CN101991680A - Application of traditional Chinese medicinal composition in preparing medicament for resisting influenza viruses - Google Patents
Application of traditional Chinese medicinal composition in preparing medicament for resisting influenza viruses Download PDFInfo
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- CN101991680A CN101991680A CN2009101684430A CN200910168443A CN101991680A CN 101991680 A CN101991680 A CN 101991680A CN 2009101684430 A CN2009101684430 A CN 2009101684430A CN 200910168443 A CN200910168443 A CN 200910168443A CN 101991680 A CN101991680 A CN 101991680A
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Abstract
The invention relates to application of a traditional Chinese medicinal preparation, in particular to the application of a traditional Chinese medicinal composition prepared from sweet wormwood herb, cape jasmine fruit and honeysuckle in preparing a medicament for resisting influenza viruses, and more particular to the application of the traditional Chinese medicinal preparation in preparing a medicament for resisting H5N1 subtype avian influenza viruses and a medicament for resisting H1N1 influenza A viruses.
Description
Technical field:
The present invention relates to a kind of purposes of Chinese medicine pharmaceutical preparation, be specifically related to the application of a kind of Chinese medicine composition in the preparation anti-influenza virus medicament.
Technical background:
Influenza is that a kind of infectiousness is strong, the disease that spread speed is fast.Caused complication of influenza and death are very serious.According to the bulletin of World Health Organization (WHO) issue, global annual influenza case is 600,000,000～1,200,000,000 examples, dead 500,000～1,000,000 people, and visible influenza is very harmful to human beings'health.
Bird flu (avian influenza, AI) be by bird flu virus (avian influenzavirus, AIV) birds that cause, birds deadly infectious disease are made of two kinds of different glycoproteins, and a kind of is hemagglutinin (hemagglutinin, H or Hp), another kind is neuraminic acid (neuraminidase, N or Np), and H has 1～15 hypotype, N has 1～9 hypotype, and H and N combination different have formed dissimilar virus.Wherein the H5N1 type is accredited as the pathogenic influenza virus of main height at the popular bird flu in Asia in 2004.
The drug main of influenza in the market will be based on Western medicine, but Western medicine has toxic and side effects in various degree usually, is subjected to certain limitation in clinical practice.And tcm clinical is prevented and treated influenza and is had effect unique, and toxic and side effects is little, and the medicine source is abundant, cheap, suppress virus replication by regulating whole immunologic function, stop pathological changes caused by virus, it is fast etc. to improve clinical symptoms, is demonstrating special advantages aspect the treatment influenza.
The crude drug of the Chinese medicine composition that the present invention relates to consists of: Herba Artemisiae Annuae 6～25 weight portions, Flos Lonicerae 3～15 weight portions, Fructus Gardeniae 3～12 weight portions.This Chinese medicine composition function cures mainly and is heat clearing away, dispelling wind, and detoxifcation is used for diseases such as hyperpyrexia, micro evil wind due to the upper respiratory tract infection (external wind heat syndrome) is cold, a general pain, cough, expectorant Huang.Relate to contents such as composition, preparation method and the dosage form of this Chinese medicine composition and method of quality control at Chinese invention patent 02114995.X, 200410000134.X has comparatively detailed description in 200510134299.0.This medicine has obtained State Food and Drug Administration's approval list marketing, and the adopted name of medicine is the pyretic toxicity injection for curing, and the approval number of the drug is the accurate word Z20050217 of traditional Chinese medicines.
The inventor is surprised to find that in clinical practice this Chinese medicine composition has very good resisiting influenza virus effect, and the inventor has carried out pharmacodynamic study to this medicine, proves its determined curative effect.
Summary of the invention:
The invention provides the application of a kind of Chinese medicine composition of forming by Herba Artemisiae Annuae, Fructus Gardeniae, Flos Lonicerae (being named as " pyretic toxicity injection for curing " in the present invention) in the preparation anti-influenza virus medicament.
Described pyretic toxicity injection for curing is mainly used in the application in anti-H5N1 subtype avian influenza virus medicine of preparation and the anti-H1N1 influenza A virus medicament.
In order to understand the present invention better, the inventor provides following research data, and technique effect of the present invention is described, is not limited to the present invention but following experiment is used to further specify.
Experiment one: the pyretic toxicity injection for curing is to the inhibiting experimentation of bird flu virus
1. experiment material
1.1 be subjected to the reagent thing: the pyretic toxicity injection for curing, produce by Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov
1.2 Strain: the H5N1 subtype avian influenza virus is provided by the Ministry of Agriculture of Harbin Veterinary Medicine Inst., China Academy of Agriculture animal influenza emphasis open laboratory.
1.3 Embryo Gallus domesticus: 10 age in days SPF Embryo Gallus domesticus are provided by the Chinese Academy of Agricultural Sciences's Harbin veterinary institute Experimental Animal Center.
2. experimental technique
2.1 viral EID
50Mensuration
With 10 times of serial dilutions of H5 subtype avian influenza virus, inoculate 10 age in days SPF Embryo Gallus domesticus, 5 pieces of Embryo Gallus domesticus of each dilution factor (0.1ml/ embryo) are measured viral 50 3nfective dose (EID
50).
2.2 medicine is to the maximal non-toxic dosage of Embryo Gallus domesticus
Pyretic toxicity injection for curing stock solution, 1: 2,1: 4,1: 8,1: 16,1: 32,1: 64 normal saline diluent are inoculated 10 age in days SPF Embryo Gallus domesticus respectively, each dilution factor inoculation 5 pieces of Embryo Gallus domesticus (0.1ml/ embryo).The Embryo Gallus domesticus of inoculation put in 37 ℃ of incubators cultivated 96 hours.Embryo Gallus domesticus with interior death removed in 24 hours, record chicken embryo death situation.
Get 10 respectively
8.25EID
50H5N1 subtype avian influenza virus suspension mixes by 1: 9 with pyretic toxicity injection for curing stock solution, 1: 5,1: 10,1: 20 normal saline diluent, and after acting on 10 minutes and 30 minutes under 20 ± 1 ℃ of conditions, the reuse sterile saline is done 10 times of dilutions of going forward one by one.Matched group replaces the pyretic toxicity injection for curing with sterile saline, handles with method; Diluent is inoculated 10 age in days SPF Embryo Gallus domesticus, each dilution factor inoculation 5 pieces of Embryo Gallus domesticus (0.1ml/ embryo).The Embryo Gallus domesticus of inoculation is put in 37 ℃ of incubators and cultivated, record chicken embryo death situation.Embryo Gallus domesticus with interior death removed in 24 hours, and later Embryo Gallus domesticus in time took out in 24 hours, all took out to 96 hours, got allantoic fluid one by one and did blood clotting (HA) test, and hemagglutination test positive person is judged to Embryo Gallus domesticus and infects.
According to the result that Embryo Gallus domesticus infects, the positive rate, the sample that calculate test group and the infection of matched group Embryo Gallus domesticus by following equation contain EID
50The logarithm and the suppression ratio of amount.
Sample contains EID
50Logarithm=the L-d (S-0.5) of amount
(L is the logarithm of minimum extension rate; D is logarithmic poor between dilution factor; S is each dilution row positive rate sum)
3. experimental result
3.1 medicine is to the maximal non-toxic dosage of Embryo Gallus domesticus
Pyretic toxicity injection for curing stock solution, all none death of normal saline diluent group Embryo Gallus domesticus in 1: 2,1: 4,1: 8,1: 16,1: 32,1: 64.The results are shown in Table 1.
Table 1 pyretic toxicity injection for curing is to the maximal non-toxic dosage of Embryo Gallus domesticus
3.2 medicine is tested viral inhibition
Pyretic toxicity injection for curing stock solution, 1: 5,1: 10,1: 20 normal saline diluent and 10
8.25EID
50H5N1 subtype avian influenza virus suspension effect 10 minutes, viral suppression ratio is respectively 99.82%, 43.76%, 0,0; With 10
8.25EID
50H5N1 subtype avian influenza virus suspension effect 30 minutes, viral suppression ratio is respectively 100%, 99.94%, 32.39%, 0.The results are shown in Table 2.
Table 2 variable concentrations pyretic toxicity injection for curing is to the inhibitory action (n=5) of N1 subtype avian influenza virus
4. conclusion
The ultimate principle of Chinese herbal medicine resisiting influenza virus is strengthening vital QI to eliminate pathogenic factors or gets rid of evils and set upright.Because every flavor Chinese medicine constituent more complicated interacts between each component, and it is more complicated to form its mechanism of action of Chinese medicinal formulae.At present, the antiviral approach of Chinese medicine mainly contains 2: (1)) directly suppress viral.Mainly contain the blocking virus reproductive process absorption, penetrate, duplicate, a certain link in the maturation, thereby reach the purpose of viral infection resisting; (2) suppress virus indirectly.Because behind the viral infection human body, must colonize in the cell of human body and could survive, breed, some drugs is in the antiviral while, can also improve body's immunological function, reach the purpose that suppresses virus by specificity and the non-specific immunity that promotes body, or by promoting inducement interferon effect etc. to reach antiviral purpose.
The pyretic toxicity injection for curing is pure Chinese medicine injection, forms by Herba Artemisiae Annuae, Flos Lonicerae etc., and the tool wind-dispelling heat-dissipating, the effect of heat-clearing and toxic substances removing is mainly used in the hyperpyrexia that respiratory tract infection is drawn.This test finds that the pyretic toxicity injection for curing can suppress the propagation of bird flu virus on Embryo Gallus domesticus external, thereby has guaranteed the survival of Embryo Gallus domesticus, has the effect of certain anti-avian influenza virus.
Experiment two: the pyretic toxicity injection for curing is to influenza virus inhibitory action experimentation
1. experiment material
1.1 cell and virus
Mdck cell (Madin-Darby canine kidney(cell line)), influenza virus A/PR/8/34 (H1N1) (influenza A virus) provides by Beijing Jindike Biological Technology Inst. of State Engineering Research Center of Virobiological Technology.
1.2 medicine and reagent
(1) pyretic toxicity injection for curing, Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov;
(2) ribavirin injection, Tianjin Pharmaceutical Group Xinzheng Co., Ltd.; Specification: 1 milliliter: 10 milligrams * 10;
(3) oseltamivir phosphate capsule, Shanghai Roche Group company limited, specification: 75mg;
(4) DMEM culture medium, the Beijing Qingdatianyi Bioisystech Co., Ltd;
(5) calf serum, Hangzhou Ilex purpurea Hassk.[I.chinensis Sims pharmaceutical agent company limited;
(6) pancreatin, tetramethyl azo azoles orchid (MTT) are all available from Amresco company;
(7) DMSO is available from Sigma company.
1.3 animal subject
BALB/c mouse, the cleaning level, body weight: 14～16g, available from Beijing Vital River Experimental Animals Technology Co., Ltd., the certification of fitness: SCXK (capital) 2007-0001.
1.4 instrument
(1) 96 porocyte culture plate, Corning company; Superclean bench, Beijing Rui Zekang bio tech ltd;
(2) CO
2Constant incubator, Japanese SANYO company;
(3) inverted microscope, Japanese OLYMPUS company;
(4) microplate reader, Bio Rad Laboratories.
2. experimental technique
2.1 medicine pair cell toxicity test
2.1.1 cytopathy is observed
Every hole inoculates about 5 * 10 in the 96 porocyte plates
4Individual mdck cell, in 37 ℃, 5%CO
2Overnight incubation in the incubator discards cell culture fluid, with pyretic toxicity injection for curing (20ul, concentration 2.6mg/ml) the liquid of keeping is replaced culture fluid, set up the negative control of virazole (concentration 40mg/ml) and no medicine simultaneously, continue to cultivate observation of cell form under the optical microscope 5 days.
2.1.2 cell proliferation experiment (mtt assay)
Under above-mentioned condition of culture, in the 5th day every hole, add 10 μ L 5mg/mL MTT solution, continue to cultivate 4h, add 100 μ L DMSO after discarding culture fluid, 37 ℃ of vibration 10min measure OD
570nmValue.Each concentration sets up 4 holes parallel, gets 4 optical density meansigma methodss and calculates, the toxicity of evaluation pyretic toxicity injection for curing pair cell.
2.2 extracorporeal antivirus effect experiment
2.2.1 the antiviral pathological changes is observed
Add influenza virus (A/PR/8/34) 5TCID50 on the mdck cell of in 24 orifice plates, cultivating, at 33 ℃ of 5%CO
2Hatched in the incubator 1 hour, Hanks liquid flush away virus liquid adds cell maintenance medium (pyretic toxicity injection for curing: 2.6mg/ml, 100ul; Virazole: 40 μ g/ml, 100ul), establish virus control, cell contrast simultaneously, put in 33 ℃ of 5%CO2 incubators and cultivate, observed continuously 5 days.
2.2.2 forming, plaque suppresses experiment
The monolayer mdck cell that has just covered with in six orifice plates discards growth-promoting media, and with Hanks liquid washing twice, according to the preliminary experiment plaque titration results of influenza virus A/PR/8/34, every hole adds 10
-6The influenza virus 0.5ml of dilution doubly, in 33 ℃, 5%CO
2Hatched in the incubator 1 hour, flush away virus liquid, add an amount of virazole (final concentration 40 μ g/ml, 20 μ g/ml, 10 μ g/ml, 5 μ g/ml, 2.5 μ g/ml, one hole is a blank), pyretic toxicity injection for curing (final concentration 260 μ g/ml, 130 μ g/ml, 65 μ g/ml, 32.5 μ g/ml, 16.25 μ g/ml, a hole is a blank), the DMEM agarose ground floor covering liquid that adds the pancreatin contain 1% agarose, 5ug/ml TPCK and to handle, 2.5%Herps buffer, pH7.2-7.4 covers cell, continuation is at 33 ℃, 5%CO
2Hatch in the incubator.Cultivate after 72 hours, add the dyeing of spending the night of the second layer covering liquid contain 3.33% dimethyl diaminophenazine chloride, the direct observation plaque.Calculate the suppression ratio of each concentration according to the plaque number, calculate its IC50 with respect to negative control.Form the active positive reference of inhibition with virazole as plaque.
2.3 interior resisting virus experiment
At first do preliminary experiment and measure the LD50 of influenza virus (A/PR/8/34).80 mices are divided into 4 groups at random, 20 every group.Be respectively: (1) normal control group: irritate stomach and give distilled water, (2) model group: irritate stomach and give distilled water, (3) positive group: irritate stomach and give oseltamivir phosphate capsule aqueous solution (20mg/kg), (4) pyretic toxicity injection for curing: irritate stomach and give pyretic toxicity injection for curing (6.76g crude drug/kg); Irritate the long-pending 10ml/kg of being of body of stomach.Respectively at successive administration behind 4h before the counteracting toxic substances and the counteracting toxic substances 5 days, every day 1 time.Behind the etherization, 10LD
50Dosage intranasal inoculation influenza virus 50 μ l.Getting 10 for every group observed 14 days continuously, record dead mouse situation (calculating mortality rate and mean survival time), other 10 respectively at mice being put to death in 4,8 days after the first administration, the aseptic lung of getting, measure Mus lung weight in wet base, the Mus lung is measured TCID50 after grinding to form the lung suspension, makees tissue slice after the Mus lung meridian formaldehyde fixed, the lung tissue morphological change is observed in HE dyeing, light microscopic down.Result's SPSS statistics software processes.
3. experimental result
3.1 medicine pair cell toxicity test
3.1.1 cytopathy is observed
The microscope observing cell pathological changes, the result shows that under this experiment condition, during pyretic toxicity injection for curing 20ul, cell state is consistent with negative control group, on cell proliferation is influence not.
3.1.2 cell proliferation experiment (mtt assay)
The microscope observing cell pathological changes, the result shows that under this experiment condition, during pyretic toxicity injection for curing 20ul, cell state is consistent with negative control group, does not have cytotoxicity.
3.2 extracorporeal antivirus effect experiment
3.2.1 the antiviral pathological changes is observed
The results are shown in Table 3, the result shows that the pyretic toxicity injection for curing is at external cytopathic effect with certain inhibition influenza virus.
Table 3 pyretic toxicity injection for curing is to the influence of pathological changes caused by virus effect
Annotate :-acellular pathological changes; ± delay cytopathy; + 1/4 following cytopathy; ++ the cell of 1/4-1/2 has pathological changes; ++ the cell of+1/2-3/4 has pathological changes; ++ ++ the cell more than 3/4 has pathological changes.
3.2.2 forming, plaque suppresses experiment
The results are shown in Table 4, the result shows, under this experiment condition, and the IC of pyretic toxicity injection for curing
50Be 53.0mg/ml, illustrate external and can suppress the influenza virus growth to have certain resisiting influenza virus effect.
Table 4 pyretic toxicity injection for curing forms the inhibition experiment to the plaque of influenza virus
3.3 interior resisting virus experiment
3.3.1 influence to mice survival rate and mean survival time
The 5th day beginning of model group behind counteracting toxic substances is dead, is 0 to the 10th day survival rate, and the mean survival time is 7.1 days; The beginning in behind counteracting toxic substances the 6th day of pyretic toxicity injection for curing group was dead, dropped to minimumly to the 9th day survival rate, and survival rate is 50%, relatively has significant difference (P＜0.01) with model group, and the mean survival time is 10.6 days; Positive group and normal control group survival rate are 100%, and the mean survival time relatively had significant difference (P＜0.01) greater than 14 days with model group.The results are shown in Table 5.
Table 5 pyretic toxicity injection for curing is to the influence (n=10) of mice survival rate and mean survival time
Compare with model group: * * P＜0.01.
3.3.2 pathological changes situation in the mouse lung of contamination back
The results are shown in Table 6, experimental result shows, compares with model group, and the mouse lung weight in wet base of pyretic toxicity injection for curing group and Pneumovirinae titre be significantly decline (P＜0.05) all.
Table 6 pyretic toxicity injection for curing is to the protective effect of 10LD50 influenza virus contamination mice
Compare with model group: * P＜0.05, * * P＜0.01.
4. conclusion
This result of the test shows that the pyretic toxicity injection for curing is under finite concentration, without any cytotoxicity; The pyretic toxicity injection for curing can suppress the influenza virus growth external, has certain resisiting influenza virus effect; In mice counteracting toxic substances protection experiment, show that by a plurality of parameter evaluations such as survival rate, mean survival time, Mus pulmonary consolidation, Mus lung inner virus TCID50 titre and the section of Mus lung pathology the pyretic toxicity injection for curing has certain anti-influenza virus activity.
Can prove that by above two experiments the pyretic toxicity injection for curing has the effect of resisiting influenza virus, wherein have the very strong anti-H5N1 subtype avian influenza virus and the effect of anti-H1N1 influenza A virus.
Description of drawings
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The specific embodiment:
[nomenclature of drug] pyretic toxicity injection for curing
[composition] Herba Artemisiae Annuae, Flos Lonicerae, Fructus Gardeniae.
[function cures mainly] heat clearing away, dispelling wind, detoxifcation.Be used for diseases such as hyperpyrexia, micro evil wind due to the upper respiratory tract infection (external wind heat syndrome) is cold, a general pain, cough, expectorant Huang, and H5N1 subtype avian influenza, influenza A H1N1.
[usage and dosage] intravenous drip.A 20ml (2) instils with 5% glucose injection or 0.9% normal saline solution 250ml dilution posterior vein, drip speed for 30-60 drip/minute, 1 time/day, 3 days courses of treatment.Or follow the doctor's advice.
Claims (3)
1. a Chinese medicine composition of being made up of Herba Artemisiae Annuae, Fructus Gardeniae, Flos Lonicerae is characterized in that the application of this Chinese medicine composition in the preparation anti-influenza virus medicament.
2. anti-influenza virus medicament according to claim 1 is characterized in that the application of this medicine in the anti-H5N1 subtype avian influenza virus medicine of preparation.
3. anti-influenza virus medicament according to claim 1 is characterized in that the application of this medicine in the anti-H1N1 influenza A virus medicament of preparation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910168443A CN101991680B (en) | 2009-08-20 | 2009-08-20 | Application of traditional Chinese medicinal composition in preparing medicament for resisting influenza viruses |
| HK11104042.7A HK1150340A1 (en) | 2009-08-20 | 2011-04-21 | The use of a traditional chinese medicine composition in the preparation of anti-influenza virus drugs |
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| CN200910168443A CN101991680B (en) | 2009-08-20 | 2009-08-20 | Application of traditional Chinese medicinal composition in preparing medicament for resisting influenza viruses |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102233021A (en) * | 2010-04-30 | 2011-11-09 | 江苏康缘药业股份有限公司 | Content measurement method for Chinese patent medicine prepared from sweet wormwood, honeysuckle and gardenia jasminoides fruit |
| CN102961467A (en) * | 2012-12-06 | 2013-03-13 | 河北神威药业有限公司 | Medicine composition capable of clearing heat and removing toxicity and preparation method of stock solution thereof |
| CN111643649A (en) * | 2020-05-20 | 2020-09-11 | 博奥生物集团有限公司 | Chinese medicinal materials for preventing and treating immune related diseases and composition thereof |
| CN113288943A (en) * | 2020-02-21 | 2021-08-24 | 江苏康缘药业股份有限公司 | Application of traditional Chinese medicine composition in preparation of medicine for treating or preventing coronavirus infection |
| WO2022022020A1 (en) * | 2020-07-30 | 2022-02-03 | 江苏康缘药业股份有限公司 | Traditional chinese medicine composition, preparation method and use |
| CN114224940A (en) * | 2021-12-31 | 2022-03-25 | 常德集智生物科技有限公司 | Toxin dissolving bionic enzyme for inactivating influenza virus and preparation method thereof |
-
2009
- 2009-08-20 CN CN200910168443A patent/CN101991680B/en active Active
-
2011
- 2011-04-21 HK HK11104042.7A patent/HK1150340A1/en unknown
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102233021A (en) * | 2010-04-30 | 2011-11-09 | 江苏康缘药业股份有限公司 | Content measurement method for Chinese patent medicine prepared from sweet wormwood, honeysuckle and gardenia jasminoides fruit |
| CN102233021B (en) * | 2010-04-30 | 2013-05-01 | 江苏康缘药业股份有限公司 | Content measurement method for Chinese patent medicine prepared from sweet wormwood, honeysuckle and gardenia jasminoides fruit |
| CN102961467A (en) * | 2012-12-06 | 2013-03-13 | 河北神威药业有限公司 | Medicine composition capable of clearing heat and removing toxicity and preparation method of stock solution thereof |
| CN102961467B (en) * | 2012-12-06 | 2014-08-06 | 河北神威药业有限公司 | Medicine composition capable of clearing heat and removing toxicity and preparation method of stock solution thereof |
| CN113288943A (en) * | 2020-02-21 | 2021-08-24 | 江苏康缘药业股份有限公司 | Application of traditional Chinese medicine composition in preparation of medicine for treating or preventing coronavirus infection |
| WO2021164401A1 (en) * | 2020-02-21 | 2021-08-26 | 江苏康缘药业股份有限公司 | Use of traditional chinese medicine composition in preparation of drugs for treating or preventing coronavirus infection |
| CN111643649A (en) * | 2020-05-20 | 2020-09-11 | 博奥生物集团有限公司 | Chinese medicinal materials for preventing and treating immune related diseases and composition thereof |
| WO2022022020A1 (en) * | 2020-07-30 | 2022-02-03 | 江苏康缘药业股份有限公司 | Traditional chinese medicine composition, preparation method and use |
| CN114224940A (en) * | 2021-12-31 | 2022-03-25 | 常德集智生物科技有限公司 | Toxin dissolving bionic enzyme for inactivating influenza virus and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101991680B (en) | 2012-09-05 |
| HK1150340A1 (en) | 2011-12-09 |
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