CN101023997A - Anti-fowl-plague Chinese medicine and preparing method - Google Patents
Anti-fowl-plague Chinese medicine and preparing method Download PDFInfo
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- CN101023997A CN101023997A CNA2007100719522A CN200710071952A CN101023997A CN 101023997 A CN101023997 A CN 101023997A CN A2007100719522 A CNA2007100719522 A CN A2007100719522A CN 200710071952 A CN200710071952 A CN 200710071952A CN 101023997 A CN101023997 A CN 101023997A
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Abstract
The present invention provides a Chinese medicine for resisting avian influenza virus and its preparation method. Said Chinese medicine is made up by using 9 Chinese medicinal materials of aspidium, isatis root, astragalus root, bupleurum root, forsythia fruit and others through a certain preparation process. Said invention also provides the concrete steps of its preparation process.
Description
(1) technical field
What the present invention relates to is a kind of medicine for animals, specifically a kind of Chinese medicine for resisting avian influenza virus.
(2) background technology
Bird flu (Avian Influenza) is a kind of acute contagious disease of the serious harm poultry husbandry that caused by A type influenza virus.The bird flu of high and low pathogenicity all brings enormous economic loss for the aviculture of countries in the world.(Avian Influenza Virus, AIV) antigen has multiformity to bird flu virus, lacks strong cross-protection between each hypotype.Because anti-avian influenza virus medicine commonly used clinically for example amantadine hydrochloride descends laying hen output, AIV easily produces drug resistance to it simultaneously, and virazole, moroxydine not only make egg production descend, and are the shared medicines of people and animals, have limited them in Clinical Application.Because the drug residue problem limited the outlet of China's animal derived food, so develop a kind of green, anti-avian influenza virus medicine pollution-free, safe, effective, that poultry are special-purpose is extremely urgent.The development of this medicine not only can solve the immune problem that is in sub-clinical state chicken group, can be one of approach of preventing bird flu effectively.
Chinese herbal medicine is abundant in china natural resources, and is cheap.For chemical synthetic drug, Chinese herbal medicine has plurality of active ingredients, not only have toxicity low, be difficult for having side effects, many target spots, pathogenic microorganism be difficult for producing chemical sproof advantage to it, and in anti-bacteria and anti-virus, can also promote growth of animal, strengthen the immune function of animal, improve body disease-resistant power.At present, the resisiting influenza virus Chinese herbal medicine great majority of domestic research are diaphoretic medicine and antipyretic and are the Chinese patent medicine that principal agent is formed with them, receive the concern of Chinese scholars.
Bird flu and human influenza have close dependency, and present isolating bird flu virus almost covers two kinds of surface antigens of whole human influenza viruses, from the internal energy human influenza virus of being separated to of poultry body that infects influenza and can detect corresponding antibody.In recent years, bird flu has brought enormous economic loss to countries in the world, Chinese herbal medicine is the big class medicine that China most possibly obtains independent intellectual property right, has stronger competitiveness and market potential, thus carry out the research of anti-avian influenza virus Chinese herbal medicine imperative.
(3) summary of the invention
The object of the present invention is to provide a kind of egg production that do not influence, the Chinese medicine for resisting avian influenza virus that side effect is little.
The object of the present invention is achieved like this: it is that weight ratio is that Rhizoma Osmundae 8-10 part, Radix Isatidis 8-10 part, Radix Astragali 15-20 part, Radix Bupleuri 8-10 part, Fructus Forsythiae 5-8 part, Rhizoma Coptidis 8-10 part, Flos Lonicerae 8-10 part, Radix Scutellariae 8-10 part and Radix Glycyrrhizae 4-6 part are made.
Product of the present invention is to adopt such method to make: by weight the ratio for Rhizoma Osmundae 8-10 part, Radix Isatidis 8-10 part, Radix Astragali 15-20 part, Radix Bupleuri 8-10 part, Fructus Forsythiae 5-8 part, Rhizoma Coptidis 8-10 part, Flos Lonicerae 8-10 part, Radix Scutellariae 8-10 part and Radix Glycyrrhizae 4-6 part each raw material is mixed, added water retting 30 minutes, decoct 2 times, each one hour, gradation was filtered, merging filtrate, be concentrated into relative density 1.2, put coldly, add ethanol and make and contain alcohol amount and reach 75%, fully stir, left standstill 12 hours.Get supernatant, reclaim ethanol to tasteless, add 3-4 times of water gaging, fully stir and be heated to boiling, static 48 hours, the leaching supernatant was concentrated into relative density 1.10, filtered, and added 0.07% ethylparaben, embedding.
Product of the present invention is a kind of compound Chinese medicinal preparation, and Chinese medicine compound is principal mode and the means that tcm clinical practice is cured the disease, and is the important component part of Chinese medicine pharmacy.Chinese medicine compound is the development of single medication, and the effect of compound recipe obviously is better than single medicinal material.Behind the avian influenza, very easily concurrent bacterial infection causes the variation of whole body organ hemorrhage in addition, causes immunosuppressant, thus select for use some anti-avian influenza virus effective, and the Chinese medicine of antibacterial action is arranged.The present invention is directed to that hyperpyrexia, hemorrhage, diarrhoea appear in the morbidity chicken, the diet desire reduces or general character such as useless exhausted, hypoimmunity, select the herbal mixture of forming by many groups such as Rhizoma Osmundae, Radix Isatidis, Radix Astragali Chinese medicine for use, play the effect that antiviral prevents secondary infection.From experimental result as can be seen, this medicine has certain preventive and therapeutic effect to the bird flu of highly pathogenicity, and has safe and reliablely, does not influence the advantages such as egg production of laying hen.The experiment proved that product of the present invention has following characteristics:
1, the effect of anti-outer bird flu virus
Can tentatively infer the effect of bird flu virus on the MDCK and on the Embryo Gallus domesticus by compound medicine: product of the present invention not only has direct deactivation to bird flu virus, can also enter the antiviral effect of performance in the cell.
2, safe and reliable
According to the foreign compound acute toxicity grading criteria that The World Health Organization (WHO) is recommended, dosage is greater than 40000mg/kg.b.w, for nontoxic behind oral administered compound Chinese medicine of white mice.Its median lethal dose(LD 50) CD50 is 425.6mg/kg.b.w behind the lumbar injection product of the present invention.Belong to low toxicity.CD behind the white mice oral administered compound Chinese medicine
50With CD behind the lumbar injection
50Differ greatly.Possible medicine is through behind the digestive tract, and noxious substance is broken down into innocuous substance and enters blood circulation.And behind the lumbar injection, medicine directly enters blood circulation with the form of prototype, causes the whole body poisoning.
3, to the chickling Immune Effects
(1) influence that the chickling immune organ is grown
Under the normal physiological situation, the immune organ weight of animal body increases, and is main because of these immune organs self cell growth promoter, division growth formation; And weight reduces, and then is that the sophisticated cell of these organs is released into blood or self cell and stops growing due to growth, the division growth.Immune organ indexes such as administration group chickling thymus, fabricius bursa and spleen all are higher than the blank group, illustrate that immune system is ripe very fast.So this product of the present invention can promote the growth of SPF chickling immune organ and ripe rapidly, and the SPF chicken immuological function is strengthened, and resists various cause pathogeny imcrobe infections, improves anti-stress ability.
(2) to the lymphocytic influence of SPF chicken T
The present patent application is tentatively carried out chickling and is taken behind the herbal mixture influence to the t lymphocyte subset group.CD3
+Be to be present in all mature T lymphocytic cell surfaces.Identify and the lymphocytic main surface marker of difference T that it is representing the state of whole machine body cellular immune function.In this experiment, the CD3 of administration group and vaccine group+administration group
+T lymphocyte content is apparently higher than matched group, so chickling after 1 age in days is taken this herbal mixture, can significantly improve chicken group's cellular immunization.CD4
+The T lymphocyte has the effect of replying with enhancing human body immunity of inducing, and can secrete the multiple immunocompetent lymphokine that has, and stimulates activation, propagation and the generation specific antibody of bone-marrow-derived lymphocyte.The avian influenza antibody of administration group in this experiment ,+immune group also proves this theory than immune group height.CD8
+The main mediated cell toxic action of T lymphocyte.The CD8 of administration group+immune group
+The lymphocytic degree of T is than blank group height, illustrate increase body by the cell toxic action begin to resist with scavenger cell in cause of disease, thereby certain defensive measure is played in the infringement of virus, and this also is the immune mechanism of the antivirus action of proof product of the present invention.The used compound preparation of this experiment can promote the propagation of immunologically competent cell in the immune organ, improves body cell immunity and humoral immunization function.This research report with other Chinese medicines is consistent.
4, study product of the present invention in the intravital pharmacokinetics of white mice with the medicine method of cumulative scale
Method with medicine method of cumulative scale research pharmacokinetics has two kinds.First kind with the animal dead rate as observation index " method of cumulative scale ": press the different intervals administration promptly for many treated animals, obtain the different time body and deposit percentile dynamic change situation, thus the calculating pharmacokinetic parameters.Second kind with curative effect of medication as observation index " method of cumulative scale ".The drug accumulation method is applicable to the pharmacokinetic studies with certain toxic Chinese medicine compound.Utilize the dynamic change in vivo of drug accumulation method research medicine, calculate relevant pharmacokinetic parameters, its advantage is to embody the globality of compound compatibility, the basic theories of medical drugs in meeting.But it mainly is applicable to the Chinese medicine compound that toxicity and pharmacodynamics effect are produced by same component, and can only reflect the pharmacokinetics rule of toxic component to a certain extent.In this experiment, (v) value is 10.039 to product of the present invention, illustrates that this medicine and blood plasma and histone adhesion are very high at the intravital apparent volume of distribution of white mice.The ka value of product of the present invention is 4.9078/hrs, and it is fast to illustrate that this medicine absorbs.Behind lumbar injection, the clinical symptoms of poisoning can appear in 10 minutes, and dead clinical manifestation appearred after half an hour, enter blood very soon from clinical symptoms explanation medicine, play a role, the two is consistent.Kel is an elimination rate constant, and its size is represented medicine elimination speed in vivo.Kel is 0.1501 in this experiment, and it is slow more that the expression medicine is eliminated speed in vivo, and drug level descends also slow, and medicine retention time in vivo is long, and drug effect is also long.In sum, behind this medicine lumbar injection, have absorption soon, eliminate slow characteristics at the white mice internal metabolism.
5, control SPF chicken infects the experimental study of bird flu virus
(1) product treatment SPF chicken of the present invention infects the research of bird flu virus
Find out that from the protective rate result of each group amantadine hydrochloride plays the certain protection effect.From the chicken mean survival time, also can confirm this point.Because amantadine hydrochloride suppresses the m of influenza virus
2Gene, thus make m
2Gene can not be exercised ion channel function, suppresses duplicating of virus.But the amantadine hydrochloride of low concentration and influenza virus during co-cultivation, very easily produce the virus of salt tolerant symmetrel under the test chamber condition.The virus of these sudden changes is very stable in heredity, and not only salt tolerant symmetrel strain very easily is separated in the fowl body, and also very easily is separated to the virus of salt tolerant symmetrel in human body.After the salt tolerant symmetrel occurred, amantadine hydrochloride lost effect very soon, and salt tolerant symmetrel strain becomes local epidemic isolates, so, after salt tolerant symmetrel strain occurs, not only can cause the treatment failure, but also can in the susceptible host body, propagate.
China screened the Chinese herbal medicine of nearly thousand kinds resisiting influenza virus since 58 years at experiment in vitro, but the also rare of sure inhibition influenza virus arranged in animal body.Has following reason: at first, the Chinese herbal medicine extract component content is very complicated, in experiment in vitro, can influence virus absorption and duplicate, but some effective ingredient is decomposed in kinetoplast, perhaps the effective ingredient of influenza is not absorbed, do not reach corresponding blood drug level even absorb, so do not reach the effect of treatment yet.Product of the present invention has a strong anti-avian influenza virus effect external, and when testing in vivo, the result is not satisfactory, may be that its reason awaits further discussion because the working concentration of this herbal mixture is low slightly.
(2) prevention SPF chicken infects the research of bird flu virus
From experimental result as can be seen, no matter herbal mixture prevention group is in the counteracting toxic substances group, still be that its dead protective rate and mean survival time are all than product treatment group height of the present invention in same group's infected group.So this herbal mixture suppresses just aspect of virus, the more important thing is and transfer the inherent motility of body, eliminate the histiocyte damage, recover homeostasis, inside and outside lay equal stress on.
(3) the viral separation case of infective virus group, same group's infected group
In test, the chicken group toxin expelling amount of Chinese medicine medication group and amantadine hydrochloride medication group is all low than matched group, illustrates that product of the present invention can suppress duplicating of virus.Under square one, it is lower than the viral level that is separated to from cotton examination of larynx to be separated to viral level from cotton examination of cloaca.This may be because product of the present invention and amantadine hydrochloride all are the mode administrations with drinking-water, and intestinal inner virus content is reduced.So according to this result, can infer, if this compound medicine, can suppress combining of influenza virus and pulmonary epithelial cells with the form administration of spray, curative effect can be better.
These characteristics draws by following test:
1. materials and methods
1.1 for having a try agent and virus
Product of the present invention is the transparent supernatant liquid of yellowish-brown.The matched group amantadine hydrochloride is purchased in Dongbei Pharmaceutical General Factory; The bird flu virus of H5N1 and H9N2 hypotype, mdck cell are provided by Chinese Academy of Agricultural Sciences animal influenza center; Tissue Culture Plate (24,96 hole), DMEM purchase the company in Gibco; SPF Embryo Gallus domesticus, SPF chicken are provided by Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center; Lymphocyte separation medium is available from Inst. of Hematology, Chinese Academy of Medical Sciences; The CD3 of FITC labelling is available from Southern BiotechnologyAssociates.Inc company, the CD4 of FITC labelling, CD8, TCR by Qiao pass tinkling of pieces of jade post-doctor, doctor Jiang Yongping is so kind as to give.H9 subtype avian influenza oil emulsion inactivated vaccine is provided by the Ministry of Agriculture of Harbin Veterinary Medicine Inst., China Academy of Agriculture animal influenza open laboratory, and FACScanTM (Flow cytometer BDIS, San Jose, CA), Beckman centrifuge etc.
1.2 the medicine non-toxic is measured
1.2.1 medicine is to the maximal non-toxic dosage of Embryo Gallus domesticus
Get a plurality of dilution factors inoculation 5 instar chicken embryos on the 10th (0.2ml/ embryo) of product of the present invention, establish the sterile saline contrast simultaneously, discarded with interior dead Embryo Gallus domesticus in 24 hours, dead Embryo Gallus domesticus in time takes out freezingly after 24 hours, and the live chickens embryo continues hatching until hatching.
1.2.2 medicine is to Madin-Darby canine kidney(cell line) (MDCK) toxicity test
Get above-mentioned different dilution product of the present invention with cell maintenance medium dilution, every dilution factor is inoculated 4 holes, every pipe 1ml, and establish the normal control pipe, every day the observation of cell pathological changes, observed result.
1.3 product of the present invention is the anti-avian influenza virus test on Embryo Gallus domesticus
1.3.1 the direct deactivation of product of the present invention and bird flu virus
With the sample of variable concentrations respectively with equivalent 10
5EID
50H5N1 and 10
4EID
50The H9N2 mixing, behind room temperature effect 1h, be inoculated in 9-10 age in days chick embryo allantois intracavity immediately respectively, 4 pieces every group, hatched 96 hours for 37 ℃, measure the hemagglutinative titer respectively organize in the chick embryo allantoic liquid respectively.Establish normal saline simultaneously, virus and room temperature are placed the virus control behind the 1h.
1.3.2 product of the present invention on Embryo Gallus domesticus to the therapeutical effect of bird flu virus
With the grouping of 9-10 day instar chicken embryo, 4/group, inoculate 10 respectively earlier
4EID
50H9N2 and 10
5EID
50H5N10.1ml is inoculated in the allantoic cavity, and 37 ℃ of hatchings were inoculated each dilution product of the present invention after 1 hour, hatches 96 hours for 37 ℃.Measure the hemagglutinative titer of virus in every group of chick embryo allantoic cavity.
1.3.3 product of the present invention on Embryo Gallus domesticus to the preventive effect of bird flu virus
Earlier inoculate medicine in accordance with the law, back virus inoculation, operational approach is the same.
1.3 product of the present invention is the anti-avian influenza virus experiment on MDCK
1.3.1 mixing mutually with virus, product of the present invention adds cell immediately
With different dilution factors, different product of the present invention (on maximal non-toxic dosage basis) and equal-volume 10TCID
50Behind H9N2 and the H5N1 immixture 1h, inoculating cell immediately, and establish virus control simultaneously, normal cell and medicine contrast.
1.3.2 product of the present invention on MDCK to the preventive effect of bird flu virus
With behind different dilution factors, 37 ℃ of processing of the different product of the present invention cell 1h, press method adding virus among the 1.3.1 earlier.Establish virus control simultaneously, normal cell and medicine contrast.
1.3.3 product of the present invention on MDCK to the therapeutical effect of bird flu virus
Inoculate 10TCID earlier
50H9N2 and H5N1 virus infection cell at 37 ℃ of hatching 1h, add the different Chinese medicines of each dilution factor by method among the 1.3.1.Establish virus control simultaneously, normal cell and medicine contrast.
1.3.4 the result judges: in Embryo Gallus domesticus, can suppress the minimum dose of blood clotting titre more than 32 times, be active drug concentration.In the mdck cell system, do not cause that cytopathic drug level is effective drug level
[2]
1.4 acute toxicity testing-median lethal dose(LD 50) (LD
50) mensuration
Take oral and two kinds of route of administration of lumbar injection.Oral dose is 330mg/10g.b.w, and the dosage ratio is 1: 0.8 between oral group of group, and test is divided into 7 groups, and wherein one group is matched group, 10/group; Observed 7 days continuously, and weigh in.Lumbar injection group injected dose is 50mg/10g.b.w, and the dosage ratio is 1: 0.75 between group, and test is 6 groups altogether, and wherein one group is matched group, 10/group.Behind the lumbar injection, dead animal in time cut open inspection and core, liver,spleen,kidney, lung do the pathology shear, observes its pathological change.Adopt and simplify the computing method calculating LD of probit
50
1.5 the research of product pharmacokinetics of the present invention
1.5.1 determining of dosage and administration time:
Get CD
5-CD
35Dosage in the scope.CD is got in this experiment
11Dosage be twice same amount medication of 125mg/kg.b.w.Get 0.5,1,2,4,8,6 time point rechallenges of 12h.
1.5.2 animal grouping
By the principle of correspondence between groups such as sex, body weight mice is divided into 6 groups, 20 every group, the mortality rate (no longer occurring till the death) after twice medication is observed in different time medication for the second time after the administration for the first time.Lumbar injection medicinal liquid capacity is 0.5ml, 125mg/kg.b.w.
2.5.3 computational methods: find the probit of each mortality rate by table, find out the corresponding dosage of mortality rate, press the body of column count dose and deposit % by the D-P straight line
Body is deposited consumption * 100% of %=(the corresponding function dosage of mortality rate-second time consumption)/for the first time
For the first time and for the second time consumption equates in this experiment in this formula.With regard to blanking time and body deposit rate, the log body is deposited the rate mapping, promptly draws the dynamic change that body is deposited % behind a drug.This changes if meet the first order kinetics rule, and curve when then pressing MCPKP program match medicine is just deposited the data of % and time with body and carried out match through the MCPKP program
[4]
1.6 product of the present invention is to the influence of SPF chicken immuological function
1 age in days, 80 plumage SPF chickens are divided into 4 groups at random, every group 20 plumage, the unified identical complete feed of feeding.Two groups of 40 chickens are from 1 age in days, 0.5% product drinking-water of the present invention, wherein 20 bird flu oil emulsion inactivated vaccines that chicken 15 ages in days are exempted from the H9 hypotype.All the other 20 as the administration group; In all the other 40 chickens, 20 15 ages in days are exempted from the bird flu oil emulsion inactivated vaccine of H9 hypotype, are the vaccine matched group; Other 20 is the blank group.Since 15 ages in days, randomly drawed normotrophic 5 plumage chickens in 22 days, 29 days, butcher according to a conventional method, separate and measure the index (immune organ weight is tried to achieve each organ index divided by corresponding chicken body weight) of thymus, spleen, fabricius bursa immune organ.Understand immune organ production developmental state.At 21 ages in days, random choose is 5 from 4 groups of experimental chickens, and heart is adopted the 2ml heparin anti-coagulating.On FAC, measure the content of CD3, CD4, CD8 and gamma delta T CR.Adopt blood clotting to suppress (HI) method and detect avian influenza antibody titre in the serum.
1.7 product control SPF chicken of the present invention infects the experimental study of bird flu virus
Test is divided into 5 groups, and the I group is the treatment by Chinese herbs group, totally 18 SPF chickens.18 chickens give 0.5% Chinese medicine drinking-water simultaneously.Wherein 8 administration while merits are hit H
5N
1Subtype avian influenza virus, counteracting toxic substances dosage are 10
4EID
50/ 0.1ml, intramuscular injection 0.2ml.Other 10 counteracting toxic substances not are so that the treatment situation after simulation chicken house chicken group's natural infection and the administration.
The II group is the amantadine hydrochloride matched group, totally 17 SPF chickens.17 chickens give 0.1% amantadine hydrochloride drinking-water simultaneously.Wherein 7 counteracting toxic substances administrations simultaneously, counteracting toxic substances dosage is 10
4EID
50/ 0.1ml, intramuscular injection 0.2ml.Other 10 counteracting toxic substances not are so that the treatment situation of simulation chicken house chicken group after giving amantadine hydrochloride.
The III group is the Chinese herb on the prevention group.12 SPF chickens of chicken are used in experiment, and 12 chickens all gave 0.5% Chinese medicine in preceding 15 days at counteracting toxic substances.6 counteracting toxic substances wherein, counteracting toxic substances dosage is 10
4EID
50/ 0.1ml, intramuscular injection 0.2ml/ are only.Other 6 counteracting toxic substances not are so that prevention and the treatment situation of simulation chicken house chicken group after giving product of the present invention.
Begin to observe 14 days continuously from counteracting toxic substances, observe and write down the death toll of chicken, calculate mortality rate then, protective rate, mean survival time.Gathered cloaca, cotton examination of larynx in 3,5,7 days behind counteracting toxic substances, cotton examination of collection is made into 10 with sterile saline
-1-10
-8Suspension, allantoic cavity are seeded in 5 SPF Embryo Gallus domesticus of 9-10 age in days, hatch 48 hours at 37 ℃, according to micro-red cell agglutination method, check hemagglutination activity in the chick embryo allantoic liquid.As have hemagglutination activity, under the prerequisite of getting rid of newcastle disease virus infection, the existence of bird flu virus is described by hemagglutination inhibition test.
The IV group is a matched group.3 groups of contrast components.One group is the counteracting toxic substances group, and counteracting toxic substances dosage is organized with other, totally 8 SPF chickens, other one group be 7 SPF chickens with group's infected group, last group uninfecting virus matched group is made up of 4 SPF chickens.
2 results
2.1 product maximal non-toxic of the present invention dosimetry
Medicine is respectively 1000mg/ml, 62.5mg/ml to the non-toxic of Embryo Gallus domesticus, MDCK.
2.2 the effect of product extracorporeal antivirus effect of the present invention
2.2.1 product of the present invention in Embryo Gallus domesticus to the effect of bird flu virus
Product of the present invention is respectively 31.25mg/ml, 125mg/ml and 250mg/ml to effective deactivation concentration of H5N1 subtype avian influenza virus, treatment concentration and prevention concentration in Embryo Gallus domesticus, is respectively 31.25mg/ml, 15.625mg/ml and 62.5mg/ml to effective deactivation concentration, treatment concentration and the prevention concentration of H9N2 subtype avian influenza virus.
2.2.2 product of the present invention on MDCK to the effect of bird flu virus
Product of the present invention effective deactivation concentration, treatment concentration and prevention concentration to the H5N1 subtype avian influenza virus in Embryo Gallus domesticus is respectively 31.25mg/ml, 125mg/ml, and treatment concentration is not measured.To effective deactivation concentration of H9N2 subtype avian influenza virus, treatment concentration and prevention concentration be 15.62 5 respectively, 31.25mg/ml and 31.25mg/ml.
2.3 security of products research of the present invention
2.3.1 product oral group of the present invention is to the acute toxicity testing of white mice
Behind the product oral of the present invention, do not cause the death of white mice.But the trend that loses weight is arranged when high dose.
2.3.2 behind the product lumbar injection of the present invention to the acute toxicity testing of white mice
Behind the mice by intraperitoneal injection Chinese medicine 10min, promptly show excited uneasiness, spasm is cuddled up in a heap, dyspnea, and 0.5h occurs dead after administration.Each internal organs of dead Mus are cutd open inspection: liver volume is enlargement slightly, the tunicle anxiety, and unsharp border, the surface is dark violet redness, and tangent plane is kermesinus, the quality deliquescing.Spleen, kidney volume all increase.According to the minimizing of dosage, the also corresponding minimizing of mortality rate, and also organ disease also reduces gradually.As can be seen, the pathological change of each internal organs and dosage are certain relation behind histology's pathological section.Calculate CD with simplifying probit computing method (also claiming and Er Enfa)
50This product of the present invention is behind lumbar injection, to the CD of white mice
50Be 425.6mg/kg.b.w.
2.4 product pharmacokinetics experiment of the present invention
Calculate body according to the mice dying rate and deposit medication amount, by the MCPKP program body is deposited percentage rate and match blanking time after, the calculating pharmacokinetic parameter and medicine-time equation.This product of the present invention white mice intravital through the time process be one-compartment model.In its,, equation was in medicine-time: C=650.66e
-4.9078t, pharmacokinetic parameters sees Table 1:
Table 1: product of the present invention is in the intravital apparent pharmacokinetic parameters of white mice
Table 1：Pharmacoketics Parameter of combination herbs in mouse
| Parameter (pameters) | Unit | Value |
| Co, (initial concentration) K α, (absorption rate constant) kel, (elimination rate constant) T1/2, (absorbing the phase half-life) T1/2 β, (eliminating the phase half-life) Tp | ug/ml l/nrs l/nrs hrs hrs hrs | 622.547 4.9078 0.1501 0.1412 4.6176 0.733 |
| Cmax, (reaching peak concentration) AUC, (area under curve) Lagtime, (lag time) Tcp, (ther) V, (apparent volume of distribution) D, (elimination factor) | ug/ml Mg/l.hrs hrs hrs L/orl/ng | 557.69 4148.1 0 70.592 10.039 1.507 |
2.5 product of the present invention is to the influence of SPF chicken immuological function
2.5.1 product of the present invention is to the exponential influence of immune organ
The fabricius bursa index of product drinking-water group of the present invention, index and spleen index, thymus index at 14 ages in days apparently higher than matched group.
2.5.2 product of the present invention is to the influence of avian influenza antibody titre in the chickling serum
Two weeks after immunity, the avian influenza antibody titre of administration group+immune group is than high 1.6 titres of immune group.
2.5.3 product of the present invention is to the influence of SPF chicken body weight change
The body weight of administration group is apparently higher than matched group, and wherein weight differential is remarkable in the time of the 21st day.
2.5.4 CD3 in the different peripheral bloods
+, CD4
+, CD8
+, TCR
+The T lymphocyte changes.
CD3
+, CD4
+, CD8
+, TCR
+The lymphocytic situation of change of T sees Table 2.
Table 2. product of the present invention is to the lymphocytic influence of T in the peripheral blood (percentage ratio)
Table 2：Influence on Tlymphocyte in peripheral blood of combination herbs(percentage)
| Group | Number of animals | CD3 + | CD4 + | CD8 + | TCR + |
| I II III IV | 5 5 5 5 | 31.25±5.68 36.57±4.56 63.61±7.65 64.01±4.32 | 21.49±3.42 30.74±1.98 29.69±6.79 27.39±5.79 | 14.56±4.57 28.12±9.16 35.58±8.17 45.05±6.32 | 11.46±1.39 26.27±2.56 33.186±5.66 32.19±4.47 |
The I group is the blank group; The II group is the vaccine immunity group; The III group is vaccine+administration group; The IV group is the administration group.
2.6 product control SPF chicken of the present invention infects the experimental study of bird flu virus
2.6.1 protective effect
Except that the uninfecting virus matched group, the infective virus group all has chicken death.
2.6.1.1 Chinese medicine is to the protective effect of infective virus group
The experimental group protective rate that gives medicine at counteracting toxic substances simultaneously all is lower than with group's infection back administration group.Product treatment group protective rate of the present invention is 0, illustrates that it can not suppress bird flu virus fully and duplicate and breed in that chicken is intravital.And the protective rate of amantadine hydrochloride group is 14.3%, illustrates that amantadine hydrochloride can play the certain protection effect.
From mean time to death, the death of viral infection matched group chicken is the fastest, and secondly be treatment by Chinese herbs group, Chinese herb on the prevention group, be the amantadine hydrochloride group once more, be matched group at last.
2.6.1.2 with the protective effect of group's infected group
Except that not infecting the bird flu virus matched group, other groups all have chicken death.Protective rate for the highest, reaches 33.4% with amantadine hydrochloride, secondly reaches 30% for product prevention group of the present invention, is that product treatment group of the present invention is 20% once more.
2.6.2 viral separation case
2.6.2.1 the viral separation case of infective virus group
Chicken to the infective virus group separated virus at 3,5,7 days.Cotton examination of cloaca, the sub-isolated viral situation of the cotton examination of larynx see Table 3.
Cotton examination of table 3. infection group and viral infection group cloaca, the sub viral separation case of the cotton examination of larynx
Table 3 Cultivating avian influenza virus of fecal and clocal
| The sub viral separation case of the cotton examination of cloaca | The sub viral separation case of the cotton examination of larynx |
| The virus separation rate | EID 50 | The virus separation rate | EID 50 | |
| Virus control product treatment of the present invention group product prevention of the present invention group amantadine hydrochloride group | 100％ 100％ 80％ 75％ | 5.5 3.5 3.0 2.5 | 100％ 100％ 82.8％ 85.7％ | 5.5 4.00 3.50 3.02 |
2.6.2.2 separation case with group's infected group virus
Chicken with group's infected group was separated virus at 3,5,7 days.Gather cotton examination of cloaca and cotton examination of larynx respectively, the results are shown in Table 4.
Table 4. is with the sub viral separation case of the cotton examination of the cloaca of group infected group
Table 4 Cultivating avian influenza virus of fecal
| The sub viral separation case of the cotton examination of cloaca | The sub viral separation case of the cotton examination of larynx | |||
| The virus separation rate | EID 50 | The virus separation rate | EID 50 | |
| Virus control product treatment of the present invention group product prevention of the present invention group amantadine hydrochloride group | 100％ 70％ 50％ 60％ | 4.75 4.00 2.5 2.0 | 100％ 80％ 66.6％ 70％ | 4.5 4.00 3.0 2.5 |
(4) specific embodiment
For example the present invention is done in more detail below and describes:
By weight the ratio that is Rhizoma Osmundae 8-10 part, Radix Isatidis 8-10 part, Radix Astragali 15-20 part, Radix Bupleuri 8-10 part, Fructus Forsythiae 5-8 part, Rhizoma Coptidis 8-10 part, Flos Lonicerae 8-10 part, Radix Scutellariae 8-10 part and Radix Glycyrrhizae 4-6 part individual raw material is mixed, added water retting 30 minutes, decoct 2 times, each one hour, gradation was filtered, merging filtrate, be concentrated into relative density 1.2, put coldly, add ethanol and make and contain alcohol amount and reach 75%, fully stir, left standstill 12 hours.Get supernatant, reclaim ethanol to tasteless, add 3-4 times of water gaging, fully stir and be heated to boiling, static 48 hours, the leaching supernatant was concentrated into relative density 1.10, filtered, and added 0.07% ethylparaben, and product is made in embedding.
By weight the ratio that is Rhizoma Osmundae 9g, Radix Isatidis 9g, Radix Astragali 18g, Radix Bupleuri 9g, Fructus Forsythiae 6g, Rhizoma Coptidis 9g, Flos Lonicerae 9g, Radix Scutellariae 9g and Radix Glycyrrhizae 5g individual raw material is mixed, added water retting 30 minutes, decoct 2 times, each one hour, gradation was filtered, merging filtrate, be concentrated into relative density 1.2, put coldly, add ethanol and make and contain alcohol amount and reach 75%, fully stir, left standstill 12 hours.Get supernatant, reclaim ethanol to tasteless, add 3-4 times of water gaging, fully stir and be heated to boiling, static 48 hours, the leaching supernatant was concentrated into relative density 1.10, filtered, and added 0.07% ethylparaben, embedding.
Claims (3)
1, a kind of Chinese medicine for resisting avian influenza virus is characterized in that: it is that weight ratio is that Rhizoma Osmundae 8-10 part, Radix Isatidis 8-10 part, Radix Astragali 15-20 part, Radix Bupleuri 8-10 part, Fructus Forsythiae 5-8 part, Rhizoma Coptidis 8-10 part, Flos Lonicerae 8-10 part, Radix Scutellariae 8-10 part and Radix Glycyrrhizae 4-6 part are made.
2, Chinese medicine for resisting avian influenza virus according to claim 1 is characterized in that: it is that weight ratio is that Rhizoma Osmundae 9g, Radix Isatidis 9g, Radix Astragali 18g, Radix Bupleuri 9g, Fructus Forsythiae 6g, Rhizoma Coptidis 9g, Flos Lonicerae 9g, Radix Scutellariae 9g and Radix Glycyrrhizae 5g make.
3, a kind of manufacture method of Chinese medicine for resisting avian influenza virus, it is characterized in that: by weight being Rhizoma Osmundae 8-10 part, Radix Isatidis 8-10 part, Radix Astragali 15-20 part, Radix Bupleuri 8-10 part, Fructus Forsythiae 5-8 part, Rhizoma Coptidis 8-10 part, Flos Lonicerae 8-10 part, the ratio of Radix Scutellariae 8-10 part and Radix Glycyrrhizae 4-6 part is mixed individual raw material, added water retting 30 minutes, decoct 2 times, each one hour, gradation was filtered, merging filtrate, be concentrated into relative density 1.2, put coldly, add ethanol and make and contain alcohol amount and reach 75%, fully stir, left standstill 12 hours, and got supernatant, reclaim ethanol to tasteless, add 3-4 times of water gaging, fully stir and be heated to boiling, static 48 hours, the leaching supernatant, be concentrated into relative density 1.10, filter, add 0.07% ethylparaben, embedding makes product.
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