| Objective:To evaluation of the acute toxicity of sinomenine hydrochloride（SH）in normal male and female rats and mice.and then study and compare the tolerance differences of SH in rats of different genders by a newly established liquid chromatography-mass spectrometry（UPLC-MS/MS）analysis and detection method.Methods:1.Using stratified random grouping method,220 male and female KM mice were assigned into 22 groups according to five administration routes of gavage,abdominal cavity,tail vein,muscle and subcutaneous,10 mice per group.And then the mice in each group were fasted with free access to water for 24 hours,and the corresponding dose of SH was given to the mice in each group on the second day.After administration of each group,the behavioral characteristics of mice were observed for 3 consecutive days and the number of mice died in each group was recorded.The median lethal dose(LD50)of SH to KM mice in different administration routes was calculated by SPSS21.0 software.72 male and female SD rats were randomly assigned into 9 groups,8 rats per group,and each group was administered the corresponding dose of SH by gavage.The behavioral characteristics and maximum tolerated dose（MTD）of male and female rats were observed and compared according to the same method to calculate the LD50of SH on normal male and female rats was calculated.2.Twelve male and female SD rats were assigned into two groups（male group,female group,6 rats per group）.After fasting for 24 hours,the rats in both groups were given the same dose of SH by gavage at 25 mg/kg（both groups were administered at 10 ml/kg and the concentration of SH was 2.5 mg/ml）.Then,blood samples were collected from the jugular vein of the rats at different time points using heparin sodium anticoagulation blood collection tubes.72 male and female SD rats were randomly assigned into 12 groups（6 male groups,6 female groups,6 rats per group）.After intragastric administration of 25 mg/kg of SH,the tissues and organs of rats were collected at 0 min（before administration）and 15 min,30 min,45 min,90min,and 120 min after administration.The established UPLC-MS/MS analysis method was used to determine the concentrations of sinomenine in plasma,heart,liver,spleen,lung,kidney,brain,testis or uterus,ankle joint respectively,and the main pharmacokinetics parameters were calculated by the Phoenix Win Nonlin 8.1 software non-compartmental model.Graph Pad Prism 8.0 software was used to draw drug concentration-time curves and histograms of the distribution of organs in each tissue,and SPSS 21.0 software was used to compare the pharmacokinetic parameters and the content of sinomenine in each tissue between groups.Results:1.After SH was administered by different routes,KM mice in each group developed acute toxic reactions of varying degrees within 5-30 minutes,mainly manifested as prone lying,slow movement,dark purple limbs,muscle fasciculations,and eventually death due to respiratory failure.The LD50 of different administration routes were gavage administration:474mg/kg,intraperitoneal administration:277mg/kg,tail vein administration:162mg/kg,muscle administration:422mg/kg and subcutaneous administration:455mg/kg.At the same time,both male and female SD rats were in poor condition,lethargic,and slow to move 10 min after intragastric administration.The rats in the high-dose group had obvious poisoning reactions within 10-20 minutes,The tail and limbs were dark purple and the limbs were accompanied by edema,and breathing was difficult.After 30 min,the body began to experience convulsions and rapid breathing,resulting in death.The MTD of male and female rats（male rat is 25 mg/kg,female rat 400 mg/kg）differed by 16 times,and the LD50of male and female rats were 72 mg/kg and 806 mg/kg,respectively.2.At the same drug dose,the main pharmacokinetric parameters of male and female rats were peak time(Tmax):30.0±0.0,30.0±0.0 min;peak concentration(Cmax):2935.8±90.5,1970.0±67.9 ng/ml;area under the drug time curve(AUC（0-t）):482944.2±5648.8,193040.8±4130.6 ng·min/ml;AUC（0-∞）:505087.8±5940.7,196801.6±4739.1 ng·min/ml;half-life(T1/2):106.5±2.5,75.9±3.1 min;clearance rate（CL/F）:49.5±0.6,127.1±3.1 ml/min/kg;apparent distribution volume（V/F）:7601.6±159.0,13904.5±368.8 ml/kg.There were statistically significant differences in the pharmacokinetic parameters between the two groups（P<0.01）,And at each time point on the plasma concentration-time curve,the concentration of sinomenine in the plasma of female rats was higher than that of male rats.The results of the distribution of various tissues and organs showed that the content of sinomenine in each tissue at different time points after administration in female rats was significantly higher than that in male rats（P<0.01 or P<0.05）,the distribution concentration of sinomenine in each tissue was in the order of kidney,liver,spleen,lung,heart,brain,ankle,testis or uterus,and the kidney and liver are the two organs with higher distribution concentration in male and female rats,it is speculated that they may be mainly responsible for the metabolism and excretion of SH.Conclusion:Sinomenine hydrochloride has obvious acute toxicity reaction in normal rats and mice.The poisoning reaction of KM mice was the most rapid in the tail vein administration route,and the LD50of different administration routes were in the order oftail vein administration,intraperitoneal administration,intramuscular administration,subcutaneous administration,and intragastric administration.The tolerance of SD rats to SH shows significant gender differences,which is female rats are more tolerant than males.At the same dose,there were significant differences in the blood pharmacokinetics and tissue distribution in male and female rats,but the leakage of sinomenine in female rats was significantly higher than that in male rats.Therefore,only the absorption and distribution of SH in rats cannot reasonably explain the gender tolerance difference of SH,so it is necessary to further study the elimination process of SH in male and female SD rats to verify the inducement of gender tolerance differences. |
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