Study On The Development And Toxicology Of Traditional Chinese Medicine "WuHuangHu" Mixture

Actinobacillus pleuropneumoniae is a Gram-negative bacterium that mainly exists in the lungs of pigs. It is the most common pathogenic bacteria in clinic and causes different age porcine infectious pleuropneumonia. This disease mainly caused the upper respiratory tract infection of pig, and often mixed infection with porcine reproductive and respiratory syndrome virus, also characterized by the acute and chronic inflammation, causing pulmonary dysfunction, and eventually death. The current clinical treatment of pig infectious pleuropneumonia mainly used antibiotics, can have an effective treatment effect but the abuse of western medicine in clinic, not only brings the problems such as drug resistance, drug residues, may also damage the organs in the process of pig growth, affect the growth and development. And traditional Chinese medicine demostrated that it has the advantages of economy, low toxicity and no residue. This experiment extract the antibacterial activity aganist A.pleuropneumoniae from 20 kinds of traditional Chinese medicine, and conduct joint bacteriostatic experiment. Screening with additive and synergistic action of traditional Chinese medicine to formula and carries on the toxicological evaluation. This study provided theoretical basis and toxicology for the development of new veterinary drugs. This research main content and the results are as follows:1. Study on antibacterial activity-of crude extract from 20 kinds of traditional Chinese medicines against A.pleuropneumoniae in vitro. The against A.pleuropneumoniae activities of twenty kinds of traditional Chinese medicine were evaluated in this study in vitro. The against A.pleuropneumoniae activities of twenty kinds of traditional Chinese medicine like Fructus mume were extracted by the methods of ethanol reflux extraction, water decoction, ultrasonic wave and so on. The antibacterial activities of twenty kinds of traditional Chinese medicine against A.pleuropneumoniae were determined by the doubling dilution. And then the joint bacteriostatic activities of a few kinds of traditional Chinese medicine in vitro were evaluated in vitro. The results showed that the MIC of the extracts obtained from Fructus mume, Coptidis rhizoma, Fructus Chebulae,Cortex fraxini, Sanguisorba and cuspidatum against Actinobacillus Pleuropneumoniae was range from 6.25 mg/mL to 50 mg/mL; the MIC of the extracts obtained from Folium isatidis, Herba artemisiae capillaries, Locorice and Cortex dictamni was range from 50 mg/mL to 100 mg/mL; the MIC was greater than 100 mg/mL of the extracts obtained from Phellodendron amurense, Eucommia ulmoides oliv, Honeysuckle, Radix Bupleuri, Dandelion, Radix isatidis, Gardenia jasminoides Ellis and Radix astragal and so on. The results of joint bacteriostatic test showed that the fractional inhibitory concentration index (FICI) of Fructus mume, Rhizoma coptidis, Fructus Chebulae, and Cuspidatum are less than or equal to 2 and the fractional inhibitory concentration index (FICI) of Fructus mume,Cortex fraxini and Cuspidatum was greater than 2. Fructus mume, Rhizoma coptidis, Fructus Chebulae and Cuspidatum had good antimicrobial activities against APP. Fructus mume, Rhizoma coptidis, Fructus Chebulae and Cuspidatum showed independent, additive or synergy action and Cortex fraxini, Fructus mume and Cuspidatum showed antagonistic action.2. Optimization of extraction process of 3 Traditional Chinese Medicines. This study was designed to obtain the optimized extracting process for the extraction of active substance from 3 traditional Chinese medicines with antibacterial activity against Actinobacillus pleuropneumoniae. The orthogonal array design was used with several factors, including solvent concentration, solid-liquid ratio, extraction frequency, extraction time and extraction temperature and the extract yield, MIC and MBC were selected as indexes.The results showed that the optimal extraction conditions for Rhizoma Coptidis were as follows:1:8 of ratio of raw material to liquid,70% of ethanol concentration, and 2 h extraction time, the optimal extraction conditions for Fructus Mume were as follows:1:10 of ratio of raw material to liquid,95% of ethanol concentration,90 ℃ extraction temperature and 8 h extraction time, the optimal extraction conditions for PoLygonum cuspidatum were as follows:1:6 of ratio of raw material to liquid,60% of ethanol concentration,90℃extraction temperature,2 h extraction time and reflux extraction for 3 times. Under the optimal extraction conditions,the MIC values of 3 traditional Chinese medicines were 15.60 mg/mL,10.20 mg/mL and 15.60 mg/mL, respectively, and the MBC values were 26.00 mg/mL,18.20 mg/mL,15.60 mg/mL, respectively.3."Wuhuanghu"Mixture is preparation and quality control. After screening, selected formula materials, and then determined by clarity, stability influence factors such as dosage of complementary makings, ultimately selected the best formula. In order to determine whether the original medicinal materials, traditional Chinese medicine extract, "Wuhuanghu" mixture and mixture according with pharmacopoeia standard, the thin layer chromatography taste were applied. Using high performance liquid chromatography (HPLC) to determine the main components in the mixture content, establish a system of content determination of standard. Results showed that:The preparation technology of Mixture is feasible, the original medicinal herbs in pharmacopoeia standard and the content of Mixture can be controled. The HPLC results showed that:Liquid chromatographic conditions acetonitrile 0.5%ammonium biphosphate (3:97) (PH=3)as the mobile phase;The detection wavelength was 210nm. column temperature was 25 ℃; flow rate was 1.0mL·min-1, the citric acid polydatin concentration in the 0.16mg·mL-1-5.00mg·L-1 and the peak area was a good linear relationship, the average content of citric acid was 14.73mg·mL-1 by 3 times tested. Liquid chromatographic conditions acetonitrile-water(23:77) as the mobile phase; The detection wavelength was 306nm, column temperature was 25℃; flow rate was 1.0m·min-1, the citric acid polydatin concentration in the 6.25μg·L-1 100.00μg·mL-1 and the peak area was a good linear relationship, the average content of citric acid was 1832.71 μg·mL-1 by 3 times tested. Conclusion:Mixture preparation technology is simple and feasible, the original medicinal materials, extract extract conform to the requirement of pharmacopoeia and meet the requirements and the range of the predetermined quality standards.4. The toxicology study of Traditional Chinese medicine formula "Wuhuanghu" Mixture. According to veterinary technology research guidelines for prescription medicine extracts rats fed acute test, toxicological evaluation including the acute toxicity test and 30 days of rate by gastric chronic toxicity test. Acute toxicity test showed that when the drug doses greater than 5000 mg/kg, no any abnormal reaction in mice, no death, namely determining LD50 in animals> 5000 mg/kg, prove formula for actual non-toxic material mixture. The chronic toxicity test results show that the "Wuhuanghu" Mixture high dose group (6.25 g/kg), middle dose group (5.00 g/kg), low dose group (3.75 g/kg) in rats after 30 d lavage for medicine, does not appear obvious toxic effects. Experimental group rats to gather the food, water and so on are all normal.30 d after the treatment by measured the hematology indexes of rats and normal control group, there is no obvious difference, in addition to the concentration of high, middle dose group of "Wuhuanghu" Mixture of blood biochemical indexes and the change of some indicators of normal control group, the rest were no obvious difference. Each viscera histopathological examination results show that high doses of "Wuhuanghu" Mixture can lead to liver, kidney, spleen, lung, in reversible degeneration changes of testis and ovaries, recoverable after medication has stopped, in 2.5 times of clinical dosage (6.25 g/kg) and below, "Wuhuanghu" Mixture lavage for medicine is safe. Conclusion:"Wuhuanghu" Mixture as a new non-toxic and effective traditional Chinese medicine formula preparation, the toxicological evaluation results for the preparation of offer the theoretical basis for clinical application.