动物造模,亚慢性毒性 z-bo 2020-09-02 363

　　Background: There are about 12,000 kinds of Chinese herbal medicines in clinical use, most of which are crude medicines. The origin and development of Chinese herbal medicines are the work experience and medical practice of working people. However, the safety and effectiveness of many commonly used herbs have not been determined. In order to ensure the safety of Kampo medicines, it is necessary to conduct subchronic toxicity and drug safety tests. Jinsei granules are composed of mixed extracts, which contain a certain proportion of golden and green fruits. In the early trials, Jin Guolan was effective in the early prevention and treatment of Helicobacter pylori infection. Jinqing granules are used to treat various diseases, but the safety of long-term use is still questionable. About 10% of people in the world suffer from gastric ulcers, and about 1% of ulcers develop into gastric cancer. Therefore, Jinsai pellets have a large market. In an early acute toxicity study, it was found that the median lethal dose of zinc protein particles in rats was 5 g/kg. Method: Jinqing granules: The medicinal materials of Jin Guolan and Qingguo were collected from Chengdu. Some auxiliary ingredients are added to the formula of golden fruit orchid and green fruit to make golden green granules. The granules were dissolved in sterile distilled water and mixed at dosage concentrations of 16 g/mL, 8 g/mL, 4 g/mL, and 2 g/mL. Animals: Healthy male and female SPF SD rats, 4-8 weeks old. The animal room is maintained at a temperature of 20-25 degrees and a humidity of 55 ± 5%. Artificial lighting 12-12 brightness adjustment. Before the experiment, the rats were allowed to drink water and adapt for 7 days.

　　Safety pharmacology experiment: The safety pharmacology experiment is carried out in accordance with the "Regulations on Veterinary Drug Control". 50 SD rats were randomly divided into 5 groups (5 in each group, 5 females). The first group, the normal control group (distilled water); the second group, the Jinqing granules 2g/kg; the third group, the Jinqing granules 4g/kg; the fourth group, the Jinqing granules 8g/kg; the fifth group, the gold 16g Feed pellet animals at a volume of 10 ml/kg at 9 am every day for 7 days. The animal behavior, posture, salivation, pupil changes, muscle tremor, hair and feces were observed for 14 days from 10 am to 16 pm. Carry out an autopsy for the deceased and the deceased in time to prevent the animal from eating tragically due to the accumulation of drugs in the body after death. Before and 24 hours after the completion of the administration, the rats were placed in a whole body motion recorder and acclimatized in the environment for 30 minutes. Observe the rat's movement (number of exercises per minute) and record the movement to determine whether the central nervous system is normal. The pole climbing test is used to evaluate the exercise ability of rats after multiple doses. Twenty-four hours after the last dose, a smooth metal rod was erected and fixed to the bottom. Place the mouse on the metal rod so that it faces down. Their ability to coordinate activities is divided into level 0, normal upgrade, level 1, taxiing, level 2, not hanging on the pole, level 3 and discordant period. Anesthesia was performed 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 can record animal heart rate, electrocardiogram, breathing depth and breathing rate. Subchronic toxicity study: 120 rats were randomly divided into 6 groups (10 males and 10 females in each group). The first group, the normal group; the second group, the normal saline control group; the third group, the Jinqing granules 2g/kg; the fourth group, the Jinqing granules 4g/kg; the V group, the Jinqing granules 8g/kg; the sixth group In the group, Jinqing granules were 16g/kg, and the rat dose was 10ml/kg. The animals were fed at a volume of 10 ml/kg at 9 am every day for 30 days. In order to analyze the cause of death in rats, it is necessary to perform anatomical analysis on dead rats. Observe the rats' behavior, attitude, saliva presence, pupil changes, muscle tremor, hair texture, food intake, water intake, feces, poisoning and death for 30 days every day from 10 am to 16 pm. The weight is recorded every 3 days. Urine samples of all animals were collected at the end of dosing. It can measure pH, ascorbic acid, specific gravity, blood sugar, white blood cell count, nitrite, protein, ketones, urobilinogen and bilirubin. Collect approximately 2 ml of blood in an anticoagulation tube containing sodium citrate to determine hematological indicators. Another non-heparinized test tube of 2 ml blood was used to measure serum biochemistry. After blood collection, the rats were sacrificed to observe possible pathological changes. Record the weight of the liver, heart, spleen, lungs, kidneys, brain, ovaries and testes. The relative weight of the organ is calculated according to the following formula: organ coefficient = organ weight/body weight x 100%. Soaked in 10% formalin buffer (pH 7.4) for 3 days, dehydrated, embedded, sectioned, deparaffinized and HE stained. Nikon 80i optical microscope can evaluate pathological changes. Results: Safety pharmacology experiment: In the safety pharmacology experiment, no deaths occurred, and all animals in all groups had no abnormal fur, posture, movement, feces, salivation and muscle tremor. .. Behaviors controlled by the central nervous system, such as posture, changes in pupil size, salivation, gait, and muscle tremor are not abnormal. There was no difference in different indexes of Jinqing Granules animal activity index in each dose group. Jinjing granules have no obvious effect on the spontaneous activities of rats. In the pole climbing test, the rats in the treatment group and the control group were all down to level 0. It has been confirmed that Jinqing granules have no obvious effect on animal coordination. Before medication, the heart rate and respiratory rate measured on the day of medication and on the 8th day after medication were not significantly different between the control group and the Jinqing granule dosage group. At the same time, there was no significant difference in heart rate and breathing rate of the same group at three different times. Therefore, it can be concluded that Jinqing Granules have no significant effect on the cardiovascular and respiratory systems of rats. The figure shows the curve of the effect of Golden Respiratory Particles on the breathing time of rats. Subchronic toxicity study: In a 30-day gavage study, 6 male animals died, of which 2 male rats and 1 female rat died. The autopsy results of these animals showed that they died due to discomfort after tube feeding. Oral Jinqing granules have no side effects. In each treatment group, no abnormalities were observed in the animal's fur, movement, posture, pupil changes, diet, salivation, muscle tremor, and stool. The table shows the consumption of food and drinking water. There was no significant difference between the drinking water control group and the oral Jinqing granule group. Except for the slightly lower consumption of 16 g/kg (group VI), there was no significant difference among 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 between the III-V groups (2 g/kg? 8 g/kg Jinqing granules orally). Compared with the control group, the weight of male rats in the VI group (16 g/kg Jinqing Granules orally) lost some weight. From day 19 to day 31, the weight gain of the sixth group of female rats similarly decreased. The indicator in the urine sample showed a high negative rate of 90% for ascorbic acid. The pH, specific gravity, blood sugar, white blood cells and nitrite of all animals were normal. Compared with the control group, the bilirubin value of the VI group (oral 16 g/kg Jinqing granules) slightly increased. Occult blood, protein, ketone bodies and urobilinogen levels increased slightly, but did not reach statistical significance. Except for the VI group (16 g/kg gold particles orally), the lymphocyte count was slightly higher, and there was no significant difference in the other dose groups. Serum biochemical indicators, VI group (oral 16g/kg Jinqing granules) only increased aspartate aminotransferase and total bilirubin, there was no significant difference in other groups. Only the VI group (oral Jinqing granules 16 g/kg) had slightly higher liver and ovarian organ values, and there was no significant difference in the other groups. Take histopathological pictures of the heart, liver, spleen, lung, kidney, stomach, intestine, brain, testis, and ovary of three rats in each group. Animals with a dose of 16 g/kg Jinqing granules showed tissue damage. Each group had complete capsules, ventricles and ventricular muscle contours, and no obvious pathological damage. Compared with the control group, the animals in the VI group had a dilated liver sinusoid, and the degree of vacuolar degeneration of hepatocytes was different. In the spleen, the control capsule is intact, the white and red serous is clearly separated, and the lymphocyte count is normal. Compared with the control group, rats in the VI group had fewer lymphocytes and increased macrophages. The lung tissues of each group were intact and all bronchus (first, second and third bronchus) were placed well. There is no obvious pathological change in kidney tissue. The renal capsule is intact, the cortex and medulla are clearly differentiated, and the renal tubules and collecting tubes are arranged neatly and the density is normal. The structure of the monolayer columnar epithelium, mucosal muscle layer and appropriate muscle layer of groups 1-6 has been completed. The structure of the intestinal mucosa, submucosa, muscle layer and adventitia is complete. The nerve fiber layer, cerebral cortex and hippocampal pyramidal cells in the brain are well placed without inflammatory infiltration or bleeding. In ovarian tissue, compared with the control group, the morphology and number of ovarian primordial follicles, hyperplastic follicles and mature follicles of the first to sixth groups were all normal. The seminiferous tubules of the testes are complete, and the sperm cells of the testes are arranged neatly and compactly. Compared with the control group, the number of sperm in the seminiferous tubules of the sixth animal group was reduced.

　　Conclusion: The safety pharmacology and sub-chronic toxicity studies of high-dose Jinqing granules (16 g/kg) in rats have shown that it has almost no toxicity to the animal nervous system, cardiovascular system, respiratory system, renal system and blood. The highest dose caused slower weight gain in animals, smaller histological changes, decreased spermatogenic tubule sperm count, and increased serum aspartate aminotransferase and bilirubin. In a 30-day feeding study, 3 rats at a dose of 16 g/kg died. This may be due to gavage injury rather than drug toxicity. SD rats took Jinqing Granules orally at a safe granule dose of 8 g/kg or less for 30 days, but it turns out that higher doses are not safe.