Study On The Interaction Of Mangiferin And Norathyriol Based On Biological Zebrafish

Objective:Zebrafish was used as the research object to study the mangiferin and norathyriol in biological samples and the interaction between them.The results are compared with traditional research methods,provide research data for zebrafish for non clinical drug research.Methods:(1)Establishing HPLC method of mangiferin and norathyriol in biological samples:The content of mangiferin and norathyriol in plasma and zebrafish of SD rats were investigated,including linear relationship,recovery rate,precision,repeatability and stability.(2)Study on the binding rate of plasma protein in SD rats with mangiferin and norathyriol:After collecting blood from the aorta of SD rats,after preparing the plasma,the mangiferin were set up three concentrations low(2.50?g/ml),medium(7.50?g/ml),and high(15.00?g/ml).The norathyriol were set up three concentrations low(10.0?g/ml),medium(25.0?g/ml),and high(50.0?g/ml).The plasma protein binding rate of mangiferin and norathyriol as well as the binding rate of plasma protein after their interaction were measured by ultrafiltration.(3)Study on the interaction between mangiferin and norathyriol in zebrafish:First,the best point of zebrafish sampling time was determined by preliminary experiment.Zebrafish is placed in a certain concentration of liquid respectively,mangiferin and norathyriol set up three concentrations low(50.0?g/ml),medium(100.0?g/ml)and high(150.0?g/ml)respectively.After 10h culture,respectively remove to death,deal with protein after series of methods,according to the"HPLC research method of mangiferin and norathyriol in biological samples"determination of mangiferin and norathyriol alone in the content of zebrafish and their interaction in zebrafish content changes.(4)The comparison between the two models and the investigation of influencing factors:Taking experimental consumables,cost and adaptation scope as indicators,we compare the differences between the two research methods.In addition,we investigated the factors such as temperature,p H,light time and so on,and discussed the effect of the change of factors on the drug content in fish.Result:(1)The chromatographic conditions for determination of mangiferin and norathyriol in plasma were:Detection wavelength at 258nm;Column temperature in 30°C;Injection volume 10?l;The mobile phase consisted of methanol(A)and 0.1%formic acid(B)solution for gradient elution.The chromatographic conditions for determination of mangiferin and norathyriol in zebrafish were:Detection wavelength at258nm;Column temperature in 30°C;Injection volume 20?l;The mobile phase consisted of acetonitrile(A)and 0.1%formic acid(B)solution for gradient elution.The analytical column was Shimadzu ODS C-18(250×4.6 mm,5?m).The linear equation of mangiferin and internal standard in SD rats plasma was:A=4.40×10~6C+1.33×10~2,r2=0.9984,the standard curve of mangiferin in the range of 0.10-0.52?g/ml had good linear relation.norathyriol and internal standard in SD rats plasma was A=6.12×10~5C+1.30×10~2,r2=0.9987,the standard curve of norathyriol the range of 0.20-3.00?g/ml had good linear relation.The linear equation of mangiferin and internal standard in zebrafish was:,the standard curve of mangiferin the range of 0.20-10.02?g/ml had good linear relation.norathyriol and internal standard in zebrafish wasstandard curve of norathyriol the range of 1.00-100.01?g/ml had good linear relation.The recovery rate of the ultrafiltration membrane of the solution of mangiferin and norathyriol in the plasma of SD rats were all90-115%,the recovery rate of mangiferin and norathyriol in zebrafish was 45-85%.The RSD%values of intraday precision,repeatability and stability were less than 15%.(2)The results showed that the plasma protein binding rate of three concentrations of low,medium and high levels of mangiferin were94.45±0.025%,97.06±0.010%and 97.93±0.025%respectively.The plasma protein binding rates of three concentrations of low,medium and high levels of norathyriol were 94.23±0.015%,95.81±0.170%and 96.78±0.025%respectively.After two drug interactions,the plasma protein binding rates of mangiferin in the low,medium and high concentrations of mangiferin were respectively:98.76±0.18%,98.13±0.03%,95.07±0.05%.98.33±0.08%,96.79±0.13%,92.80±0.08%.97.19±0.11%,94.81±0.11%,86.26±0.07%.For norathyriol in the low,medium and high concentrations of mangiferin were respectively:98.80±0.08%,97.40±0.07%,96.58±0.08%.98.79±0.06%,97.76±0.15%,95.53±0.04%.98.72±0.07%,95.16±0.16%,83.82±0.15%.(3)It was found by experiments that when the incubation time was 10h,the content of body medicine in zebrafish reached the highest.The distribution of mangiferin and norathyriol in the zebrafish was determined by HPLC.The content of mangiferin and norathyriol in fish was calculated by standard curve.The results showed that the contents of three concentrations of mangiferin in low concentration,medium and high concentrations were?g/g,?g/g,?g/g.The contents of three concentrations of norathyriol in low concentration,medium and high concentrations were?g/g,?g/g,?g/g.After two drug interactions,the distribution of mangiferin in zebrafish decreased with the increase of norathyriol concentration.For norathyriol,the distribution of norathyriol in zebrafish decreased with the increase of the concentration of mangiferin.(4)Zebrafish in vivo research method(A)and ultrafiltration(B)were compared:The study object obtains the degree of ease A>B.Research cost A<B.Experimental period A>B.Physiological activity of A can increase p H,temperature and other factors.Through the investigation of various factors,it was found that the changes of temperature,p H and light time had great effects on the content of drugs in fish.When the temperature is equal to 35?,p H equals 4 and the illumination time equals 5h,zebrafish has the highest drug content.Conclusion:(1)The RP-HPLC analysis method of mangiferin and norathyriol in the biological samples established by this experiment is sensitive,fast and stable,which accords with the basic requirements of biological sample determination.(2)The study method of zebrafish in vivo is similar to the results of ultrafiltration,and can be used in the research of drug protein binding rate and drug interaction,and the application of zebrafish in the field of pharmacokinetics study is expanded.The changes of temperature,p H and light time affect the contents of drugs in zebrafish.These factors should be taken into consideration in future studies.