CN103381162A - Applications of Chukrasone A in medicines used for treating dengue virus infection - Google Patents
Applications of Chukrasone A in medicines used for treating dengue virus infection Download PDFInfo
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Abstract
The invention discloses applications of Chukrasone A in medicines used for treating dengue virus infection. Chukrasone A is capable of inhibiting dengue virus multiplication effectively, and possesses little toxicity on cells, so that Chukrasone A can be further used for preparation of medicines used for treating diseases caused by dengue virus infection, and the application prospect is promising. The applications of Chukrasone A in preparation of medicines used for treating or preventing dengue virus infection are disclosed for the first time. The molecular skeleton is novel, so that inhibitory activity on dengue virus is surprisingly strong, and it is impossible to estimate the inhibitory activity according to the activities of other compounds. Chukrasone A possesses outstanding substantiality, and the applications of Chukrasone A in treatment and prevention of dengue virus infection possess significant advantages.
Description
Technical field
The invention belongs to biomedicine field, more specifically relate to the application of a kind of Chukrasone 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 is separated to dengue virus in the mosquito body.But, also there is no 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 Compound C hukrasone A that the present invention relates to is one and delivered (Liu in 2012, H. B. et al., 2012. Chukrasones A and B:Potential Kv1.2 Potassium Channel Blockers with New Skeletons from Chukrasia tabularis. Organic Letters 14 (17), 4438 – 4441.) New skeleton compound, this compound has brand-new framework types, present purposes only relates to potassium-channel and suppresses active (Liu, H. B. et al., 2012. Chukrasones A and B:Potential Kv1.2 Potassium Channel Blockers with New Skeletons from Chukrasia tabularis. Organic Letters 14 (17), 4438 – 4441.), belong to open first for the purposes of the Chukrasone 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 compounds, possesses 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 Chukrasone A in treating as preparation or preventing the dengue virus infection medicine, Chukrasone A depressed rate to DENV under 10.0uM concentration is 94.95%.And Chukrasone A is under the 100uM condition in concentration, and the survival rate of Vero cell is 92.93%.Chukrasone 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 Compound C hukrasone A structure is as shown in formula I:
Formula I
The purposes of the Chukrasone 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 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, the toxicity test of Chukrasone A to the Vero cell:
The Vero cell is the permissive cell of DENV.Therefore, at first this experiment detects Chukrasone A to the cytotoxicity of Vero cell, Chukrasone A with variable concentrations acts on the Vero cell, understanding at cell survival rate is the maximum working concentration of the Chukrasone A more than 90%, and for follow-up Chukrasone A, the inhibitory action of DENV being tested provides reference data.
2, the inhibition experiment of Chukrasone A to DENV:
Chukrasone A with variable concentrations acts on the Vero cell that has infected DENV, obtains Chukrasone A to the suppression efficiency of virus.The working concentration of Chukrasone 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 Chukrasone A and the analysis of experimental data is exerted an influence.
(inhibition) of Chukrasone A in treatment or prevention dengue virus infection medicine used, and its basic process is:
A. the toxicity test of Chukrasone A to the Vero cell:
Vero cell (African green monkey kidney cell) is the permissive cell of DENV.Vero cell in experiment is bought the Shanghai cell data center of Sheng Ke institute in 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 100ul;
2: Cultivation of Vero: at 37 ℃, 5%(v/v) CO
2Under condition of culture, cultivated 2 days;
3: add Chukrasone A: inhale the DMEM culture medium of abandoning in each hole, adding 100 ul 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) Chukrasone 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 condition of culture 4 hours, and then added the Formazan lysate, at 37 ℃, 5%(v/v) CO
2Continued to hatch under condition of culture 4 hours, and found that Formazan all dissolved until observe under ordinary optical microscope;
(1): measure: measure absorption value at 570nm.
The toxicity test of the Chukrasone A of table 1 variable concentrations to the Vero cell
B. the inhibition experiment of Chukrasone A to 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, after 24 hours, cell grows to monolayer (area at the bottom of the cell coverage hole is about 80% ~ 90%), the sucking-off culture medium, access virus sample 200ul, 37 ℃ adsorbed 2 hours.After absorption is completed, inhale the virus liquid of abandoning in each hole, wash away the not virus of absorption with the DMEM culture medium.Add with containing 10%(v/v) the Chukrasone 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, after 24 hours, cell grows to monolayer (area at the bottom of the cell coverage hole is about 80% ~ 90%), the culture medium in each hole is abandoned in suction, access is with containing 3%(v/v) virus sample 200 ul of 10 times of serial dilutions of DMEM culture medium of hyclone, 37 ℃ of absorption 2 hours.After absorption is completed, the supernatant plate in each hole is inhaled and is abandoned, use 10%(v/v) the DMEM culture medium of FBS washes away the 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.Be dissolved in 10%(v/v with crystal violet (U.S. AMRESCO) dyeing (2%(w/v) crystal violet after plaque formation) 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 Chukrasone 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 Chukrasone A DENV is had good inhibitory action, have application background widely from having proved in essence this medicine treatment DENV catches.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 Chukrasone 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 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 Compound C hukrasone A involved in the present invention is referring to document (Liu, H. B. et al., 2012. Chukrasones A and B:Potential Kv1.2 Potassium Channel Blockers with New Skeletons from Chukrasia tabularis. Organic Letters 14 (17), 4438 – 4441.).
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 Compound C hukrasone A tablet involved in the present invention:
Get 20 and digest compound Chukrasone A, add conventional adjuvant 180 grams that prepare tablet, mixing, conventional tablet machine are made 1000.
Embodiment 2: the preparation of Compound C hukrasone A capsule involved in the present invention:
Get 20 and digest compound Chukrasone A, add the conventional adjuvant such as starch 180 grams that prepare capsule, mixing is encapsulatedly made 1000.
Further illustrate its pharmaceutically active below by pharmacodynamic experiment.
Experimental example 1:
Chukrasone A uses as (inhibition) in preparation treatment or prevention dengue virus infection medicine, and its basic process is:
A. the toxicity test of Chukrasone A to the 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 condition of culture, cultivated 2 days;
3: add Chukrasone 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) Chukrasone 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 condition of culture 4 hours, and then added the Formazan lysate, at 37 ℃, 5%(v/v) CO
2Continued to hatch under condition of culture 4 hours, and found that Formazan all dissolved until observe under ordinary optical microscope;
5: measure: measure absorption value at 570nm.
6: experimental result sees Table 1.
B. the inhibitory action of Chukrasone A to 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, after 24 hours, cell grows to monolayer (area at the bottom of the cell coverage hole is about 80% ~ 90%), the sucking-off culture medium, and access virus sample 200ul, 37 ℃ adsorbed 2 hours.After absorption is completed, inhale the virus liquid of abandoning in each hole, wash away the not virus of absorption with the DMEM culture medium.Add with containing 10%(v/v) the Chukrasone 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, after 24 hours, cell grows to monolayer (area at the bottom of the cell coverage hole is about 80% ~ 90%), 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 completed, the supernatant plate in each hole is inhaled and is abandoned, use 10%(v/v) the DMEM culture medium of FBS washes away the 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.Be dissolved in 10%(v/v with crystal violet (U.S. AMRESCO) dyeing (2%(w/v) crystal violet after plaque formation) 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: Chukrasone A has very strong inhibitory action to dengue virus, and safety, so Chukrasone A has application background widely in treatment dengue virus infection disease.
Claims (1)
1. the Chukrasone A application in treatment or prevention dengue virus infection medicine, described Compound C hukrasone A structure as
Formula IShown in:
Formula I.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1342156A (en) * | 1998-08-25 | 2002-03-27 | 耶鲁大学 | Inhibition and treatment of hepatitis B virus and flavivirus by helioxanthin and its analogs |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1342156A (en) * | 1998-08-25 | 2002-03-27 | 耶鲁大学 | Inhibition and treatment of hepatitis B virus and flavivirus by helioxanthin and its analogs |
Non-Patent Citations (1)
| Title |
|---|
| HONG-BING LIU等: "Chukrasones A and B: Potential Kv1.2 Potassium channel blockers with new skeletons from chukrasia tabularis", 《ORGANIC LETTERS》, vol. 14, no. 17, 31 December 2012 (2012-12-31), pages 4438 - 4441 * |
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Application publication date: 20131106 |