CN105832755B - Hyperoside and Oseltamivir combine the application in preventing H7N9 type flu pharmaceuticals - Google Patents
Hyperoside and Oseltamivir combine the application in preventing H7N9 type flu pharmaceuticals Download PDFInfo
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- CN105832755B CN105832755B CN201610348142.6A CN201610348142A CN105832755B CN 105832755 B CN105832755 B CN 105832755B CN 201610348142 A CN201610348142 A CN 201610348142A CN 105832755 B CN105832755 B CN 105832755B
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
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Abstract
Hyperoside and Oseltamivir combine the application in preventing H7N9 type flu pharmaceuticals, belong to application in TCM field, the present invention is implemented by the following technical programs：H7N9 wild types or saltant type influenza neuraminidase liquid and Hyperoside and Oseltamivir mixed liquor is obtained first to be incubated 30 minutes, substrate is added to be detected, the experimental results showed that two kinds of Drug combinations are to H7N9 wild types and saltant type influenza neuraminidase is active inhibiting effect, the anti influenza that Hyperoside is used in combination on a cellular level with Oseltamivir is then had detected to act on, it was found that the two, which is used in combination, to play therapeutic effect to the cell of infection H7N9 wild types and saltant type influenza virus, finally, both have detected the influence being used in combination to mouse influenza models, it was found that after Hyperoside and Oseltamivir mixture is added, the mitigation of influenza mouse weight can be relieved, mouse is set to be gradually restored to the general level of the health, and it can significantly reduce the Lung Exponent of influenza mouse.
Description
Technical field
The invention belongs to application in TCM fields, and in particular to Hyperoside and Oseltamivir are combined in prevention H7N9 type influenzas
Application in virus drugs.
Background technology
Influenza (hereinafter referred to as influenza) is a kind of acute viral caused by influenza virus by orthomyxoviridae family
Respiratory infectious disease.Its infectiousness is strong, prevalence is wide, incidence is high.Worldwide, the annual prevalence of influenza causes about 300
Ten thousand to 5,000,000 people's illness are seriously and about 250,000 to 500,000 people are dead.It is also easiest to cause generation currently, influenza A virus is most common
It is very popular and area prevalence on boundary.Now, only two class antiviral drugs have been approved by the fda in the United States for influenza prevention and have controlled
It treats, is M2 inhibitors of ion channels and neuraminic acid enzyme inhibitor respectively.However, according to U.S.'s Disease Control and Prevention Center report
Road, 99.6% seasonal H1N1 viruses there is drug resistance, 100% seasonal H3N2 viruses to have to adamantane Oseltamivir
Drug resistance.
2013, novel type A avian influenza virus --- the H7N9 that can infect the mankind, neural ammonia have been broken out in China
Sour enzyme mutant type H7N9 mutant virus strain is more than 10000 times of wild-type strain to the tolerance of Oseltamivir.Drug resistance
The clinical effectiveness for generally occurring reducing Tamiflu to treatment of influenza of strain.In view of to neuraminidase inhibitor class medicine
There is object the side effect of the appearance and Tamiflu of drug-resistant viral, many scientists to be dedicated to a variety of antiviral drugs groups
Close use, to can reduce the usage amount of Tamiflu and improve treatment influenza effect.
In China, the antiviral history for having thousands of years of traditional Chinese medicine is had accumulated big in terms of using Chinese herb prevention influenza
Measure valuable theory and practice experience.Natural resources of Chinese medicinal materials is very abundant, uses Chinese medicine influenza significant in efficacy from ancient times to the present.Spun gold
Peach glycosides is the reactive compound isolated from azalea, research shows that Hyperoside has the works such as anti-oxidant, anticancer, hypoglycemic
With.Recently, also some researches show that it anti influenza.Present invention research finds Hyperoside and Tamiflu Ao Sita
Wei is used in combination plays synergy to the neuraminidase inhibition of H7N9 wild types and H7N9 saltant types, and can reduce drug
Usage amount.
Invention content
The purpose of the present invention is to provide Hyperosides and Oseltamivir to be used in combination in preventing H7N9 type flu pharmaceuticals
Application.Hyperoside is used in combination with carboxylic acid oseltamivir can effectively inhibit H7N9 wild types and saltant type influenza virus
The activity of neuraminidase to inhibit the breeding of influenza virus in vivo, and then mitigates or cures flu victims.The present invention couple
Hyperoside and carboxylic acid oseltamivir have carried out external zymetology inhibition to H7N9 wild types and saltant type neuraminidase
Detection finds half-inhibition concentration (IC50) difference of the carboxylic acid oseltamivir to H7N9 wild types and saltant type neuraminidase
For 1.75nmol/L and 26.52 μm of ol/L；Hyperoside distinguishes the IC50 of H7N9 wild types and saltant type neuraminidase
For 117.4 μm/L and 127.7 μm ol/L.It has then carried out Hyperoside and external zymetology inhibition is used in combination with carboxylic acid Oseltamivir
The detection of property, the function and effect of drug combination are indicated with association index value：Association index value shows that two kinds medication combined make less than 1
It is acted synergistically with playing.The experimental results showed that two kinds of drugs play synergistic effect to the inhibition of H7N9 wild type neuraminidases,
And it is more obvious to the synergy of H7N9 saltant type neuraminidases inhibition.We are again to both H7N9 influenza infections
Mdck cell is simultaneously added Hyperoside and carboxylic acid Oseltamivir mixture and has carried out the measurement of cell survival rate, finds Hyperoside
The survival rate of H7N9 cells following viral infections can be improved with carboxylic acid Oseltamivir mixture.Finally, we to Hyperoside with
Oseltamivir mixture carries out the changes of weight of mouse, lethality and Lung Exponent H7N9 wild types and saltant type influenza virus
It measures, it is found that Hyperoside and Oseltamivir mixture can be relieved symptom of the H7N9 types influenza virus to mouse.This hair
It is bright to provide basis by the new drug of target spot of neuraminidase for exploitation, it is used in combination applied to people with Oseltamivir for Hyperoside
The treatment of parainfluenza provides reference.
Description of the drawings
Fig. 1 is that various concentration Hyperoside is used in combination with carboxylic acid Oseltamivir to H7N9 wild type influenza virus nerve ammonia
The inhibiting effect of sour enzyme.
Fig. 2 is that various concentration Hyperoside is used in combination with carboxylic acid Oseltamivir to H7N9 saltant type influenza virus nerve ammonia
The inhibiting effect of sour enzyme.
Fig. 3 is that various concentration Hyperoside is used in combination with carboxylic acid Oseltamivir to infecting H7N9 wild type influenza virus
The therapeutic effect of mdck cell.
Fig. 4 is that various concentration Hyperoside is used in combination with carboxylic acid Oseltamivir to infecting H7N9 saltant type influenza viruses
The therapeutic effect of mdck cell.
Fig. 5 is the shadow for the mouse weight that Hyperoside is used in combination with Oseltamivir to infecting H7N9 wild type influenza virus
It rings.
Fig. 6 is the shadow for the mouse weight that Hyperoside is used in combination with Oseltamivir to infecting H7N9 saltant type influenza viruses
It rings.
Specific implementation mode
Below by way of the specific implementation modes of example forms, the present invention is described in further detail.
Embodiment 1
10 μ L Hyperosides of various concentration or carboxylic acid oseltamivir are separately added into H7N9 wild types or the mutation of 40 μ L
In type neuraminidase solution, 30min is placed in 37 DEG C of incubators, and 50 μ L20 μM MU-NANA substrate buffer solutions are then added,
It is detected in microplate reader, excitation wavelength 360nm, launch wavelength 450nm, detection duration 8min.Testing result is：Carboxylic acid
Oseltamivir is respectively 1.75nmol/L and 26.52 μm of ol/L to the IC50 of H7N9 wild types and saltant type neuraminidase；
Hyperoside is respectively 117.4 μm/L and 127.7 μm ol/L to the IC50 of H7N9 wild types and saltant type.
Embodiment 2
According to the Hyperoside of measurement with carboxylic acid Oseltamivir to the IC50 of H7N9 wild types and saltant type neuraminidase
To determine the addition concentration of Hyperoside and carboxylic acid Oseltamivir.Hyperoside and the concentration of carboxylic acid Oseltamivir are selected as respectively
0.25,0.5,1,2 and 4 times of the IC50 concentration relative to H7N9 wild types and saltant type neuraminidase.And it is added
The ratio of the concentration of Hyperoside and the concentration of carboxylic acid Oseltamivir is to remain consistent, is the ratio of its IC50 value.We are first
The H7N9 wild types after the dilution of 30 μ l or saltant type neuraminic acid enzyme solution are first added in 96 hole elisa Plates, then in each hole
The carboxylic acid Oseltamivir of the middle Hyperoside and 10 μ l corresponding concentrations that 10 μ l various concentrations are added, each concentration do 4 repetitions.?
After being incubated 30min at 37 DEG C, the NA specific substrate MU-NANA of 50 μ l20 μM are added, detect H7N9 wild types and saltant type
The fluorescence intensity of neuraminidase.Data are recorded, the association index (CI of both drugs is then calculated with CalcuSyn softwares
Value), it is as a result as follows：
<Note>：ED50, ED75 and ED90 indicate that enzymatic activity is suppressed 50%, 75% and 90% respectively.
CI values are medication combined less than 1 two kinds of explanation to play synergistic effect, and the smaller synergy of CI values is better.By number in table
According to understanding that two kinds of Drug combinations play synergistic effect to the inhibition of H7N9 neuraminidases.
Embodiment 3
In order to detect whether Hyperoside and carboxylic acid Oseltamivir mixture have anti-H7N9 wild types on mdck cell
With the effect of saltant type influenza virus, we have chosen the mdck cell progress bed board in logarithmic phase, per 10000, hole, 37
It is cultivated for 24 hours in the cell incubator of DEG C 5%CO2.Culture for 24 hours afterwards discards DMEM culture mediums, is then cleaned 2~3 times with DMEM.
It is dense with the H7N9 wild types of 100 μ l or saltant type influenza virus allantoic fluid (a concentration of 100 times of TCID50 concentration) and difference again
The lower Hyperoside of degree with mix 2h at 100 μ l37 DEG C of carboxylic acid Oseltamivir mixture, Hyperoside and carboxylic acid Oseltamivir
0.25,0.5,1,2 and 4 times of the IC50 that concentration is selected as respectively relative to H7N9 wild types and saltant type neuraminidase is dense
The ratio of degree, the concentration for the Hyperoside being added and the concentration of carboxylic acid Oseltamivir remains consistent, is the ratio of its IC50 value.
Then mixed liquor is added in mdck cell in cell incubator and is incubated, mixed liquor is sucked out after 2h, 2~3 are cleaned with DMEM
It is secondary, the DMEM culture solutions culture containing 1 μ g/ml pancreatin and 2%FBS is then added for 24 hours.After for 24 hours, trained with same culture solution
It supports 3 days, culture solution is sucked out, cleaned 2~3 times with DEME, 100 μ lDMEM and 15 μ lMTT (5mg/ml) then are being added, gently
Mixing is swung in microseism, and 4h is then stood in cell incubator.Then discard the DMEM containing MTT, with PBS clean to cleaning solution without
Color adds 100 μ lDMSO, slightly shakes mixing.Its ultraviolet absorptivity at 570nm is detected in 30min.Pass through
OD570 calculates the survival rate of cell.
As a result as shown in Figure 3 and Figure 4, under the conditions of different disposal, Hyperoside and carboxylic acid Oseltamivir mixed liquor is added
When with H7N9 wild types or saltant type influenza virus, with the increase of drug concentration, cell survival rate also increases as.Illustrate gold
Silk peach glycosides can inhibit H7N9 wild types and saltant type influenza virus with Oseltamivir mixed liquor, protect mdck cell from virus
It destroys, and dose dependent is presented also with the increase of Hyperoside and carboxylic acid Oseltamivir mixture concentration in protective effect.
Embodiment 4
In order to detect Hyperoside and Oseltamivir mixture to infected H7N9 wild type influenza virus mouse whether
There is therapeutic effect, we have chosen the Female ICR mice 50 of about 20g, are then anaesthetized with ether, then the side for passing through collunarium
The H7N9 wild type influenza virus dilutions of 10 times of LD50 concentration are instilled in mouse nasal cavity and establish mouse influenza models by formula, with drop
Enter the mouse of 50 μ lPBS buffer solutions as a control group.50 mouse are randomly divided into 5 groups by us, this 5 groups are respectively：Control group
(PBS groups)；Viral group (10 times of LD50H7N9 wild type influenza virus)；Hyperoside group (10 times of LD50H7N9 wildtype influenzas
Virus+25mg/kg Hyperosides)；Oseltamivir group (10 times of LD50H7N9 wild type influenza virus+25mg/kg Oseltamivirs)；
Hyperoside and Oseltamivir mixture group (10 times of LD50H7N9 wild type influenza virus+25mg/kg Hyperosides and 25mg/
Kg Oseltamivirs mixture).Start to be administered before 2 days viral to mouse inoculation, 2 times a day, continuous gavage 10 days, every mouse one
Secondary administration 0.2ml.The weight and death condition of every group of mouse are recorded daily.At the 15th day, after the 8h that cuts off the water supply jejunitas to mouse, into
Row dislocation is put to death, and is taken out the lungs of mouse and is weighed.It is calculated according to formula：
The results are shown in Figure 5, can alleviate the mitigation of H7N9 wild type influenza virus infecting mouse weight, improve mouse
Survival condition makes mouse be gradually restored to the general level of the health.
The results are shown in table below, and it is small to illustrate that Hyperoside with Oseltamivir mixed liquor can significantly reduce H7N9 wildtype influenzas
The Lung Exponent of mouse.
Result above illustrates that Hyperoside mixes liquid energy with Oseltamivir and risen to the mouse for infecting H7N9 wildtype influenzas
To the effect for the treatment of, mouse is made to be gradually restored to the general level of the health.
Embodiment 5
In order to detect Hyperoside and Oseltamivir mixture to infected H7N9 saltant type influenza viruses mouse whether
There is therapeutic effect, we have chosen the Female ICR mice 50 of about 20g, are then anaesthetized with ether, then the side for passing through collunarium
The H7N9 saltant type influenza virus dilutions of 10 times of LD50 concentration are instilled in mouse nasal cavity and establish mouse influenza models by formula, with drop
Enter the mouse of 50 μ lPBS buffer solutions as a control group.50 mouse are randomly divided into 5 groups by us, this 5 groups are respectively：Control group
(PBS groups)；Viral group (10 times of LD50H7N9 saltant types influenza viruses)；Hyperoside group (10 times of LD50H7N9 saltant type influenzas
Virus+25mg/kg Hyperosides)；Oseltamivir group (10 times of LD50H7N9 saltant type influenza virus+25mg/kg Oseltamivirs)；
Hyperoside and Oseltamivir mixture group (10 times of LD50H7N9 saltant types influenza virus+25mg/kg Hyperosides and 25mg/
Kg Oseltamivirs mixture).Start to be administered before 2 days viral to mouse inoculation, 2 times a day, continuous gavage 10 days, every mouse one
Secondary administration 0.2ml.The weight and death condition of every group of mouse are recorded daily.At the 15th day, after the 8h that cuts off the water supply jejunitas to mouse, into
Row dislocation is put to death, and is taken out the lungs of mouse and is weighed.It is calculated according to formula shown in embodiment 4.
Shown in result figure 6, the mitigation of H7N9 saltant type influenza infection mouse weights can be alleviated, improve the life of mouse
State is deposited, mouse is made to be gradually restored to the general level of the health.
The results are shown in table below, and it is small to illustrate that Hyperoside with Oseltamivir mixed liquor can significantly reduce H7N9 saltant type influenzas
The Lung Exponent of mouse.
Result above illustrates that Hyperoside mixes liquid energy with Oseltamivir and risen to the mouse for infecting H7N9 saltant type influenzas
To the effect for the treatment of, mouse is made to be gradually restored to the general level of the health.
Claims (3)
1. Hyperoside and Oseltamivir, which are combined, is preparing the application in preventing H7N9 type flu pharmaceuticals.
2. application as described in claim 1, which is characterized in that the Hyperoside and Oseltamivir are combined with the side of oral medication
Formula application.
3. application as described in claim 1, which is characterized in that the Hyperoside and Oseltamivir are combined to be administered to prescription
Formula application.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101891785A (en) * | 2010-08-16 | 2010-11-24 | 江西山香药业有限公司 | Extraction method of hyperin and application thereof in medicament preparation |
| KR20130071664A (en) * | 2011-12-21 | 2013-07-01 | 한국생명공학연구원 | Flavonoid comprising anti-virus activity |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101891785A (en) * | 2010-08-16 | 2010-11-24 | 江西山香药业有限公司 | Extraction method of hyperin and application thereof in medicament preparation |
| KR20130071664A (en) * | 2011-12-21 | 2013-07-01 | 한국생명공학연구원 | Flavonoid comprising anti-virus activity |
Non-Patent Citations (3)
| Title |
|---|
| Inhibitory potency of flavonoid derivatives on influenza virus neuraminidase;Christin Rakers等;《Bioorganic & Medicinal Chemistry Letters》;20140718;第4312-4317页 * |
| SCREENING OF POTENTIAL ANTI-INFLUENZA AGENTS FROM JUGLANS MANDSHURICA MAXIM. BY DOCKING AND MD SIMULATIONS;Y. YANG等;《Digest Journal of Nanomaterials and Biostructures》;20150331;第10卷(第1期);第43-57页 * |
| 抗H7N9禽流感病毒药物研究进展;张耘实等;《人民军医》;20150101;第58卷(第1期);第105-106页 * |
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