Screening And Formulating, And Its Action Mechanism And Clinical Application Of Anti-IBDV Traditional Chinese Medicine(TCM)

IBD is an acute, extremely contagious infectious disease caused by IBDV.Nowadays, it is wild spread in the world, especially in those countries developing inraising poultry. IBD happens everywhere in our country and causes serious economic loss,its incidence rate reaches 80 percent exceedingly in some areas and mortality ratio rangesfrom 30 percent to 70 percent, which is regarded as one of three serious diseases toinfluence poultry industry in the world.IBDV holds better resistence to environment, and can exist for a long time in thecircumstance; however, it is hard to be eradicated in case of infection. At present, weprevent birds from IBD mainly by inoculating vaccine, whereas its virulence andantigenicity are changing because of excessive dose and being used in too large area,resulting in producing variant IBDV and very virulent IBDV strain. Due to antigendifference of different serotype IBDV and variant strain, it is hard to acquire a perfectimmune effect by means of vaccination, even inducing IBD on occasion. IBDV mainlyinvades birds'central immune organ-bursa of Fabricius and leads to immunosuppressionor incomplete immune. Therefore, it is important to search new drugs for preventing andcuring IBD.Up to now, no special western medicine can treat IBD; the curative effect of someantivirus drugs is not assured, and furthermore they cause some problems such aspoisonous and side effect, drug tolerance, and food safety etc. With regard to earlier sickchicken, we often introduce yolk antibody or high valence immune serum which showcertain effect, but manifest many deficiency, such as stress in injection, emission of someplague source diseases, and irregular therapy impact caused by astatic product quality. According to articles and much clinical practice, the results reveal some Chineseherb medicines are capable of preventing and curing IBD with higher curative ratio;nevertheless, we select these prescriptions with experience and definite subjectivity, nostrict screening, no systematic effect and safety evaluation, and clarifying anti-IBDVmechanism inadequately, so these reasons hold back its clinical application. Thereby, based on available data and clinical demand, we discover the most effctivedrugs by screening 80 anti-virus Chinese herb medicines systematicly, futher formulateprescription of anti-IBDV in term of chinese veterinary pharmacological theory anddetermination of treatment based on the differentiation of syndromes, and penetrate intoits mechanism.1 Detecting safety concentration of eighty Chinese herb medicines on CEF(chickenembryo fibroblast): Eighty Chinese herb medicines (entirely distilled granulated preparation, boughtfrom Guangdong Yifang Pharmaceutical Co. Ltd.) were prepared into 1mg/ml liquor withdeionized distilled water as mother liquor, and diluted in ten times gradiently. Afteradding the liquors onto CEF at 12 h, 24 h, and 48 h, we observed cell conditions andobtained the intervals of safety concentration. Then diluting the liquors in two timesgradiently within the intervals, and treating CEF as above described method, we finallygot the safety concentrations. The results showed that the safety concentrations ofsixty-one Chinese herb medicines vary from 0.25 mg/ml to 5.00 mg/ml, accounting for76.25 percent among the tested drugs; rhubarb and coptis root affect CEF most, and thesafety concentration of gypsum on CEF is highest, 125 mg/ml.2 Screening anti-IBDV Chinese herb medicines on CEF: Add both the liquors, within safety concentration, and IBDV onto CEF monolayerby three manners, drugs following virus, virus following drugs, and both virus and drugssimultaneously. By MTT assay, twenty-four anti-IBDV Chinese herb medicines weredefined, as follows: bupleurum root, gypsum, snakegourd root, gardenis fruit, forsythiafruit, isatis root, isatis leaf, honeysuckle flower, honeysuckle stem, dandelion,andrographis herb, scutellaria root, phellodendron bark, dried fresh rehmannia root, poria,umbellate pore fungus, aspidium, pomegranate bark, imperata root, ginseng leaf, piloseasiabell root, astragalus root, white atractylodes rhizome, and licorice root. Thereintogypsum has the best anti-IBDV effect.3 Screening anti-IBDV Chinese herb medicines with chicken embryo: The above described twenty-four anti-IBDV Chinese herb medicines were perparedinto 1 mg/ml liquor with phosphate buffered solution (PBS) as mother liquor, and thendiluted into 1:2, 1:4, 1:8 liquors. IBDV was prepared into 100 EID50 with PBS. IBDV andliquors were inoculated through allantoic cavity by three manners, drugs following virus,virus following drugs, and both virus and drugs simultaneously. Until 160 h, elevenchinese herb medicines were sorted out, such as gypsum, gardenia fruit, forsythia fruit,isatis root, isatis leaf, honeysuckle flower, ginseng leaf, pilose asiabell root, astragalusroot, white atractylodes rhizome, and licorice root. The protective ratio of gypsum onchicken embryo is beyond 80 percent.4 Preventing IBD experiment and clinical application: Based on the theory of traditional Chinese veterinary medicine, these elevenChinese herb medicines are divided into two groups. One group consists of antipyreticdrugs, dispelling pathogenic factors, the other group consists of invigorative drugs,supporting healthy qi. We formulate the prescriptions respectively. First prescription isfuzhengyin, consisting of invigorative drugs. Second prescription is quxieyin, consistingof heat-clearing and detoxifying drugs. Third prescription quxiefuzhengyin, consisting ofboth heating and detoxifying drugs and invigorative drugs. Fourth prescription consists ofgypsum. And control group is treated with yolk antibody. By inoculating chicks withIBDV artificially, the preventive test was carried out. The results revealed that thirdprescription, quxiefuzhengyin, has the highest protective ratio, the weakest pathologicchange, and significant weight gain, better than yolk antibody and gypsum. In the clinicalapplication test, 3830 birds were given the prescription, 3790 birds were cured, thecurative ratio is from 98.86% to 99.14%, and the average is 98.96%. The times of birdsgiven drugs is from two to six, the average is three point four.5 Safety evaluation of quxiefuzhengyin: In acute toxic experiment, we undiscover toxic reaction without a dead mouse, soLD50 exceed 40g/kg when inoculating kunming mice with 400mg/10g ofquxiefuzhengyin for seven days. Thus, it is safe and nontoxic.6 Immune functional research of quxiefuzhengyin: Effect of quxiefuzhengyin on immune organ of mice and chicken is assayed, theresults show that immune organ coefficient, such as thymus and spleen etc, augmentobviously, bursal coefficient is significant compared with control group (P<0.05).At 100 μg/ml concentration of quxiefuzhengyin, it promotes proliferation clearly of Tlymphocyte induced by ConA and B lymphocyte induced by LPS. It strengthens mousephagocytic cell action to some extent. At 1000μg/ml concentration, it enhancesproliferation of control group and extra-hemolymph T cell stimulated by ConA; whenconcentration reaching 100μg/ml, it obviously promotes proliferation of chickenextra-hemolymph B lymphocyte stimulated by LPS; its action weaken and even dissolvewhile being up to 500μg/ml. The prescription of quxiefuzhengyin enhances serum IFNvalence to some extent, but influences release of IL-2 without statistical significance.7.Study on quxiefuzhengyin inhibiting apoptosis of CEF induced by IBD virus: Flow cytometry, and agarose gel electrophoresis of DNA were used to study onquxiefuzhengyin inhibiting apoptosis of CEF induced by IBD virus. The result shows thatquxiefuzhengyin can to some extent inhibit apoptosis of CEF induced by IBD virus. In order to find efficient anti-IBD Chinese herb medicines or prescriptions, a large...