Study On Pharmacokinetics And Inhibition Of Gastric Cancer Cell For Aconitum Extract And Its Binary Sustained-release Capsules

Aconitum binary sustained-release capsule is transformed from Aconitum injection composed by the raw Chuanwu and raw Caowu for treatment of serious pain in the terminal gastric and liver cancer,which is composed of normal capsule released rapidly and sustained-release capsule in the intestine.Stomach cancer and liver cancer in terminal stage are serious with a kind of continuously sharp pain.Therefore,the injection is transformed into a sustained-release capsules which is composed of normal capsule released rapidly in the stomach and sustained-release capsule sustainably released in the intestine.By effective and slow binary release,drug concentrations in the stomach and liver can be increased and stabilized,which is rational,effective and practical dosage form changes.Based on the previous research,in this article,the method for determining the content of whole alkaloids substance and aconitine,mesaconitine,hypaconitine,benzoylaconitine,benzoylmesaconine,benzoylhypacoitine in vivo was established with the aconitum extract and its binary capsules as research objects.Combining with the tests of the acute toxicity,cardiac,inhibiting the proliferation of gastric cancer cells in vitro and the efficacy of analgesics,the studies on the pharmacokinetics and absolute bioavailability for aconitum and its preparation were carried out to prove that it can strengthen the heart and suppression of cancer while analgesia and define the safe and effective dosage of binary sustained-release capsule for analgesia and suppression of cancer,and to provide theoretical basis for its safe and effective clinical application.Firstly,in terms of analysis,by the method of area under absorbance-wave curve?AUAWC?determining the whole alkaloids substance in rats and Beagle dogs with the methanol precipitation protein and bromocresol green color,there were good specific properties,precision and linearity,and the linear equations were?AUAWC=1.2355C+3.2935?r=0.999?,?AUAWC=0.6992x+1.3403?r=0.9998?.The plasma sample was stable within 8 hours,and the method recovery and the relative recovery were 80.45%-107.34%and 92.13%-105.34%,respectively,which met the requirements for the determination of biological samples.The method of determining content of aconitine,mesaconitine,hypaconitine,benzoylaconitine,benzoylmesaconine,benzoylhypacoitine in plasma was established by ultra performance liquid chromatography mass spectrometry?UPLC-MS/MS?.The six kinds of aconitine alkaloids and internal standard substances could be well separated,and the analysis time was within 8 minutes,and the minimum detection limit and the minimum quantification limit were 0.05 ng·mL^-11 and 0.1 ng·mL^-1.There were good linear relationship in the range of 0.1-200 ng·mL^-1,and the linear equations were Y=0.5752C+0.9537?0.9995?,Y=0.5784C+0.8026?0.9998?,Y=0.6866C+2.4519?0.9998?,Y=0.1096C+0.5388?0.9999?,Y=0.1212C+0.6092?0.9998?,Y=0.0463C+0.16?0.9992?,respectively.And the precision of the instrument was good,and the sample was stable within 12 hours,and the matrix effect and the extraction recovery rate were76.48-102.47%and 75.09-102.98%,which could meet the requirements for the determination of biological samples.The results showed that the methods of AUAWC and UPLC-MS/MS could be used for the determination of aconitum alkaloids and individual components in plasma,respectively.At the same time,the study on pharmacokinetics for aconitum extract and its binary sustained-release capsules in rats and Beagle dogs were carried out by the methods of AUAWC and UPLC-MS/MS.The time of maximum drug concentration in plasma(T_max)of the whole alkaloids substance was 2 h,the time of half-life(t_1/2)was 4 h,and DF was 3.12,and there were difference among the individual components in vivo,so that the t_1/2/2 were different,but all of the components reached the peak concentrations within 1 h,especially aconitine,which reached the peak concentration in 0.25 h,and the volatility of concentration of aconitine and benzoylaconitine were large.It showed that the aconitine alkaloids were quickly absorbed and metabolism in vivo,and the blood drug concentration fluctuated greatly.Although there were difference in the Beagle dogs after administration of the binary sustained-release capsules and ordinary,from the drug time curve of the whole alkaloids and individual components,there was a consistent trend.The drug was absorbed quickly and gradually decreaseed after reaching the maximum blood drug concentration within 1 hour,and the blood drug concentration fluctuated greatly in the group of ordinary capsule.Howere,the drug was released quickly and could be maintained for 30 hours.When the Beagle dogs were administered the same dose of ordinary capsules and binary sustained-release capsules,the Beagle dogs had a poisoning reaction,but the ordinary capsules had a quicker poisoning reaction with dying within 1 hour than that of binary sustained-release capsules with 4 hours.The drug in plasma was determined by the method of UPLC-MS/MS when the Beagle dogs were at death,and the concentration of aconitine was high,which indicated that aconitine was directly related to toxicity.After halving the dose of the binary sustained-release capsule,and the Beagle dogs were health,and the maximum concentration of the whole aconitine alkaloid in plasma was halved,and the concentration of aconitine in plasma was less than 10 ng·mL^-1,Which indicated that the death of Beagle dogs administered the binary sustained-release capsules may be due to the overdose.The drug concentration were determined when the rats were administered different doses of aconitum extract for 1 h.It was found that the concentration of aconitine with the halved dose of the binary sustain-release capsule was significant lower than that of the original dose.The halved clinical dose of wutou injection was used as the dose of the binary sustained-release capsule,and the effective drug concentration in vivo was correspondingly reduced,and the safety was increased.In terms of absolute bioavailability test,the rats were injected with wutou injection in the tail vein and oral aconitum extract,respectively.The method of UPLC-MS/MS was used to determine the concentrations of six aconitine alkaloids in plasm,and the absolute bioavailability of six alkaloids were 28.14%,44.26%,61.48%,57.70%,50.81%,79.06%,respectively.It showed that the drug was converted into other metabolites and hydrolysates after oral administration under the action of the acidic environment in the stomach and the intestinal flora,and the effects of the first pass effect of the liver,resulting in lower absolute bioavailability.Finally,in the acute toxicity test of rats,Lethal Dose,50%(LD₅₀)of crude aconitum and processed aconitum were 3.9 g·kg^-11 and 37 g·kg^-11 respectively,equivalent to 37 times and351.7 times of the clinical dose.LD₅₀0 of the extract of processed aconitum was nine times than that of crude aconitum and the minimum poisoning dose of raw aconite is 0.25g·kg^-1?2.3 times of the clinical dose?.The liver and kidney of dead rats were dark with obvious symptoms of poisoning after dissection,while all the organs of rats were normal with the clinical and lower dose.In terms of cardiac test,compared the heart rate of rats at 0 min with that of 30th min after administration,a trend from the group of crude aconitum was found that the heart rate decreased with the 1-4 times of the clinical dose,then stabilized with the 8 times of the clinical dose,and increased finally with the 8 times of the clinical dose,while for the group of processed aconitum,the trend showed a different order with stability,decreasing and increasing in turn.The result indicated that both raw aconitum and processed aconitum could reach the cardiotonic effect,but the group of crude aconitum could slow heart rate of rats when administered with a low dose,and play a strong role in heart failure patients.The survival inhibition rate was evaluated by 3-?4,5-Dimethylthiazol-2-yl?-2,5-diphenyltetrazolium bromide?MTT?,both extracts of crude aconitum and processed aconitum had significant inhibitory effect on the proliferation of AGS gastric cancer cells,which of crude aconitum was stronger than that of processed aconitum with the same dose.The raw aconitum analgesic efficacy verification test showed that when the dose was the half of the clinical dose of wutou injection,the pain suppression rate could reach 59.26%,which was higher than that of the positive drug ibuprofen.It was concluded that the safety of raw aconitum was smaller than the processed aconitum,but it had a greater effect on slowing heart rate and suppressing cancer,that is to say raw aconite could show a better results at low doses.The analgesic efficacy test also further confirmed that the minimum effective dose was0.5 g of crude medicinal herbs per day for clinical use.In summary,it is feasible to transform the wutou injection into binary sustained-release capsule,which can avoid poisoning caused by too high drug concentration in plasma,and which can achieve the purpose of long-lasting and lasting.The clinical dose of wutou injection(0.5 g raw medicinal material·mL^-1)is 1-2mL each time,1-2 times each day,that is0.5-2 g raw medicinal material everyday,and there is a certain flexibility in the dosing regimen for analgesia of patients,but it also increases the drug-induced toxic side effects in patients with weak constitution.The clinical dose of wutou injection was used as the reference dose,which was converted into the dose of the binary sustained release capsule for Beagle dogs,however,the Beagle dog died of poisoning with the dose,and the concentration of aconitine in plasma was higher at the time of death.And the concentration was significantly reduced with the halved dose,and the Beagle dogs were health.Acute toxicity test showed that the minimum poisoning dose of aconitum extract was 2.3 times of the clinical dose,indicating that 0.5-2 times of the clinical dose?the minimum effective dose of0.5 g raw medicinal materials/day?was safe.Therefore,it was initially determined that the dose of 0.5 g raw medicinal material/day was conversed as the dose of binary sustained-release capsule,and dosing once a day,one ordinary capsule and one enteric capsule each time.