Chemicals
Py-mulin (white powder) was synthesized in our lab and the synthetic pathway and confirmations of it structure by IR, NMR and HR-MS spectrometry were described previously [22]. The purity of Py-mulin was checked by HPLC analyses with 99.2%. During the study, the test sample was stored in the dark at a temperature of 4 °C and dissolved in DMSO freshly before use. All the other chemicals and solvents used were obtained from standard vendors and used without further purification.
Animals
Adult specific pathogen free (SPF) Kunming mice (4–5 weeks old) and Sprague-Dawley (SD) rats (4–6 weeks old) were purchased from the Laboratory Animal Center of Lanzhou University (Lanzhou, China). The animals were separated by sex, housed in clean stainless steel cages (two rat per cage, three mice per cage), free access to food and water, and maintained under the 23 °C conditions with a constant 12 h light-dark cycle. Animals were acclimated for at least 7 days prior to the experiment. All experiments were carried out between 08:30 AM and 17:30 PM. The experimental procedures were performed in accordance with the Ethical Principles in Animal Research and were approved by the Committee for Ethics in the Laboratory Animal Center of Lanzhou University (number: SCXK2013–0002). The study was carried out in compliance with the ARRIVE guidelines.
Acute Oral toxicity in mice
After being stratified by weight, the SPF mice (30 male and 30 female, initial weight of 20.2–24.1 g and 19.3–21.8 g, respectively) were randomly assigned to six groups, including five treatment groups and one vehicle control group (5 female and 5 male mice for each group). The vehicle control group received only vehicle (ethyl oleate:1,2-propanediol:tween-80: sterile water = 4:10:31:55) in a volume of 10 mL/kg body weight (b.w.) by oral gavage. After mice were fasted for 3 h, Py-mulin was dissolved in vehicle and orally administered to the mice at doses of 55, 175, 550, 1750 and 5000 mg/kg b.w. (with volume of 10 mL/kg), respectively. Vehicle or Py-mulin was administered only once (on day 0) and animals were observed for next 14-days post treatment. All mice were observed twice daily for behavioural symptoms and mortality for two week. Individual body weights were recorded on days 0, 7 and 14. All of the surviving animals were euthanized by anesthetized with pentobarbital sodium 0.06 g/kg (i.p.) at the end of the observation period of 14 days, and their vital organs were individually observed for overt pathology by necropsy. Finally, the LD50 and 95% confidence interval for female and male mice were calculated by the probit weighted regression method (Bliss method) using IBM SPSS Statistics for Windows version 24.0 (SPSS Inc., Chicago, USA).
Subacute toxicity
Study design
The subacute toxicity study of Py-mulin was performed according to OECD guideline No. 407 [23]. Female and male SD rats (initial weight of 126.3–145.6 g and 141.2–158.1 g, respectively) were randomly divided into four groups after one week of acclimatization. Each group consisted of 10 female rats and 10 male rats. Doses were determined based on the result of acute oral toxicity. Py-mulin was dissolved in vehicle (ethyl oleate:1,2-propanediol:tween-80: sterile water = 4:10:31:55) and administrated to rats by oral gavage at doses of 50, 100 and 300 mg/kg bw or control (4 mL/kg bw vehicle) for 28 days (administration at the same time each day ±1 h). All the animals were observed once daily to detect toxic symptoms. During the whole experiment, body weight and food consumption were measured every week and the gavage volume was adjusted based on the last measured body weight.
Clinical observations
During the 28-day treatment, for all the rats, behavioral changes, symptoms and signs of gross toxicity, food consumption, water intake and mortality were closely observed at least once per day. The individual body weights of the rats and food consumption were measured weekly. The mean weekly body weight gain and daily food consumption were calculated for each sex animal and dose level.
Haematological and biochemical analysis
At the end of the experiment, following an overnight fast, rats were anesthetized with excess of CO2 and euthanized by exsanguination. Blood sample (5 mL) of animal in each group were collected from the femoral artery in ethylenediaminetetraacetic acid (EDTA) coated tubes and non-heparinized tubes for the haematological and biochemical analysis, respectively [24]. The haematological parameters, including the white blood cell (WBC), the hemoglobin (HGB), red blood cell (RBC) counts, hematocrit (HCT) levels, mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC) and number of platelets (PLT), were determined using a Poche-100iv Diff instrument (SYSMEX, Kakogawa, Japan). The biochemical parameters measured were alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), total bilirubin (TB), total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL), triglycerides (TG), urea nitrogen (UN), creatinine (Crea), total protein (TP), glucose (Glu), albumin (Alb), calcium (Ca) and phosphorous (P), using reagent kits and a Mindray BS-420 auto hematology analyzer (Mindray Corporation, Shenzhen, China).
Macroscopic examination and organ weights
After necropsy, each rat was examined for the external surface, visceral organs and all orifices, followed by recording the gross pathological changes. Finally, the heart, liver, spleen, lungs, kidneys, thymus, testes or ovaries were collected and weighed. The relative organ weight (organ/body-weight ratio) was calculated based on the final animal’s body weight.
Histopathologic examination
Histopathological examinations were performed on the kidneys, spleen, heart, liver, thymus, lungs, small intestines, testes or ovaries of randomly selected five female and five male rats using a routine paraffin-embedding technique. The organs were preserved in 10% neutral-buffered formalin, and their slides were prepared. Upon completion of staining with hematoxylin and eosin (H&E), microscopic examinations were performed on all the mentioned tissues.
Ames test
S. typhimurium strains, including TA97, TA98, TA100 and TA1535, were first checked for their genetic integrity. Biotin dependence, rfa marker (crystal violet), biotin and histidine dependence, histidine dependence and presence of the plasmid pKM101 (ampicillin resistance) or pAQ1 (tetracycline resistance) were then tested. Different strains were used to identify different types of mutations.
The test was performed with five concentrations of Py-mulin. After weighing accurately, 100 mg Py-mulin was dissolved in DMSO (2 mL) and the obtained solution (1 mL) was diluted by fivefold with distilled water. The 0.5 mL obtained serial dilutions were diluted with 9.5 mL melted top agar to the desired concentrations (500, 100, 20, 4 and 0.8 μg/mL). DMSO (0.5 mL) was directly incorporated into 9.5 mL melted top agar (the final concentrations was 100 μL/plate) as the vehicle control. For all bacterial strains, 2-aminofluorene and fenaminosulf (the final concentration of 10 μL/plate) were used as positive control with and without S9, respectively. As described in the literatures [25, 26], the Ames test was conducted with some modifications. Each strain (1 mL) was inoculated into 5 mL of nutrient broth, followed by incubating overnight at 37 °C. The mixture comprised respective strains, DMSO, 2-aminofluorene and drug for testing with the S9. For testing without S9, 1 × phosphate buffer saline (PBS, pH 7.4) replaced the S9 mix. The obtained mixtures were then added into the tube, mixed well and incubated (20 min at 37 °C). Then, the top agar (2 mL) was melted and added to each tube and gently mixed, followed by pouring onto the surface of glucose minimal (GM) agar plate (the final concentrations of Py-mulin were 1000, 200, 40, 8 and 1.6 μg/plate, respectively, and the final concentrations of DMSO was 100 μL/plate). The plate was then swirled to distribute the overlay agar to all surfaces of GM agar, followed by inverting and incubation (37 °C for 48 h) when the top agar solidified. The resultant colonies were counted manually as the number of revertant colonies per plate. The experiments were performed in triplicate.
Statistical analysis
We used IBM SPSS Statistics for Windows version 24.0 [27] to perform the statistical analysis. Using One-way analysis of variance (ANOVA) and Dunnett’s post-hoc tests as appropriate analyzed the data. Statistical significant difference was defined as a p < 0.05 and the extremely significant difference was defined as a p < 0.01.