Jinqing granules
Radix Tinosporae and Canarii fructus were both collected from Chengdu (Sichuan, China). The plants were identified and authenticated by Professor Qiao-jia Fan of the College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China. The mixture extract that contains Radix Tinosporae and Canarii fructus in proportions according to a longstanding formula add some excipients to make Jinqing granules. The granules were completely dissolved in sterilized distilled water and made into dosages of 16 g/mL, 8 g/mL, 4 g/mL, and 2 g/mL, according to the requirements of China’s new-drug safety evaluation.
Animals
Healthy male and female SD rats, 4 and 8 weeks old, were purchased from the specific pathogen-free facility at Chengdu Dossy Experimental Animals Co., Ltd. [License no. SCXK (Sichuan) 2008–24]. All experimental procedures involving animals were approved by Sichuan Agricultural University Animal Care and Use Committee (registration No. SCAU2016061801). Based on the principles of the International Committee on Laboratory Animals, the rats were separated equally according to gender and maintained at 20–25 °C and relative humidity of 55 ± 5%. Artificial lighting was adjusted to 12 h of light (from 08:00 to 20:00) and 12 h of darkness from 20:00 to 08:00. Conventional laboratory feed was provided by the Chengdu Dashuo Biology Science and Technology Co., Ltd. The rats were allowed to drink water freely, and they acclimated for 7 days before experiments were begun.
Safety pharmacology experiments
Safety pharmacology experiments were performed according to guidelines of the Veterinary Drugs Control [17,18,19,20].
Fifty SD rats were randomly distributed into 5 groups (5 female and 5 male in each group). The five groups were: Group I, normal control group (sterile distilled water); Group II, 2 g/kg Jinqing granules; Group III, 4 g/kg Jinqing granules; Group IV, 8 g/kg Jinqing granules; and Group V, 16 g/kg Jinging granules. Animals were treated daily at 9 a.m. with gastric infusion in a volume of 10 ml/kg given for 7 days.
We observed the animals for behavior, posture, salivation, pupil change, muscle tremors, hair, and feces were observed from 10 a.m. to 16 p.m. continually for 14 days [17,18,19,20]. In order to prevent living animals from eating animals that had died due to accumulation of drug in the body, endangered or dead rats were autopsied promptly [21].
Before and 24 h after the last administration of control water or Jinqing granules, the rats were placed in the versatile recorder of rats’ locomotor activity and allowed to adapt to the environment for 30 min. Their activity (number of movements in 1 min) was recorded by watching the rats’ movements in order to determine if this aspect of the central nervous system was normal.
The climbing pole test was performed in order to evaluate the mobility of the rats after receiving the medication for various times. Twenty-four hours after the last administration, a smooth metal bar was erected vertically and fixed at the bottom. The rats were placed at the top of the metal bar and allowed to descend head first. Their ability to coordinate this activity was graded as: Level 0, normal step-by-step climbing down; Level 1, coasting down; Level 2, inability to hang from the lever; Level 3, discoordination over a period of time.
On the day before medication was given, the day after it was given, and the eighth day after being given, 3% pentobarbital sodium was injected intraperitoneally to anesthetize the rats [17,18,19,20]. The animals’ heart rate, electrocardiogram, and the depth of breathing and respiratory rate were recorded with the BL-420F multi-channel physiological signal acquisition processing system.
Subchronic toxicity experiment
The toxicity experiments were conducted according to the Economic Cooperation and Development Guideline 407 [22].
One hundred-twenty rats were randomly distributed into 6 groups (10 female and 10 male rats in each group). The 6 groups were: Group I, normal group; Group II, saline control group; Group III, 2 g/kg; Group IV, 4 g/kg; Group V, 8 g/kg; Group VI, 16 g/kg in a volume of 10 ml/kg Jinging granules. Animals were treated daily at 9 a.m. with gastric infusion in a volume of 10 ml/kg given for 30 days. The determination of repeated dose 30-day oral toxicity was carried out according to the Organization for Economic Cooperation and Development Guideline 407 [22]. In order to avoid accumulation of drug that existed in the dead rats in the living body by eating, and analyzed the causes of death in rats, the dead rats were needed to anatomical analysis immediately. [21].
Daily from 10 a.m. to 16 p.m., we observed the rats’ behavior, attitude, presence of saliva, pupillary change, muscle tremors, hair texture, food intake, water intake, feces, poisoning, and death, continually for 30 days. Weight was recorded every three days.
At the end of medication period, urine samples were collected from all animals. The samples were sent to Chengdu Lilai Biology Science and Technology Co., Ltd., for determination of pH, ascorbic acid, specific gravity, glucose, leukocyte counts, nitrite, protein, ketones, urobilinogen, and bilirubin.
About 2.0 mL of blood was collected in an anti-coagulation tube containing sodium citrate to measured hematologic indices, and another 2.0 mL blood was collected in non-heparinized tubes to measured serum biochemistry.
Rats were sacrificed after blood was collected and were opened surgically for evaluation of possible pathological changes. The weights of liver, heart, spleen, lung, kidney, brain, ovaries, and testes were recorded. The relative weight of organs was calculated according to the formula: organ coefficient = organ weight / body weight ×100%) [23, 24].
Organs were soaked in 10% solution of buffered formalin (pH 7.4) for three days, followed by dehydration, embedding, sectioning, dewaxing, and staining with hematoxylin-eosin. Pathologic alterations were assessed with a Nikon 80i optical microscope. Photomicrographs were taken for reference.
Statistical analysis
Results were analyzed with SPSS 19.0 statistical software. The mean and standard deviation were expressed. One-way analysis of variance (ANOVA) and Newman-Keuls post hoc test were performed for statistical analyses. Statistically significant difference was defined as p < 0.05.