The Mechanism Research About Gelsemium Elegans Compatibility With Mussaenda Pubescens To Reduce The Toxicity

Objective:To discuss the detoxification mechanism of Gelsemium elegans combining with Mussaenda pubescens, clarifing the scientific connotation of detoxification, and providing the experiment data and theoretical bases to the safe and rational clinical application of Gelsemium elegans.Method:1. The correlation experiment of toxicity/efficacy:The extraction solvent best compatible ratio、toxic part and detoxification part of Gelsemium elegans was optimized with the death of mice as index. The analgesic effect before and after compatibility of Gelsemium elegans was studied by three kinds of analgesic experiments of acetic acid stretching, hot plate and formalin.2. Effects on drug transport:The stability of toxicity/efficacy part of Gelsemium elegans was studied by HPLC. The toxicity/efficacy part of Gelsemium elegans was identified by UPLC-MS/MS. Three kinds of experiments of bidirectional transmembrane transport、P-gp transport and MRP2 transport were investigated in toxicity/efficacy part of Gelsemium elegans by Caco-2 cells monolayers model.3. Effects on biotransformation:It may clarify the phase Ⅰ metabolites mechanism of Gelsemium elegans before and after compatibility by gene mRNA expression of CYP2E1 and CYP1A2 with RT-PCR, protein expression of CYP2E1 and CYP1A2 with ELISA or WesternBlot. It may clarify the phase Ⅱ metabolites mechanism of Gelsemium elegans before and after compatibility by gene mRNA expression of GST-pi and GSTml with RT-PCR, protein expression of GST-pi and GSTm1 with WesternBlot, and activity of GST enzyme.Results:1. The correlation experiment of toxicity/efficacy(1) Detoxification experiment ①Extraction solvent screening:A gradual increase in toxicity of Gelsemium elegans was found with the increase of ethanol concentration. The mice death rate was 100% in group of 90% alcohol extract. Thus, the extraction solvent was intended as 90% ethanol. ②Optimal proportions experiment:The mice death rate declined with the increase of combination rate of Gelsemium elegans and mussaenda pubescens. When the combination rate was more than 1:40, the mice death rate no longer reduced. Thus, the optimal proportion was intended as 1:40, and the mice average death rate was 23.33±5.78%. ③Toxicity/efficacy part experiment:The mice death rate was 100% after dosing dichloromethane part from 90% alcohol extract of Gelsemium elegans. The dichloromethane part was toxicity part. When the toxicity part was combined with four extraction parts of mussaenda pubescens respectively, the mice death rate all declined. Aqueous part was detoxification part for its low death rate. The average mortality was remarkably decreased after toxicity part combined with detoxification part(P<0.05).(2) Analgesic experiment ①Acetic acid stretching experiment:As compared with the blank group, the twisting times was markedly reduced within 15 min in each treated group. The analgesic rate was dose-dependent after combined use of Gelsemium elegans and mussaenda pubescens. ②Hot plate experiment:After treatment 30min, the pain thresholds was very significantly different from that in the blank group(P<0.05). The effect remained after treatment 60min and 90min in high dosage group(P<0.05). ③Formalin experiment:Compared with the blank group, duration of pain response in the phase Ⅱ was remarkably decreased in each treated group(P<0.01). Compared with the blank group, duration of pain response in the phase Ⅰ was remarkably decreased in high and middle dosage group(P<0.01).2. Effects on drug transport(1) Stability study before drug transport ①Stability study by HPLC:The RSD of relative retention time of common peak in toxicity/efficacy part was 0.28%-0.91%, and RSD of relative peak areas was 0.35%-4.02%. The toxicity/efficacy part was stabilized within 48h. The analysis method of 9 common peaks had good repeatability, stability and characterization. The similarity of chromatographic fingerprint was at 0.93 upwards. ②Identification by UPLC-MS/MS:6 kinds of indole alkaloids, gelsemine、koumine、humantenidine、 gelsenicine、gelsevirine and humantenine, were respectively indentified in toxicity/efficacy part by UPLC-MS/MS.(2) Caco-2 model experiment:①bidirectional transmembrane transport:The Papp value of AP→BL direction was decreased from toxicity/efficacy part, particularly humantenidine、 gelsenicine、gelsevirine and humantenine. But the Papp value of BL→AP direction was increased. The y value of humantenidine、gelsenicine and gelsevirine was at 2 upwards. ②P-gp transport:The absorption and efflux of 6 kinds of indole alkaloids were all increased after combined use of verapamil and toxicity part. After verapamil was combined with toxicity part and detoxification part, the efflux rate of humantenidine decreased by 43.69 percent. And that of gelsenicine, gelsevirine and humantenine decreased by 41.42,36.00 and 8.90 percent, respectively. ③MRP2 transport:The absorption and efflux of 6 kinds of indole alkaloids were all increased after combined use of ciclosporin and toxicity part. After ciclosporin was combined with toxicity part and detoxification part, the efflux rate of humantenidine decreased by 42.32 percent. And that of gelsenicine, gelsevirine and humantenine decreased by 40.59,34.00 and 15.07 percent, respectively.3. Effects on biotransformation(1) Effects on phase Ⅰ metabolites ①Effects on gene expression:As compared with the blank group, the content of CYP2E1 gene expression was markedly reduced in toxicity part groups(P<0.01). But it was increased after combined use of toxicity part and detoxification part, compared with the high toxicity part group(P<0.05). As compared with the blank group, the content of CYP1A2 gene expression was reduced in toxicity part group(P <0.05). It was increased after combined use of toxicity part and detoxification part, but there was no significant statistic difference. ②Effects on protein expression:As compared with the blank group by WesternBlot, the content of CYP2E1 protein expression was markedly reduced in toxicity part groups(P<0.05). But it was increased after combined use of toxicity part and detoxification part, compared with the high toxicity part group(P<0.05). As compared with the blank group by WesternBlot, the content of CYP1A2 protein expression was reduced in high and middle toxicity part group(P<0.05). It was increased after combined use of toxicity part and detoxification part, but there was no significant statistic difference. It showed positive staining cells appeared tan or brown by immunohistochemical method. As compared with the blank group, the areal and number density of CYP2E1 were markedly reduced in toxicity part groups(P<0.01 or P<0.05). But those were increased after combined use of toxicity part and detoxification part, compared with the high toxicity part group(P<0.01 or P<0.05). As compared with the blank group, the areal and number density of CYP1A2 were reduced in toxicity part groups(P<0.01 or P<0.05). But those were increased after combined use of toxicity part and detoxification part, compared with the high toxicity part group(P<0.05).(1) Effects on phase Ⅱ metabolites ① Effects on gene expression:As compared with the blank group, the content of GSTml gene expression was markedly reduced in toxicity part groups(P<0.01). But it was increased after combined use of toxicity part and detoxification part, compared with the high toxicity part group(.P<0.05). As compared with the blank group, the content of GST-pi gene expression was reduced in high toxicity part group(P< 0.05). But there was no significant statistic difference in other groups. ②Effects on protein expression:As compared with the blank group, the content of GSTm1 protein expression was markedly reduced in toxicity part groups(P<0.05). But it was increased after combined use of toxicity part and detoxification part, compared with the high toxicity part group(P< 0.05). As compared with the blank group, the content of GST-pi protein expression was reduced in toxicity part groups(P<0.05). It was increased after combined use of toxicity part and detoxification part, but there was no significant statistic difference. ③Effects on enzyme activity:As compared with the blank group, the content of GST enzyme activity was markedly reduced in toxicity part groups(P<0.01 or P<0.05). But it was increased after combined use of toxicity part and detoxification part, compared with the high toxicity part group(P<0.05).Conclusion:1. The dichloromethane part of Gelsemium elegans not only reduces toxicity, but also keeps the analgesic efficacy after combined with detoxification part of Mussaenda pubescens.2. The reducing absorption and increasing efflux of indole alkaloids are founded in Caco-2 model after combined use of toxicity part and detoxification part. The existence of MRP2 and MRP2 transporters revulsant has been proposed as possible mechanisms.3. The purpose of detoxification basically achieve through up-regulation gene expression of CYP2E1、CYP1A2 in phase I metabolites and enhancing GST enzyme activity in phase II metabolites after combined use of toxicity part and detoxification part.