Study On The Dose-toxicrelationship Of Panax Ginseng And Veratrum Nigrum Component Compatibility

"Eighteen Incompatibilities" is the core content of the incompatibility of Chinese herb drugs.Veratrum nigrum L.(VN),also called lilu,bitter in taste,cold-natured,it had good antihypertensive effect,so with the extensive application of clinical asprevalence of antihypertensive drugs.In recent years,scholars at home and abroad application of modern science and technology,had a large number of research on veratridine toxicity problem,Achieved a proud achievement.Toxicity is one of the characteristics of traditional Chinese medicine basic,But if used correctly,could cure mang complicated treatments,obtain special effect.The effect of veratridine on anti-tumor,anti thrombosis strange has been confirmed by many physicians.With the value of in-depth study and extensive significance.But because its security window is narrow,it is difficult to grasp its therapeutic dose and the toxic dose,so the clinical application is limited VN and ginseng as a pair of incompatible partners,, included in list of the Eighteen Incompatibilities,but the combined application of VN and ginseng drugs on the medical records have been recorded as early as in ancient times,and heritage so far.The compatibility of VN and PG in the treatment of complicated diseases is significant in several ways,If we use modern science and technology.Additional research of the toxicological mechanism of the compatibility of VN and ginseng by the material basis, dose toxicity relationship,toxic effect mechanism,illuminate the incompatible biological mechanism,can draw on the advantages and avoid disadvantages,play a pharmacodynamics.And throμgh the study of typical combination drugs, found the incompatible drug on some of the common law,to find the best combination of safe and effective,which provides an important basis for realizing the compatibility of traditional Chinese medicine safe and effective drugs in clinical development.This study deals with VN and its incompatible partner Panax ginseng (PG), by cytochrome P450 enzymes as the breakthrough point,using HepG2 cell as the toxicity evaluation model,examined the effect of enhancement of toxicity on cell level in vitro by VN and PG. HepG2 cells contained numerous mitochondria,and has rich phase Ⅰ and Ⅱ metabolizing enzymegenes,is considered to be an ideal cell line to evaluate foreign compounds toxicity.Firstly,veratridine treated HepG2 cells after 24 h, the detection of cell viability,cell morphology was observed in HepG2 cells, detect the changes in mitochondrial membrane potential,the level of oxidative stress,cell apoptosis,as well as detect apoptosis related gene by Q-PCR,to determine the toxicity of different concentrations of veratridine model caused by injury and apoptosis different levels of HepG2 cells,and then use the ginsenoside as the main components of PG and veratridine fixed concentration in different proportions deal with HepG2 cells,detected cell viability,observe the morphological changes by DAPI staining,to detect the changes of mitochondrial membrane potential, apoptosis,and the Caspase 3 mRNA to determine the level of increased toxicity at the cellular level. Through the detection of P450 enzyme activity,gene expression and protein level in HepG2,to reveal the incompatible effect mechanism.Finally,through the compatibility of ginsenoside and veratrine in human liver microsomes,corresponding with five subtypes of probe substrate incubated,detected specific metabolites to determine the effect on the metabolic enzymes by LC/MS-MS,further verify the effects of combination of VNand PG on the enzyme activity of cytochrome P450.The results showed that HepG2 cell viability was significantly reduced following exposure to VH 0.1 ～ 0.5 g·L-1.The LDH release rate,ROS and apoptosis rate of HepG2 cells was significantly increased after exposure to VH 0.1～0.5 g·L-1 for 24h,and the mitochondrial membrane potential markedly declined.The expression of apoptosis related gene were increased.0.1 g·L-1 veratridine and ginsenoside compatibility combination,compared with the single use of veratridine,decreased cell viability,mitochondrial membrane potential decreased significantly,the apoptosis rate was significantly increased. Real time fluorescence quantitative PCR detection of Caspase 3 mRNA expression was significantly increased.To detect the expression of HepG2 cell CYP450 enzyme,CYP3A4 in the gene expression,activity and protein levels were suppressed,and has certain effect on CYP1A induced.The activity of human liver microsomal enzyme test results of veratridine and ginsenoside treatment shows,it has a significant inhibitory effect on CYP450.This experiment is based on the cell level, and reveals the possible mechanism of the action of the opposite of the veratridine and ginsenoside, and provides some guidance for the clinical safety.