-1 and polysaccharides,irrelated with the metabolic enzyme system.4.The interaction between the four polysaccharides and Tryptophan-P-1Isothermal titration calorimetry(ITC)and binding experiments in vitro were adopted to investigate the interaction between Tryptophan-P-1 and polysaccharides.The ITC binding curve indicated that there was no obvious interaction between Arabic gum and Tryptophan-P-1,but the other three polysaccharides had interaction with Tryptophan-P-1.Analysis of thermodynamic parameters found that the interaction between Tryptophan-P-1 and xanthan gum was mainly driven by entropy change.The interaction between Tryptophan-P-1 with carrageenan and CMC was mainly driven by enthalpy change.In the binding experiment in vitro,the concentration of polysaccharides was expressed as polysaccharide:Tryptophan-P-1,which was 0.1:1,0.2:1,0.5:1,1:1,10:1,and 500:1,respectively.The resultes showed that:(1)The binding temperature(25 ? or 37?)showed no significant effects on the binding ratio of the four polysaccharides to Tryptophan-P-1.(2)Different concentrations of polysaccharides showed different effects on the ratio of Tryptophan-P-1 binding with polysaccharides.The ratio of Tryptophan-P-1 binding with Arabic gum or xanthan gum was not significantly changed with the concentration of polysaccharides.The ratio of Tryptophan-P-1 binding with Arabic gum was<5%at all concentrations.And the ratio of Tryptophan-P-1 binding with xanthan gum was stable at all concentrations,ranging from 77.45%to 85.50%.The ratio of Tryptophan-P-1 binding with CMC or carrageenan grew with the concentration of polysaccharides at beginning and then stabilized.When the concentration ratio of CMC to Tryptophan-P-1 changed from 1:1 to 500:1,the ratio of Tryptophan-P-1 binding with CMC became stable,ranging from 79.72%to 84.16%.When the concentration ratio of carrageenan to Tryptophan-P-1 was increased from 10:1 to 500:1,the ratio of Tryptophan-P-1 binding with carrageenan became stable,ranging from 81.9%to 86.8%.In conclusion,this study found that at low concentrations(<1%,w/v),Arabic gum had no significant effect on the intestinal absorption and mutagenicity of Tryptophan-P-1;xanthan gum,carrageenan and CMC reduced Tryptophan-P-1 absorption in cell monolayer,intestinal mucosa,and mice,as well as inhibited the mutagenicity of Tryptophan-P-1 against TA98.The reduction of absorption and the inhibition rate of Tryptophan-P-1's mutagenicity were related to polysaccharides' concentration.The involved mechanisms was related with the interaction between Tryptophan-P-1 and polysaccharides,irrelated with the metabolic enzyme system.The finding provided scientific support for the application of the idea to reduce absorption and the mutagenicity of Tryptophan-P-1 by polysaccharides.