CN103784538A - Traditional Chinese medicinal compound drug for effectively preventing and treating swine influenza - Google Patents
Traditional Chinese medicinal compound drug for effectively preventing and treating swine influenza Download PDFInfo
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Abstract
The invention discloses a traditional Chinese medicinal compound drug for effectively preventing and treating swine influenza, which comprises the following raw materials in parts by weight: Herba Ephedrae 20-40 parts, Cinnamomum cassia twig 15-30 parts, Semen Armeniacae Amarum 20-35 parts, and processed Radix Glycyrrhizae 10-25 parts. In vivo and in vitro experiments prove that the traditional Chinese medicinal compound drug disclosed by the invention can effectively prevent and treat swine influenza.
Description
Technical field
The invention belongs to veterinary Chinese medicine field, relate in a kind of body and the Chinese medicine compound of vitro inhibition swine influenza virus.
Background technology
Swine flue (swine influenza, SI) claim again swine influenza, swine influenza virus (the swine influenza virus by the A of orthomyxovirus section type Influenza Virus, SIV) one that causes is acute, hot, height contact respiratory infectious disease, clinical take burst, high heat, cough, dyspnea, repeatedly show effect, sickness rate is high, mortality rate is low as feature.This disease has generation all over the world, harm is serious, and economic loss is huge, and the mankind's health is constituted a threat to.Its outstanding epidemic characteristic is often exist with subclinical form and often cause other respiratory tract antibacterials and viral secondary or mixed infection, as pig breeding and breathing syndrome, Porcine circovirus desease and porcine contagious pleuropneumonia etc., make epidemic situation become complicated, mortality rate increases.
In the control of clinical practice swine flue, the feature due to antibiotic with control complication is widely used, and coordinates a large amount of medicines such as antipyretic-antalgic, glucocorticoids simultaneously.These processing modes have also increased the weight of immunosuppressant in suppressing body inflammatory reaction, cause the state of an illness more to worsen.Chinese herbal medicine, as natural product, has medicine source abundant, and residual hazard is lower or without residual hazard, side effect is little, is difficult for producing the advantages such as drug resistance; Can regulate body's immunity, suppress virus replication, improve clinical symptoms, on treatment disease of viral infection, be widely used simultaneously.Therefore, application herbal control swine flue, prospect is very wide.
Make a general survey of the antiviral experimentation of present Chinese medicine compound, many to the research of influenza virus A hominis and bird flu virus, and less to the experimentation of swine influenza virus.And mostly adopt respectively the method independent studies of in vivo test or in vitro tests, effect that can not thoroughly evaluating Chinese medicine compound anti-swine flu virus.
For addressing the above problem, in the urgent need to filtering out the Chinese medicine compound of effectively preventing and treating swine flue.And adopt in vitro tests and in vivo test, the effect of comprehensive study Chinese medicine compound anti-swine flu virus from the mechanism of action and clinical practice, for clinical prevention provides theoretical and actual foundation simultaneously.
Summary of the invention
The object of this invention is to provide a kind of Chinese medicine compound of effectively preventing and treating swine flue.
Chinese medicine compound for anti-swine flu virus provided by the present invention, it consists of: Herba Ephedrae 20 ~ 40 weight portions, Ramulus Cinnamomi 15 ~ 30 weight portions, Semen Armeniacae Amarum 20 ~ 35 weight portions, Radix Glycyrrhizae Preparata 10 ~ 25 weight portions.
As the Chinese medicine compound of more specifically implementing, can be formed by the Chinese medicine of following weight portion: comprise following medicine: Herba Ephedrae 30 weight portions, Ramulus Cinnamomi 25 weight portions, Semen Armeniacae Amarum 30 weight portions, Radix Glycyrrhizae Preparata 15 weight portions.
Described compound recipe refers to contained Chinese medicine kind and Chinese medicine proportion thereof.
Weight portion described in the application's book refers to that various medicines in compound recipe account for the part by weight of whole compound recipe.
As another object of the present invention, also comprise a kind of extracting method of the Chinese medicine compound for anti-swine flu virus, adopt water decocting method slightly to carry, concrete steps are:
(1) by Herba Ephedrae 30 weight portions, Ramulus Cinnamomi 25 weight portions, Semen Armeniacae Amarum 30 weight portions, Radix Glycyrrhizae Preparata 15 weight ratios, take each Chinese medicine, composition 100g Chinese medicine compound;
(2) 100g Chinese medicine compound is crushed to 20 orders, soaks 1h, decocting 2 times boils 30 min at every turn, merges 2 times decocting liquid;
(3) by decocting liquid at normal-pressure vacuum sucking filtration and concentrated, be settled to 200mL decocting liquid, every milliliter is equivalent to 0.5g crude drug (being 500mg/mL);
(4) decocting liquid is carried out to autoclaving, it is for subsequent use in 4 ℃ of refrigerators being saved to temperature.
The Herba Ephedrae that the present invention adopts: warm in nature, acrid in the mouth, micro-hardship, have relieving the exterior syndrome by diaphoresis, relieving asthma, the effect such as diuretic; Being usually used in treating wind and cold table disease, measles initial stage and thoroughly sending out the diseases such as smooth, cough and asthma, is one of medicine that tcm clinical practice is conventional; Research through invention shows: Herba Ephedrae has blocking-up, suppresses and direct killing action H1N1 and H3N2 hypotype swine influenza virus.
The Ramulus Cinnamomi that the present invention adopts: Ramulus Cinnamomi, warm in nature, acrid in the mouth, sweet, GUIXIN, lung, urinary bladder channel; There is the effects such as antiinflammatory, antiviral, diuresis, blood vessel dilating, promotion diaphoresis, antipyretic, analgesia, calmness, convulsion, antiinflammatory, antiallergic
The Semen Armeniacae Amarum that the present invention adopts: micro-hardship, warm in nature, micro-poison, have expelling phlegm for arresting cough, relieving asthma, intestine moistening and the therapeutic method to keep the adverse QI flowing downwards open the effects such as numbness.
The Radix Glycyrrhizae Preparata that the present invention adopts: property is flat, and sweet in the mouth, returns 12 warps; For the root and rhizome of dicotyledon pulse family Radix Glycyrrhizae, Glycyrrhiza inflata Bat. or Glycyrrhiza glabra L., main effect is invigorating the spleen and replenishing QI, heat-clearing and toxic substances removing, expelling phlegm for arresting cough, relieving spasm to stop pain;
Beneficial effect of the present invention:
Chinese medicine compound provided by the invention is anti-swine flu virus effectively, and can effectively prevent and treat swine flue clinically.
The specific embodiment
Experimental technique in following embodiment, if no special instructions, is conventional method.
For preventing and treating a Chinese medicine compound for swine flue disease, comprise following medicine: Herba Ephedrae 20 ~ 40 weight portions, Ramulus Cinnamomi 15 ~ 30 weight portions, Semen Armeniacae Amarum 20 ~ 35 weight portions, Radix Glycyrrhizae Preparata 10 ~ 25 weight portions.
Described compound recipe refers to contained Chinese medicine kind and Chinese medicine proportion thereof.
Described weight portion refers to that various medicines in compound recipe account for the part by weight of whole compound recipe.
In following embodiment, experiment material used is as follows:
(1) experimental animal
9 ~ 11 age in days SPF Embryo Gallus domesticus are purchased from Guangdong great Hua Nong fowl egg company limited; SPF level 15g ± 2g BALB/c mouse is purchased from Guangdong Medical Lab Animal Center.
(2) solution or reagent preparation
1. the preparation of PBS: take NaCl 8.0g, Na2HPO4 3.63g, KCl 0.2g KH2PO4 0.24g is dissolved in appropriate ultra-pure water, adjust pH to 7.4, is settled to 1000mL; 100ml/ bottle subpackage, 15 pounds of autoclaving 30min, 4 ℃ save backup.
2. the preparation of 1% chicken erythrocyte suspension: first draw 3.8% liquor sodii citratis (1/5 that its consumption is required blood volume) with sterilizing syringe, from chicken wing vein haemospasia to required blood volume, put in sterilizing centrifuge tube, adding sterilizing PBS is anticoagulant 2 times, with the centrifugal 10min of 2000r/min, abandon supernatant, then add normal saline suspension blood cell, the same method centrifugation, so by red blood cell washing three times, finally according to institute's expense, be made into 1% suspension with sterilizing PBS, now with the current.
3. the extraction of Chinese medicine: adopt water decocting method slightly to carry, concrete steps are:
(1) by Herba Ephedrae 30 weight portions, Ramulus Cinnamomi 25 weight portions, Semen Armeniacae Amarum 30 weight portions, Radix Glycyrrhizae Preparata 15 weight ratios, take each Chinese medicine, composition 100g Chinese medicine compound;
(2) 100g Chinese medicine compound is crushed to 20 orders, soaks 1h, decocting 2 times boils 30 min at every turn, merges 2 times decocting liquid;
(3) by decocting liquid at normal-pressure vacuum sucking filtration and concentrated, be settled to 200mL decocting liquid, every milliliter is equivalent to 0.5g crude drug (being 500mg/mL);
(4) decocting liquid is carried out to autoclaving, it is for subsequent use in 4 ℃ of refrigerators being saved to temperature.
The In Vitro Anti swine influenza virus effect of embodiment 1 compound Chinese medicinal preparation
1. the mensuration of SIV to Embryo Gallus domesticus median infective dose (EID50)
Virus liquid is made to 10 times of doubling dilutions successively, be inoculated in 10 age in days chick embryo allantois intracavity, each dilution factor inoculation 0.2ml/ piece, does 4 repetitions, establishes normal Embryo Gallus domesticus contrast simultaneously.Put hatching 72h in 37 ℃ of calorstats, discard dead embryo in 24h, after results 24h, the allantoic fluid of dead embryo and 72h embryo alive, carries out the mensuration that HA tires.Press Reed-Muench Liang Shi method and calculate EID50.
2. the mensuration of the maximum safe concentration of compound Chinese medicinal preparation to Embryo Gallus domesticus
Chinese medicine extraction liquid is made to 2 times of doubling dilutions successively, be diluted to 7 concentration such as original content, 1:2,1:4,1:8,1:16,1:32,1:64, be then inoculated in 10 age in days chick embryo allantois intracavity, each dilution factor inoculation 0.2ml/ piece, do 4 repetitions, establish normal saline contrast simultaneously.Put 37 ℃ of calorstats and hatch 72 h, observe survival condition every day, discard dead embryo in 24h.The Embryo Gallus domesticus initial concentration that all the highest drug level of survival is formal test, i.e. the maximum safe concentration of Chinese medicine to Embryo Gallus domesticus.
3. the test of compound Chinese medicinal preparation anti-swine flu virus in Embryo Gallus domesticus
Embryo Gallus domesticus is divided into three groups at random, adopts three kinds of dosing methods:
Mode one, first add Chinese medicine and add again virus (blocking effect of Chinese medicine to SIV): first each Chinese medicine extraction liquid is inoculated in to instar chicken embryo on the 10th, 0.2ml/ piece, do 4 repetitions, put 37 ℃ of calorstats and hatch 2h, then inoculate 100EID50 virus liquid 0.2ml/ piece, 37 ℃ of constant temperature culture 72h, measure the blood clotting (HA) of chick embryo allantoic liquid and tire.
Mode two, first add virus and add again Chinese medicine (inhibitory action of Chinese medicine to SIV): first 100EID50 virus liquid is inoculated in to instar chicken embryo on the 10th, 0.2ml/ piece, do 4 repetitions, put 37 ℃ of calorstats and hatch 2h, then inoculate each Chinese medicine extraction liquid 0.2ml/ piece, 37 ℃ of constant temperature culture 72h, measure the blood clotting (HA) of chick embryo allantoic liquid and tire.
Mode three, Chinese medicine virus add (the direct killing action of Chinese medicine to SIV) after mixing together: after first each Chinese medicine extraction liquid being mixed with equivalent 100EID50 virus liquid, put after 37 ℃ of 2h, be inoculated in instar chicken embryo on the 10th, 0.2ml/ piece, does 4 repetitions.37 ℃ of constant temperature culture 72h, measure the blood clotting (HA) of chick embryo allantoic liquid and tire.
Set up virus control and negative control simultaneously.
4. date processing
Data represent with x (-) ± S.E, with the analysis of SPSS17.0 software statistics.
As shown in Table 1:
The effect of table 1 compound Chinese medicinal preparation anti-swine flu virus in Embryo Gallus domesticus
Note: above data are for recording HA meansigma methods; Compared with virus control, * represents P ﹤ 0.01, and difference is extremely remarkable.
Table 1 shows Chinese medicine compound of the present invention anti-swine flu virus effect research in Embryo Gallus domesticus, and result shows that Chinese medicine compound is compared with virus control group, and difference is extremely remarkable, has obvious anti-swine flu virus effect.
The prevention effect of embodiment 2 Chinese medicine compound to mouse infection swine influenza virus
1. dead protective effect and the life-saving effect of Chinese medicine compound to swine influenza virus infecting mouse
(1) laboratory animal grouping
BALB/c mouse is divided into Normal group, model control group, virazole group, Chinese medicine compound group at random, totally 4 groups, 10 every group.
(2) laboratory animal administration
Each group mice is all with gastric infusion, and 2 times/d, 0.2 ml/ time.Each group of Chinese medicine starts administration, continuous 5 d in infecting front 1 d.Meanwhile, Normal group, model control group give equivalent distilled water
(3) laboratory animal counteracting toxic substances
Mice, with after etherization, except Normal group, is respectively organized to collunarium and inoculates 10 LD50 influenza virus 50 μ l, and Normal group is inoculated 50 μ l sterilizing PBS.
(4) experimental observation
Day by day observe mouse invasion situation, record death toll, time-to-live, calculate mortality rate=(respectively organizing death toll/each group sum × 100%); Dead protective rate=(model control group mortality rate-administration group mortality rate)/model control group mortality rate × 100%]; Life-saving rate=(administration group mean survival time-model control group mean survival time)/model control group mean survival time × 100%, refers to table 2:
Dead protective effect and the life-saving effect of table 2 compound Chinese medicinal preparation to swine influenza virus infecting mouse
Note: above data are compared with model control group, and * represents P ﹤ 0.01, and difference is extremely remarkable.
2. the impact of Chinese medicine compound on swine influenza virus infecting mouse pneumonia
(1) laboratory animal grouping, administration, the same 1(1 of modeling) (2) (3)
(2) infect rear the 5th day (more than fasting 8h) and weigh, pluck eyeball sacrificed by exsanguination mice, the aseptic lungs of winning, weigh respectively lung weight with PBS flushing and after sucking surperficial unnecessary liquid with aseptic filter paper.
Lung index and lung index inhibition percentage computing formula are as follows:
Lung index=lungs weight (g)/mice weight (g) × 100
Lung index inhibition percentage=(matched group lung index mean-experimental group lung index mean)/matched group number × 100%
(3) statistical analysis: analyze by SPSS17.0 software statistics, refer to table 3:
The impact of three kinds of compound Chinese medicinal preparation of table 3 on swine influenza virus infecting mouse pneumonia
Note: above data are compared with model control group, and * represents P ﹤ 0.01, and difference is extremely remarkable.
Chinese medicine compound on swine influenza virus infecting mouse after the impact of Blood Cytokines content
(1) laboratory animal grouping, administration, the same 1(1 of modeling) (2) (3)
(2) preparation of serum
Infect latter the 5th day, eyeball blood sampling, is placed in sterilizing centrifuge tube, room temperature blood natural coagulation 2 ~ 3 hours, centrifugal about 20 minutes (2000-3000 rev/min).Carefully collect supernatant, in preservation process, precipitate as occurred, answer recentrifuge.
(3) adopt ELISA test kit to detect
1. the dilution of standard substance and application of sample: establish 10 holes, standard substance hole on the coated plate of enzyme mark, add respectively standard substance 100 μ l in first, second hole, then add standard substance diluent 50 μ l in first, second hole, mix; Then from the first hole, the second hole, respectively get 100 μ l and be added to respectively the 3rd hole and the 4th hole, then add respectively standard substance diluent 50 μ l in the 3rd, the 4th hole, mix; Then in the 3rd hole and the 4th hole, first respectively get 50 μ l and discard, respectively get 50 μ l and be added to respectively in the 5th, the 6th hole, then add respectively standard substance diluent 50ul in the 5th, the 6th hole, mix; After mixing, from the 5th, the 6th hole, respectively get that 50 μ l are added to respectively the 7th, in octal, again the 7th, add respectively standard substance diluent 50 μ l in octal, after mixing from the 7th, get respectively 50 μ l octal and be added in the 9th, the tenth hole, add respectively standard substance diluent 50 μ l in the 90 hole again, after mixing, from the 90 hole, respectively get 50 μ l and discard.(after dilution, each hole application of sample amount is all 50 μ l, and concentration is respectively 2400 pg/mL, 1600 pg/mL, 800 pg/mL, 400 pg/mL, 200 pg/mL).
2. application of sample: establish respectively blank well (blank hole does not add sample and enzyme marking reagent, and all the other each step operations are identical), testing sample hole.On the coated plate of enzyme mark, in testing sample hole, first add sample diluting liquid 40 μ l, and then to add the final dilution factor of testing sample 10 μ l(sample be 5 times).Sample is added on bottom, ELISA Plate hole by application of sample, do not touch hole wall as far as possible, rocks and mix gently.
3. incubation: use the rearmounted 37 ℃ of incubations of shrouding film shrouding 30 minutes.
4. dosing: by 20 times of 30(48T) times concentrated cleaning solution 20 times of distilled water 30(48T) doubly dilution is rear for subsequent use.
5. washing: carefully take shrouding film off, discard liquid, dry, cleaning mixture is filled it up with in every hole, leaves standstill and discards after 30 seconds, so repeats 5 times, pats dry.
6. enzyme-added: every hole adds enzyme marking reagent 50 μ l, except blank well.
7. incubation: operation is with 3.
8. washing: operation is with 5.
9. colour developing: every hole first adds developer A50 μ l, then adds developer B50 μ l, and light shaking mixes, 37 ℃ of lucifuges develop the color 15 minutes.
10. stop: every hole adds stop buffer 50 μ l cessation reaction (the now blue vertical yellow that turns).
0 11 measure: with blank air-conditioning zero, 450nm wavelength is sequentially measured the absorbance (OD value) in each hole.Mensuration should be carried out in 15 minutes adding after stop buffer.
(4) statistical analysis
Analyze by SPSS17.0 software statistics, refer to table 4.
The impact (x (-) ± S.E, ng/mL) of table 4 compound Chinese medicinal preparation on swine influenza virus infecting mouse cytokine
Note: above data are compared with model control group, and * represents P ﹤ 0.05, significant difference; * represents P ﹤ 0.01, and difference is extremely remarkable.
Result demonstration, Chinese medicine compound of the present invention is compared with virus model group, and difference is extremely remarkable.
4. the clinical symptoms of Chinese medicine compound after on mouse infection swine influenza virus and the impact of pulmonary lesion
(1) laboratory animal grouping, administration, the same 1(1 of modeling) (2) (3)
(2) observe after infecting mice the mental status, breathe, search for food and the situation such as drinking-water, within the 5th day, (more than fasting 8h) weighs, and plucks eyeball sacrificed by exsanguination mice, the aseptic lungs of winning are observed pulmonary lesion.
Clinical symptoms is as follows: model group mice occurs lassitude on the 2nd day connecing after poison, bradykinesia, and wrinkle hair, the back of a bow, appetite declines and rapid breathing, likes to flock together, and rolls up, and perpendicular hair, trembles; Within the 3rd day, occur death, 5th ~ 6 days all dead.There is not clinical symptoms in Normal group mice, the clinical symptoms of herbal mixture group mice has alleviating in various degree.
Pulmonary lesion is as follows, and mice lungs pathological changes substantially is respectively organized in perusal: Normal group mice lungs are rose pink, and gassiness, without consolidation district, without abnormal finding.The visible infecting mouse lungs of viral infection model group volume increases, and great majority all have the above consolidation district in blocks of 2 lobes of the lung, present kermesinus outward appearance, and herbal mixture group pulmonary lesion has alleviating in various degree.
Claims (5)
1. for a Chinese medicine compound for anti-swine flu virus, comprise following medicine: Herba Ephedrae 20 ~ 40 weight portions, Ramulus Cinnamomi 15 ~ 30 weight portions, Semen Armeniacae Amarum 20 ~ 35 weight portions, Radix Glycyrrhizae Preparata 10 ~ 25 weight portions.
2. Chinese medicine compound as claimed in claim 1, is characterized in that, comprises following medicine: Herba Ephedrae 30 weight portions, Ramulus Cinnamomi 25 weight portions, Semen Armeniacae Amarum 30 weight portions, Radix Glycyrrhizae Preparata 15 weight portions.
3. a kind of as described in claim 1 ~ 2 is characterized in that for preventing and treating the Chinese medicine compound of swine flue, and described compound recipe refers to contained Chinese medicine kind and Chinese medicine proportion thereof.
4. a kind of for preventing and treating the Chinese medicine compound of swine flue as described in claim 1 ~ 2, described weight portion refers to that various medicines in compound recipe account for the part by weight of whole compound recipe.
5. claimed in claim 1ly it is characterized in that for preventing and treating the extracting method of Chinese medicine compound for swine flue, adopt water decocting method slightly to carry, concrete steps are:
(1) by Herba Ephedrae 30 weight portions, Ramulus Cinnamomi 25 weight portions, Semen Armeniacae Amarum 30 weight portions, Radix Glycyrrhizae Preparata 15 weight ratios, take each Chinese medicine, composition 100g Chinese medicine compound;
(2) 100g Chinese medicine compound is crushed to 20 orders, soaks 1h, decocting 2 times boils 30 min at every turn, merges 2 times decocting liquid;
(3) by decocting liquid at normal-pressure vacuum sucking filtration and concentrated, be settled to 200mL decocting liquid, every milliliter is equivalent to 0.5g crude drug (being 500mg/mL);
(4) decocting liquid is carried out to autoclaving, it is for subsequent use in 4 ℃ of refrigerators being saved to temperature.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104189120A (en) * | 2014-09-19 | 2014-12-10 | 天农大（天津）生物科技有限公司 | Traditional Chinese medicine composition for treating swine influenza and preparation method thereof |
| CN105362400A (en) * | 2014-08-29 | 2016-03-02 | 李丹 | Traditional Chinese medicine composition for treating cold fever |
| CN106237050A (en) * | 2016-07-06 | 2016-12-21 | 佛山科学技术学院 | A kind of Chinese medicine compound of effective preventing and treating H1N1 and H3N2 swine flue and pig blue-ear disease and preparation method thereof |
| CN106421329A (en) * | 2016-09-27 | 2017-02-22 | 佛山科学技术学院 | Traditional Chinese medicine compound prescription for simultaneously resisting swine infuenza and porcine reproductive and respiratory syndrome and extraction method thereof |
| CN108902507A (en) * | 2018-08-06 | 2018-11-30 | 无为县旺盛饲料加工厂 | A kind of sweetening agent of preventing swine influenza |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102716221A (en) * | 2012-05-24 | 2012-10-10 | 肖文辉 | Preparation for mahuang decoction and oral preparation of adjusted formula thereof, and clinic application |
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- 2013-12-12 CN CN201310672928.XA patent/CN103784538A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102716221A (en) * | 2012-05-24 | 2012-10-10 | 肖文辉 | Preparation for mahuang decoction and oral preparation of adjusted formula thereof, and clinic application |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105362400A (en) * | 2014-08-29 | 2016-03-02 | 李丹 | Traditional Chinese medicine composition for treating cold fever |
| CN104189120A (en) * | 2014-09-19 | 2014-12-10 | 天农大（天津）生物科技有限公司 | Traditional Chinese medicine composition for treating swine influenza and preparation method thereof |
| CN106237050A (en) * | 2016-07-06 | 2016-12-21 | 佛山科学技术学院 | A kind of Chinese medicine compound of effective preventing and treating H1N1 and H3N2 swine flue and pig blue-ear disease and preparation method thereof |
| CN106421329A (en) * | 2016-09-27 | 2017-02-22 | 佛山科学技术学院 | Traditional Chinese medicine compound prescription for simultaneously resisting swine infuenza and porcine reproductive and respiratory syndrome and extraction method thereof |
| CN108902507A (en) * | 2018-08-06 | 2018-11-30 | 无为县旺盛饲料加工厂 | A kind of sweetening agent of preventing swine influenza |
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Application publication date: 20140514 |