Experimental animals
A total of 263 ICR female mice (7 ~ 8-week-old, 26 ~ 30 g) were used. They were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in individually ventilated cages and had free access to food and water. A 12 h light/dark cycle was used in the room. The room temperature and humidity were 20 ~ 22 °C, 50 ~ 70%, respectively. Before the start of the study, the animal experiments were approved by the Division of Animal Control and Inspection, Department of Food and Animal Inspection and Control, Instituto para os Assuntos Cívicos e Municipais (IACM), Macao (AL020/DICV/SIS/2018).
In the experiment, each mouse was weighed and fasted 4 h with drink water freely before administration. For oral administration of nicotine and sinomenine hydrochloride, 0.2 ml was given for every 10 g of mice body weight. And 0.4 ml of berberine hydrochloride was given for every 10 g of mice body weight. After administration, the mice were fasted for 1 h with drink water freely. The survival or death of two consecutive animals is called reversal. For the main test, the testing stops when one of the following stopping criteria is occurred: (a) 3 subsequent animals survive at the highest dosage; (b) 5 reversals occur in any 6 subsequent animals administered; (c) at least 4 animals have followed the first reversal and the specified likelihood-ratios exceed the critical value.
When the experiment was stopped, all the survived mice were humanely killed and necropsied after a 14-day observation. Observed and recorded the pathological changes of organs.
Materials
Nicotine (purity > 99%, CAS number: 54–11-5) and berberine hydrochloride (purity > 99%, CAS number: 2086-83-1) were obtained from Sigma Chemical Company (St. Louis, MO, USA). Sinomenine hydrochloride (purity > 99%, CAS number: 115–53-7) was kindly provided by Hunan Zhengqing Pharmaceutical Group Limited (Huaihua, Hunan Province, China).
The acute toxicity assay of nicotine in mice by iUDP
According to previous literature results, nicotine was a highly toxic substance. Therefore, the estimated initial LD50 dosage was 20 mg/kg. Sigma was 0.2, slope was 5, and T was 1.6. Calculated the dosage by AOT425StatPgm. The sequential dosages were 2000, 1260, 800, 500, 320, 200, 126, 80, 50, 32, 20, 12.6, 8, 5, 3.2, 2 mg/kg. The first dosage of 12.6 mg/kg was given to the first mouse. Symptoms of poisoning were recorded within 24 h. If it was survived, 20 mg/kg was given as the second dosage. If it died, 8 mg/kg was chosen. Follow the experimental sequence until the standard stopping rules appeared.
The acute toxicity assay of sinomenine hydrochloride in mice by iUDP
According to previous literature results, sinomenine hydrochloride was moderately toxic with a significant dosage-response relationship [30, 33]. Therefore, the estimated initial LD50 was 175 mg/kg. Sigma was 0.2, slope was 5, and T was 1.6. Calculated the dosage by AOT425StatPgm. The sequential dosages were 2000, 1100, 700, 440, 280, 175, 110, 70, 44, 28, 17.5, 11, 7, 4.4, 2.8, 1.75 mg/kg. The first dosage of 175 mg/kg was given to the first mouse. Symptoms of poisoning were recorded within 24 h. If it was survived, 280 mg/kg was given as the second dosage. If it died, 110 mg/kg was chosen. Follow the experimental sequence until the standard stopping rules appeared.
The acute toxicity assay of berberine hydrochloride in mice by iUDP
According to previous literature results, berberine hydrochloride was a low or non-toxic compound. Therefore, the estimated initial LD50 dosage was 2500 mg/kg. Sigma was 0.5, slope was 2, and T was 3.16. Calculated the dosage by AOT425StatPgm. The sequential dosages were 5000, 2500, 790, 250, 79, 25, 7.9, 2.5, 0.79 mg/kg. The first dosage of 790 mg/kg was given to the first mouse. Symptoms of poisoning were recorded within 24 h. If it was survived, 2500 mg/kg was given as the second dosage. If it died, 250 mg/kg was chosen. Follow the experimental sequence until the standard stopping rules appeared.
The acute toxicity assay of nicotine in mice by mKM
Twenty-four ICR female mice were randomly divided into 4 groups. The dosage ratio was 0.7, and oral dosage was 14, 20, 28.5, 40.8 mg/kg. The lowest dosage with 100% mortality (Dm = 40.8 mg/kg) and the highest dosage with 0% mortality (14 mg/kg) were obtained to provide references for subsequent experiments.
Fifty ICR female mice were randomly divided into 5 groups. The lowest and highest dosage were selected (16 mg/kg, 39.1 mg/kg, respectively). And 0.8 was chosen as the dosage ratio. After dosing, symptoms of poisoning, number of survival and dead mice were recorded. All mice were subjected to gross necropsy.
The acute toxicity assay of sinomenine hydrochloride in mice by mKM
Twenty-four ICR female mice were randomly divided into 4 groups. The dosage ratio was 0.7, and oral dosage was 350, 500, 665, 715 mg/kg. Obtained the lowest dosage of 100% mortality (Dm = 665 mg/kg) and the highest dosage of 16% mortality (350 mg/kg). To obtain the highest dosage with 0% mortality (Dn), 300 mg/kg was added.
Fifty ICR female mice were randomly into 5 groups. The lowest and highest dosage were selected (300 mg/kg, 665 mg/kg, respectively). And 0.82 was chosen as the dosage ratio. After dosing, symptoms of poisoning, number of survival and dead mice were recorded. All mice were subjected to gross necropsy.
The acute toxicity assay of berberine hydrochloride in mice by mKM
Twenty-four ICR female mice were randomly divided into 4 groups. The dosage ratio was 0.5, and oral dosage was 1000, 2000, 4000, 8000 mg/kg. The lowest dosage with 90% mortality (8000 mg/kg) and the highest dosage with 16.7% mortality (1000 mg/kg) were obtained. Then 11,428 (100% mortality) and 700 mg/kg (0% mortality) were carried out.
Fifty ICR female mice were randomly into 5 groups. The lowest and highest dosage were selected (703 mg/kg, 11,250 mg/kg, respectively). And 0.5 was chosen as the dosage ratio. After dosing, symptoms of poisoning, number of survival and dead mice were recorded. All mice were subjected to gross necropsy.
Statistical analyses
In iUDP, the dosage and numbers of all survival and dead mice were recorded. The computational formula are as follows:
$$ {\mathrm{LD}}_{50}=\sum \left(\mathrm{Xi}\right)/\mathrm{N}+\left(\mathrm{A}+\mathrm{C}\right)\ast \mathrm{d}/\mathrm{N}, $$
(1)
$$ \mathrm{SE}=\mathrm{SD}\ast \surd \left(2/\mathrm{N}\right), $$
(2)
Wherein, Xi was the dosage level, N was the total number of animals, A and C values were obtained from Dixon’s tables [30], which were obtained from the number of O and X in N trials. And d was lgDn minus lgD(n + 1), SE was the standard error, SD was the standard deviation of all dosages in N trails.
In mKM, mortality rate of each group was calculated, and then values were substituted into formulas to obtain LD50 [34]. The computational formula are as follows:
$$ {\mathrm{LgLD}}_{50}=\mathrm{LgDmax}-\left(\mathrm{LgDN}-\mathrm{LgD}\left(\mathrm{N}+1\right)\right)\ \left(\sum \mathrm{p}-0.5\right), $$
(3)
$$ {\mathrm{SE}}_{50}=\mathrm{I}\ast \surd \left(\left(\sum \mathrm{p}-\sum \mathrm{p}\hat{\mkern6mu} 2\right)/\left(\mathrm{n}-1\right)\right), $$
(4)
$$ \mathrm{d}=\pm 4.5\ast {\mathrm{LD}}_{50}\ast {\mathrm{SE}}_{50}, $$
(5)
$$ \mathrm{CI}\ \mathrm{of}\ 95\%={\mathrm{LD}}_{50}\pm \mathrm{d}, $$
(6)
Wherein, m was LgLD50, D was the dosage of each group, Dmax was maximum dosage level, DN was the dosage of N group, D(N + 1) was the dosage of (N + 1) group, p was the mortality of each group of animals, and d was the standard error (σ), I was LgDN minus LgD (N + 1), and n was the number of animals in each group.
Data of organ indexes were plotted in GraphPad Prism (7.0) using One-way ANOVA and Dunnett’s multiple comparisons test. The data were presented in mean ± SD, *P < 0.05 vs Normal, **P < 0.01 vs Normal.