CN103638147A - Traditional Chinese medicine compound for resisting swine influenza virus - Google Patents
Traditional Chinese medicine compound for resisting swine influenza virus Download PDFInfo
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- CN103638147A CN103638147A CN201310649172.7A CN201310649172A CN103638147A CN 103638147 A CN103638147 A CN 103638147A CN 201310649172 A CN201310649172 A CN 201310649172A CN 103638147 A CN103638147 A CN 103638147A
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Abstract
The invention discloses a traditional Chinese medicine compound for resisting a swine influenza virus. The traditional Chinese medicine compound comprises traditional Chinese medicine species and content, and is prepared from the following drugs in parts by weight: 20-30 parts of ephedra, 10-15 parts of bitter apricot kernel, 10-15 parts of fried licorice root, 15-25 parts of gypsum and 10-15 parts of fructus forsythia. In vivo and in vitro experiments show that the traditional Chinese medicine compound preparation disclosed by the invention can effectively resist the swine influenza virus.
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 of the Shi You orthomyxovirus A of section type Influenza Virus, SIV) a kind of acute, hot, the height contact respiratory infectious disease that cause, clinical take burst, high heat, cough, dyspnea, repeatedly show effect, sickness rate is high, mortality rate is low is 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 with subclinical form, exist 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 the medicines such as a large amount of antipyretic-antalgics, glucocorticoids simultaneously.Yet when suppressing body inflammatory reaction, also deepened immunosuppressant, caused 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 effective anti-swine flu virus.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 ~ 30 weight portions, Semen Armeniacae Amarum 10 ~ 15 weight portions, Radix Glycyrrhizae Preparata 10-15 weight portion, Gypsum Fibrosum 15 ~ 25 weight portions, Fructus Forsythiae 10 ~ 15 weight portions.
Chinese medicine compound as more specifically implementing, can consist of the Chinese medicine of following weight portion: comprise following medicine: Herba Ephedrae 30 weight portions, Semen Armeniacae Amarum 15 weight portions, Radix Glycyrrhizae Preparata 15 weight portions, Gypsum Fibrosum 25 weight portions, Fructus Forsythiae 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, Semen Armeniacae Amarum 15 weight portions, Radix Glycyrrhizae Preparata 15 weight portions, Gypsum Fibrosum 25 weight portions and Fructus Forsythiae 15 weight ratios, take each Chinese medicine, form 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 standby 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 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;
The Gypsum Fibrosum that the present invention adopts: cold in nature, sweet in the mouth is pungent, enters lung, stomach warp.Having heat clearing away removing water retention by purgation, the relieving restlessness effect such as quench the thirst, is one of modal mineral Chinese medicine.
The Fructus Forsythiae that the present invention adopts: cold in nature, acrid in the mouth is bitter, enters lung, the heart, gallbladder meridian.There is heat-clearing and toxic substances removing, mass dissipating and swelling eliminating, the effect of dispelling wind and heat pathogens, is clinical conventional traditional heat-clearing and detoxifying herb.Fructus Forsythiae has blocking-up and direct killing action to H1N1 hypotype swine flue, and H3N2 hypotype swine influenza virus is had to blocking effect.
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 a Chinese medicine compound for anti-swine flu virus, comprise following medicine: Herba Ephedrae 20 ~ 30 weight portions, Semen Armeniacae Amarum 10 ~ 15 weight portions, Radix Glycyrrhizae Preparata 10-15 weight portion, Gypsum Fibrosum 15 ~ 25 weight portions, Fructus Forsythiae 10 ~ 15 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, with sterilizing PBS, be made into 1% suspension, 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, Semen Armeniacae Amarum 15 weight portions, Radix Glycyrrhizae Preparata 15 weight portions, Gypsum Fibrosum 25 weight portions and Fructus Forsythiae 15 weight ratios, take each Chinese medicine, form 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 standby 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 compound Chinese medicinal preparation to the maximum safe concentration of 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, the i.e. 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, by SPSS17.0 software statistics, analyze.
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; Compare 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 demonstration Chinese medicine compound is compared with virus control group, and difference is extremely remarkable, has obvious anti-swine flu virus effect.
The preventive and therapeutic effect of embodiment 2 Chinese medicine compound to swine influenza virus infecting mouse
1. Chinese medicine compound is to the dead protective effect of swine influenza virus infecting mouse and life-saving effect
(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 organizes mice 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
By mice, with after etherization, except Normal group, each is organized 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:
Table 2 compound Chinese medicinal preparation is to the dead protective effect of swine influenza virus infecting mouse and life-saving effect
Note: above data are compared with model control group, * represents P ﹤ 0.01, 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, * represents P ﹤ 0.01, difference is extremely remarkable.
3. the impact of Chinese medicine compound on swine influenza virus infecting mouse cytokine
(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, as occurred, precipitate, answer recentrifuge.
(3) adopt ELISA test kit to detect
1. the dilution of standard substance and application of sample: on the coated plate of enzyme mark, establish 10 holes, standard substance hole, in first, second hole, add respectively standard substance 100 μ l, then in first, second hole, add standard substance diluent 50 μ l, 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, in the 90 hole, add respectively standard substance diluent 50 μ l 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).Application of sample is added on bottom, ELISA Plate hole by sample, does not touch hole wall as far as possible, rocks and mixes gently.
3. incubation: with rearmounted 37 ℃ of incubations of shrouding film shrouding 30 minutes.
4. dosing: by 20 times of 30(48T) times concentrated cleaning solution is with 20 times of distilled water 30(48T) doubly dilution is rear standby.
5. washing: carefully take shrouding film off, discard liquid, dry, cleaning mixture is filled it up with in every hole, discards after standing 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 with interior for 15 minutes after adding stop buffer.
(4) statistical analysis
By SPSS17.0 software statistics, analyze, refer to table 4:
The impact of table 4 compound Chinese medicinal preparation on swine influenza virus infecting mouse cytokine
Note: above data are compared with model control group, * 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, significant difference.
Claims (5)
1. for a Chinese medicine compound for anti-swine flu virus, comprise following medicine: Herba Ephedrae 20 ~ 30 weight portions, Semen Armeniacae Amarum 10 ~ 15 weight portions, Radix Glycyrrhizae Preparata 10-15 weight portion, Gypsum Fibrosum 15 ~ 25 weight portions, Fructus Forsythiae 10 ~ 15 weight portions.
2. Chinese medicine compound as claimed in claim 1, is characterized in that, comprises following medicine: Herba Ephedrae 30 weight portions, Semen Armeniacae Amarum 15 weight portions, Radix Glycyrrhizae Preparata 15 weight portions, Gypsum Fibrosum 25 weight portions, Fructus Forsythiae 15 weight portions.
3. a kind of Chinese medicine compound for anti-swine flu virus as described in claim 1 ~ 2, is characterized in that, described compound recipe refers to contained Chinese medicine kind and Chinese medicine proportion thereof.
4. a kind of Chinese medicine compound for anti-swine flu virus 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. for an extracting method for the Chinese medicine compound of anti-swine flu virus, it is characterized in that, adopt water decocting method slightly to carry, concrete steps are:
(1) by Herba Ephedrae 30 weight portions, Semen Armeniacae Amarum 15 weight portions, Radix Glycyrrhizae Preparata 15 weight portions, Gypsum Fibrosum 25 weight portions and Fructus Forsythiae 15 weight ratios, take each Chinese medicine, form 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 standby in 4 ℃ of refrigerators being saved to temperature.
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Cited By (4)
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| 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 |
| CN113209189A (en) * | 2021-06-17 | 2021-08-06 | 河北正农牧业有限公司 | Traditional Chinese medicine preparation for preventing porcine respiratory diseases and preparation method thereof |
| CN115192678A (en) * | 2021-09-16 | 2022-10-18 | 广州智园生物科技有限公司 | Natural antiviral atomization composition and preparation method thereof |
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Cited By (4)
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| 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 |
| CN113209189A (en) * | 2021-06-17 | 2021-08-06 | 河北正农牧业有限公司 | Traditional Chinese medicine preparation for preventing porcine respiratory diseases and preparation method thereof |
| CN115192678A (en) * | 2021-09-16 | 2022-10-18 | 广州智园生物科技有限公司 | Natural antiviral atomization composition and preparation method thereof |
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Application publication date: 20140319 |