CN103127050A - Application of salvianolic acid B in preparing H5N1 influenza virus resisting medicine - Google Patents
Application of salvianolic acid B in preparing H5N1 influenza virus resisting medicine Download PDFInfo
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- CN103127050A CN103127050A CN201110394891XA CN201110394891A CN103127050A CN 103127050 A CN103127050 A CN 103127050A CN 201110394891X A CN201110394891X A CN 201110394891XA CN 201110394891 A CN201110394891 A CN 201110394891A CN 103127050 A CN103127050 A CN 103127050A
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- salvianolic acid
- influenza virus
- albumen
- activity
- rna polymerase
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Abstract
The invention provides application of salvianolic acid B in preparing H5N1 influenza virus medicine which is used for preventing or curing H5N1 influenza virus. A structural formula of the salvianolic acid is shown as following. The salvianolic acid can effectively restrain activity ribonucleic acid (RNA) polymerase subunit polyacrylonitrile (PAN) protein of H5N1 influenza virus.
Description
Technical field
The present invention relates to pharmaceutical field, be specifically related to the application of salvianolic acid B.
Background technology
Influenza is that a class is propagated, pathogenic, variability is stronger, and involves the wider epidemic disease of scope, is infected by the different subtype Strain to cause, mainly is divided into A type, Type B and C type three major types, especially take the A type as main.A type influenza virus RNA polymerase PA subunit N end protein (PA
N) play a crucial role participating in stablizing the function aspects such as PA structure, endonuclease activity, cap-like structure combination, promoter combination, because of its high conservative in A type, Type B, C type strain RNA sequence, become an important target spot of research and development broad spectrum type influenza virus inhibitor at present simultaneously.Wherein highly pathogenic bird flue virus H 5 N 1 subtype just belongs to A type influenza virus, although H5N1 can directly not be transmitted to the mankind by birds at present, and the report of existing human infection's bird flu and many people death, its hazardness is quite serious.
More existing influenza compound medicine action target spots are indefinite, and the action component complexity is various.And also there is certain drug resistance at present in existing medicine such as Buddha's warrior attendant gastral cavity amine, the oseltamivir phosphate capsule etc. that are used for the treatment of influenza in clinical practice.RNA polymerase PA
NAlbumen is brand-new as one, the target spot of popular research broad spectrum type influenza virus inhibitor, and is at present less for the inhibitor of this target spot, and without the finished product marketed drug.The inhibitor that the natural product of having reported is originated also cans be counted on one's fingers.
It is long-standing that natural product and derivant thereof are used to treat various diseases.With synthetic drug Comparatively speaking, natural product has structure diversity, functional diversity, the advantage such as side effect is low, wherein Chinese medicine is gone through medical treatment checking in several thousand especially, very likely therefrom filters out the compound of the anti-H5N1 influenza virus of novel structure, model of action uniqueness.
Salvianolic acid B (Salvianolic acid B) is a kind of native compound that extracts from plant amedica (as Radix Salviae Miltiorrhizae), can remove oxygen-derived free radicals, suppress lipid peroxidation, and be one of the strongest natural product of present known antioxidant activity; Simultaneously but its anticoagulant also, suppress thrombosis.The organs such as the heart, brain, liver, kidney had the valuable pharmacological effect.
Liu's equalitys in 2000 open salvianolic acid B magnesium in patent CN1270809A has the effect of anti peroxidation of lipid anti-liver injury, the function that alleviates extent of liver fibrosis is arranged, and point out can be used for treatment and the diseases such as prevention hepatic fibrosis, liver cirrhosis and fatty liver, salvianolic acid B magnesium can be used to prepare the medicament for the treatment of chronic hepatopathy; The open pressure differential self in patent CN1378837A such as Zhang Juntian in 2002 has the effect that suppresses the mammal HIV (human immunodeficiency virus) infection; Thanked to day training in 2003 and wait the application of open salvianolic acid B in preparation medicine for treating tumor thing in patent CN1408353A, and point out that salvianolic acid B and its esters (iron ion, sodium ion, potassium ion, calcium ion and magnesium ion) have lethal effect to tumor cell, and experimental results show that to have antitumous effect in animal body; The prosperous grades of Xiao Ming in 2005 open salvianolic acid B in patent CN1578668A can be used as the purposes of the medicament of the disease that treatment or prevention OxLDL ELISA cause; Wang Sheng in 2008 opens etc. that the pharmaceutical composition of open salvianolic acid B and its esters (potassium ion, sodium ion, calcium ion and magnesium ion) has the anti-parkinson purposes in patent CN101239057A; Ni Li army in 2009 etc. discloses derivant and the synthetic method thereof of structure modified salvianolic acid B in patent CN101362741A; He disclosed again the application of structure modified outcome of salvianolic acid B in pharmacy in patent CN101627988A in 2010, and pointed out that these modified outcomes can be used as medicaments for resisting myocardial ischemia, anti-cerebral ischemia, atherosclerosis, antithrombotic, antitumor, defying age, anti-liver cirrhosis and anti-hepatic fibrosis medicines; Zhang Zuoguang open salvianolic acid B in patent CN101375844A can be used as the raw material of antidepressant drug; Fruit Dean etc. discloses a kind of purposes of compound in preparation MMP-9 activity inhibitor with salvianolic acid B mother nucleus structure in patent CN101496798A; The open salvianolic acid B in patent CN101530409A such as Han Jingyan can extend the thrombosis zero-time, suppresses the mesentery thrombosis that photochemistry causes, and has antithrombotic and uses.
Although salvianolic acid B and derivant thereof have stronger pharmacological action at aspects such as antioxidation, anti-liver injury, antitumor, antidepressant, defying age and antithrombotics, there is no its application aspect prevention or treatment H5N1 influenza virus.
Summary of the invention
The present invention provide salvianolic acid B for the preparation of the application in the medicine of prevention or treatment H5N1 influenza virus, wherein said salvianolic acid B has following structural:
In described application, the molecular formula of salvianolic acid B is C
36H
30O
16, molecular weight is 718.62.
In described application, salvianolic acid B can prepare by the whole bag of tricks known in the art or alternative separation and Extraction, synthetic or semisynthetic method from plant, also can be directly commercially available.
In described application, salvianolic acid B is used for suppressing the activity of H5N1 influenza virus RNA polymerase.
In described application, salvianolic acid B is used for suppressing H5N1 influenza virus RNA polymerase subunit PA
NThe activity of albumen.Wherein, described H5N1 influenza virus RNA polymerase subunit PA
NAlbumen is one of target spot that suppresses the H5N1 influenza virus.
The present invention chooses the expressed PA of H5N1 type influenza virus RNA polymerase hypotype PA subunit N terminal nucleotide sequence
NAlbumen is lived high flux as the activity rating method of screening as target proteins with vitro enzyme, finds that salvianolic acid B has obvious inhibition PA
NProtein active is measured its IC
50Value is 16.3 μ M.Salvianolic acid B can suppress the activity of H5N1 influenza virus RNA polymerase.Can be for the preparation of the medicine of prevention or treatment H5N1 influenza virus.
Description of drawings
When reading in conjunction with the accompanying drawings, the present invention may be better understood according to the following detailed description.
Fig. 1 shows the salvianolic acid B standard solution (50mg/mL) of embodiments of the invention and negative control, positive control to H5N1 type influenza virus RNA polymerase hypotype PA
NThe protease activity curve.
Fig. 2 shows according to an embodiment of the invention, and salvianolic acid B suppresses H5N1 type influenza virus RNA polymerase hypotype PA
NThe IC of albumen
50The value curve chart.
The specific embodiment
For the present invention is described better, below in detail specific embodiments of the present invention will be described in detail.
The present invention chooses the expressed PA of H5N1 type influenza virus RNA polymerase hypotype PA subunit N terminal nucleotide sequence
NAlbumen is as target proteins, and the endonuclease activity that utilizes this albumen to have is lived high flux as the activity rating method of screening with vitro enzyme, and the existing natural product monomer of this laboratory storehouse is screened, and finds that salvianolic acid B is suppressing PA
NHaving outstanding performance in the protein active aspect, can develop into the medicine of anti-H5N1 influenza virus.
The pharmacological evaluation part
1. the enzyme test philosophy of living
Utilize FRET (fluorescence resonance energy transfer) (fluorescence resonance energy transfer, FRET) technical measurement for PA
NThe activity of the inhibitor of albumen.According to PA
NThe endonuclease activity that albumen has, design one section nucleic acid fragment as substrate: 5 ' FAM--AA--3 ' BHQ1, when inhibitor to PA
NAlbumen is not made used time, PA
NAlbumen can be brought into play the activity of its Cobra venom endonuclease substrate is cut, and quenching group (BHQ1) after fluorophor absorbs the utilizing emitted light of 494nm, inspires the light wave of 526nm, by the variation of detector fluorescence intensity away from fluorophor (FAM); If inhibitor is to PA
NAlbumen has inhibitory action, and quenching group can not cut, and the emission light wave rear portion energy that fluorophor absorbs 494nm can be transferred to quenching group, and the excitation light wave intensity of 526nm will weaken, and detects inhibitor to PA with this
NThe inhibition of albumen.
2. the enzyme method of testing of living
The vitro enzyme that the present invention adopts appraisement system composition alive sees Table 1:
Table 1. vitro enzyme appraisement system alive
Wherein, PA
NAlbumen is bird flu virus A/goose/Guangdong/1/1996 (H1N5) strain RNA polymerase PA subunit 1-256 amino acids residue, and PAN albumen storage solutions is: 20mM TrispH 8.0,150mM NaCl, pH=8.0, final PA
NProtein concentration is 1mg/mL; Substrate is 5 ' FAM--AA--3 ' BHQ1 (is purchased from Shanghai and gives birth to work biological engineering company limited), is dissolved in buffer (20mM Tris pH 8.0,150mM NaCl, pH=8.0), and concentration is 50 μ M; The concentration of EDTA is 200mM, and its preparation method is: get 14.97 gram EDTA and be dissolved in 150 milliliters of distilled waters, then be settled to 200 milliliters; In experimental group, the concentration of standard solution of salvianolic acid B is 50.00mg/mL, can obtain successively the salvianolic acid B solution of variable concentrations with the qdx dilution.
Appraisement system is set the negative and positive contrast.Positive controls is for to add 1.00 μ L 200mMEDTA solution in system, negative control group is for to add 1.00 μ L 200mM Tris (pH=8.0) solution in system.Experimental group is for adding the salvianolic acid B solution of 1.00 μ L variable concentrations in the system.
With the described system of table 1 after hatching 10 minutes under 37 ℃, utilize microplate reader (FluoroskanAscent, Thermo Scientific company, optical filter is that machine carries), measure the exciting light and the utilizing emitted light that adopt and be respectively 494nm and 526nm, recorded the first order fluorescence strength values every 30 seconds, record altogether 200 times.Draw enzyme curve alive according to obtaining fluorescence intensity numerical value, the enzyme that standard salvianolic acid B solution and positive control and negative control are measured curve alive is seen Fig. 1, and calculates PA
NThe residual activity of albumen (RA), parallel repetition 3 times is averaged.
3. the computational methods of residual activity (RA):
In enzyme was lived curve, getting negative control slope of curve maximum value was V
0, medicine slope of curve maximum is V
1, PA
NThe residual activity RA=V of albumen
1/ V
0
The following example only is used for illustrating the present invention, to the present invention without any restriction.
Embodiment 1 salvianolic acid B is to H5N1 type influenza virus RNA polymerase subunit PA
NAlbumen suppresses active mensuration
5.00mg salvianolic acid B (available from Chengdu Man Site reference substance company limited, HPLC analyzes content＞98%) is dissolved in the double steaming solution of 100.00 μ L 95%DMSO fully, is mixed with the standard solution that concentration is 50.00mg/mL.
The salvianolic acid B standard solution of getting 10.00 μ L carries out with 95% DMSO the gradient dilution that 2 times of amounts increase progressively, and is diluted to altogether variable concentrations as shown in table 2 solution to be measured.Adopt the enzyme curve alive of enzyme as described in pharmacological evaluation part test determines alive solution to be measured, and calculate PA
NThe residual activity of albumen, parallel repetition 3 times.The residual activity of measuring the results are shown in Table 2.
Divided by the molecular weight of respective compound and get the denary logarithm value as X-axis, corresponding residual activity is Y-axis, maps and calculates corresponding IC with GraphPad Prism 5 with the compound concentration of dilution
50Value.IC
50The value curve chart is seen Fig. 2.The IC that measures
50Value is 16.3 μ M.
The residual activity of table 2. salvianolic acid B under different diluted concentrations
| Test concentrations (mg/mL) | Residual activity | Test concentrations (mg/mL) | Residual activity |
| 1.00 | 0.002896 | 0.015625 | 0.3597 |
| 0.50 | 0.04234 | 0.007813 | 0.6117 |
| 0.25 | 0.05906 | 0.003906 | 0.8417 |
| 0.125 | 0.08515 | 0.001953 | 0.9574 |
| 0.0625 | 0.08398 | 0.000977 | 0.9625 |
| 0.03125 | 0.2011 | 0.000488 | 0.9647 |
According to above-described embodiment, can understand salvianolic acid B and have and suppress well PA
NThe effect of protein active, its IC
50Value is 16.3 μ M.Given this, salvianolic acid B can suppress the activity of H5N1 influenza virus RNA polymerase, thereby can be for the preparation of the medicine of prevention or treatment H5N1 influenza virus.
For clear and understandable purpose, explanation and embodiment have described foregoing invention in detail by way of example.Can change and revise in the scope of subsidiary claim, this will be readily apparent to persons skilled in the art.Therefore, be appreciated that top description is intended to for explanation rather than for restriction.Therefore, scope of the present invention should not determine with reference to above-mentioned description, and should determine with reference to the determined four corner of doctrine of equivalents that following claims and these claim are enjoyed.
Claims (7)
1. salvianolic acid B is for the preparation of the application in the medicine of prevention or treatment H5N1 influenza virus, and wherein said salvianolic acid B has following structural:
2. application according to claim 1, the molecular formula of wherein said salvianolic acid B are C
36H
30O
16, molecular weight is 718.62.
3. separation and Extraction or synthetic or semisynthetic method acquisition from plant are adopted in application according to claim 1, wherein said salvianolic acid B.
4. application according to claim 1, wherein said salvianolic acid B is used for suppressing the activity of H5N1 influenza virus RNA polymerase.
5. application according to claim 1, wherein said salvianolic acid B are used for suppressing H5N1 influenza virus RNA polymerase subunit PA
NThe activity of albumen.
6. application according to claim 4, wherein said H5N1 influenza virus RNA polymerase subunit PA
NAlbumen is one of target spot that suppresses the H5N1 influenza virus.
7. application according to claim 4, wherein said salvianolic acid B suppress H5N1 influenza virus RNA polymerase subunit PA
NThe IC of protein active
50Value is 16.3 μ M.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110394891.XA CN103127050B (en) | 2011-12-02 | Salvianolic acid B application in preparing anti-H5N1 influenza virus medicine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110394891.XA CN103127050B (en) | 2011-12-02 | Salvianolic acid B application in preparing anti-H5N1 influenza virus medicine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103127050A true CN103127050A (en) | 2013-06-05 |
| CN103127050B CN103127050B (en) | 2016-12-14 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112516133A (en) * | 2020-05-18 | 2021-03-19 | 南方医科大学 | Application of salvianolate for injection in preparing antiviral drug |
| CN112807300A (en) * | 2021-01-27 | 2021-05-18 | 西安交通大学 | Pharmaceutical application and medicine of combination of salvianolic acid A, salvianolic acid B and salvianolic acid C for resisting 2019-nCov virus |
| WO2021233239A1 (en) | 2020-05-18 | 2021-11-25 | 南方医科大学 | Application of active ingredient of root of ligulilobe sage or pharmaceutically acceptable salt thereof in preparing antiviral drug |
| CN115040499A (en) * | 2022-06-08 | 2022-09-13 | 北京农学院 | Application of tanshinol in preparing medicine for treating or preventing H9N2 subtype avian influenza virus |
| CN116570583A (en) * | 2023-06-20 | 2023-08-11 | 广东医科大学 | Application of salvianolic acid B in preparation of rotavirus resisting preparation |
Citations (4)
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| CN1312722A (en) * | 1998-06-25 | 2001-09-12 | 乔治敦大学 | Compounds obtained from i(salvia) species having antiviral activity |
| CN1378837A (en) * | 2001-04-09 | 2002-11-13 | 中国医学科学院药物研究所 | Application of danshinolic acid compounds in preparing medicines |
| CN1651433A (en) * | 2004-12-10 | 2005-08-10 | 山东大学 | Salviolic acid berberine double salt, its preparation method and application |
| US20090074716A1 (en) * | 2006-02-17 | 2009-03-19 | Virologik Gmbh | method for treating influenza virus infection |
Patent Citations (4)
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|---|---|---|---|---|
| CN1312722A (en) * | 1998-06-25 | 2001-09-12 | 乔治敦大学 | Compounds obtained from i(salvia) species having antiviral activity |
| CN1378837A (en) * | 2001-04-09 | 2002-11-13 | 中国医学科学院药物研究所 | Application of danshinolic acid compounds in preparing medicines |
| CN1651433A (en) * | 2004-12-10 | 2005-08-10 | 山东大学 | Salviolic acid berberine double salt, its preparation method and application |
| US20090074716A1 (en) * | 2006-02-17 | 2009-03-19 | Virologik Gmbh | method for treating influenza virus infection |
Non-Patent Citations (1)
| Title |
|---|
| PUWEI YUAN ET AL.: "Crystal structure of an avian influenza polymerase PAN reveals an endonuclease active site", 《NATURE》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112516133A (en) * | 2020-05-18 | 2021-03-19 | 南方医科大学 | Application of salvianolate for injection in preparing antiviral drug |
| WO2021233239A1 (en) | 2020-05-18 | 2021-11-25 | 南方医科大学 | Application of active ingredient of root of ligulilobe sage or pharmaceutically acceptable salt thereof in preparing antiviral drug |
| EP4154878A4 (en) * | 2020-05-18 | 2024-01-03 | Univ Southern Medical | Application of active ingredient of root of ligulilobe sage or pharmaceutically acceptable salt thereof in preparing antiviral drug |
| CN112807300A (en) * | 2021-01-27 | 2021-05-18 | 西安交通大学 | Pharmaceutical application and medicine of combination of salvianolic acid A, salvianolic acid B and salvianolic acid C for resisting 2019-nCov virus |
| CN115040499A (en) * | 2022-06-08 | 2022-09-13 | 北京农学院 | Application of tanshinol in preparing medicine for treating or preventing H9N2 subtype avian influenza virus |
| CN116570583A (en) * | 2023-06-20 | 2023-08-11 | 广东医科大学 | Application of salvianolic acid B in preparation of rotavirus resisting preparation |
| CN116570583B (en) * | 2023-06-20 | 2024-02-09 | 广东医科大学 | Application of salvianolic acid B in preparation of rotavirus resisting preparation |
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