CN102988352A - Application of Aphanamixoid A in medicine for treating dengue virus infection - Google Patents
Application of Aphanamixoid A in medicine for treating dengue virus infection Download PDFInfo
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- CN102988352A CN102988352A CN2012104691688A CN201210469168A CN102988352A CN 102988352 A CN102988352 A CN 102988352A CN 2012104691688 A CN2012104691688 A CN 2012104691688A CN 201210469168 A CN201210469168 A CN 201210469168A CN 102988352 A CN102988352 A CN 102988352A
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- aphanamixoid
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses application of Aphanamixoid A in a medicine for treating dengue virus infection. Aphanamixoid A is capable of effectively inhibiting proliferation of dengue virus, but has low toxicity on cells; and therefore, Aphanamixoid A can be further developed into a medicine for treating diseases caused by dengue virus infection and has wide application prospect. The application of Aphanamixoid A for preparing the medicine for treating or preventing dengue virus infection is disclosed for the first time; because the skeleton type belongs to a brand new skeleton type and the dengue virus inhibitory activity of Aphanamixoid A is strong unexpectedly, the possibility that other compounds give any inspiration does not exist; Aphanamixoid A has outstanding substantive characteristics; and simultaneously, Aphanamixoid A has obvious progress for preventing and treating dengue virus infection.
Description
Technical field
The invention belongs to biomedicine field, more specifically relate to the application of a kind of Aphanamixoid A in preparation treatment or prevention dengue virus infection medicine.
Background technology
Dengue virus (Dengue virus, DENV) can cause dengue fever, and it is propagated by yellow-fever mosquito, and the severe infections person dengue hemorrhagic fever occurs and steps on leather shock syndrome, and case fatality rate is high.This virus has four serotypes, and serum 2 types are popular diseased plants.Primary disease is mainly in Southeast Asia, and is popular in the Perenniporia martius country in the Pacific region and America.Chinese Guangdong is economized, Hainan Province, and Guangxi province once had the dengue prevalence of 14 different scales.The main communication media of China's dengue fever is Aedes aegypti and aedes albopictus.The former is distributed in the coastal south, and extensively there is the North and South in latter at the North and South.There is report to show and in the mosquito body, is separated to dengue virus.But, also do not have clinically at present effective medicine.
Therefore, we should pay much attention to the research to dengue virus, strengthen the Research intensity to it, carry out the deposit of relevant knowledge and medicine, for China is making certain contribution aspect the prevention and control dengue virus infection.
The compd A phanamixoid A that the present invention relates to is one and delivered (Cai in 2012, J. Y. et al., 2012. Aphanamixoid A, a Potent Defensive Limonoid, with a New Carbon Skeleton from Aphanamixis polystachya. Organic Letters 14 (10), 2524 – 2527.) New skeleton compound, this chemical compound has brand-new framework types, present purposes only relates to insect antifeedant activity (Cai, J. Y. et al., 2012. Aphanamixoid A, a Potent Defensive Limonoid, with a New Carbon Skeleton from Aphanamixis polystachya. Organic Letters 14 (10), 2524 – 2527.), belong to open first for the purposes of the Aphanamixoid A that the present invention relates in preparation treatment or prevention dengue virus infection medicine, because framework types belongs to brand-new framework types, and it suppresses active unexpectedly strong for dengue virus, there is not the possibility that is provided any enlightenment by other chemical compounds, possess outstanding substantive distinguishing features, the control that is used for simultaneously dengue virus infection obviously has significant progress.
Summary of the invention
The objective of the invention is to be to provide the application of a kind of Aphanamixoid A in conduct preparation treatment or prevention dengue virus infection medicine, Aphanamixoid A depressed rate to DENV under 10.0 uM concentration is 94.95%.And Aphanamixoid A is under the 100 uM conditions in concentration, and the survival rate of Vero cell is 91.8%.Aphanamixoid A can suppress the propagation of dengue virus effectively, but very little to cytotoxicity, can further be developed as the medicine for the treatment of dengue virus infection disease, is with a wide range of applications.
Described compd A phanamixoid A structure is shown in formula I:
The purposes of the Aphanamixoid A that the present invention relates in preparation treatment or prevention dengue virus infection medicine belongs to open first, because framework types belongs to brand-new framework types, and it suppresses active unexpectedly strong for dengue virus, there is not the possibility that is provided any enlightenment by other chemical compounds, possess outstanding substantive distinguishing features, the control that is used for simultaneously dengue virus infection obviously has significant progress.
To achieve these goals, the present invention is by the following technical solutions:
1, Aphanamixoid A is to the toxicity test of Vero cell:
The Vero cell is the permissive cell of DENV.Therefore, this experiment at first detects Aphanamixoid A to the cytotoxicity of Vero cell, Aphanamixoid A with variable concentrations acts on the Vero cell, understanding at cell survival rate is the maximum working concentration of the Aphanamixoid A more than 90%, provides reference data for follow-up Aphanamixoid A tests the inhibitory action of DENV.
2, Aphanamixoid A tests the inhibition of DENV:
Aphanamixoid A with variable concentrations acts on the Vero cell that has infected DENV, obtains Aphanamixoid A to the suppression efficiency of virus.The working concentration of Aphanamixoid A in this experiment all makes the Vero cell in the scope of the concentration of 90% above survival rate, therefore, can get rid of the excessive concentration of Aphanamixoid A and the analysis of experimental data is exerted an influence.
(inhibition) of Aphanamixoid A in treatment or prevention dengue virus infection medicine used, and its basic process is:
A. Aphanamixoid A is to the toxicity test of Vero cell:
Vero cell (African green monkey kidney cell) is the permissive cell of DENV.Vero cell in the experiment is bought in cell data center of Shanghai Sheng Ke institute of the Chinese Academy of Sciences; The MTT test kit is purchased from green skies biotechnology research institute; Hyclone (Fetal Bovine Serum, FBS) is bought the GIBCO company in the U.S.; Tissue Culture Plate is bought the company in German Greiner bioone; DMEM culture medium and MEM culture medium are bought the GIBCO company in the U.S..
Experimental procedure is as follows:
1: inoculation Vero cell: with containing 10%(v/v) the DMEM culture medium of hyclone is made into the individual cells suspension, is inoculated into 96 porocyte culture plates with every hole 1000-10000 cell, every hole inoculation volume 100 ul;
2: Cultivation of Vero: at 37 ℃, 5%(v/v) CO
2Under the condition of culture, cultivated 2 days;
3: add Aphanamixoid A: inhale the DMEM culture medium of abandoning in each hole, adding 100ul in each hole with containing 10%(v/v) the DMEM culture medium of hyclone is diluted to respective concentration (0 uM, 0.4 uM, 1.2 uM, 3.7 uM, 11 uM, 33 uM, 100 uM, 300 uM) Aphanamixoid A, control wells adds and contains 10%(v/v) the DMEM culture medium 100ul of hyclone;
4: colour generation: cultivate after 48 hours, every hole adds MTT solution 10ul, at 37 ℃, and 5%(v/v) CO
2Continued to hatch under the condition of culture 4 hours, and then added the Formazan lysate, at 37 ℃, 5%(v/v) CO
2Continued to hatch under the condition of culture 4 hours, and found that Formazan all dissolved until under ordinary optical microscope, observe;
(1): measure: measure absorption value at 570nm.
The Aphanamixoid A of table 1 variable concentrations is to the toxicity test of Vero cell
B. Aphanamixoid A tests the inhibition of DENV:
Take the Vero cell as cultivating the cell of viral DENV, MOI is 0.1, and experimental procedure is as follows:
Access Vero cell in 24 porocyte culture plates, cell grows to monolayer (area at the bottom of the cell coverage hole is about 80% ~ 90%) after 24 hours, the sucking-off culture medium, access virus sample 200ul, 37 ℃ adsorbed 2 hours.After absorption is finished, inhale the virus liquid abandon in each hole, the virus of not adsorbing with DMEM culture medium flush away.Add with containing 10%(v/v) the Aphanamixoid A of the prescribed concentration (0 uM, 0.04 uM, 0.12 uM, 0.37 uM, 1.1 uM, 3.3 uM, 10.0 uM) of the DMEM culture medium dilution of hyclone, in 37 ℃, 5%(v/v) CO
2Incubator in cultivated 42 hours, collect the supernatant in each hole, do viral plaque experiment.
Virus plaque experiment: access Vero cell in 24 orifice plates, cell grows to monolayer (area at the bottom of the cell coverage hole is about 80% ~ 90%) after 24 hours, the culture medium in each hole is abandoned in suction, access is with containing 3%(v/v) the virus sample 200ul of 10 times of serial dilutions of DMEM culture medium of hyclone, 37 ℃ of absorption 2 hours.After absorption is finished the supernatant plate in each hole inhaled and abandons, use 10%(v/v) the DMEM culture medium flush away of the FBS virus of not adsorbing.Add 45 ℃ of fresh preheatings semifixed culture medium [the A composition: 3%(v/v) FBS, 2 * MEM culture medium, penicillin (U.S. AMRESCO) and streptomycin (U.S. AMRESCO) final concentration are respectively 100U/ml, 0.1mg/ml; B composition: agarose 1%(w/v) (French Biowest).The A composition: the B composition=1:1(v/v)], in 37 ℃, 5%(v/v) CO
2Incubator in cultivated 48 ~ 72 hours.Behind plaque formation, be dissolved in 10%(v/v with crystal violet (U.S. AMRESCO) dyeing (2%(w/v) crystal violet) formaldehyde), wash away crystal violet and agarose with flowing water after 2 hours, at the cell plaque of microscopically to the generation of every hole, calculate viral titre (PFU/ml, PFU:plaque formation unit plaque forming unit) according to plaque number and extension rate.PFU=viral dilution degree * P/V, (P: cavitation corrosion speckle number; V: inoculum concentration).Test 2 different times.
The Aphanamixoid A of table 2 variable concentrations reduces and inhibitory action the DENV titre
The present invention compared with prior art, have the following advantages and effect: the present invention by to the inhibition experiment confirm of DENV Aphanamixoid A DENV is had good inhibitory action, treatment DENV catches, have widely application background from having proved in essence this medicine.This invention can provide for the disease that clinical treatment is caused by DENV good drug candidate, has very good application prospect.
The purposes of the Aphanamixoid A that the present invention relates in preparation treatment or prevention dengue virus infection medicine belongs to open first, because framework types belongs to brand-new framework types, and it suppresses active unexpectedly strong for dengue virus, there is not the possibility that is provided any enlightenment by other chemical compounds, possess outstanding substantive distinguishing features, the control that is used for simultaneously dengue virus infection obviously has significant progress.
The specific embodiment
The preparation method of compd A phanamixoid A involved in the present invention is referring to document (Cai, J. Y. et al., 2012. Aphanamixoid A, a Potent Defensive Limonoid, with a New Carbon Skeleton from Aphanamixis polystachya. Organic Letters 14 (10), 2524 – 2527.).
The present invention is further detailed explanation by the following examples, but protection scope of the present invention is not subjected to any restriction of specific embodiment, but limited by claim.
Embodiment 1: the preparation of compd A phanamixoid A tablet involved in the present invention:
Get 20 and digest compound Aphanamixoid A, add conventional adjuvant 180 grams of preparation tablet, mixing, conventional tablet machine are made 1000.
Embodiment 2: the preparation of compd A phanamixoid A capsule involved in the present invention:
Get 20 and digest compound Aphanamixoid A, add conventional adjuvant such as starch 180 grams of preparation capsule, mixing is encapsulatedly made 1000.
Further specify its pharmaceutically active below by pharmacodynamic experiment.
Experimental example 1:
Aphanamixoid A uses as (inhibition) in preparation treatment or prevention dengue virus infection medicine, and its basic process is:
A. Aphanamixoid A is to the toxicity test of Vero cell:
Vero cell (African green monkey kidney cell) is the permissive cell of DENV.
Experimental procedure is as follows:
1: inoculation Vero cell: with containing 10%(v/v) the DMEM culture medium of hyclone is made into the individual cells suspension, is inoculated into 96 porocyte culture plates with every hole 1000-10000 cell, every hole inoculation volume 100ul;
2: Cultivation of Vero: at 37 ℃, 5%(v/v) CO
2Under the condition of culture, cultivated 2 days;
3: add Aphanamixoid A: inhale the DMEM culture medium of abandoning in each hole, adding 100ul in each hole with containing 10%(v/v) the DMEM culture medium of hyclone is diluted to respective concentration (0uM, 0.4uM, 1.2uM, 3.7uM, 11uM, 33uM, 100uM, 300uM) Aphanamixoid A, control wells adds and contains 10%(v/v) the DMEM culture medium 100ul of hyclone;
4: colour generation: cultivate after 48 hours, every hole adds MTT solution 10ul, at 37 ℃, and 5%(v/v) CO
2Continued to hatch under the condition of culture 4 hours, and then added the Formazan lysate, at 37 ℃, 5%(v/v) CO
2Continued to hatch under the condition of culture 4 hours, and found that Formazan all dissolved until under ordinary optical microscope, observe;
5: measure: measure absorption value at 570nm.
6: experimental result sees Table 1.
B. Aphanamixoid A is to the inhibitory action of DENV:
Take the Vero cell as cultivating the cell of viral DENV, MOI is 0.1, and experimental procedure is as follows:
1. access the Vero cell in 24 porocyte culture plates, cell grows to monolayer (area at the bottom of the cell coverage hole is about 80% ~ 90%) after 24 hours, the sucking-off culture medium, and access virus sample 200ul, 37 ℃ adsorbed 2 hours.After absorption is finished, inhale the virus liquid abandon in each hole, the virus of not adsorbing with DMEM culture medium flush away.Add with containing 10%(v/v) the Aphanamixoid A of the prescribed concentration (0 uM, 0.04 uM, 0.12 uM, 0.37 uM, 1.1 uM, 3.3 uM, 10.0 uM) of the DMEM culture medium dilution of hyclone, in 37 ℃, 5%(v/v) CO
2Incubator in cultivated 42 hours, collect the supernatant in each hole, do viral plaque experiment.
2. viral plaque experiment: access Vero cell in 24 orifice plates, cell grows to monolayer (area at the bottom of the cell coverage hole is about 80% ~ 90%) after 24 hours, the culture medium in each hole is abandoned in suction, access is with containing 3%(v/v) the virus sample 200ul of 10 times of serial dilutions of DMEM culture medium of hyclone, 37 ℃ of absorption 2 hours.After absorption is finished the supernatant plate in each hole inhaled and abandons, use 10%(v/v) the DMEM culture medium flush away of the FBS virus of not adsorbing.Add 45 ℃ of fresh preheatings semifixed culture medium [the A composition: 3%(v/v) FBS, 2 * MEM culture medium, penicillin and streptomycin final concentration are respectively 100U/ml, 0.1mg/ml; B composition: agarose 1%(w/v) (French Biowest).The A composition: the B composition=1:1(v/v)], in 37 ℃, 5%(v/v) CO
2Incubator in cultivated 48 ~ 72 hours.Behind plaque formation, be dissolved in 10%(v/v with crystal violet (U.S. AMRESCO) dyeing (2%(w/v) crystal violet) formaldehyde), wash away crystal violet and agarose with flowing water after 2 hours, at the cell plaque of microscopically to the generation of every hole, calculate viral titre (PFU/ml, PFU:plaque formation unit plaque forming unit) according to plaque number and extension rate.PFU=viral dilution degree * P/V, (P: cavitation corrosion speckle number; V: inoculum concentration).
3. experimental result sees Table 2.
Conclusion: Aphanamixoid A has very strong inhibitory action to dengue virus, and safety, so Aphanamixoid A has widely application background in treatment dengue virus infection disease.
Claims (1)
1. the application of Aphanamixoid A in preparation treatment or prevention dengue virus infection medicine, described compd A phanamixoid A structure is shown in formula I:
Formula I.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2011002635A1 (en) * | 2009-06-30 | 2011-01-06 | Siga Technologies, Inc. | Treatment and prevention of dengue virus infections |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011002635A1 (en) * | 2009-06-30 | 2011-01-06 | Siga Technologies, Inc. | Treatment and prevention of dengue virus infections |
Non-Patent Citations (1)
| Title |
|---|
| JIE-YUN CAI, ET AL.: "Aphanamixoid A, a Potent Defensive Limonoid, with a New Carbon Skeleton from Aphanamixis polystachya", 《ORGANIC LETTERS》 * |
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Application publication date: 20130327 |