| Background:Aconiti Lateralis Radix Praeparata(Fuzi)is the processed lateral root of Aconitum carmichaelii Debx.It has the effects of rescuing from collapse by restoring Yang,which is known as the most effective medicine.Fuzi has an efficient therapeutic effect on cardiovascular diseases.However,Fuzi also shows strong neurotoxicity and cardiotoxicity.Improper use of fuzi will cause arrhythmia,coma,and even death.The main toxic components of fuzi are diester alkaloids and monoester alkaloids.The difference of metabolic kinetics of multiple alkaloids at multidose is essential,because the therapeutic window was narrow and most adverse reactions were caused by improper use of dosage.Heart failure is the final stage of the development of various heart diseases.Its morbidity and mortality are extremely high,which mainly manifested as dyspnea and fatigue.However,there is no very effective treatment plan at present.The combination of fuzi and Radix Ginseng could be used to improve blood circulation in the body for the heart failure and other diseases.However,the characteristics of metabolic dynamics of aconite and ginseng combined in animals with heart failure are not clear.Therefore,the metabolic characteristics of fuzi single use or combined with ginseng in pathological state need to be further explored to provide a basis for clinical safety.Objective:To establish a UPLC-MS/MS method with strong specificity,high sensitivity and convenience for quantitative analysis of six aconitum alkaloids in rat plasma samples,and then verify the methodo.The characteristics of pharmacokinetic parameters after single intragastric administration of 0.5 g/kg,1.0 g/kg and 2.0g/kg fuzi in normal rats were measured and analyzed.The pharmacokinetic parameters between groups need to be compared.The rats with heart failure was established and evaluated.The pharmacokinetic difference of fuzi between the normal and heart failure rats was measured and analyzed.Besides,the effect of continuous administration of Panax ginseng on the metabolic behavior of fuzi in the rats with heart failure was determined.Methods:The biological samples were pretreated by protein precipitation method,and the UPLC-MS/MS method of quantitative analysis for six substances in rat plasma was established.The gradient elution separation was performed with 0.1%formic acid water-0.1%formic acid acetonitrile.The positive ion mode of multi-reaction monitoring was used for quantitative detection.In addition,the specificity,sensitivity,precision and accuracy,matrix effect,extraction recovery and stability of the method were verified according to the requirements of Chinese Pharmacopoeia 2020.The method of pharmacokinetic study of fuzi in different doses:24 SD rats(half male and half female)were randomly divided into three groups:high(2.0 g/kg),middle(1.0 g/kg)and low(0.5g/kg)dose groups.The blood samples were collected from the fossa orbitalis vein at 0.08,0.25,0.5,1,2,4,8,12,24 and 48 h after oral administration of fuzi extracts.Using UPLC-MS/MS for quantitative detection after sample pretreatment,the collected data were analyzed and processed by Excel,Graph Pad software and SPSS software.The pharmacokinetic parameters were calculated by DAS pharmacokinetic program with using non-compartment method.The animal model with heart failure was established by ligating the descending branch of left coronary artery in rats,which would lead to myocardial ischemia.The changes of markers,such as BNP,CTNT AST,CK and CKMB in the rats serum,ECG,carotid artery pressure,left ventricular pressure,heart coefficient and corresponding pathological sections of the rats with heart failure were used to evaluate the success of establishing animal models with heart failure.The experimental methods of fuzi in normal animals and heart failure model animals were as follows.The experimental rats were divided into three groups(each group eight rats):The normal,model,model plus Panax ginseng group.After two weeks of recovery of myocardial infarction model rats,the model plus Panax ginseng group were given 1.0g/kg extract of Panax ginseng for seven consecutive days,and the other two groups were given 1.0m L/kg distilled water at the same time.After three weeks,the rats in each group were orally administered with 1.0g/kg fuzi extract,and 0.30 m L plasma samples were taken from the fundus venous plexus at 0,0.25,0.5,1,2,4,8,12,24 and 48 h after administration.UPLC-MS/MS was used to detect the concentration of samples.Excel,Graph Pad and SPSS software were used to analyze and process the collected data.DAS software was used to calculate and analyze the pharmacokinetic parameters.Results and discussion:A method for simultaneous determination of six aconitine alkaloids in rat plasma was established.The standard curve range of this method was 0.1-10 ng·m L-1,which could detect the plasma concentration of six aconitine alkaloids in 5min.The specificity,linearity,accuracy and precision,stability,extraction recovery and matrix effect of this method were in line with the verification guideline of biological analysis method in Chinese Pharmacopoeia 2020.The main pharmacokinetic parameters of different dosages of fuzi in normal rats showed that the AUC(0-48h)of six substances in low dose group was 13.47±3.28,62.90±26.89,26.39±5.42,16.21±5.43,0 and 6.85±2.40?g/L*h,while in high dose group was 23.82±7.77,185.72±64.72,85.82±20.71,50.54±19.45,17.29±6.18 and44.20±18.82?g/L*h,which showed that the AUC(0-48h)of high dose group was significantly higher than that of low dose group,indicating that the absorption was better.The Vd of six aconitine alkaloids in the high dose group was 166.81±80.09,80.41±35.49,87.22±43.08,800.57±396.01,383.54±177.00,616.61±290.20 L/kg,while the low dose group was 14.01±4.33,12.75±6.88,33.10±25.06,193.64±116.81,0,207.34±75.96 L/kg,which could be seen that the six aconitine alkaloids in the high dose group could be distributed to more extensive tissues and organs.The t1/2 of six substances in high dose group was 11.47±4.30,11.92±4.59,6.04±3.85,13.67±4.26,8.75±7.14 and 19.14±8.84 h,respectively,and those in the low-dose group were1.90±0.31,1.83±0.64,1.72±0.25,4.48±0.64,0 and 4.53±3.84 h,indicating that the duration of drug action in the high-dose group was longer.The model rats with heart failure were obtained by ligating the descending branch of the left coronary artery.It was found that using ventilator,electric blanket and moving down the ligation site during operation could significantly improve the survival rate of rats.The results of the evaluation model showed that compared to the model group rats,the levels of markers,such as BNP,CTNT,CK,AST and CKMB in the normal group,sham operation group and model plus Panax ginseng group were significantly decreased.The ECG of rats in the model group showed an increase in ST segment.The LVSP and+dp/dtmax of rats in the model group decreased significantly,while LVEDP and-dp/dtmax showed an obvious upward trend,but there was no significant difference in HR and MAP.According to the state of hearts,it was observed that the heart coefficient,myocardial hypertrophy and myocardial infarction of the rats in the model group was significantly increased.The pathological section of the heart of the rats with heart failure showed that there was a large area of myocardial cell death and the gap between myocardial fibers was widened.Compared to the model group,the heart failure markers in the model plus Panax ginseng group were significantly decreased,the ECG and cardiac function indexes were improved,the death area of myocardial tissue cells was smaller,and the fiber gap of myocardial cells was narrowed.The pharmacokinetic results of fuzi in normal and heart failure rats showed that Tmax of AC and HA in model group were 2.25±1.73,2.00±1.64 h,in the normal group were4.57±1.51,5.33±3.27 h,while in the model plus Panax ginseng group were 5.14±1.95,5.33±2.07 h,respectively.The AUC(0-48h)of AC and HA in model group were3.96±1.47,54.58±19.35?g/L,in the normal group were 8.25±3.51,138.42±33.39?g/L,while in the model plus Panax ginseng group 8.29±3.10,15±59.46?g/L,respectively.The results showed that the absorption of two substances in model group was relatively low,and it could reach the peak in a short time.After continuous administration of Panax ginseng,the absorption value of two substances increased significantly,which achieved the effect of promoting its absorption,and the peak concentration decreased.There was no significant difference in the apparent distribution volume among the three groups,indicating that the distribution of the two alkaloids in the rats with heart failure was similar to that in normal rats.The t1/2 of AC and HA were4.48±1.72 and 4.17±0.34 h in the model group,10.99±4.25 and 9.16±3.30 h in the normal group and 17.83±8.78 and 7.63±0.78 h in the model plus ginseng group.It can be seen that compared to the model group,the half-life of AC and HA in the normal group and the model plus ginseng group was significantly prolonged.The Clz/F of AC and HA in the model group was 23.08±6.46,7.59±2.29 L/h/kg,the normal group was13.20±3.27,3.26±0.74 L/h/kg,and the model plus Panax ginseng group was9.01±3.11,3.49±1.26 L/h/kg.It can be found that the clearance rate of AC and HA in the model group was significantly increased,indicating that the model group had faster elimination rate and shorter time.Conclusion:A rapid,sensitive and reproducible UPLC-MS/MS method to simultaneously detect the concentration of six aconitine alkaloids in rat plasma has been developed,validated and then employed to the pharmacokinetics study successfully.The pharmacokinetic characteristics of six aconitum alkaloids after oral administration of fuzi extracts in SD rats have shown that the Cmax,t1/2 and AUC(0-48h)of the six compounds were increased with multiplicative dosages,and there were significant differences between the high and low dose group and the six aconitum alkaloids in the high group could be more quickly absorbed,more slowly eliminated and more widely distributed.The rat model of heart failure was established by ligating the descending branch of the left coronary artery,which met the requirements.It also showed that Panax ginseng could improve the heart function of the rats with heart failure to some extent.Finally,compared to normal rats,the absorption of fuzi extract in heart failure model was poorer,and metabolic rate was accelerated.After given 1.0 g/kg extract of Panax ginseng for seven consecutive days,the absorption of AC and HA in model rats was increased and the elimination rate was slower,which further illustrates that fuzi and Panax ginseng used together could improve effect and decrease toxicity.The pharmacokinetics data could provide valuable information for the clinical safety application of fuzi. |
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