CN1197570C - Application of porcelain ampelopsis in preparation of medicine for preventing and treating AIDS - Google Patents

Abstract

The present invention relates to an application of porcelain ampelopsis in the preparation of an AIDS prevention and cure medicament. The in vitro anti-HIV-1 function of porcelain ampelopsis, and the regulation function of the porcelain ampelopsis to a cellular immunity function in an immunity inhibition model of a mouse are researched by the present invention. The protective functions of the porcelain ampelopsis to an HIV-1 infection human PBMCs cell and an MT-4 cell, and the inhibition function of the porcelain ampelopsis to the accessory receptor CXCR4 of HIV-1 are observed, which indicates that the porcelain ampelopsis has the in vitro anti HIV function. The CXCR4 at a PBMCs surface can be lowered, and the immunity function of the immunity inhibition model of the mouse can be enhanced, which indicates that the porcelain ampelopsis has the function for preventing and curing AIDS. The porcelain ampelopsis can be used for preparing a medicament for preventing and curing the AIDS.

Description

Ampelopsin is prevented and treated application in the AIDS-treating medicine in preparation

Technical field

The present invention relates to a kind of medicine of preventing and treating acquired immune deficiency syndrome (AIDS), specifically ampelopsin is prevented and treated application in the AIDS-treating medicine in preparation.

Background technology

Acquired immune deficiency syndrome (AIDS) is that acquired immune deficiency syndrome (AIDS) (AIDS) is the disease that is caused by human immunodeficiency virus (HIV).World Health Organization (WHO) estimates recently, ends to the end of the year 2000, and the whole world has added up total infected by HIV number 5,790 ten thousand, and the number of dying from AIDS has reached 2,180 ten thousand, and new infected by HIV in 2000 has 5,300,000, dies from AIDS and reaches 3,000,000.China estimates that at present existing 60～1,000,000 people infect, and have now entered the quick rise period.AIDS is a kind of Social disease, and involvement aspect is quite wide, and is very big to its prevention and control difficulty.Though adopted highly active antiretroviral therapy in recent years, AIDS patient's symptom after taking medicine is improved, the cd4 cell number rises, and virus load reduces, and treatment costs an arm and a leg, and these medicines have following shortcoming: (1) induces the HIV variant that develops immunity to drugs; (2) short-term and secular toxic and side effects; (3) can not block the early infection process of HIV.Because the HIV variation is fast, the strain of different regions also may be different, makes the development difficulty of vaccine very big.Therefore, seeking to have and prevent the high-efficiency low-toxicity medicine that HIV infects, control HIV duplicates, treats the AIDS pathological changes, is the urgent problem of global anti-HIV/AIDS research.

Ampelopsin has another name called dihydromyricetin, belongs to flavone compound, is the main composition of Vitaceae ampelopsis (as Radix Ampelopsis Cantoniensis, Ampelopsis grossedentata etc.).Radix Ampelopsis Cantoniensis is the root or the full root of ampelopsis cantoniensis [Ampelopsiscantoniensis (HooK.et Arn.) Planch], and main product is one band in China south China, is the folk tradition medication." Chinese medicine voluminous dictionary " record, the function of its tool eliminating inflammation and expelling toxin, it is swollen etc. to cure mainly osteomyelitis, acute lymphoblastic inflammation, acute parotitis, pustule disease, wet disease erysipelas, scabies.Ampelopsis grossedentata is the goods of Ampelopsis grossedentata [Ampelopsis grossedentata (Hand.-Mazz.) W.T.Wang].Begin ampelopsin from the seventies and cause people's attention gradually, it is extracted purification, isolation identification, structural analysis etc. carried out more complete research.Chinese patent application 00117225.5 has reported that it has antineoplastic action, and Chinese patent application 01109641.1 has been reported Ampelosis general flavone composition, and wherein the Fructus Vitis viniferae element accounts for 10～70%, has hepatoprotective effect.Up to now, do not see have ampelopsin to prevent and treat the report of using in the AIDS-treating medicine in preparation.

Summary of the invention

Purpose of the present invention is exactly in order to provide prevention, treatment acquired immune deficiency syndrome (AIDS) is effective, toxicity is little ampelopsin to prevent and treat application in the AIDS-treating medicine in preparation.

The present invention is achieved in that

Ampelopsin can adopt Radix Ampelopsis Cantoniensis to extract according to a conventional method, also can take the mode of synthetic to prepare.

Ampelopsin of the present invention can adopt any dosage form that allows on the pharmaceutics, comprises peroral dosage form such as tablet, capsule, oral liquid etc., also can adopt injection, as transfusion, powder pin etc., can also adopt exterior-applied formulation, as ointment etc.When adopting peroral dosage form and exterior-applied formulation, ampelopsin can adopt the mode of the not purified mixed extract of the water extract of Radix Ampelopsis Cantoniensis or ethanol extract.Also can adopt other to contain the extract of the plant of ampelopsin.

Ampelopsin of the present invention can be made compound preparation with other medicines of preventing and treating acquired immune deficiency syndrome (AIDS), as the AZT of drugs approved by FDA listing.

The use amount adult who adopts can be 10mg～20mg/ time, every day two～three times.

Only express CD ₄Non-human archeocyte film but not hiv-1-infected or that virus envelope protein mediation do not take place merge this prompting people CD ₄Still be not enough to cause the HIV-1 target cell infection.Along with suppressing HIV-1, RANTES chemotactic factors (chemokine) such as (regulated upon activation normal T cell expressedand secreted) infects CD ₄ ⁺The discovery of cell, people begin to turn one's attention to chemotactic factor and receptor thereof.Very fast Feng etc. utilizes functional cDNA clone's method, and the receptor of having found to be called fusin is that HIV-1 enters the essential accessory receptor of cell (co-receptor).People found its unique ligands specific SDF-1 (stromal cell-derived factor 1) afterwards, and it is included into chemokine receptors family, called after CXCR4.This discovery becomes the milestone of acquired immune deficiency syndrome (AIDS) research, a few years subsequently, and people find successively that again a series of chemokine receptors such as CCR5, CCR2 play a key effect in HIV infects.It has been recognized that at present the HIV initial infection is a macrophage preferendum virus (M-tropic), it mainly is accessory receptor with CCR5, along with HIV changes T cell tropism (T-tropic) and two preferendum strain (Dual-tropic) into, the accessory receptor that it utilized also is converted into based on CXCR4.This just provides new thinking for setting about preventing and treating AIDS from the initial stage approach of HIV invasion cell.

Chemotactic factor is by its corresponding receptor functionating.Not only in the removing of host to pathogenic factor, inflammatory reaction and anaphylactic disease, aspects such as the growth of immunocyte, differentiation and immune homeostasis all play an important role, and work at cardiovascular cell development, injury repairing and tumor growth and deterioration.Micromolecular compounds such as chemotactic factor and derivant thereof, chemokine receptors monoclonal antibody, AMD3100 are by the mediation receptor down-regulated or directly block HIV-1 and receptors bind, and play the effect that anti-HIV-1 infects.

More clearly based on the process of HIV being invaded target cell, mainly tend in the world at present study from HIV is infected commitment.As with the core texture of HIV envelope protein gp41, the CD of HIV ₄Molecular receptor and CXCR4 and CCR5 accessory receptor etc. are as the novel targets of new drug research, gene therapy and vaccine development.The accessory receptor of HIV intrusion target cell is different with the difference in the stage of infection.At the HIV-1 of initial infection is macrophage preferendum (M-tropic) virus (R5), and it mainly is accessory receptor with the chemokine receptor CCR 5, infects macrophage and former generation CD ⁺ ₄The T cell, but can not in the T cell that transforms, duplicate.Along with after HIV changes T cell tropism (T-tropic) strain (X4) and two preferendum strain (R5X4) into, its accessory receptor also transfers to based on CXCR4.Have been found that β class chemotactic factor RANTES, MIP-1 α, MIP-1 β can suppress the intrusion of M-tropic virus, the ligands specific SDF-1 of CXCR4 then can strong inhibition HIV-1 infection of PBMCs s and HeLa-CD4 cell.Nearest useful transformed chemotactic factor Aop-RANTES, Met-SDF-1 β unite as the inhibitor of CCR5 or CXCR4 makes the mixed infection that is used for blocking R5 and X4 type HIV-1.But chemotactic factor can cause inflammatory reaction, and the host is produced adverse effect.The gp120 of all HIV viruses all has and CD ₄With the bonded conservative site of CCR5, this just provides possibility for the infection of the multiple Strain of preparation blocking-up simultaneously.As utilizing natural chemotactic factor, polypeptide small molecule etc. and corresponding receptors bind, receptor capable of blocking combines with gp120/41.The albumen Trichosanthin (TCS) that extracts from the Chinese medicine Radix Trichosanthis can suppress HIV duplicating in T lymphocyte that has infected and mononuclear phagocyte, has been used as the medicine for the treatment of AIDS.Recently find that TCS can improve RANTES and the chemotaxis and the G protein active of SDF-1 α in lymphocyte widely, TCS plays mediation by the interaction with CCR5 or CXCR4.And for example newfound several micromolecular compounds are the CCR5 inhibitor that selectivity suppresses R5 type HIV-1 as TAK-779, the Ca of blocking-up CCR5 mediation ²⁺Signal; And AMD3100 is the inhibitor of X4 type HIV-1 invasion target cell.Exclusive inhibitor peptide T140 and analog thereof have been synthesized recently to CXCR4.Therefore accessory receptor one chemokine receptors of HIV can be used as an effective novel targets of anti-HIV of research and AIDS medicine.

Based on this, the present invention has investigated the effect of the external anti-HIV-1 of ampelopsin, investigated effect to HIV-1 accessory receptor CXCR4, the regulatory function of pair cell immunologic function in mouse immune inhibition model, show that ampelopsin possesses the effect of external anti-HIV, possess the effect of downward modulation CXCR4, can strengthen the immunologic function that mouse immune suppresses model.Show that ampelopsin has the effect of prevention, treatment acquired immune deficiency syndrome (AIDS).

The specific embodiment

The invention will be further described below in conjunction with the specific embodiment.

Following examples be to ampelopsin prevent and treat specifying of acquired immune deficiency syndrome (AIDS), but not to the restriction of its concrete effect.

Embodiment 1, the preparation of ampelopsin.

Get the root of Vitaceae ampelopsis ampelopsis cantoniensis, adopt conventional extracting method to prepare ampelopsin, and can be distributed into the powder pin ampoule that 10mg/ props up.

Embodiment 2

Get ampelopsin 10g and AZT 10g, mixing is distributed into 1000 ampoules, makes compound preparation.

Embodiment 3, ampelopsin extracorporeal antivirus effect (HIV) experiment.

1, human peripheral blood mononuclear cell's (PBMCs) separation and cell culture

Get the healthy people's whole blood of sodium citrate anticoagulant, the PBS two-fold dilution is behind the centrifugal 10min of 1500r/min, discard blood plasma, hanged hemocyte, with 2: 1 ratio, slowly be added to the upper strata of lymphocyte separation medium, the centrifugal 20min of 2200r/min, the PBMC layer of sucking-off between PBS and lymphocyte separation medium, wash twice with PBS, at last with containing 10%FBS, 0.5%Hepes, PHA-P 10 μ g/ml, the RPMI 1640 of the two anti-100U of blue or green chain trains liquid fully and hangs, and adjusting cell concentration is 1 * 10 ⁶/ ml places 5%CO ₂, cultivate stand-by in 37 ℃ of incubators.MT-4 cell strain, H9 cell strain be with containing 10%FBS, and 0.5%Hepes, the RPMI 1640 of the two anti-100U of blue or green chain train liquid fully and cultivate and go down to posterity.

2, the cultivation of HIV-1 and titer determination

The MT-4 cell of trophophase of taking the logarithm washes twice with PBS, and the RPMI 1640 that contains 10%FBS with 1ml hangs, and adds 1ml HIV-1 _SF33Liquid is in CO ₂Behind the incubator 2h,, train the resuspended cultivation of liquid fully with RPMI 1640 again with the virus that the PBS flush away does not adsorb.The centrifuging and taking supernatant is done 10 times of serial dilutions, 10 when treating that pathological changes is obvious ^-1～10 ^-6Each dilution factor is established 4 multiple holes, establishes the normal cell contrast simultaneously, continues to cultivate.Measure the P24 antigen levels in P24 antigenic reagent box on the 4th, press the Spearman-Karber formula and calculate TCID ₅₀, at last it is diluted to 200 TCID ₅₀With 0.45 μ m filtering with microporous membrane, stand-by (the Dimitrov D of liquid nitrogen cryopreservation, et al.Microculture assay for isolation of HumanImmunodeficiency Virus type 1 and for titration of infected peripheralblood mononuclear cells.J Clin Micro.1990,28:734～737).HIV-1 _GD-1Contact 2h with the PBMCs of exponential phase, train liquid fully with the RPMI 1640 that contains IL-2 10u/ml and cultivate, and regular replenishment adds the normal lymphocyte that PHA-P stimulates.The same HIV-1 of titer determination _SF33(Scarlatti G, et al.In vivo evolution of HIV-1 co-receptorusage and sensitivity to chemokine-mediated suppression.Nat Med.1997,3:1259～1265).

3, antiviral experiment

3.1 medicine is to HIV-1 _SF33The influence of adsorption process

The MT-4 cell or the centrifugal rear overhang of PBMCs of exponential phase are risen, adjust concentration to 1 * 10 ⁶Individual/ml.Then add each concentration ampelopsin and 200 TCID respectively ₅₀HIV-1 _SF33Virus liquid is set up virus-positive contrast (PC) and normal cell negative control (NC) simultaneously, puts incubator and educates 1.5h altogether.Medicine in the centrifugal supernatant discarded and the virus of not adsorbing add PBS and wash one time, have hanged cell again, add 96 orifice plates, establish 3 multiple holes.The 4th with mtt assay (Cotter R, et al.Regulation of Human Immunodeficiency Virus type 1infection, β-chemokine production, and CCR5 expression inCD40L-stimulated macrophages:immune control of viral entry.J Virol.2001,75 (9): 4308～4320) measure absorbance (A), with (A with microplate reader 540nm _Drug-A _PC)/(A _NC-A _PC) * 100% calculates corresponding cytoprotective rate.Get cells and supernatant 100 μ l simultaneously, detect the expression (MooreJP of HIV-1 antigen P24 with the P24 antigenic reagent box, et al.Virions of primary Human Immunodeficiency Virus type 1isolates resistant to soluble CD4 (sCD4) neutralization differ in sCD4binding and glycoprotein GP 120 retention from sCD4-sensitive isolates.JVirol.1992,66:235～243), measure absorbance (A) at microplate reader 450nm wavelength, with (A _PC-A _Drug)/A _PC* 100% calculates corresponding P24 antigen suppression ratio (Ditzel HJ, etal.The CCR5 receptor acts as an alloantigen in CCR5 △ 32 homozygousindividuals:identification of chemokine and HIV-1 blocking humanantibodies.Proc Natl Acad Sci USA, 1998,95:5241～5245).

3.2 medicine is to HIV-1 _SF33The protection of educating altogether with cell

Get the variable concentrations ampelopsin, do the positive drug contrast, establish PC, NC with AZT.Each point is all established 3 multiple holes.Add MT-4 cell or PBMCs, put make medicine and cell preact 2h in the incubator after, except that NC (adding culture fluid), each hole adds 200 TCID ₅₀HIV-1 _SF33Virus liquid is put incubator and is cultivated.Added training liquid 40 μ l on the 3rd.Measured corresponding cytoprotective rate on 4th with mtt assay; detect the HIV-1 antigen P24 of culture supernatant with the P24 antigenic reagent box; calculate suppression ratio (Trkola A; et al.Potent; broad-spectruminhibition of Human Immunodeficiency Virus type 1 by the CCR5monoclonal antibody PRO140.J Virol; 2001,75 (2): 579～588).

3.3 medicine is to the protective effect of actute infection cell

Get MT-4 cell or PBMCs, add with volume 200 TCID ₅₀HIV-1 _SF33Virus liquid is put incubator and is educated 2h altogether.Centrifugal back supernatant discarded, PBS washes one time, and is resuspended to train liquid fully.To infected by HIV-1 _SF33The abundant mixing of MT-4 cell after, add the variable concentrations ampelopsin, do the positive drug contrast with AZT, establish PC, NC, each point is all established 3 multiple holes.Put CO ₂Cultivate in the incubator.Measured corresponding cytoprotective rate on 4th with mtt assay; detect and calculate HIV-1 antigen P24 suppression ratio (Zheng Y T with the P24 antigenic reagent box; et al.Alpha-monorcharin inhibits HIV-1 replication in acutely but notchronically infected T-lymphocytes.Acta Pharmacol Sin.1999,20 (3): 239～243).

4, result: ampelopsin all has significant protective effect to absorption, actute infection and chronic infection MT-4 cell and the PBMCs cell of HIV-1

HIV-1SF33 is inoculated in the MT-4 cell strain, can induce syncytium to form, and is SI type Strain, and its cell tropism is T cell tropism (T-tropic), and the accessory receptor of utilization is CXCR4.This result observes consistent (Verschoor E J with forefathers, et al.Efforts tobroaden HIV-1-specific immunity by boosting with heterologous peptidesor envelope protein and the influence of prior exposure to virus.J MedPrimatol.1999,28 (4): 224～232).HIV-1 _GD-1One from Guangdong Province HIV-1 positive patient of separation also can induce syncytium to form its Virus Type and HIV-1 when cultivating altogether with PBMCs _SF33Identical.These two kinds of HIV-1 Strain reproduction speeds are fast, can see obvious cytopathy on 3rd under mirror in inoculation, and huge syncytium forms the degeneration of many cell cavity samples, part cracking death.Measure the drug toxicity of ampelopsin to the MT-4 cell strain, the result shows that ampelopsin has certain toxic action to MT-4 when high concentration, reduces with concentration, and cell survival rate rises.CC ₅₀Be 8.364mg/ml.Equally, measure the drug toxicity of ampelopsin, CC to PBMCs ₅₀Be 6.147mg/ml.

Ampelopsin is to HIV-1 _SF33The influence of absorption MT-4 cytosis sees Table 1.The mtt assay measurement result shows that the cytoprotective rate of each concentration of ampelopsin is all very high, apparently higher than AZT group (P＜0.01).The ELISA method is measured P24 antigen result and is shown that each concentration of ampelopsin all can obviously suppress the antigenic generation of P24 (P＜0.01).The prompting ampelopsin can disturb HIV-1 _SF33The process of absorption MT-4 cell, cytoprotective rate and P24 antigen suppression ratio show that AZT is to HIV-1 _SF33Absorption MT-4 impact cell is little.Ampelopsin is to HIV-1 _SF33Absorption MT-4 cell and to the antigenic inhibiting EC of P24 ₅₀Be respectively 0.175mg/ml and 0.024mg/ml.

Ampelopsin is to HIV-1 _SF33The influence of educating altogether with the MT-4 cell sees Table 2.Ampelopsin and AZT all show the protective effect to the MT-4 cell, P24 antigen is produced reduce.Ampelopsin is to HIV-1 _SF33Absorption MT-4 cell and to the antigenic inhibiting EC of P24 ₅₀Be respectively 0.229mg/ml and 0.197mg/ml.

Ampelopsin is to actute infection HIV-1 _SF33The effect of MT-4 cell see Table 3.Ampelopsin and AZT can suppress P24 antigen and produce all to the MT-4 cell tool protective effect of actute infection.Ampelopsin is to HIV-1 _SF33Absorption MT-4 cell and to the antigenic inhibiting EC of P24 ₅₀Be respectively 0.179mg/ml and 0.348mg/ml.

Equally, ampelopsin is to HIV-1 _SF33Absorption PBMCs cell, HIV-1 _SF33Educate actute infection HIV-1 altogether with the PBMCs cell _SF33The PBMCs cell also show stronger cytoprotective and P24 inhibitory action.Its ED of cytoprotection with the mtt assay detection ₅₀Be respectively 0.146mg/ml, 0.183mg/ml, 0.100mg/ml; Its ED of P24 inhibitory action with the detection of ELISA method ₅₀Be respectively 0.169mg/ml, 0.321mg/ml, 0.249mg/ml.Utilize HIV-1 _GD-1Or the H9 cell strain, can be observed analog result.

Table 1 ampelopsin is to HIV-1 _SF33The influence of absorption MT-4 cytosis

ELISA method mtt assay

Processing factor P24 antigen suppression ratio cytoprotective rate

A value (n=3) A value (n=3)

(％) (％)

Ampelopsin

0.255±0.015 80.052 0.947±0.017 87.252

1.000mg/ml

Ampelopsin

0.426±0.010 66.632 0.923±0.025 80.264

0.500mg/ml

Ampelopsin

0.475±0.034 62.768 0.904±0.052 74.882

0.250mg/ml

Ampelopsin

0.789±0.153 38.198 0.859±0.032 62.134

0.125mg/ml

Ampelopsin

1.012±0.088 20.757 0.832±0.034 54.674

0.050mg/ml

AZT

0.739±0.090 42.141 0.780±0.052 39.849

10μg/ml

PC 1.277±0.036 0.000 0.639±0.022 0.000

NC 0.033±0.007 0.992±0.047

Table 2 ampelopsin is to HIV-1 _SF33The influence of educating altogether with the MT-4 cell

ELISA method mtt assay

Processing factor P24 antigen suppression ratio cytoprotective rate

A value (n=3) A value (n=3) is (%) (%)

Ampelopsin

0.920±0.086 73.724 1.058±0.128 80.189

1.000mg/ml

Ampelopsin

0.977±0.084 72.086 0.943±0.093 68.169

0.500mg/ml

Ampelopsin

1.503±0.088 57.067 0.772±0.047 50.175

0.250mg/ml

Ampelopsin

2.143±0.162 38.762 0.698±0.034 42.453

0.125mg/ml

Ampelopsin

2.406±0..191 31.248 0.582±0.038 30.259

0.050mg/ml

AZT

1.318±0.064 62.352 0.894±0.040 63.033

10μg/ml

PC 3.500±0.000 0.000 0.293±0.052 0.000

NC 0.043±0.007 1.247±0.103

Table 3 ampelopsin is to the effect of the MT-4 cell of actute infection HIV-1SF33

ELISA method mtt assay

Processing factor P24 antigen suppression ratio cytoprotective rate

A value (n=3) A value (n=3)

(％) (％)

Ampelopsin

0.348±0.014 72.932 1.071±0.041 83.223

1.000mg/ml

Ampelopsin

0.490±0.053 61.887 1.002±0.051 71..816

0.500mg/ml

Ampelopsin

0.752±0.066 41.535 0.927±0.042 59.358

0.250mg/ml

Ampelopsin

0.875±0.023 31.942 0.812±0.027 40.199

0.125mg/ml

Ampelopsin

0.957±0.146 25.538 0.775±0.052 34.164

0.050mg/ml

AZT

0.410±0.070 68.110 1.006±0.078 72.481

10μg/ml

PC 1.286±0.053 0.000 0.570±0.026 0.000

NC 0.028±0.006 1.172±0.071

5, analyze

After HIV-1 infects responsive host cell, can cause cell death, so detection of drugs is one of common method of primary dcreening operation inverase to the protective effect of infection cell.P24 is one of protein ingredient that constitutes HIV-1 taper core, and the RNA chain of HIV-1 is played stable and protective effect.In the experiment in vitro, can judge the levels of replication of virus by P24 antigen levels in the detection culture, this also is one of index (the Viscidi R of the detection of drugs HIV (human immunodeficiency virus)-resistant activity used always, et al.Enzyme immunoassay for detection of humanimmunodeficiency virus antigens in cell cultures.J Clin Microbiol.1988,26 (3): 453～458).In this experimentation, adopted CD to the HIV-1 sensitivity ₄ ⁺T lymphocyte: human PBMC s, MT-4 cell and H9 cell.Measure the cytotoxicity of ampelopsin to PBMCs and MT-4 with mtt assay, finding increases with dosage in the toxicity of ampelopsin pair cell.But the ampelopsin toxic action is not high when 1mg/ml.

International standard strain HIV-1 _SF33With endemicity strain HIV-1 _GD-1Be and duplicate fast the SI type HIV-1 virus of high titre.Observing the experiment of ampelopsin anti-HIV-1 carries out from 3 aspects: ampelopsin is to the influence of viral adsorption target cell; Virus and cell are educated altogether; Ampelopsin is to the effect of actute infection cell.Measure the cytoprotective rate of medicine with mtt assay, and measure P24 antigen inhibitory action simultaneously, both results have good concordance.Ampelopsin and HIV-1 and cell are educated 1.5h altogether, this is the material time that virus absorption enters cell, the centrifugal then virus of removing medicine and not adsorbing, if there is the factor of viral interference absorption to exist, then the quantity of infection cell and virus replication level can be lower than virus-positive matched group.The result confirms: the cell survival of each concentration group of ampelopsin increases, and the P24 level reduces, and observed syncytium number also reduces.Plasmodial formation is infection cell and the CD that does not infect ₄ ⁺The result of cell fusion, relevant with cell death (Cao J with the cytopathy that virus causes, et al.Moleculardeterminants of acute single-cell lysis by human immunodeficiency virustype 1.J Virol.1996,70 (3): 1340～1354).Ampelopsin 1mg/ml reaches 77.5% and 87.2% respectively to the cytoprotective rate of PBMCs and MT-4, and P24 antigen suppression ratio reaches 78.1% and 80.0%, EC ₅₀About about 0.2mg/ml.The above results shows the obviously adsorption process of viral interference of ampelopsin.The syncytium number that AZT (10 μ g/ml) produces is a little less than the virus-positive matched group, and its cytoprotective rate and P24 suppression ratio are about 30% only, illustrates that AZT can not influence the adsorption process of virus.The main action target spot of AZT belongs to nucleoside analog at the reverse transcriptase of HIV, and by activity in vivo metabolisable form competitive inhibition reverse transcriptase, thereby blocking-up HIV duplicates.

HIV-1 _SF33, the result that educates altogether of MT-4 and ampelopsin shows that ampelopsin can be protected permissive cell, suppress P24 antigen and produce.Similar effect also shows on the PBMCs.Utilize HIV-1 _GD-1The antivirus action of same susceptible of proof ampelopsin.AZT (10 μ g/ml) has significant protective effect to these two kinds of cells, and can suppress the expression therein of P24 antigen, has further confirmed the effect characteristics of AZT.MT-4 actute infection HIV-1 _SF33Or HIV-1 _GD-1After educate altogether with ampelopsin immediately, the result shows that ampelopsin also has significant protective effect and P24 antigen inhibitory action to the MT-4 cell of actute infection.AZT (10 μ g/ml) is equally to actute infection tool inhibitory action.Like this, by infect at HIV-1 multi-form in contrast not same-action characteristics of AZT and ampelopsin, we find that ampelopsin has a significant anti-HIV-1 effect external, its antiviral mechanism at least with viral interference absorption, suppress P24 antigen, to suppress virus replication relevant.

Embodiment 4, experiment of ampelopsin chemotaxis and cells were tested by flow cytometry experiment

1, chemotactic assay

24 hole Transwell chemotactic cells (the poly-carbonic acid film 5 μ m apertures that contain PVP), last chamber adds 0.1ml PBMCs (5 * 10 ⁶/ ml), following chamber adds 0.6ml variable concentrations ampelopsin medicine and/or chemotactic factor RANTES, SDF-1 α, and each concentration is established 3 multiple holes.In 5%CO ₂, 2h in 37 ℃ of incubators.Take off synthetic membrane with blade, 70% methanol is 1min fixedly, and coomassie brilliant blue staining 5min, optical microscope count film superficial cell number down down.The cell number in 5 different visuals field during every film is counted up and down.Chemotactic activity is represented with chemotactic index CI: CI=sample wandering cell number/negative control (Loyda Y, et al.Synthetic full-lengthand truncated RANTES inhibit HIV-1 infection of primary macrophages.AIDS.1998,12:977～984).

2, cells were tested by flow cytometry

PBMCs is resuspended to 1 * 10 with PBS 1ml (containing 1%BSA) ⁶/ ml.Select the ampelopsin medicine and/or the SDF-1 α of variable concentrations, educate 30min altogether at 37 ℃ with PBMC.PBS flush away medicine with ice is divided into two parts of equivalent with cell, a anti-CXCR4 monoclonal antibody that adds the FITC labelling, another part adds homotype contrast (isotypecontrol) mouse-anti people CXCR4 monoclonal antibody of FITC labelling, ice bath 1h is with cold PBS (1%BSA, 0.05%NaN ₃) wash 2 times, re-suspended cell is with the flow cytometer counting 10 of band FACS ⁴The average fluorescent strength of individual cell (MI).If negative cells matched group (NC).Each concentration is established 3 multiple holes.The result represents with the suppression ratio of expression of receptor.Receptor suppression ratio (%)=1-(MI _Drug-MI _Isotype)/(MI _Nc-MI _Isotype).In order to separate the model of action of medicine and receptor, with above-mentioned method, detect respectively in the time of 4 ℃, ampelopsin and SDF-1 α are to the influence (Torre of expression of receptor, VS, et al.Variable sensitivity of CCR5-Tropic HumanImmunodeficiency Virus type 1 isolates to inhibition by RANTESanalogs.J Virol.2000,74 (10): 4868～4876).

3, ampelopsin strengthens cytokine mediated PBMCs chemotactic motion

Chemotactic factor has induces target cell to produce the characteristic of chemotactic motion, and the chemotactic experiment is usually used in learning active at the observation in vitro drug effect in the associated biomolecule of chemokine receptors.PBMCs expresses HIV-1 accessory receptors such as CCR5, CXCR4, and its main part is respectively RANTES and SDF-1 α.RANTES induces the PBMCs chemotaxis migration of fresh separated, increase with RANTES concentration, be typical dose dependent bell-shaped curve, optimum concentration is 100ng/ml (CI 4.30), and ampelopsin during this concentration (1mg/ml) can significantly strengthen PBMCs, and (CI 8.17 to the chemotaxis migration of RANTES; P＜0.01).SDF-1 α has similar chemotaxis, and chemotactic effect bell-shaped curve reaches maximum (CI 4.20) at 100ng/ml, and ampelopsin (1mg/ml) makes its chemotactic effect significantly improve that (CI 7.70 equally; P＜0.01).Ampelopsin itself also demonstrates certain chemotaxis, but low (CI 3.29 during 1mg/ml than RANTES and SDF-1 α; P＜0.01).The RANTES of 100ng/ml and SDF-1 α all can significantly strengthen the chemotactic effect of ampelopsin, and (CI is respectively 8.17 and 7.70; P＜0.01).The results are shown in Table 4-8

4, the CXCR4 on ampelopsin downward modulation PBMCs surface

SDF-1 α is the part of HIV-1 accessory receptor CXCR4, can combine with the CXCR4 specificity and block of the utilization of X4 type HIV-1 strain accessory receptor, thereby play the effect that suppresses the poisoning intrusion target cell, its part blocking effect also produces by making the CXCR4 internalization reduce cell surface accessory receptor quantity.The mouse-anti people CXCR4 monoclonal antibody of FITC labelling can specific recognition target cell surface CXCR4, the average fluorescent strength that records with flow cytometer has reflected the acceptor quantity of cell surface.In the time of 37 ℃, SDF-1 α 100ng/ml and 1000ng/ml group all can significantly reduce the mark fluorescent intensity (P＜0.01) on PBMCs surface; In the time of 4 ℃, because the receptor internalization is stoped by temperature, this effect is significantly suppressed (P＞0.05).Ampelopsin 0.1mg/ml and 1mg/ml group can significantly reduce mark fluorescent intensity (P＜0.01) equally in the time of 37 ℃, significantly suppressed (P＞0.05) in the time of 4 ℃.Ampelopsin and SDF-1 α (10ng/ml) use simultaneously, and the inhibitory action of generation obviously strengthens (P＜0.05).The result under 37 ℃ and the 4 ℃ of conditions relatively, when the receptor internalization was suppressed, the effect of ampelopsin almost disappeared, and showed that its effect is by with the CXCR4 effect and cause that receptor down-regulated realizes.The results are shown in Table 9-10.

The chemotactic result of table 4.RANTES

Group	Drug level (ng/ml)	Migrating cell number (individual/visual field) (X ± SD)	Chemotactic index (CI)	P value (with solvent control group ratio)
					RANTES group RANTES group RANTES group RANTES group RANTES group RANTES group RANTES group solvent control group	0.1 1 10 50 100 500 1000 —	3.87±1.55 4.93±1.83 6.27±2.05 9.20±2.81 14.07±4.74 11.27±3.43 9.87±3.16 3.27±1.79	1.18 1.52 1.92 2.81 4.30 3.45 3.01 —	P＞0.05 P＜0.05 P＜0.01 P＜0.01 P＜0.01 P＜0.01 P＜0.01 —

The chemotactic result of table 5.SDF-1 α

Group	Drug level (ng/ml)	Migrating cell number (individual/visual field) (X ± SD)	Chemotactic index (CI)	P value (with solvent control group ratio)
					SDF-1 α group SDF-1 α group SDF-1 α group SDF-1 α group SDF-1 α group SDF-1 α group SDF-1 α group solvent control group	0.1 1 10 50 100 500 1000 —	3.73±1.58 5.00±1.56 6.60±1.80 9.07±2.86 13.73±3.73 11.40±3.60 9.93±2.58 3.27±1.62	1.14 1.53 2.02 2.77 4.20 3.49 3.04 —	＞0.05 ＜0.05 ＜0.01 ＜0.01 ＜0.01 ＜0.01 ＜0.01 —

The chemotactic result of table 6. ampelopsin

Group	Drug level (ng/ml)	Migrating cell number (individual/visual field) (X ± SD)	Chemotactic index (CI)	P value (with solvent control group ratio)
					Ampelopsin group ampelopsin group ampelopsin group ampelopsin group ampelopsin group ampelopsin group ampelopsin group solvent control group	0.1 1 10 100 500 1000 10000	4.13±1.51 4.80±1.26 4.87±1.55 7.13±3.09 10.07±3.22 11.20±4.11 10.53±3.23 3.40±1.45	1.22 1.41 1.43 2.10 2.96 3.29 3.10	P＞0.05 P＜0.01 P＜0.05 P＜0.01 P＜0.01 P＜0.01 P＜0.01

The chemotactic result of table 7. ampelopsin+RANTES (100ng/ml)

Group	Ampelopsin concentration (ng/ml)	Migrating cell number (individual/visual field) (X ± SD)	Chemotactic index (CI)	P value (with solvent control group ratio)
					Ampelopsin+R group ampelopsin+R group ampelopsin+R group ampelopsin+R group ampelopsin+R group ampelopsin+R group ampelopsin+R group solvent control group	0.1 1 10 100 500 1000 10000	18.33±4.17 19.47±3.70 19.87±5.42 22.73±5.90 25.40±5.57 26.13±7.04 25.33±5.09 3.20±1.15	5.73 6.08 6.21 7.10 7.94 8.17 7.92	P＜0.01 P＜0.01 P＜0.01 P＜0.01 P＜0.01 P＜0.01 P＜0.01

The chemotactic result of table 8. ampelopsin+SDF-1 α (100ng/ml)

Group	Ampelopsin concentration (ng/ml)	Migrating cell number (individual/visual field) (X ± SD)	Chemotactic index (CI)	P value (with solvent control group ratio)
					Ampelopsin+S group ampelopsin+S group ampelopsin+S group ampelopsin+S group ampelopsin+S group ampelopsin+S group ampelopsin+S group solvent control group	0.1 1 10 100 500 1000 10000	20.47±5.36 21.60±4.44 23.20±6.41 25.13±5.32 27.67±5.51 28.73±4.93 28.33±6.63 3.73±1.49	5.48 5.79 6.21 6.73 7.41 7.70 7.59	P＜0.01 P＜0.01 P＜0.01 P＜0.01 P＜0.01 P＜0.01 P＜0.01

Table 9.CXCR4 receptor down-regulated experiment (37 ℃)

Sample	Fluorescence intensity	The homotype contrast	Difference	Meansigma methods	Suppression ratio	P value (with the blank ratio)
							SDF-1 (10ng/1)	0.744 0.729 0.645	0.200 0.210 0.204	0.544 0.519 0.441	0.501± 0.054	10.0％	＞0.05
SDF-1 (100ng/ml)	0.435 0.521 0.489	0.212 0.201 0.212	0.223 0.320 0.277	0.273± 0.049	51.0％	＜0.01
							SDF-1 (1000ng/ml)	0.330 0.343 0.312	0.203 0.214 0.214	0.127 0.129 0.098	0.118± 0.017	78.8％	＜0.01
Amp(10ng/ml)	0.689 0.805 0.749	0.207 0.214 0.203	0.482 0.591 0.546	0.540± 0.055	3.0％	＞0.05
							Amp (100ng/mml)	0.576 0.570 0.544	0.203 0.214 0.205	0.373 0.356 0.339	0.356± 0.017	36.1％	＜0.01
Amp (1000ng/ml)	0.368 0.415 0.385	0.210 0.220 0.216	0.158 0.195 0.169	0.174± 0.019	68.8％	＜0.01
							Amp(10ng/ml) + SDF-1 (10ng/ml)	0.512 0.612 0.561	0.201 0.217 0.199	0.311 0.400 0.362	0.358± 0.045	35.7％	＜0.01
Amp (100ng/ml) + SDF-1 (10ng/l)	0.503 0.398 0.499	0.212 0.197 0.211	0.291 0.201 .0288	0.260± 0.051	53.3％	＜0.01
							Amp (1000ng/ml) + SDF-1 (10ng/ml)	0.324 0.293 0.300	0.207 0.203 0.209	0.117 0.090 0.091	0.099± 0.015	82.2％	＜0.01
The blank group	0.735 0.775 0.777	0.205 0.203 0.208	0.530 0.272 0.569	0.557± 0.023	—	—

Annotate: Amp represents ampelopsin

Table 10.CXCR4 receptor down-regulated experiment (4 ℃)

Sample	Fluorescence intensity	The homotype contrast	Difference	Meansigma methods	Suppression ratio	P value (with the blank ratio)
							SDF-1 (10ng/ml)	0.742 0.729 0.745	0.203 0.210 0.208	0.533 0.519 0.537	0.530± 0.009	2.0％	＞0.05
SDF-1 (100ng/ml)	0.775 0.723 0.741	0.219 0.205 0.209	0.556 0.518 0.532	0.535± 0.019	1.1％	＞0.05
							SDF-1 (1000ng/ml)	0.706 0.802 0.721	0.217 0.221 0.205	0.489 0.581 0.516	0.529± 0.047	2.2％	＞0.05
Amp (10ng/ml)	0.727 0.769 0.740	0.212 0.214 0.203	0.515 0.555 0.537	0.536± 0.020	0.9％	＞0.05
							Amp (100ng/ml)	0.752 0.757 0.721	0.227 0.210 .0211	0.525 0.547 0.510	0.527± 0.019	2.6％	＞0.05
Amp (1000ng/ml)	0.783 0.742 0.735	0.223 0.206 0.211	0.560 0.536 0.524	0.540± 0.018	0.2％	＞0.05
							The blank group	0.765 0.703 0.774	0.207 0.201 0.211	0.558 0.502 0.563	0.541± 0.034		＞0.05

Annotate: Amp represents ampelopsin

5, analyze

Chemotactic factor is on the one hand by combining the binding site that seals HIV-1 specifically with accessory receptor, can reduce the expression of accessory receptor on the other hand, so chemotactic factor and derivant thereof are one of key components of accessory receptor antagonist on HIV-1 permissive cell surface.Chemokine receptors mainly is distributed in the immune cell surface, belongs to the g protein coupled receptor family of striding film for 7 times.Chemokine receptors normal function in vivo is to combine with corresponding chemotactic factor, by coming transmission information with heterotrimeric G protein binding, inducing immune cells moves (Patricia D to the position, source of chemotactic factor, et al.Current evidence and futuredirections for Targeting HIV entry.JAMA.2000,284 (2): 215～222).Ampelopsin has the chemotactic inducibility to PBMCs, and collaborative with the chemotaxis generation of RANTES and SDF-1 α, demonstrates ampelopsin the chemokine receptors tool is had certain effect.Thereby make us infer that the antiviral mechanism of ampelopsin is relevant with chemokine receptors.Utilize fluorescently-labeled CXCR4 antibody, observed the effect of ampelopsin to the HIV-1 accessory receptor, the result has confirmed this supposition.The receptor internalization of known cell surface is prevented from 4 ℃, and ampelopsin has lost the inhibition to CXCR4 and antibodies under this temperature, shows that ampelopsin shows as the expression of downward modulation receptor on the target cell surface to the effect of CXCR4.The blocking-up of ampelopsin to HIV-1 adsorption target cell can be partly explained in this effect.CXCR4 accounts for critical role in the disease process that HIV-1 infects, change the T cell into and bite sexually transmitted disease (STD) poison (T-tropic) when HIV-1 bites sexually transmitted disease (STD) poison (M-tropic) by the macrophage of early infection, the accessory receptor of its utilization is correspondingly by with CCR5 being the main CXCR4 that transfers to.Though the mice of the mice of SDF-1 gene delection or CXCR4 gene knockout, its B cell, medullary cell are grown damaged, and the migration of cerebellum neuron is disorderly, finally causes death.With blocking-up CXCR4 is that the anti-HIV-1 medicine of target spot is not seen similar effect (the Proudfoot AE to the people in clinical experiment, et al.Thestrategy of blocking the chemokine system to combat disease.ImmunolRev.2000,177:246～256).Show that CXCR4 can be used as one of target spot of anti-HIV-1.The downward modulation CXCR4 effect of ampelopsin makes it to become invades the T lymphocyte in early days from HIV-1 and sets about, the novel drugs of control AIDS.

Embodiment 5, and ampelopsin is to the influence experiment of mouse cell function.

1, modelling and cell preparation

The BALA/C mice is divided into 5 groups at random, be respectively ampelopsin height (400mg/kg), in (200mg/kg), low (100mg/kg) dosage group, hydrocortisone (HC) group, normal control group (NC).At first use intramuscular injection HC 0.5mg/20g, be administered once every day, and continuous 5 days, to set up the immunosuppressant model.Lumbar injection ampelopsin then, HC group and NC organize injecting normal saline.Be administered once every day, continuous 10 days.Put to death mice on the 2nd day after the drug withdrawal, get spleen and grind, 200 mesh sieves filter, and the centrifugal supernatant that goes is used 0.83%Tris-NH ₄The Cl splitting erythrocyte.With RPMI 1640 complete culture solutions 2 * 10 ⁶/ ml cultivates stand-by.

2, NK cells in mice is killed and wounded vitality test

Get the NK cells in mice action effect cell (E) of above-mentioned splenocyte suspension; The take the logarithm YAC-1 cell (T) 1 * 10 of trophophase ⁵/ ml is as target cell.Add target cell and each 100 μ l of effector lymphocyte at 96 orifice plates, target is imitated than being 20: 1.While laying effect cell and target cell control wells.Each group is all established 3 multiple holes.In CO ₂Incubator is measured each hole A value with mtt assay after cultivating 24h.NK cytoactive (%)=[1-(A _E+T-A _E)/(A _T)] * 100% (Zhang Zhiqiang, Tian Zhigang, Cui Zhengyan, etc.The MTT reducing process detects NK and the active methodology of LAK is inquired into.Chinese experimental clinical immunology magazine, 1994,12 (6): 356～358).

3, mice spleen T lymphopoiesis functional examination

The extracting spleen cell suspension adds 10 μ l ConA (final concentration is 10 μ g/ml) positive contrast (PC) in 96 orifice plates, does not contain the negative contrast in ConA hole (NC).In CO ₂Incubator is measured each hole A value with mtt assay after cultivating 48h.With Δ A=(A _PC-A _NC) represent T lymphopoiesis function (Xue Bin.The immunotoxicology experimental technique.Combined publication society of China Concord Medical Science University of Beijing Medical University.October nineteen ninety-five, front page).

4, mice IL-2 Determination on content

Detect with the IL-2 detection kit, microplate reader 450nm wavelength is measured the A value.Set up standard curve with series concentration IL-2 standard substance, by IL-2 concentration (pg/ml) in the spleen cell cultures supernatant of regression equation calculation ConA stimulation.

5, ampelopsin is to the enhancing of mouse cell immunologic function

To the relatively demonstration of the general situation of mice, after the immunosuppressant modelling, lethargy and depilation phenomenon appear in mice, the fur low in glossiness.Behind the injection ampelopsin, each dosage group mice mental status and depilation phenomenon all have improvement in various degree, and the fur glossiness is recovered, and mice all survives.HC group mice lethargy, fluffy, the depilation of fur, dead 2; NS group mice all survives generally in order.

The killing activity (%) of mouse T lymphocyte propagation function status (Δ A), NK cell and in the mouse boosting cell suspension that ConA stimulates content (pg/ml) difference of IL-2 make one factor analysis of variance (ANOVA) respectively, P＜0.01 illustrates between each group to have significant difference; Carry out the multiple comparisons (as table 11) of mean again with the LSD method.3 indexs finding HC group mice all are lower than NS group (P＜0.05), show that HC can obviously suppress the mouse cell immunologic function; 3 indexs of each dosage group mice of ampelopsin all are higher than HC group (P＜0.05), and the prompting ampelopsin can strengthen the cellular immune function that is suppressed by HC.Dosage group immunological enhancement is the most remarkable in the ampelopsin, and its T lymphopoiesis function and IL-2 level even be higher than NS group all have significant difference (P＜0.05) with each group; The killing activity of its NK cell is compared zero difference (P＞0.05) with high dose group, but is significantly higher than other each groups (P＜0.05).

The content of table 11 mouse T lymphocyte propagation function (Δ A), NK cell activity (%) and IL-2

Number of mice NK kill rate T lymphopoiesis

Amount of packets (only) is function IL-2 (pg/ml) (%)

(ΔA)

Ampelopsin is high by 5 59.480 ± 6.578 ^Cd0.572 ± 0.057 ^Bcde50.927 ± 6.343 ^Bd

In the ampelopsin 8 65.838 ± 6.017 ^Cde0.769 ± 0.075 ^Acde67.699 ± 8.839 ^Acde

Ampelopsin low 7 46.143 ± 9.027 ^Abde0.429 ± 0.071 ^Abd44.446 ± 5.346 ^Bd

HC organizes 6 33.117 ± 3.220 ^Abce0.261 ± 0.017 ^Abce34.673 ± 4.714 ^Abce

NS organizes 6 56.100 ± 7.588 ^Bcd0.492 ± 0.048 ^Abd49.356 ± 4.693 ^Bd

ANOVA F 23.026 68.878 25.461

P 0.000 0.000 0.000

LSD P P＜0.05 P＜0.05 P＜0.05

Annotate: HC is the hydrocortisone group; NS is the normal saline group; The high, medium and low dosage of ampelopsin is respectively 0.4,0.2,0.1g/kg; ^AbcdeRepresent the high, medium and low dosage group of ampelopsin, HC group, NS group respectively

The LSD check.

6, analyze

This experiment is at the research ampelopsin during to the mouse cell Immune Effects, primary part observation the influence of ampelopsin to NK cell killing activity, T lymphopoiesis function and the IL-2 level (pg/ml) of BALA/C mice.

Near the intravital immune functional state of HIV the infected, set up the immunosuppressant animal model for more with the method for intramuscular injection hydrocortisone (HC).By observing ampelopsin to the immunosuppressive condition effect of immunologic function, statistical analysis shows, the immunological enhancement of ampelopsin does not become dose-effect relationship, the most remarkable with the effect of middle dosage group, its T lymphopoiesis function and IL-2 level and each group all have significant difference, and its NK cell killing activity is close with high dose group.Experiment shows ampelopsin in range of doses, can significantly strengthen cellular immune function, and excessive its effect of dosage weakens on the contrary.Ampelopsin is to the potentiation of immunosuppressive condition immune function of mice, and the immunologic hypofunction due to prompting is infected HIV has potentiation.

Ampelopsin of the present invention is a kind of plant extract, can invade target cell initial stage approach from HIV, is target spot with HIV accessory receptor and part chemotactic factor thereof, is developed to the inhibition HIV infection of high-efficiency low-toxicity and the medicine of treatment AIDS.

Claims (5)

1, ampelopsin is prevented and treated application in the AIDS-treating medicine in preparation.

2, ampelopsin according to claim 1 is prevented and treated application in the AIDS-treating medicine in preparation, it is characterized in that adopting any peroral dosage form that allows on the pharmaceutics.

3, ampelopsin according to claim 1 is prevented and treated application in the AIDS-treating medicine in preparation, it is characterized in that adopting any injection type that allows on the pharmaceutics.

4, ampelopsin according to claim 1 is prevented and treated application in the AIDS-treating medicine in preparation, it is characterized in that ampelopsin and other prevent and treat the medicine of acquired immune deficiency syndrome (AIDS) and make compound preparation.

5, ampelopsin according to claim 2 is prevented and treated application in the AIDS-treating medicine in preparation, it is characterized in that ampelopsin adopts the not purified extract of Radix Ampelopsis Cantoniensis.