CN105832755A - Application of combination of hyperoside and oseltamivir to drug for prevention and treatment of H7N9 influenza - Google Patents
Application of combination of hyperoside and oseltamivir to drug for prevention and treatment of H7N9 influenza Download PDFInfo
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- CN105832755A CN105832755A CN201610348142.6A CN201610348142A CN105832755A CN 105832755 A CN105832755 A CN 105832755A CN 201610348142 A CN201610348142 A CN 201610348142A CN 105832755 A CN105832755 A CN 105832755A
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- 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
The invention relates to application of the combination of hyperoside and oseltamivir to a drug for prevention and treatment of H7N9 influenza and belongs to the field of traditional Chinese medicine application. The invention is implemented by the following technical scheme: firstly, H7N9 wild type or mutant influenza virus neuraminidase liquid and hyperoside and oseltamivir mixed liquid are incubated for 30 minutes, a substrate is added for detection, and an experimental result indicates that the combined use of the two drugs has an inhibiting effect on H7N9 wild type and mutant influenza virus neuraminidase activity; then, the anti-influenza effect of the combined use of the hyperoside and the oseltamivir on cellular levels is detected to discover that the combined use of the hyperoside and the oseltamivir can play a role in treating cells infected by an H7N9 wild type or mutant influenza virus; finally, the effect of the combined use of the hyperoside and the oseltamivir on a mouse influenza model is detected to discover that after a hyperoside and oseltamivir mixture is added, the weight of a influenza mouse can be obviously reduced, so that the mouse can gradually regain health, and the lung indexes of the influenza mouse can be obviously reduced.
Description
Technical field
The invention belongs to application in TCM field, be specifically related to Hyperoside and Oseltamivir combines the application in preventing and treating H7N9 type influenza virus medicine.
Background technology
Influenza (hereinafter referred to as influenza) is a kind of acute viral respiratory infectious disease caused by the influenza virus of orthomyxoviridae family.Its infectiousness
By force, popular wide, the incidence of disease is high.Worldwide, influenza the most popular cause about 300 ten thousand to 500 ten thousand people ill serious and about 250,000 to
500000 people are dead.At present, most common being also easiest to of influenza A virus causes pandemics and area popular.Now, only two class antiviral agent
Thing has been approved by the fda in the United States for influenza prevention and treatment, is M2 inhibitors of ion channels and neuraminic acid enzyme inhibitor respectively.But,
According to CDC of the U.S., the seasonal H1N1 virus of 99.6% has drug resistance to Oseltamivir, the seasonal H3N2 of 100%
Virus has drug resistance to adamantane.
2013, having broken out the novel type A avian influenza virus H7N9 that can infect the mankind in China, its neuraminidase saltant type H7N9 is dashed forward
Modification Strain is more than 10000 times of wild-type strain to the tolerance of Oseltamivir.Generally the occurring of Drug resistance strain reduces Tamiflu
Clinical effectiveness to treatment of influenza.In view of neuraminidase inhibitor class medicine is had the appearance of drug-resistant viral and the side effect of Tamiflu,
Many scientists are devoted to be applied in combination multiple antiviral drugs, to reducing the usage amount of Tamiflu and improve the effect for the treatment of influenza
Really.
In China, the traditional Chinese medicine history of antiviral existing thousands of years, in terms of utilizing Chinese herb prevention influenza, have accumulated a large amount of valuable theory and practice warp
Test.Natural resources of Chinese medicinal materials is the abundantest, evident in efficacy by Chinese medicine influenza from ancient times to the present.Hyperoside is isolated active ingredient from azalea
Thing, research shows that Hyperoside has the effects such as anti-oxidant, anticancer, hypoglycemic.Recently, also there are some researches show that it has the effect of anti influenza.This
Bright research finds that Hyperoside is used in combination the neuraminic acid enzyme level to H7N9 wild type and H7N9 saltant type with Tamiflu oseltamivir and rises
To synergy, and the usage amount of medicine can be reduced.
Summary of the invention
It is an object of the invention to provide Hyperoside and Oseltamivir is used in combination the application in preventing and treating H7N9 type flu pharmaceutical.Hyperoside and carboxylic
Acid oseltamivir is used in combination and can effectively suppress H7N9 wild type and the activity of saltant type influenza neuraminidase, thus suppresses influenza sick
Poison breeding in vivo, and then alleviate or cure flu victims.The present invention to Hyperoside and carboxylic acid oseltamivir to H7N9 wild type and sudden change
Type neuraminidase has carried out the detection of external zymetology inhibition, finds that carboxylic acid oseltamivir is to H7N9 wild type and saltant type neuraminidase
Half-inhibition concentration (IC50) it is respectively 1.75nmol/L and 26.52 μm ol/L；Hyperoside is to H7N9 wild type and saltant type neuraminic acid
The IC of enzyme50It is respectively 117.4 μm/L and 127.7 μm ol/L.Then carry out Hyperoside and external zymetology suppression has been used in combination with carboxylic acid Oseltamivir
Property detection, the action effect association index value of drug combination represents: association index value shows that less than 1 two kinds of Drug combinations play collaborative work
With.Test result indicate that synergy is played in the suppression of H7N9 wild type neuraminidase by two kinds of medicines, and to H7N9 saltant type neuraminic acid
The synergy of enzyme level becomes apparent from.We are again to both H7N9 influenza infection mdck cells and to add Hyperoside and carboxylic acid difficult to understand
Si Tawei mixture has carried out the mensuration of cell survival rate, after finding that Hyperoside and carboxylic acid Oseltamivir mixture all can improve the infection of H7N9 virus
The survival rate of cell.Finally, we to Hyperoside and Oseltamivir mixture to H7N9 wild type and saltant type influenza virus the body weight to mouse
Change, fatal rate and Lung Exponent are determined, and find that Hyperoside and Oseltamivir mixture can substantially alleviate H7N9 type influenza virus to mouse
Symptom.The present invention provides basis for developing the new drug with neuraminidase as target spot, is used in combination for Hyperoside and Oseltamivir and is applied to the mankind
The treatment of influenza provides reference.
Accompanying drawing explanation
Fig. 1 is that variable concentrations Hyperoside is used in combination the inhibitory action to H7N9 wild type influenza virus neuraminidase with carboxylic acid Oseltamivir
Fig. 2 is that variable concentrations Hyperoside is used in combination the inhibitory action to H7N9 saltant type influenza neuraminidase with carboxylic acid Oseltamivir
Fig. 3 is that variable concentrations Hyperoside is used in combination the treatment to the mdck cell infecting H7N9 wild type influenza virus with carboxylic acid Oseltamivir
Effect
Fig. 4 is that variable concentrations Hyperoside is used in combination the treatment to the mdck cell infecting H7N9 saltant type influenza virus with carboxylic acid Oseltamivir
Effect
Fig. 5 is that Hyperoside is used in combination the impact on the Mouse Weight infecting H7N9 wild type influenza virus with Oseltamivir
Fig. 6 is that Hyperoside is used in combination the impact on the Mouse Weight infecting H7N9 saltant type influenza virus with Oseltamivir
Fig. 7 is that Hyperoside is used in combination infecting the impact of virus load in H7N9 wild type or saltant type influenza virus mouse lung with Oseltamivir
Detailed description of the invention
Below by way of the detailed description of the invention of example forms, the present invention is described in further detail.
Embodiment 1
10 μ L Hyperosides of variable concentrations or carboxylic acid oseltamivir are separately added into H7N9 wild type or the saltant type neuraminidase solution of 40 μ L
In, 37 DEG C of incubators are placed 30min, is subsequently adding 20 μMs of MU-NANA substrate buffer solutions of 50 μ L, ELIASA detects, swash
Sending out wavelength is 360nm, and a length of 450nm of transmitted wave detects duration 8min.Testing result is: carboxylic acid oseltamivir to H7N9 wild type and
The IC of saltant type neuraminidase50It is respectively 1.75nmol/L and 26.52 μm ol/L；Hyperoside is to H7N9 wild type and the IC of saltant type50
It is respectively 117.4 μm/L and 127.7 μm ol/L.
Embodiment 2
According to the Hyperoside measured and the carboxylic acid Oseltamivir IC to H7N9 wild type Yu saltant type neuraminidase50Determine Hyperoside and carboxylic
The addition concentration of acid Oseltamivir.Hyperoside is chosen as each relative to H7N9 wild type and saltant type nerve ammonia with the concentration of carboxylic acid Oseltamivir
The IC of 0.25,0.5,1,2 and 4 times of acid enzyme50Concentration.And the ratio of the concentration of the concentration of the Hyperoside added and carboxylic acid Oseltamivir
It is to remain consistent, for its IC50The ratio of value.First we add the H7N9 wild type after the dilution of 30 μ l or sudden change in 96 hole ELISA Plates
Type neuraminidase liquid, then adds Hyperoside and the carboxylic acid Oseltamivir of 10 μ l corresponding concentration of 10 μ l variable concentrations in each hole, each
Concentration does 4 repetitions.After hatching 30min at 37 DEG C, add the NA specific substrate MU-NANA of 50 μ l 20 μMs, detect H7N9
Wild type and the fluorescence intensity of saltant type neuraminidase.Record data, then use CalcuSyn software to calculate the association index (CI of both medicines
Value), result is as follows:
<note>: ED50, ED75 and ED90 represent that enzymatic activity is suppressed 50%, 75% and 90% respectively.
CI value is less than 1 two kinds of medication combined synergies that play of explanation, and the least synergy of CI value is the best.Two kinds of medicine connection are understood by data in table
Close and use the suppression to H7N9 neuraminidase to play synergy.
Embodiment 3
In order to detect whether Hyperoside and carboxylic acid Oseltamivir mixture have anti-H7N9 wild type and saltant type influenza virus on mdck cell
Effect, the mdck cell that we have chosen in logarithmic phase carries out bed board, and 10000, every hole, at 37 DEG C of 5%CO2Cell culture incubator in
Cultivate 24h.After cultivating 24h, DMEM culture medium is discarded, then clean 2～3 times with DMEM.Again by the H7N9 wild type of 100 μ l
Or saltant type influenza virus allantoic fluid (concentration is the TCID of 100 times50Concentration) and variable concentrations under Hyperoside mix with carboxylic acid Oseltamivir
Mixing 2h at thing 100 μ l 37 DEG C, Hyperoside and the concentration of carboxylic acid Oseltamivir are chosen as each neural with saltant type relative to H7N9 wild type
The IC of 0.25,0.5,1,2 and 4 times of propylhomoserin enzyme50The scale dimension of the concentration of concentration, the concentration of the Hyperoside added and carboxylic acid Oseltamivir
Hold consistent, for its IC50The ratio of value.Then mixed liquor is added in mdck cell and hatch in cell culture incubator, by mixed liquor sucking-off after 2h,
Clean 2～3 times with DMEM, be subsequently adding the DMEM nutrient solution containing 1 μ g/ml pancreatin and 2%FBS and cultivate 24h.After 24h, use
Same nutrient solution is cultivated 3 days, by nutrient solution sucking-off, cleans 2～3 times with DEME, is then adding 100 μ l DMEM and 15 μ l MTT
(5mg/ml), slightly shake mixing, in cell culture incubator, then stand 4h.Then discard the DMEM containing MTT, use PBS
Colourless to cleaning fluid, add 100 μ l DMSO, slightly shake mixing.Its ultraviolet absorptivity at 570nm is detected when 30min.Logical
Cross OD570Calculate the survival rate of cell.
Result as shown in Figure 3 and Figure 4, under the conditions of different disposal, adds Hyperoside and carboxylic acid Oseltamivir mixed liquor and H7N9 wild type or prominent
During modification influenza virus, along with the increase of drug concentration, cell survival rate also increases as.Illustrate that Hyperoside can press down with Oseltamivir mixed liquor
H7N9 wild type processed and saltant type influenza virus, protection mdck cell is from viral subversive, and protective effect is also with Hyperoside and carboxylic
The increase of acid Oseltamivir mixture concentration presents dose dependent.
Embodiment 4
In order to detect whether Hyperoside has therapeutic action with Oseltamivir mixture to the mouse having infected H7N9 wild type influenza virus, we choose
The Female ICR mice 50 of about 20g, is then anaesthetized with ether, then by 10 times of LD by the way of collunarium50The H7N9 of concentration is wild
Raw type influenza virus dilution sets up mouse influenza models, to instill the mouse of 50 μ l PBS as a control group in instilling mouse nasal cavity.I
50 mouse are randomly divided into 5 groups, these 5 groups are respectively as follows: control group (PBS group)；Virus group (10 times of LD50H7N9 wildtype influenza
Virus)；Hyperoside group (10 times of LD50H7N9 wild type influenza virus+25mg/kg Hyperoside)；Oseltamivir group (10 times of LD50H7N9
Wild type influenza virus+25mg/kg Oseltamivir)；Hyperoside and Oseltamivir mixture group (10 times of LD50H7N9 wild type influenza virus
+ 25mg/kg Hyperoside and 25mg/kg Oseltamivir mixture).To mouse inoculation virus 2 days before start be administered, every day 2 times, fill continuously
Stomach 10 days, every mouse single administration 0.2ml.Every day, body weight and the death condition of mouse often organized in record.When the 15th day, jejunitas disconnected to mouse
After water 8h, carry out dislocation and put to death, take out the lungs of mouse and weigh.Calculate according to formula:
Result as it is shown in fig. 7, explanation Hyperoside and Oseltamivir mixed liquor can significantly reduce the Lung Exponent of H7N9 wild type influenza virus infecting mouse,
Alleviating of H7N9 wild type influenza virus infecting mouse body weight can be alleviated, improve the survival condition of mouse, make mouse be gradually restored to the general level of the health.
Result is as shown in the table, illustrates that Hyperoside and Oseltamivir mixed liquor can significantly reduce the Lung Exponent of H7N9 wildtype influenza mouse.
Result above all illustrates, the mouse infecting H7N9 wildtype influenza can be played the effect for the treatment of with Oseltamivir mixed liquor by Hyperoside, makes little
Mouse is gradually restored to the general level of the health.
Embodiment 5
In order to detect whether Hyperoside has therapeutic action with Oseltamivir mixture to the mouse having infected H7N9 saltant type influenza virus, we choose
The Female ICR mice 50 of about 20g, is then anaesthetized with ether, then by 10 times of LD by the way of collunarium50The H7N9 of concentration dashes forward
Modification influenza virus dilution sets up mouse influenza models, to instill the mouse of 50 μ l PBS as a control group in instilling mouse nasal cavity.I
50 mouse are randomly divided into 5 groups, these 5 groups are respectively as follows: control group (PBS group)；Virus group (10 times of LD50H7N9 saltant type influenza
Virus)；Hyperoside group (10 times of LD50H7N9 saltant type influenza virus+25mg/kg Hyperoside)；Oseltamivir group (10 times of LD50H7N9
Saltant type influenza virus+25mg/kg Oseltamivir)；Hyperoside and Oseltamivir mixture group (10 times of LD50H7N9 saltant type influenza virus
+ 25mg/kg Hyperoside and 25mg/kg Oseltamivir mixture).To mouse inoculation virus 2 days before start be administered, every day 2 times, fill continuously
Stomach 10 days, every mouse single administration 0.2ml.Every day, body weight and the death condition of mouse often organized in record.When the 15th day, jejunitas disconnected to mouse
After water 8h, carry out dislocation and put to death, take out the lungs of mouse and weigh.Calculate according to formula shown in embodiment 4.
Shown in result Fig. 7, illustrate that Hyperoside and Oseltamivir mixed liquor can significantly reduce the Lung Exponent of H7N9 saltant type influenza infection mouse,
Alleviating of H7N9 saltant type influenza infection Mouse Weight can be alleviated, improve the survival condition of mouse, make mouse be gradually restored to the general level of the health.
Result is as shown in the table, illustrates that Hyperoside and Oseltamivir mixed liquor can significantly reduce the Lung Exponent of H7N9 saltant type influenza mouse.
Result above all illustrates, the mouse infecting H7N9 saltant type influenza can be played the effect for the treatment of with Oseltamivir mixed liquor by Hyperoside, makes little
Mouse is gradually restored to the general level of the health.
Claims (3)
1. Hyperoside and oseltamivir combine the application in preventing and treating H7N9 type flu pharmaceutical.
2. as in claim 1 Hyperoside and oseltamivir combine the application with Oral administration.
3. as in claim 1 Hyperoside and oseltamivir combine with the application of drug administration by injection mode.
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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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- 2016-05-24 CN CN201610348142.6A patent/CN105832755B/en not_active Expired - Fee Related
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 |
|---|
| CHRISTIN RAKERS等: "Inhibitory potency of flavonoid derivatives on influenza virus neuraminidase", 《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS》 * |
| Y. YANG等: "SCREENING OF POTENTIAL ANTI-INFLUENZA AGENTS FROM JUGLANS MANDSHURICA MAXIM. BY DOCKING AND MD SIMULATIONS", 《DIGEST JOURNAL OF NANOMATERIALS AND BIOSTRUCTURES》 * |
| 张耘实等: "抗H7N9禽流感病毒药物研究进展", 《人民军医》 * |
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