| Gelsemium elegans is a kind of traditional Chinese medicine commonly used for the treatment of nerve pain and anti-tumor,and it is poisonous to people and has growth-promoting effects on foodstuffs such as pigs and sheep.Because of the species difference between humans and animals,there has been no repore on whether there is tissue residue in animals and whether it affects the food safety of animal origin.It was proved by modern medicine that Gelsemium alkaloids was the mian cative parts of Gelsemium elegans.Up till now,there has no any research on the material basis of Gelsemium elegans,determine dynamic changes in vivo of chemical components in plasma or residue depletion.On basis of the the material basis of Gelsemium elegans effect on the whole and chemical component spectra in the early stage of the laboratory and LC-MS/MS analysis technology was mainly used,and on the whole the pharmacokinetics and the law of residue elimination in pigs were studied,and the pharmacokinetics characteristics,dynamic changes and the law of elimination of Gelsemium elegans alkaloids.The relevant findings will lay the foundation for development of Gelsemium elegans in veterinary medicine,the clinical rational use,studies on the quality standard and the pharmacological action,and also provide important scientific reference for animal food safety evaluation.1.Quantitative confirmative study on three kind of Gelsemium elegans alkaloids in pig plasma and tissueIn this paper,an LC-MS/MS assay method was established to simultaneously detect Gelsemine,Koumine and Humantenine in pig plasma and tissue,and its specificity,matrix effect,linear and linear relationship,LOD and LOQ,accuracy and precision,and stability were investigated,results show that the Gelsemine,Koumine and Humantenine in the pig plasma and muscle,liver and kidney is good of specificity,the linear relationship is greater than 0.99,the LOD was 0.2μg/L in pig plasma and was 1.0μg/L in pig muscle,liver and kidney,accuracy between 80-120%,precision <15%,standards comply with FDA regulation of biological sample analysis,the method was also applied to the determination of three kinds of Gelsemium elegans alkaloids in pig bile,urine and other tissue.2.Quantitative confirmatory study on total Gelsemium elegans alkaloids in pig plasma and tissueCreated the multicomponent Gelsemium elegans alkaloids overall quantitative confirmed analysis strategy,adopt semi-quantitative technology,established a multicomponent Gelsemium elegans alkaloids in pig plasma and tissue confirmed quantitative methods solved the multicomponent Gelsemium elegans alkaloids without reference substance situation quantitative analysis of the technical problem at the same time,and the multicomponent Gelsemium elegans alkaloids in pig blood and muscle,liver,kidney,respectively investigates the methodology,the results showed that in the pig plasma method,a total of 27 kinds of Gelsemium elegans alkaloids have better detection results;in pig muscle,liver and kidney,24 kinds of Gelsemium elegans alkaloids have good results.The method was applied to the determination of total alkaloids in pig bile,urine and other tissue.3.Pharmacokinetics of Gelsemium elegans alkaloids in pigs3.1.Pharmacokinetics study on single administration of three standard drug and total alkaloidsThe pharmacokinetics study on the single and multiple administration of Gelsemium elegans alkaloids in pig was carried out using the established quantitative methods of three Gelsemium elegans alkaloids and multi-component Gelsemium elegans total alkaloids in pig plasma.The main pharmacokinetic parameters of single administration pharmacokinetics were calculated by using the non-atrioventricular model.The results showed that the Cmax of Gelsemine,Koumine and Humantenine were 128.67±85.16ng/mL,46.00±36.38 ng/mL and 128.85±74.17 ng/mL,respectively.AUC0-t was 1068.87±837.96 ng/mL*h,272.72±194.04 ng/mL*h and 337.95 ng/mL*h,respectively,and the trend of the drug time curve of the corresponding GA-4,GA-2 and GA-6 in the total alkaloids was consistent.The peaking time of the three alkaloids was 0.92±0.38 h,0.75±0.27 h and 0.5±0h,respectively,indicating that the Gelsemium elegans alkaloids could be absorbed quickly after administration.In addition,27 Gelsemium elegans alkaloids in the total Gelsemium elegans alkaloids were well absorbed.3.2.Pharmacokinetic study of three standard drugs and multiple administration of total alkaloidThe pharmacokinetics study on the single administration of Gelsemium elegans alkaloids in pig was carried out using the established quantitative methods of three Gelsemium elegans alkaloids and multi-component Gelsemium elegans total alkaloids in pig plasma.Non-atrioventricular model was used to calculate the main pharmacokinetics parameters of single dose,the results showed the peak time of 27 kinds of Gelsemium elegans alkaloids was between 0.46 to 2.5h,and all 27 kinds of Gelsemium elegans detected were absorbed at a rapid rate and could reach the peak concentration rapidly.At the same time,their elimination half-life(T1/2)was relatively long,between 8.0 and 72.1h,indicating that the Gelsemium elegans alkaloids had a relatively long retention time in pig,revealing that the Gelsemium elegans alkaloids may have residues and metabolic activation.GA-4,GA-2 and GA-6 in the multi-component uncinated alkaloids have similar peak time and elimination half-life compared with the precise quantitative method results of Gelsemine,Koumine and Humantenine,indicating that the simultaneous quantitative strategy for multicomponent Gelsemium elegans alkaloids without standard reference materials is correct.On this basis,the pharmacokinetics experiments were carried out,the results showed that the peaking time was short,on the one hand,the steady-state concentration of multi-component Gelsemium elegans alkaloids in blood was close to the peak concentration,on the other hand,the whole plant was fed through intestinal absorption was transferred into the blood in a very short time,indicating that the Gelsemium elegans could be quickly absorbed into the blood,and the related mechanism needs further study.In addition,there was a larger area under the curve,which was consistent with results of Gelsemium elegans maintaining a high steady-state plasma concentration.Besides it suggested that there might be residues in Gelsemium elegans.4.Study on the distribution and elimination of Gelsemium elegans alkaloidsThe whole grass of Gelsemium elegans mixture was used to feed pigs for 45 d,and Gelsemium elegans alkaloids were taken as the research object.Through integrating the methods and technologies of pig behavior observation,determination of physiological and biochemical indexs,tissue section,mass spectrometry detection and analysis,and metabolism,effects of Gelsemium elegans alkaloids on growth promotion,distribution and elimination were explored.The results showed that there was no significant difference in daily behavior between the normal control group and the 2% group.Compared with the blank control group,the 2% group was superior to the blank control group in the aspect of meat ratio.There was no significant difference between two groups in blood routine,urine routine and tissue section observation.The above phenomenon indicated that the Gelsemium elegans alkaloids had no obvious toxic effect on pigs and had growth-promoting effect.The distribution and residual elimination of three kinds of Gelsemium elegans alkaloids and multi-component Gelsemium elegans total alkaloids in pig tissues were studied by quantitative methods.The results showed that as many as 23 kinds of Gelsemium elegans alkaloids were detected.All tissues except the gonads were detected to contain Gelsemium elegans alkaloids.However,most alkaloids were not detected or below the quantitative limit after 1d,half of GA-1 detected in the small intestine was attenuated for 6.83 days and half of GA-23 in bile was attenuated for 11.77 days.Further research is needed to establish the relevant maximum residue limits. |
