Analysis For Effects Of Raw And Honey-processed Licorice On The Metabolism Of Main Components In Three Classical Prescriptions By Immunoassay

Glycyrrhiza is derived from the dry roots of licorice,which is widely used in clinical practice.There are different applications of crude licorice and honey-processed licorice in clinical,there is an obvious distinction especially in the ancient prescriptions of the application of crude licorice and honey-processed licorice,and they two could not be replaced by the other.The different applications of crude licorice and honey-processed licorice are the result of the clinical practice of ancient people,the first evidence can be found in "the theory of typhoid fever".In this paper,the immunoassay of glycyrrhizic acid,paeoniflorin and puerarin in mice blood was established by using the monoclonal antibody technique of small molecule of traditional Chinese medicine.The effects of honey on the metabolism of glycyrrhizic acid in licorice from the classic prescriptions of Peony licorice decoction,Sinisan and Gegen Qinlian decoction were studied.The metabolism of paeoniflorin between the two groups of crude licorice and honey-processed licorice were compared in the aspect of tissue and blood,effects of crude licorice and honey-processed licorice on important components in three classic prescriptions were investigated from the aspect of pharmacokinetics,providing an important scientific basis for TCM compatibility.At present,there is a lot of comparative research on the crude licorice and honey-processed licorice,however,the comparative study of different effects of crude licorice and honey-processed licorice on the important components in classic prescriptions have not been investigated based on immunoassay.Objectives:1.To establish an immunoassay for the detection of the content of three small molecules of traditional Chinese medicine(glycyrrhizic acid,paeoniflorin and puerarin)in mice blood,and to establish an immunoassay for the determination of paeoniflorin in mice tissues(Heart,Liver,Spleen,Lung,Kidney);2.To investigate the effects of honey on the pharmacokinetic characteristics of glycyrrhizic acid in mice,and then analyze the different effects of crude licorice and honey-processed licorice on the paeoniflorin or puerarin in Sini san or Gegen Qinlian decoction for the influence of the dynamic characteristics based on immunoassay.Methods:1.The method of enzyme-linked immunosorbent assay of glycyrrhizic acid based on monoclonal antibody of glycyrrhizic acid was established and the methodological investigation was carried out,including linearity,specificity,precision,accuracy,recovery and stability.Monoclonal antibody and quantum dots were conjugated to study the effect of quantum dots on glycyrrhizin enzyme-linked immunoassay and to establish a quantum dot-based glycyrrhizin-linked immunoassay for the first time;2.The changes of metabolic kinetic parameters of glycyrrhizic acid were studied by comparing differences between licorice extract and glycyrrhizic acid with and without honey respectively.The main components of honey(glucose,fructose,sucrose and the mixture)were investigated separately on the effects on the metabolism of glycyrrhizic acid;3.The method of enzyme-linked immunosorbent assay(ELISA)was used to study the effects of paeoniflorin based on paeoniflorin monoclonal antibody in mouse blood and tissue(liver,spleen and lung),including linear,specificity,precision,accuracy,recovery and stability.The pharmacokinetic parameters of paeoniflorin were evaluated based from the aspect of pharmacokinetics in the tissues and blood.The effect of crude and honey-processed licorice was compared on the analgesic effect of paeoniflorin after combined with peony was evaluated from the aspect of pharmacology;4.The method of enzyme-linked immunosorbent assay(ELISA)was used to study the effects of paeoniflorin on the blood of mice,including linearity,specificity,precision,accuracy,recovery and stability.Effects of crude and honey-processed licorice on the pharmacokinetics of paeoniflorin in Sini san was investigated and compared;5.The method of-enzyme-linked immunosorbent assay(ELISA)was established by using puerarin-based puerarin monoclonal antibody.The method was verified,including the linearity,specificity,precision,accuracy,recovery and stability.Effects of crude and honey-processed licorice on the pharmacokinetics of-puerarin was investigated and compared.Results:1.The linear range of glycyrrhizic acid was 7.81 ng/mL-1000 ng/mL,and the regressionequation was y =-0.143 In(x)+1.2551(R2 = 0.9918).The accuracy is good,the recovery rate is 100.52%-104.02%,suitable for the detection of biological samples,the detection stability is good;2.The linear range of glycyrrhizic acid based on monoclonal antibody of glycyrrhizic acid was established in the blood of mice.The linear range was from 7.8 ng/mL to 250 ng/mL,and the regression equation was y =-0.141 In(x)+ 0.9268(R2 = 0.9935),the recovery rate was 98.04%-103.25%,and the quantum dot-based glycyrrhizin-based immunoassay was established for the first time.The quantum dot was diluted in the ratio of 1:1000,and its linear range has a good precision and accuracy,the recovery rate is 101.6%-113.4%,the linear range of quantum dot-based glycyrrhizin-based immunoassay was from 31 ng/mL to 4000 ng/mL,and the regression equation was y =-0.0741n(x)+ 1.0074(R2 = 0.9921).Both methods had a good accuracy,suitable for the detection of biological samples with a good stability for detection;3.The peak time of glycyrrhizic acid in blood was 40min after the administration of licorice extract alone,and the peak time of glycyrrhizic acid was 240min after the administration of licorice extract with honey.After administration of glycyrrhizic acid in mice,the peak time of glycyrrhizic acid in blood was 45min,and the peak concentration of glycyrrhizic acid was delayed to 600min after the administration adding with honey.Given glycyrrhizic acid and glucose to the mice,the peak time of glycyrrhizic acid in blood was 45min,and the peak time of glycyrrhizic acid was 720min after the administration of fructose with glycyrrhizic acid,and the peak time of glycyrrhizic acid was 720min after given sucrose with glycyrrhizic acid.After the administration of glycyrrhizic acid with the mixture of three kinds of sugar,the peak time of glycyrrhizic acid was 630min;4.Immunoassay of paeoniflorin based on paeoniflorin monoclonal antibody in mouse blood,and tissues were established.The linear range was from 3.13 ng/mL to 1600 ng/mL,the precision and good accuracy demonstrate the method was suitable for the detection of biological samples,the detection stability is good.(1)The results showed that the 'maximum plasma concentration of paeoniflorin was 14.33ug/mL and the peak time was 35min in group of HRL(P<0.05).The results showed that the content of glycyrrhizic acid in HPL group was significantly higher than that in the control group.The maximum plasma concentration of paeoniflorin in HRL group was 9.08 ug/mL,the peak time was 35min,the maximum plasma concentration of paeoniflorin in MRL group was 15.37 ug/mL,the peak time was 35min,The maximum plasma concentration of paeoniflorin in MPL group was 12.42 ug/mL,the peak time was 35min,the maximum plasma concentration of paeoniflorin in LRL group was 11.10ug/mL,the peak time was 35min,the maximum plasma concentration of the low dose of honey-processed licorice LPL group was 7.49 ug/mL and the peak time was 240 min.(2)The content of paeoniflorin in the high dose group(HRL group)was 0.55ug/mg,and the content of paeoniflorin in honey-processed licorice high dose group(HPL group)was 0.06ug/mg,which was significantly different.In other tissues,the content of paeoniflorin was also revealed differences;(3)Hot plate test showed that the longest response latency time of the mice was 15.08s and it was in the middle dose group of honey-processed licorice(MPL group),response latency time of the correspondingly honey-processed licorice group(MPL group)was 12.69s and the time points reaching the longest response latency were all at 60 min in most of groups while it was at 90 min in the low dose group of honey-processed licorice(LPL group);5.The maximum plasma concentration of paeoniflorin was 4949.12ng/mL in crude licorice group of Sini san,the peak time was 35min,while the maximum plasma concentration of paeoniflorin was 6732.46 ng/mL in the honey-processed licorice group in Sinisan decoction,and the peak time was 60min.The area under the curve of blood of honey-processed licorice was about 1.5 times of that of the crude licorice group.6.The icELISA was established in the blood of mice based on puerarin monoclonal antibody.The linear range was from 3.9 to 1000 ng · mL-1,the regression equation was y =-0.1661n(x)?1.0016(R2 = 0.9939),and the recovery range was from 104.08%to 107.25%.The maximum plasma concentration of puerarin was 74.38ng/mL in the crude licorice group and that of honey-processed licorice group was 67.77 ng/mL,and the peak time of both groups was 60 min.Conclusion:1.The pharmacokinetic parameters of glycyrrhizic acid in mice were studied after the combination of honey and licorice extract or glycyrrhizic acid.Honey could sustain or delay the peak time of maximum concentration of glycyrrhizic acid peak time,which proved the correctness of the Chinese medicine theory "sweet food may help decrease the acute pain",and this sustain effect is likely to be caused mainly by the fructose in honey;2.Set Peony licorice decoction as the template,the effects of crude and honey-processed licorice on the metabolism of paeoniflorin in blood and tissues of mice were investigated.The results showed that the honey-processed licorice may sustain the peak time of paeoniflorin in the blood and tissues,in contrast to crude licorice.In the experiment of hot plate test,the reaction time of mice after given honey-processed licorice with peony was significantly prolonged compared with crude licorice with peony group.This suggests that this effect maybe caused by honey in the processing of licorice on peony,it is a valuable data to verify the compatibility of traditional Chinese medicine in the compatibility of honey-processed licorice with peony;3.Further studies in comparing crude licorice and honey-processed licorice on the pharmacokinetics of paeoniflorin in Sinisan and puerarin in Gegen Qinlian decoction were carried out,results showed a sustaining effect of honey-processed licorice on the pharmacokinetics of paeoniflorin in Sinisan.The effect of honey-processed licorice on the peak time of puerarin in Gegen Qinlian decoction was not obvious,but it had certain influence on its plasma concentration and other metabolic parameters,which indicated that honey-processed licorice had certain effect in different Chinese herbal compound,mainly reflected in the mitigate of the drug efficacy and postpone of the peak time of important components,it is in accordance with the traditional Chinese medicine compatibility theory of honey-processed licorice has the effect of coordinating the drug actions in a prescription.