动物实验,亚慢性毒性,安全性药理 z-bo 2020-11-17 312

　　Background: There are about 12,000 kinds of Chinese medicines in clinical use, most of which are herbal medicine. The origin and development of Chinese medicine are the practical experience and medical practice of working people. However, the safety and effectiveness of many commonly used Chinese medicines have not yet been established. In order to ensure the safety of traditional Chinese medicine, it is necessary to conduct subchronic toxicity and drug safety tests. Jinqing granules are made of a mixed extract containing golden fruit and green fruit in a formula ratio. In early trials, Jin Guolan was effective in the prevention and early treatment of Helicobacter pylori infection. Although Jinqing granules are used to treat various diseases, the safety of long-term use is still questionable. Approximately 10% of the world’s population suffers from gastric ulcers, and approximately 1% of ulcers progress to gastric cancer. Therefore, Jinqing pellets have a very large market. In the early acute toxicity experiment, we found that the median lethal dose of Jinqing granules to rats was 5 g/kg.

　　Method: Jinqing granules: Jinguolan and Qingguo medicinal materials were collected from Chengdu. Jin Guolan and Qing Guo formula add some auxiliary materials to make Jinqing granules. The granules are dissolved in sterile distilled water to make the dosage concentrations of 16 g/mL, 8 g/mL, 4 g/mL and 2 g/mL.

　　Animal: Healthy male and female SPF SD rats, 4-8 weeks old. The animal room is kept at a temperature of 20-25 degrees and a humidity of 55 ± 5%. Artificial lighting 12-12 brightness adjustment. The rats drink freely and acclimate for 7 days before the start of the experiment.

　　Safety pharmacological experiment: Safety pharmacological experiment is carried out in accordance with "Veterinary Drug Management Regulations". Fifty SD rats were randomly divided into 5 groups (5 in each group, 5 females). The first group, the normal control group (distilled water); Group II, 2 g/kg Jinqing granules; Group III, 4 g/kg Jinqing granules; IV group, 8 g/kg Jinqing granules; V group, 16 g/ kg Jinqing Granules. Give animals a gastric gavage at 9 am every day, with a volume of 10 ml/kg for 7 days. The animals’ behavior, posture, salivation, pupil changes, muscle tremor, hair and feces were observed for 14 days from 10 am to 16:00 pm. In order to prevent animals from being eaten tragically after death due to the accumulation of drugs in the body, an autopsy on dying and dead rats is carried out in time. Before the end of the administration and 24 hours after the end of the administration, the rats were placed in the whole body motion recorder and allowed to adapt to the environment for 30 minutes. Record their activities by observing the movements of the rats (the number of movements in 1 minute) to determine whether the central nervous system is normal.

　　The climbing pole test is used to evaluate the exercise ability of rats after multiple doses. 24 hours after the last dose, a smooth metal rod was erected and fixed to the bottom. Place the mouse on top of the metal rod, allowing them to face down. Their ability to coordinate activities is divided into: 0 level, normal climbing step by step; level 1, sliding; level 2, not hanging on the pole; level 3, a period of disharmony. The rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital on the day before, on the day after administration and on the 8th day after administration. BL-420F multi-channel physiological signal acquisition and processing system records the animal's heart rate, electrocardiogram, breathing depth and breathing rate.

　　Subchronic toxicity test: 120 rats were randomly divided into 6 groups (10 males and 10 females in each group). The first group, normal group; group II, normal saline control group; group III, 2 g/kg Jinqing Granules; group IV, 4 g/kg Jinqing Granules; group V, 8 g/kg Jinqing Granules; group VI , 16 g/kg Jinqing Granules, the rat administration dose is 10 ml/kg. The animals were given intragastric gavage at 9 am every day, with a volume of 10 ml/kg, for 30 days. In order to analyze the cause of death in rats, the dead rats need to be dissected and analyzed. Every day from 10 am to 16:00 pm, the rats' behavior, attitude, presence of saliva, pupil changes, muscle tremor, hair texture, food intake, water consumption, feces, poisoning and death were observed for 30 days. The weight is recorded every three days. At the end of the medication, all animal urine specimens were collected. Measure pH, ascorbic acid, specific gravity, blood sugar, white blood cell count, nitrite, protein, ketones, urobilinogen, and bilirubin. Collect about 2 ml of blood in an anticoagulation tube containing sodium citrate to determine hematological indicators. Another 2 ml blood collection non-heparinized tube measures serum biochemistry. After the blood was taken, the rats were sacrificed to observe possible pathological changes. Record the weight of liver, heart, spleen, lung, kidney, brain, ovary and testis. Calculate the relative weight of organs according to the formula: organ coefficient = organ weight/body weight × 100%). Soak in 10% formalin buffer (pH 7.4) for three days, dehydration, embedding, sectioning, deparaffinization, and HE staining. Nikon 80i optical microscope to evaluate pathological changes.

　　Result: Safety pharmacology experiment: In the safety pharmacology experiment, no deaths occurred. All animals in all groups had no abnormal fur, posture, movement, feces, salivation, and muscle tremor. The central nervous system control behaviors of rats in each dose group, such as posture, changes in pupil size, salivation, gait, and muscle tremor, were not abnormal. There was no difference in various indexes of animal activity index in each dose of Jinqing Granules. Jinqing granule has no obvious effect on spontaneous activities of rats. In the pole climbing test, the rats in the treatment group and the control group both climbed down step by step, that is, level 0. It is proved that Jinqing granules have no obvious effect on animal coordination. Heart rate and respiratory rate measured before medication, on the day of medication and on the 8th day after medication showed that there was no significant difference between the control group of rats and the dose group of Jinqing granules. At the same time, at three different time points in the same group, there was no significant difference in heart rate and respiratory rate. Therefore, it is concluded that Jinqing Granules has no significant effect on the cardiovascular and respiratory systems of rats. The effect curve of Jinqing Granules on rat breathing time is shown in the figure.

　　Subchronic toxicity test: In the 30-day gavage test, 2 male and 1 female rats in the sixth group died. The autopsy results of these animals showed death due to discomfort after gavage. No side effects occurred after oral administration of Jinqing granules. No abnormalities were found in the fur, movement, posture, pupil changes, diet, salivation, muscle tremor, and feces of the animals in each dose group. The consumption of food and drinking water is shown in the table. There was no significant difference between the drinking water control group and the oral Jinqing granule groups. Except for the consumption of 16 g/kg (group VI) which was slightly lower, there was no significant difference in the other groups. There was no significant difference between the weight gain control group (group I) and the saline group (group II). There was no significant difference in weight gain in the administration group III-V (oral 2 g/kg to 8 g/kg Jinqing granules). Compared with the control group, male rats in group VI (16 g/kg Jinqing Granules orally) gained slightly less weight. From the nineteenth to the thirty-first day, the weight gain of the sixth group of female rats also decreased similarly. The indicators of urine samples show that the negative rate of ascorbic acid is as high as 90%. The pH, specific gravity, blood sugar, white blood cells, and nitrite of all animals were normal. Compared with the control value, the value of bilirubin in the VI group (oral 16 g/kg Jinqing granules) slightly increased. The values of occult blood, protein, ketone bodies, and urobilinogen increased slightly, but did not reach statistical significance. Except for the VI group (oral 16 g/kg Jinqing granules), the lymphocyte count was slightly higher, there was no significant difference in the other dose groups. Serum biochemical indicators, only the VI group (oral 16 g/kg Jinqing granules) aspartate aminotransferase and total bilirubin increased, and there was no significant difference in the other groups. Only the VI group (16 g/kg Jinqing granules orally) had slightly higher organ values of liver and ovary, and there was no significant difference in the other groups. Three rats in each group were taken for histopathological examination of heart, liver, spleen, lung, kidney, stomach, intestine, brain, testis, and ovary. Animals receiving 16 g/kg Jinqing granules showed tissue damage. Each group had a complete capsule, ventricle and ventricular muscle contours, and no obvious pathological damage. Compared with the control group, animals in the VI group had dilated liver sinusoids and had varying degrees of vacuolar degeneration of liver cells. In the spleen, the capsule of the control group was intact, the white pulp and red pulp were clearly separated, and the lymphocyte count was normal. Compared with the control group, rats in group VI decreased lymphocytes and increased the number of macrophages. The lung tissues of each group were intact, and all bronchial tubes (first-grade bronchus, second-grade bronchus, and third-grade bronchus) were arranged neatly. The pathological changes of kidney tissue were not obvious. The renal capsule is intact, the cortex and medulla are clearly demarcated, the renal tubules and collecting ducts are neatly arranged, and the density is normal. The structure of single-layer columnar epithelium, muscularis mucosa and muscularis propria in the first group to the sixth group is complete. The structure of the intestinal mucosa, submucosa, muscle layer, and outer membrane layer is complete. The nerve fiber layer, cerebral cortex, and hippocampal pyramidal cells in the brain are arranged neatly and tightly, without inflammatory infiltration or hemorrhage. In ovarian tissue, from group I to group VI, the morphology and number of ovarian primordial follicles, proliferating follicles and mature follicles were all normal compared with the control group. The seminiferous tubules in the testis have a complete structure, and the spermatids in the testis are arranged neatly and compactly. Compared with the control group, the number of sperm in the seminiferous tubules of the sixth group of animals decreased.

　　Conclusion: The safety pharmacology and sub-chronic toxicity test of rats receiving high-dose Jinqing Granules (16 g/kg) showed low toxicity to the animal’s nervous, cardiovascular, respiratory, kidney and blood systems. The highest dose resulted in slower weight gain of animals, minor histological changes, decreased sperm count in seminiferous tubules, and increased serum aspartate aminotransferase and bilirubin. In the 30-day feeding test, 3 rats that received a dose of 16 g/kg died, possibly due to oral gavage injury, not drug toxicity. SD rats orally take Jinqing granules for 30 days at a dose of 8 g/kg or less, which are safe granules, and higher doses cannot be proven safe.