| The study is a part of "Essential study of the property of TCM base on three factors theory", which is supported by the Major State Basic Research Development Program of China(National Project 973)(No.2007CB512605). The study focuses on investigating the factors of the formation and expression of the property of TCM. A hypothesis is proposed that the property of TCM derives from its bioactivity, which is related to the status of organism and chemical constituents. Therefore, the status of organism, chemical constituents and bioactivity are considered to be three factors of the property of TCM. The organism plays a role in expression of bioactivity of drugs. This study is searching for physiological indexes characterizing the status of organism and machanism of bioactivity of drugs in different organism status.Drug metabolism is an important form of the body action to drug, the action may be affected when the body appearance changed.Aconitum carmichaeli is a kind of processed product of Aconitum carmichaeli Debx roots in the secondary root of buttercup, its functions are to cause restoration from collapse, supplement body fire and reinforce yang, dispel wind, cold and damp. The un-processed product of Aconitum carmichaili has strong toxicity; it can use when it was preprocessed to reduce toxicity.We chose representatives of Aconite as a toxic medicine, aconite aconitine contained in the new hypaconitine aconitine and its effective and toxic ingredients, but for the first two hypaconitine relatively stable in the body. Toxicity and toxic drugs for the study the relationship between body condition, to be on the different states hypaconitine poison Pharmacokinetics big difference and the distribution differences of the heart were divided into two parts:First, the establishment of hypaconitine simple, stable and high recovery of biological samples was used to determine the time point of blood in rats.Comparison of liquid-liquid extraction, alcohol precipitation and solid phase extraction method, results show that by liquid liquid extraction, the samples were extracted with diethyl ether to extract more than 70% recovery rate, easy operation, low cost, ease of clinical application and experimental study. Hypaconitine for later analysis of in vivo accuracy and sensitivity to provide protection.Rats were selected 21 hypaconitine, ig dose of 2mg·kg-1. After drug administration for 15min,25min,60min,120min,240min,420min and 660min inferior vena cava blood, hypaconitine in normal rats plasma concentration after an initial drop in the near 4h maximum plasma concentration after gradually decreased.7 hours after the blood concentration decreased to stabilize. Hypaconitine fast in the normal rat heart in the distribution, then decreased. Another absorption peak at 2h after the Department gradually decreased,7 hours later reduced plasma concentration stabilized.The results showed that hypaconitine rapid distribution of produce in the heart of the role of pharmacology and toxicology, and clinical effect reported in line quickly. While its retention in the body longer.Through this research, the preliminary draw hypaconitine in rats and the pharmacokinetic changes of heart in the target organs in the distribution of blood to determine time of 5min, 15min,30min,60min,120min,180min,210min,240min,300min,360min,480min, 720min.Carried out for the next big hypaconitine poison Pharmacokinetics of different states and different heart research basis for content distribution.Second, in rat plasma and cardiac hypaconitine LC/MS/MS analysis testing internal standard method. Study hypaconitine in Deficiency rats, and Hot rats and normal rats pharmacokinetics differences and differences in the heart of the distribution.Propafenone hydrochloride as the internal standard, rat plasma containing drug and drug-containing heart homogenate by liquid-liquid extraction using diethyl ether extraction, samples were positive electrospray ionization ion source, the adoption of triple quadrupole tandem mass spectrometer, using multiple reaction monitoring (MRM) scan mode (monitoring ions hypaconitine 616.6-105.2) plasma level and heart hypaconitine concentration. Optimization of chromatographic conditions for the Gemini C18 column (150×4.6mm,5μm),Waters C18 pre-column;methanol -0.1% formic acid as the mobile equivalent degree of elution,0.5mL·min-1 flow rate.3 min in the peaks of endogenous substances do not interfere with sample analysis; Plasma and cardiac samples hypaconitine in the 0.1～12.5 ng·mL-1 range a good linear relationship;Limit of quantification was 0.1 ng·ml-1.High, medium and low concentration of drug-containing plasma samples of three days of good precision and inter-day precision, extraction recovery and stability, samples at room temperature, frozen (-80℃), freezing, frozen residue (-20℃) conditions Stability meet requirements. The method is sensitive, specific, and the advantage of rapid,consistent with biological sample analysis requirements.Replication Deficiency model with hydrocortisone, dexamethasone copy and Hot models, normal rats treated with saline,2 weeks after the administration 5min,15min,30min,60min, 120min,180min,210min,240min,300min,360min,480min,720min blood and heart, processing, measurement, drawing the curve and distribution curve of the heart.It can be seen hypaconitine gradually absorbed in normal rats,120min Department has an absorption peak concentration after the decrease in the maximum blood concentration of 240min soon after the lower,300-480min maintained within a relatively stable level, to 720min when the lower levels. In Deficiency rats,120min after the maximum plasma concentration quickly decreased,300min Department has a small absorption peak, then gradually decreased. Rapidly absorbed in rats and Hot,30min to reach maximum plasma concentration, quickly reduce,180-360min maintained between the relatively stable level, then slowly lower. Hypaconitine and Hot Rats in the peak faster than the Deficiency rats and normal rats, rats and in Deficiency and Hot Rats maximum plasma concentration was higher than normal rats, in three types Rats have long residence time, suggesting the role of a kind hypaconitine long-term mechanism. Speculated that the phenomenon of double peaks exist enterohepatic circulation. Software to process data with DAS2.1.1 drug in normal and Deficiency of Cold and Hot models in rats by non-linear process of statistical moment method to calculate pharmacokinetic parameters AUC0-t of normal rats were 834.025 ug·L-1·min;MRT0-t for the 247.144mm; t1/2z to 145.384min.Deficiency Taishu AUC0-t were 794.75 ug·L-1·min; MRT0-t for the 224.082min;t1/2Z to 152.502min. And Hot Taishu AUC0-t were 553.625 ug·L-1·min; MRT0-t for the 249.979min; t1/2Z to 475.683min.Hypaconitine quickly in the normal distribution of rat heart,15min is the highest concentration, then decreased, a longer period in the 120-300min maintained at a stable level, then to low and stable levels within 720min. Deficiency in the rat heart in rapidly absorbed, 30min and reached the highest concentration, then decreased at 60-180min to maintain at a stable level, over time hypaconitine further reduce the content in the body, but in 210 the more stable-300min,360min has an absorption peak, then gradually decreased and stabilized. In the rat heart and Hot gradually absorbed the highest concentration in the 120min after declining in the 210-360min has an absorption peak between, then gradually decreased and stabilized. Comparison of three states hypaconitine rat heart can be seen the distribution of normal and high content of rapidly absorbed, followed by the Deficiency group and Hot group absorbed slowly and with lower content. Three states have the phenomenon of double peaks, suggesting enterohepatic circulation exists. From this distribution trend can be seen hypaconitine rapid distribution of produce in the heart of the role of pharmacology and toxicology, and clinical effect reported in line quickly. While its retention in the body longer. Hypaconitine the same content in different states with different absorption and distribution in rats, the intensity of Pharmacology and Toxicology produce different effects.Through this research, get hypaconitine rats in different states between pharmacokinetics and distribution of the differences in heart. Hypaconitine state is symptomatic treatment for the Deficiency for and Hot state is evidence to the contrary, the role of a strong performance for the normal state of toxicity. This traditional Chinese medicine theory of "syndrome differentiation, right medicine," "toxic poison is to the ordinary people, and with the patient medical treatment" consistent, rational use of drugs to prove the correctness of clinical stress.This is the first animal model used Deficiency and Hot animal model compared with the normal animal testing and heart blood concentration distribution of drugs and the relationship between the state body for the study of toxicity of traditional Chinese medicine to provide new ideas and methods. |
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