Studies On Analytical Methods Of Triptoxins And Toxicities Of Honey Produced With Tripterygium Wilfordii As Honey Plant

Bee honey is recognized as the traditional health food,however poisoning,even deaths caused by poisonous honey still consistently occurs all over the world.Plant toxins in honey not only lead to the acute food poisoning,but also may do harm to human body when the honey is consumed for the long time.Biotoxins in honey has attracted much attention in China in the recent years and has been one of the important food safety issues.As an important Chinese herbal medicine,Tripterygium wilfordii has been planted on a large scale in FuJian.However,the plant is toxic and generally considered to be toxic honey plant.It is generally believed that the honey from T.wilfordii’s nectar can cause human poisoning accident.The honey poisoning incident that caused food deaths was suspected to be related to it,so it is urgently needed to carry out research.This paper developed a analytical method to determine multiple triptoxins in honey and biological samples and studied the preparation method of honey from T.wilfordii honey,and conducted animal toxicity and toxin metabolism kinetics studies on T.wilfordii honey.The purpose is to provide a rapid fast and accurate screening technology for the occurrence of the T.wilfordii honey poisoning event and provide reliable technical support for timely rescue the patients with T.wilfordii honey poisoning.The main results were as follows:(1)An ultraperformance liquid chromatography-tandem mass spectrometry method was developed to determine 5 kinds of Triptoxins in honeyAn analytical method was developed to simultaneous determine 5 kinds of triptoxins in honey using hydrocortisone internal standard method and ultra-high performance liquid chromatography-mass spectrometry technique.The honey samples were extracted with ethyl acetate solution and cleaned up with neutral alumina solid phase extraction column,separated by an ACQUITY UPLC BEH C8(2.1×100mm,1.7μm)column with the mobile phase gradient elution of 0.1%formic acid water and 0.1%formic acid methanol.The identification and quantification were achieved by using electrospray ionization in positive ion mode(ESI+)with multiple reaction monitoring(MRM).The methodological indicators of the method met the requirements of trace analysis.The detection limits(LOD)of 5 kinds Triptoxins were between 0.1 and 2.5μg/kg;Triptolide A at 10～200 μg/kg and the others Triptoxins at 5～100 μg/kg(n=6)in honey,the average recoveries were between 78.6%and 101%,and the relative standard were less than 11.6%.The method has the advantages of simple operation and high sensitivity,and can be applied to the qualitative and quantitative analysis of multiple triptoxins in honey.(2)Development of the analytical method for quantifying 5 kinds of Triptoxins in biological samplesA solid phase extraction combined with UPLC/MS detection technology was used to establish a method for the simultaneous determination of 5 kinds of triptoxins in whole blood and urine,and the extraction conditions and solid phase extraction purification methods were optimized.The whole blood and urine samples were extracted with ethyl acetate solution and cleaned up with HLB solid phase extraction column,separated by an ACQUITY UPLC BEH C18(2.1×100mm,1.7μm)column with the mobile phase gradient elution of 0.1%formic water acid and 0.1%formic acid methanol.The determination was carried out with electrospray ion source under the positive mode and multiple reaction monitoring(MRM)mode.Under optimal experimental conditions,the detection limits(LOD)of triptolide,Triptonide,triptolide A,Wilfordine,Wilforgine in whole blood and urine were 1.0～6.0μg/L and 1.0～18μg/L,respectively.When whole blood and urine were the substrates,and 5 kinds of triptoxins at three concentrations spiked experiments,the average recoveries were between 79.5%～114%and 79.5%～102%,relative standard were less than 5.86%and 7.73%.both meet the requirements of trace analysis.The methods were used for metabolic studies and also achieved better results.(3)Study on the preparation of tripterygium honey honeyIn the Tripterygium plantation area,"free-range" and "human intervention" were adopted to raise bees,and other toxic honey plants of the tripterygium during flowering period were preliminary investigated.Under the "human intervention" approach,beekeeping has acquired the honey from T.wilfordii,and the content of triptolide was was 10～25.8μg/kg.(4)Mice acute toxicity effect of triptolide in honeyAn oral acute toxicity study was conducted on ICR mice using the exclude and include triptolide honey as the negative group and experimental group(dose 0.879μg/kg·bw)respectively,and the artificially added high-dose triptolide to honey as the positive group(1.00mg/kg·bw).The results showed that the mice in the negative group had no abnormal daily habits and pathological changes of the liver and kidney tissues.The effects of the triptolide honey in the experimental group on the mice showed some gender differences.The female mice were nore sensitive to them and their livers showed slight tissue changes and the others had no abnormalities.The female mice in the positive group showed significant body weight loss and two mice deaths after 48 hours exposure,the liver and kidney tissues of female showed severe pathological lesions and of male mice were slightly damaged.(5)Study on the Metabolic Kinetics and Toxicity of triptoxins in Honey in RatsHoney were artificially added with triptolide and 5 kinds of triptoxins to study the metabolic kinetics and toxicity of SD rats by gavage.The results showed that triptoxins was eliminated quickly in rat’s blood,and it was completely eliminated in about 8 hours,the triptolide peak time(tmax)and half-life(t1/2)of blood in triptolide(4mg/kg·bw)group were 0.54±0.15 and 2.24±0.45h,respectively.The tmax and t1/2 of triptolide,Wilfordine,and Wilforgine in T.wilfordii mixed toxins(4mg/kg·bw)group were 0.62±0.08,0.68±0.12,0.74±0.04h and 2.91±0.78,2.74±0.50,2.65 ± 1.05h,respectively.The biochemical index aspartate aminotransferase(AST)in rats of triptolide group and T.wilfordii mixed toxins group were 89.5±13.6 and 205.0士34.5U/L,and the alanine aminotransferase(ALT)activity were 205.0±24.1 and 307.0±33.0U/L.Compared with the control group,the activity of AST and ALT in the T.wilfordii mixed toxins group was significantly higher than that of the triptolide group.The pathological sections showed that the rat’s damage degree of liver and kidney tissue in triptolide group was less than that in T.wilfordii mixed toxins group.Triptoxins has severe hepatotoxicity in rats,and the degree of damage to the liver and kidney in rats has dose-response relationship.