CN102755335A - Application of triptolide in preparation of medicament for treating or preventing human immunodeficiency viruses (HIV) - Google Patents
Application of triptolide in preparation of medicament for treating or preventing human immunodeficiency viruses (HIV) Download PDFInfo
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- CN102755335A CN102755335A CN2012102438952A CN201210243895A CN102755335A CN 102755335 A CN102755335 A CN 102755335A CN 2012102438952 A CN2012102438952 A CN 2012102438952A CN 201210243895 A CN201210243895 A CN 201210243895A CN 102755335 A CN102755335 A CN 102755335A
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Abstract
The invention discloses an application of triptolide in preparation of a medicament for treating or preventing human immunodeficiency viruses (HIV). The triptolide is diterpenoid naturally existing in roots of tripterygium wilfordii hook, and can dose-dependently inhibit replication of I type HIV (HIV-1) in cells in vitro. The half inhibitory concentrations on HIV-1 inhibition in TZM-b1 cells, JurkatT lymphocytes and human peripheral blood mononuclear cells are respectively 0.32nM, 0.45nM and 1.1nM. The triptolide has a remarkable inhibiting effect on the replication of the HIV-1 in the TZM-b1 cells, the JurkatT lymphocytes and the human peripheral blood mononuclear cells; and the triptolide is an active ingredient in Chinese medicinal tripterygium wilfordii hook, so the triptolide is wide in source. The compound has a broad prospect for developing anti-HIV-1 medicaments.
Description
Technical field
The present invention relates to belong to medical technical field, more specifically relate to the application of a kind of triptolide in the preparation treatment or the cytotoxic drug that prevents AIDS.
Background technology
AIDS is claimed AIDS (acquired immunodeficiency syndrome again; AIDS); Be a kind of by HIV (human immunodeficiency virus; HIV) causing, is the infectious disease of characteristic with the systemic immune system grievous injury.2007, the whole world had 3,300 ten thousand people and carries HIV, and newly-increased the infected 2,700,000,2,000,000 people die from AIDS-related diseases.China's AIDS is the acceleration fashion trend, and HIV propagates to the general population, and the HIV the infected and the HIV sufferers of survival in 2009 reach 740,000 people.Human health and development of global economy in the popular serious threat of AIDS.
HIV is a RNA viruses, belongs to Retroviridae, lentivirus.In the course of infection of HIV, the outer membrane glycoprotein gp120 of HIV-1 combines with the CD4 receptor-specific of T4 lymphocytic cell surface and is adsorbed in cell.Under the assistance of complementary receptor CCR 5 or CXCR4, the outer membrane glycoprotein gp41 of HIV-1 merges with host cell membrane and makes in the virion entering cell, and sloughs capsid then and expose nucleic acid.Under the catalysis of viral reverse transcriptase and ribonuclease H, HIV-1 single-stranded RNA reverse transcription becomes single-stranded DNA, and and then in nucleus, is catalyzed into distrand DNA by the cell polymerase.The intergrase of HIV-1 is integrated in the chromosome of host cell the distrand DNA provirus, forms the latent infection of HIV-1.With the viral DNA of integrating is template and the mRN0A of synthetic virus translates big fusion polyprotein, and then under the virus protease effect, these polyproteins are formed sophisticated virus structural protein by montage and processing.At last, viral nucleic acid combines with structural protein, assembles the virion that makes new advances, and is discharged into the extracellular with the mode of sprouting.
Be used for the clinical treatment that HIV-1 infects first in 1987 from zidovudine (AZT); So far have the clinical use that goes through of 25 kinds of medicines; Comprising 8 kinds of nucleoside and ucleotides RTI; 4 kinds of non-nucleoside reverse transcriptases suppress 10 kinds of albumen enzyme inhibitors, 2 kinds of viral entry inhibitors and a kind of integrase inhibitor.Be prone to single antiviral agents deposits yields drug resistance to HIV, highly active antiretroviral therapy (highly active antiretroviral therapy, HAART)) becomes the method for clinical widely used treatment AIDS at present.But this kind therapy costs an arm and a leg, and toxic and side effects is big, can not thoroughly remove the intravital HIV of people, and is prone to cause that virus produces variability and drug resistance.Therefore, searching has the newtype drug that new action target spot, overriding resistance and price are low, curative effect is good, toxicity is little has become the trend that present inverase is studied.
The native compound in Chinese medicine and medicinal plants source has characteristics such as structure diversity, toxicity are lower, wide material sources, thereby special advantages and great potential is arranged aspect the AIDS preventing and treating.Researcher to native compound anti-HIV effect carried out big quantity research, and found that native compounds such as some alkaloids, coumarin, lignanoid, flavonoid, terpenoid, tannin class, polysaccharide, protein and polypeptide class have the activity of anti-HIV.Triptolide (triptolide) is one and has multiple bioactive diterpenoid-lactone; Derive from the root of Chinese medicine Radix Tripterygii Wilfordii (Tripterygium wilfordii Hook.f.), it is the main effective ingredient of preparations such as treatment rheumatoid disease Radix Tripterygii Wilfordii tablet, Glucosidorum Tripterygll Totorum.Research now shows that it has antioxidation, anti-inflammatory, effects such as immunosuppressant and antitumor.At present, triptolide anti-HIV-1 activity is not also appeared in the newspapers.
Summary of the invention
The objective of the invention is to be to provide the application of a kind of triptolide in preparation treatment HIV medicine.Triptolide has significant inhibitory effect to duplicating of HIV-1 in TZM-b1 cell, Jurkat T lymphocyte and human peripheral blood single nucleus cell; Triptolide is the effective ingredient in the Chinese medicine Radix Tripterygii Wilfordii, wide material sources; Triptolide can carry out therapeutic alliance with present existing anti-HIV-1 medicine.
In order to prove that triptolide is used to treat the feasibility of HIV infection, the present invention adopts following technical measures:
Triptolide can rely on ground inhibition HIV-1 duplicating in cell by dosage under vitro conditions.The half-inhibition concentration (EC50) that I type HIV is duplicated in TZM-b1 cell, Jurkat T lymphocyte and human peripheral blood single nucleus cell is respectively 0.32nM, 0.45nM and 1.1nM.
Described triptolide has remarkable inhibition HIV and duplicates effect; Can be used as active component and process any pharmaceutically acceptable dosage forms such as tablet, capsule, granule, oral liquid, slow releasing agent, controlled release preparation, nanometer formulation, injection, be used to treat HIV-1 the infected and HIV sufferers.Triptolide is used for antiviral therapy except processing single agent, also can in therapeutic alliance, have administration simultaneously of the active chemical compound of anti AIDS virus or separate administration with other, or comes administration through each chemical compound being combined into the combination medicine.
According to report, the chemical structural formula of triptolide is:
Wherein: the O represention oxygen atom, H represents hydrogen atom.
The present invention compared with prior art has the following advantages and effect:
1. triptolide has significant inhibitory effect to duplicating of HIV-1 in TZM-b1 cell, Jurkat T lymphocyte and human peripheral blood single nucleus cell, and half-inhibition concentration is respectively 0.32nM, 0.45nM and 1.1nM;
2. triptolide is the effective ingredient in the Chinese medicine Radix Tripterygii Wilfordii, wide material sources;
3. triptolide can carry out therapeutic alliance with present existing anti-HIV-1 medicine, has the bright prospects that are developed to the anti-HIV-1 medicine.
Description of drawings
Fig. 1 is cytotoxicity and the anti-HIV-1 active sketch map of a kind of triptolide in the TZM-b1 cell.
Variable concentrations medicine and TZM-b1 co-culture of cells 48 hours are used CellTiter Glo reagent to carry out cytoactive and are detected; The TZM-b1 co-culture of cells that variable concentrations medicine and HIV-1NL4-3 infect 48 hours uses Steady-Glo reagent to detect the virus replication level.
Fig. 2 is cytotoxicity and the anti-HIV-1 active sketch map of a kind of triptolide in the JurkatT lymphocyte.
Variable concentrations medicine and JurkatT lymphocyte were cultivated 48 hours altogether, used CellTiter Glo reagent to carry out cytoactive and detected; The JurkatT lymphocyte of variable concentrations medicine and HIV-1 single-wheel report viral infection was cultivated 48 hours altogether, used Steady-Glo reagent to detect the virus replication level.
Fig. 3 is the cytotoxicity sketch map of a kind of triptolide in human peripheral blood single nucleus cell.
Variable concentrations medicine and human peripheral blood single nucleus cell were cultivated 6 days altogether, used CellTiter Glo reagent to carry out cytoactive and detected.
Fig. 4 is the active sketch map of the anti-HIV-1 of a kind of triptolide in human peripheral blood single nucleus cell.
The human peripheral blood single nucleus cell that variable concentrations medicine and HIV-1NL4-3 infect is hatched altogether, after 2,4,6,8 days, uses p24ELISA to detect HIV-1p24 antigenic content in the culture supernatant.
The specific embodiment
Embodiment 1: cytotoxicity and the anti-HIV-1 of triptolide in the TZM-b1 cell is active
In the present embodiment, use HIV-1NL4-3 to infect TZM-b1 cell (from state-run allergy of the U.S. and infectious disease institute).The TZM-b1 cytotostatic is expressed HIV-1 receptor CD4, and co-receptor CXCR4 and CCR5, and in genome, is integrated with the reporter gene LUC Photinus pyralis LUC Photinus pyralis FL that receives HIV-1 promoter L TR control.The levels of replication that the expression of reporter gene can indicator virus behind the viral infection.In the experiment; Use Cell TiterGlo (Promega Company products) thus reagent comes the pair cell activity to carry out the cytotoxicity that detection by quantitative is confirmed triptolide, SteadyGlo (Promega Company products) detects by the expression of reporter gene in the infection cell.Concrete experimental technique is following:
Cytotoxicity experiment: trypsinization exponential phase TZM-b1 cell, adjustment cell density to 1 * 10
5/ mL, every hole 100 μ L cell suspension add 96 orifice plates.Second day, the medicine 50 μ L of 3 times of gradient dilutions of adding, 3 repetitions of every concentration, matched group does not add any medicine.After culture plate is put incubator 48h, add cytoactive detectable Cell TiterGlo 100 μ L, behind the level concussion 10min, Envision2102 multiple labeling detector detects chemiluminescence signal.The cytoactive computational methods are relatively:
Cytoactive (%)=(drug treating-acellular contrast) * 100%/(cell contrast-acellular contrast)
The antiviral activity experiment: HIV-1 cDNA clone pNL4-3 (from state-run allergy of the U.S. and infectious disease institute) transfection 293T (available from American type culture collection) cell obtains HIV-1NL4-3.Trypsinization exponential phase TZM-b1 cell, adjustment cell density to 1 * 10
5/ mL, every hole 100 μ L cell suspension add 96 orifice plates.Second day, the medicine 50 μ L of 3 times of gradient dilutions of adding, 3 repetitions of every concentration, matched group does not add any medicine.Add the NL4-3 virus storage liquid 50 μ L of suitably dilution again, guarantee that infection multiplicity is 1, do not infect matched group and do not add virus.After culture plate is put incubator 48h; 100 μ L supernatants are abandoned in every hole; And adding uciferase activity detectable SteadyGlo (Promega Company products) 100 μ L; Behind the level concussion 10min, Envision2102 multiple labeling detector (Perkin Elmer Company products) detects chemiluminescence signal.Viral relatively levels of replication computational methods are:
Virus replication (%)=(drug treating-infect contrasting) * 100%/(viral infection contrast-infect contrasting)
As shown in Figure 1, under the concentration that does not influence cytoactive, triptolide can dosage relies on to such an extent that suppress HIV-1NL4-3 duplicating in TZM-b1 (EC50=0.32nM).Under 5.6nM, Radix Tripterygii Wilfordii reaches 97.9% to the suppression ratio of virus replication.
Embodiment 2: cytotoxicity and the anti-HIV-1 of triptolide in Jurkat T lymphocyte is active
In the present embodiment, the applicant utilizes HIV-1 single-wheel dubbing system to verify that the anti-HIV-1 of triptolide is active.In this system, the HIV-1 pseudovirus that is enclosed with the VSV-G envelope protein infects Jurkat cell (available from American type culture collection).Owing to have reporter gene on the viral genome, so the levels of replication that the expression of reporter gene can indicator virus.Experimental technique is following:
Method for using according to Lipofectamine2000 (Roche Company products); PNL4-3.Luc.R-E-(from state-run allergy of the U.S. and infectious disease institute) and pVpack-VSV-G (Stratagene Company products) with the quality of 2:1 than cotransfection 293T cell; Collect supernatant behind the 48h, 0.45 μ M membrane filtration and packing are stored in-80 ℃.
The Jurkat cell is with 4 * 10
4The density in/hole is laid on 96 orifice plates, adds the chemical compound and the HIV-1 single-wheel infective virus of suitably dilution afterwards, and final volume is 200 μ L.After cultivating 48h, 100 μ L supernatants are abandoned in every hole, and add uciferase activity detectable SteadyGlo100 μ L, and behind the level concussion 10min, Envision2102 multiple labeling detector detects chemiluminescence signal.The suppression ratio computational methods are:
Suppression ratio (%)=(viral infection contrast-sample) * 100%/(viral infection contrast-infect contrasting)
In the experiment of parallel detection cell, cell is without viral infection, and other treatment conditions are consistent.The cytoactive computational methods are relatively:
Cytoactive (%)=(sample-acellular contrast) * 100%/(no medicine contrast-acellular contrast)
The result is as shown in Figure 2, and under the concentration that does not influence cytoactive, triptolide can dosage relies on to such an extent that suppress HIV-1NL4-3 duplicating in the JurkatT lymphocyte (EC50=0.45nM).Under the situation that the 5nM triptolide exists, duplicating of HIV-1 receives 87.9% inhibition.
Embodiment 3: cytotoxicity and the anti-HIV-1 of triptolide in PMNC (PBMCs) is active
In the present embodiment, the applicant has detected the antivirus action of triptolide to HIV-1NL4-3 in PBMCs, and experimental technique is following:
Use lymphocyte separation medium Ficoll-Hypaque PREMIUM1.073 (GE Company products) to separate the human peripheral blood single nucleus cell that secures good health.Cell is cultivated in the RMPI1640 (Gibco Company products) that contains 50ng/mL IL-2 (Sigma-Aldrich Company products) and 10%FBS (Gibco Company products).Before the experiment, PBMCs stimulates 72h through phytohemagglutinin (5 μ g/mL, Sigma-Aldrich Company products).HIV-1NL4-3 infection of PBMCs s, MOI=0.01.Cell is with 2 * 10
5The density in/hole is inoculated in 96 orifice plates, adds suitably medicine to the cumulative volume 200 μ L of dilution.Behind every cultivation 48h, take out 100 μ L supernatants and be used for the p24 detection, and add 100 μ L fresh mediums and continue to cultivate.
In the experiment of parallel assay toxicity of compound, cell is without viral infection, and other treatment conditions are the same.When being cultured to 6d, use CellTiter Glo to detect cytoactive.
The result shows: after triptolide and PBMCs cultivate 6 days altogether, and the survival of the following concentration pair cell of 4nM influence, when concentration reached 8nM, cytoactive was suppressed, and was 74.4% of no medicine contrast.Detecting virus replication level (p24) finds: triptolide can dosage relies on to such an extent that suppress duplicating of HIV-1.On the peak (the 6th day) of virus replication, 8nM can suppress virus replication fully.Under avirulence concentration, the p24 of HIV-1 produces and has also received remarkable inhibition (EC
50=1.1nM), the inhibition ratio of 4nM reaches more than 93%.
Claims (2)
1. the application of triptolide in the preparation treatment or the cytotoxic drug that prevents AIDS.
2. application according to claim 1 is characterized in that: described triptolide is an active constituents of medicine, processes acceptable forms on any pharmaceutics of tablet, capsule, granule, oral liquid, slow releasing preparation, controlled release preparation or injection.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102908443A (en) * | 2012-11-15 | 2013-02-06 | 宋福德 | Medicinal composition for therapy or adjuvant therapy of aids, preparation method of medicinal composition and application of medicinal composition |
| CN103083334A (en) * | 2012-12-20 | 2013-05-08 | 李太生 | Novel application of tripterygium glycosides in treatment of diseases relative to incompetent immune reconstitution and non-AIDS (Acquired Immune Deficiency Syndrome) and incompetent immune reconstitution of HIV (Human Immunodeficiency Virus)/AIDS patient |
| CN107753546A (en) * | 2017-11-27 | 2018-03-06 | 中国医学科学院北京协和医院 | Application of the tripterygium wilfordii in the medicine for preparing feature treatment of AIDS |
| CN114230630A (en) * | 2021-11-16 | 2022-03-25 | 郑州人民医院(郑州人民医院医疗管理中心) | Triptolide derivative and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1465340A (en) * | 2002-06-24 | 2004-01-07 | 成都思摩纳米技术有限公司 | Process for preparing thunder godvine nano particle |
| CN101015550A (en) * | 2006-02-08 | 2007-08-15 | 福建省医学科学研究所 | Preparing method of triptolide molecule microcapsule |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1465340A (en) * | 2002-06-24 | 2004-01-07 | 成都思摩纳米技术有限公司 | Process for preparing thunder godvine nano particle |
| CN101015550A (en) * | 2006-02-08 | 2007-08-15 | 福建省医学科学研究所 | Preparing method of triptolide molecule microcapsule |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102908443A (en) * | 2012-11-15 | 2013-02-06 | 宋福德 | Medicinal composition for therapy or adjuvant therapy of aids, preparation method of medicinal composition and application of medicinal composition |
| CN102908443B (en) * | 2012-11-15 | 2014-11-26 | 宋福德 | Medicinal composition for therapy or adjuvant therapy of aids, preparation method of medicinal composition and application of medicinal composition |
| CN103083334A (en) * | 2012-12-20 | 2013-05-08 | 李太生 | Novel application of tripterygium glycosides in treatment of diseases relative to incompetent immune reconstitution and non-AIDS (Acquired Immune Deficiency Syndrome) and incompetent immune reconstitution of HIV (Human Immunodeficiency Virus)/AIDS patient |
| CN103083334B (en) * | 2012-12-20 | 2014-12-17 | 李太生 | Novel application of tripterygium glycosides in treatment of diseases relative to incompetent immune reconstitution |
| CN107753546A (en) * | 2017-11-27 | 2018-03-06 | 中国医学科学院北京协和医院 | Application of the tripterygium wilfordii in the medicine for preparing feature treatment of AIDS |
| CN107753546B (en) * | 2017-11-27 | 2021-06-29 | 中国医学科学院北京协和医院 | Application of tripterygium wilfordii in preparation of medicine for functionally curing AIDS |
| CN114230630A (en) * | 2021-11-16 | 2022-03-25 | 郑州人民医院(郑州人民医院医疗管理中心) | Triptolide derivative and application thereof |
| CN114230630B (en) * | 2021-11-16 | 2024-03-08 | 郑州人民医院(郑州人民医院医疗管理中心) | Triptolide derivative and application thereof |
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