CN101450173B - Traditional Chinese medicine composition and preparation method and use thereof - Google Patents

Abstract

The invention discloses a traditional Chinese medicine composition for preventing and treating AIDS and preparation method thereof. The medicament composition is mainly made from the following raw material medicaments of weight proportions of: astragalus 15-20 parts, Ginseng 7-12 parts, Chinese angelica 8-13 parts, wolfberry 9-14 parts, Chinese magnoliavine 7-12 parts, Tuber of Dwarf Lilyturf 7-12 parts, trichosanthes root 8-13 parts, Poria cocos 7-12 parts, licorice 7-12 parts, radix bupleuri 3-8 parts and cimicifuga foetida 3-8 parts. The traditional Chinese medicine composition of the invention has the functions of supplementing center, boosting qi, nourishing blood and nourishing yin; it can not only resist AIDS virus, but also increase the immunization function of patient substantially, and can eliminate the common symptoms of AIDS patients.

Description

A kind of treatment acquired immune deficiency syndrome (AIDS) Chinese medicine composition and preparation method thereof and pharmaceutical applications

Technical field

The present invention relates to the field of Chinese medicines, be specifically related to a kind of Chinese medicine composition and preparation method thereof of AIDS virus resisting and in AIDS virus resisting and/or improve application aspect the immunologic function.

Background technology

HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) claims human immune deficiency sexually transmitted disease (STD) poison (HIV) again, and this sick pathological process mainly is the MOI of multisystem, many organs, many pathogen, and the patient more than 95% finally will die from various opportunistic infection or tumor.Show according to the United Nations's " report of 2005 annual global acquired immune deficiency syndrome (AIDS) ": world's acquired immune deficiency syndrome (AIDS) crisis aggravates, and global HIV (human immunodeficiency virus) infection number has been increased to 6,900 ten thousand people in 2005 from 3,500 ten thousand of calendar year 2001, and wherein 2,300 ten thousand people are dead.2005 annual global increase patients infected hiv 4,900,000 newly, have 3,100,000 people to die from acquired immune deficiency syndrome (AIDS), have been since acquired immune deficiency syndrome (AIDS) in 1981 is found, increase maximum in 1 year.China's patients infected hiv just rises with fast speeds, infection speed is only second to India, Thailand, Philippine in the Asia, infected total number of persons according to incompletely statistics in 2004 and reached 840,000 people, surpass 1,000,000 at present, and the speed that still is 20-30% increases fast.

Acquired immune deficiency syndrome (AIDS) popular formed huge AIDS-treating medicine market, and acquired immune deficiency syndrome (AIDS) is treated with Western medicine by at present a lot of countries, and mainly contain two big classes: a class is the inhibitor of HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) reverse transcription (RT); Another kind of is the inhibitor of HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) hydrolytic enzyme (protease).Though this two classes medicine can obviously suppress duplicating of HIV (human immunodeficiency virus), delay the development of the state of an illness and the life-span of prolongation aids patient, there are many problems in these medicines, as cost an arm and a leg, and toxicity is big and side effect is many, and is easy to generate drug resistance etc.The treatment by Chinese herbs acquired immune deficiency syndrome (AIDS) is a very good outlet, the treatment by Chinese herbs acquired immune deficiency syndrome (AIDS) has remarkable advantages than Western medicine, as with low cost, resource is extensive, toxic and side effects is little, and can take for a long time and the difficult drug resistance etc. that produces, and it is sure that such advantage obtains the world of medicine, but the patent medicine of treatment acquired immune deficiency syndrome (AIDS) seldom can not be met the need of market on the domestic market at present.

Summary of the invention

The technical problem to be solved in the present invention just provides a kind of Chinese medicine composition, and this Chinese medicine composition not only has the effect of AIDS virus resisting, also can significantly improve patient's immunologic function, progressively eliminates the common sympton that HIV sufferers occurs.

Another technical problem that the present invention will solve just provides a kind of preparation method of described Chinese medicine composition.

The inventor is according to the understanding of theory of Chinese medical science to epidemic situation, in conjunction with ancient medicine to the argumentation of diseases such as " pestilence ", " pestilence ", " asthenia " and relevant basic research bibliographical information, find from the angle of differentiation of tcm, to HIV sufferers, particularly based on the early metaphase HIV sufferers of type of deficiency of both QI and YIN, adopt three kinds of method of treatment (heat-clearing and toxic substances removing, supplementing QI and nourishing YIN, eliminating pathogens by diaphoresis and purgation respectively) but supplementing QI and nourishing YIN, tonification lung spleen.

Therefore, the present invention solves the technical scheme that above-mentioned first technical problem adopts, a kind of Chinese medicine composition, it can mainly be to be made by following raw medicaments in portion by weight: 3～8 parts of 15～20 parts of the Radixs Astragali, 7～12 parts of Radix Ginsengs, 8～13 parts of Radix Angelicae Sinensis, 9～14 parts of Fructus Lycii, 7～12 parts of Fructus Schisandrae Chinensis, 7～12 parts of Radix Ophiopogonis, 8～13 parts of Radix Trichosanthis, 7～12 parts in Poria, 7～12 parts in Radix Glycyrrhizae, 3～8 parts of Radix Bupleuri and Rhizoma Cimicifugaes.

Can also contain one or more pharmaceutically acceptable auxiliary additives in the above-mentioned Chinese medicine composition of the present invention, promptly can add the needed various conventional adjuvants of pharmaceutically acceptable preparation different dosage form, as disintegrating agent, lubricant, binding agent, correctives etc., be prepared into capsule, tablet, granule and oral liquid etc. with the formulation method of routine.

Preferably, described auxiliary additive can be lactose, dextrin etc., and dosage form can be granule, capsule and tablet etc.

The present invention solves above-mentioned second technical scheme that technical problem adopted: a kind of preparation method of described Chinese medicine composition can comprise the following steps:

A) get the Radix Astragali, Fructus Lycii, Radix Trichosanthis, Poria and the Radix Glycyrrhizae 4 flavor medicines of described weight proportion, the decocting that adds 7～10 times of suitable crude drug weight boils twice, and each 1～2.5 hour, collecting decoction, filter, filtrate is condensed into the clear paste that relative density is 0.9～1.1 (30～60 ℃);

B) get Radix Ginseng, Radix Angelicae Sinensis, Fructus Schisandrae Chinensis, Radix Ophiopogonis, Radix Bupleuri and the Rhizoma Cimicifugae 6 flavor medicines of described weight proportion, 50～80% (m/m) ethanol that adds 5～8 times of suitable crude drug weight, reflux or immersion secondary, each 1～2.5 hour, merge ethanol liquid, filter, decompression filtrate recycling ethanol also is condensed into the clear paste that relative density is 0.9～1.1 (30～60 ℃);

C) merge above-mentioned two clear paste, being concentrated into relative density is the extractum of 1～1.2 (30～60 ℃), is preferably further spray drying of extractum or vacuum drying, gets dry extract, is the active component of medicine of the present invention.

Granule wherein can be made by following method: gets the described dry extract that makes 1 weight portion, adds the lactose of 0.36 weight portion and the dextrin of 0.14 weight portion, and mixing, granule is made in dry-pressing, crosses 30 mesh sieves, drying, promptly.Chinese medicine composition of the present invention is hereinafter referred to as " Ai Ning ", and its granule is called " Ai Ning granule " accordingly, and its capsule is called " Chinese mugwort Yiganning capsule ", and its tablet is called " Ai Ning sheet ", and its oral liquid is called " Ai Ning oral liquid ".

Chinese medicine composition supplementing QI and nourishing YIN of the present invention, tonification lung spleen has the AIDS virus resisting effect, can significantly improve the immunologic function of HIV sufferers, be specially adapted to early metaphase HIV sufferers, can improve obviously and progressively eliminate that it is weak, low grade fever based on type of deficiency of both QI and YIN, spontaneous perspiration, night sweat, poor appetite, chronic diarrhea, cough, common sympton such as become thin.Ai Ning granule of the present invention once in the period of 1991 to 1997, was treated 126 routine HIV/AIDS patients in Tanzania, had obtained significant clinical effective rate.1998-2000 has observed 29 routine Tanzania patients and 15 routine Chinese patients (Beijing You An hospital) again, according to unified diagnostic criteria, the 44 routine HIV/AIDS patients that make a definite diagnosis are given Ai Ning granule therapy of the present invention and carry out clinical observation, 3 months time, carry out efficacy determination from aspects such as immunologic function, virus load and clinical symptoms signs, all obtained definite results.

The specific embodiment

Further specify the present invention with embodiment below, but the present invention is not limited.

Below be the preparation embodiment of Chinese medicine composition of the present invention.

Embodiment 1 Ai Ning granule

Radix Astragali 15g, Radix Ginseng 7g, Radix Angelicae Sinensis 8g, Fructus Lycii 9g, Fructus Schisandrae Chinensis 7g, Radix Ophiopogonis 7g, Radix Trichosanthis 8g, Poria 11g, Radix Glycyrrhizae 12g, Radix Bupleuri 8g, Rhizoma Cimicifugae 8g.

Preparation process:

Get the five tastes such as the above-mentioned Radix Astragali, Fructus Lycii, Radix Trichosanthis, Poria, Radix Glycyrrhizae, add the 385ml decocting to boil secondary, each 1 hour, collecting decoction filtered, and filtrate is concentrated into the clear paste that relative density is 0.9 (30～60 ℃), and is standby; Six-elements such as all the other Radix Ginsengs, Fructus Schisandrae Chinensis, Radix Angelicae Sinensis, Radix Ophiopogonis, Radix Bupleuri, Rhizoma Cimicifugae add 60% (m/m) ethanol of 225ml, the reflux secondary, each 2.5 hours, merge ethanol liquid, filter, decompression filtrate recycling ethanol also is concentrated into the clear paste that relative density is 0.9 (30～60 ℃), merge with above-mentioned clear paste, be concentrated into relative density 1.0 (30～60 ℃), spray drying gets dry extract 17.5g.Get dry extract, add lactose 6.3g, dextrin 2.4g, mixing is made granule, drying, promptly.

Embodiment 2 Ai Ning granules

Radix Astragali 20g, Radix Ginseng 12g, Radix Angelicae Sinensis 13g, Fructus Lycii 10g, Fructus Schisandrae Chinensis 10g, Radix Ophiopogonis 7g, Radix Trichosanthis 8g, Poria 7g, Radix Glycyrrhizae 7g, Radix Bupleuri 3g, Rhizoma Cimicifugae 3g.

Preparation process:

Get the five tastes such as the above-mentioned Radix Astragali, Fructus Lycii, Radix Trichosanthis, Poria, Radix Glycyrrhizae, add the 610ml decocting to boil secondary, each 2.5 hours, collecting decoction filtered, and filtrate is concentrated into the clear paste that relative density is 1.0 (30～60 ℃), and is standby; Six-elements such as all the other Radix Ginsengs, Fructus Schisandrae Chinensis, Radix Angelicae Sinensis, Radix Ophiopogonis, Radix Bupleuri, Rhizoma Cimicifugae add 60% (m/m) ethanol of 312ml, soak secondary, each 2 hours, merge ethanol liquid, filter, decompression filtrate recycling ethanol also is concentrated into the clear paste that relative density is 1.0 (30～60 ℃), merge with above-mentioned clear paste, be concentrated into relative density 1.2 (30～60 ℃), vacuum drying gets dry extract 18g.Get dry extract, add lactose 6.5g, dextrin 2.5g, mixing is made granule, drying, promptly.

Embodiment 3 Ai Ning granules

Radix Astragali 15g, Radix Ginseng 7g, Radix Angelicae Sinensis 8g, Fructus Lycii 14g, Fructus Schisandrae Chinensis 12g, Radix Ophiopogonis 12g, Radix Trichosanthis 10g, Poria 9g, Radix Glycyrrhizae 7g, Radix Bupleuri 3g, Rhizoma Cimicifugae 3g.

Preparation process:

Get the five tastes such as the above-mentioned Radix Astragali, Fructus Lycii, Radix Trichosanthis, Poria, Radix Glycyrrhizae, add the 440ml decocting to boil secondary, each 2 hours, collecting decoction filtered, and filtrate is concentrated into the clear paste that relative density is 1.0 (30～60 ℃), and is standby; Six-elements such as all the other Radix Ginsengs, Fructus Schisandrae Chinensis, Radix Angelicae Sinensis, Radix Ophiopogonis, Radix Bupleuri, Rhizoma Cimicifugae add 60% (m/m) ethanol of 270ml, the reflux secondary, each 2 hours, merge ethanol liquid, filter, decompression filtrate recycling ethanol also is concentrated into the clear paste that relative density is 1.0 (30～60 ℃), merge with above-mentioned clear paste, be concentrated into relative density 1.1 (30～60 ℃), spray drying gets dry extract 17g.Get dry extract, add lactose 6g, dextrin 2.4g, mixing, dry method is made 16～24 order granules, drying, promptly.

Embodiment 4 Chinese mugwort Yiganning capsules

1. the preparation technology of Ai Ning extract

Prescription:

Radix Astragali 15g, Radix Ginseng 9g, Radix Angelicae Sinensis 8g, Fructus Lycii 9g, Fructus Schisandrae Chinensis 7g, Radix Ophiopogonis 7g, Radix Trichosanthis 13g, Poria 12g, Radix Glycyrrhizae 10g, Radix Bupleuri 5g, Rhizoma Cimicifugae 5g.

Preparation process:

Get the five tastes such as the above-mentioned Radix Astragali, Fructus Lycii, Radix Trichosanthis, Poria, Radix Glycyrrhizae, add the 472ml decocting to boil secondary, each 2 hours, collecting decoction filtered, and filtrate is concentrated into the clear paste that relative density is 1.1 (30～60 ℃), and is standby; Six-elements such as all the other Radix Ginsengs, Fructus Schisandrae Chinensis, Radix Angelicae Sinensis, Radix Ophiopogonis, Radix Bupleuri, Rhizoma Cimicifugae add 60% (m/m) ethanol of 287ml, soak secondary, each 2 hours, merge ethanol liquid, filter, decompression filtrate recycling ethanol also is concentrated into the clear paste that relative density is 1.0 (30～60 ℃), merge with above-mentioned clear paste, be concentrated into relative density 1.2 (30～60 ℃), spray drying gets dry extract 17.5g.

The Chinese mugwort Yiganning capsule preparation technology

Prescription:

Ai Ning extract 17.5g

Dextrin 4.4g

Starch 4.4g

Lactose 2.6g

Magnesium stearate 0.5g

Method for making:

In the prescription ratio, get dry extract, the adjuvant of Ai Ning extract, mixing, wet granulation, 60 ℃ of following aeration-drying 3 hours obtains the granule of good fluidity, uses automatic filling machine fill capsule, and aluminium foil plate machinery is packing automatically, promptly.

Embodiment 5 Ai Ning sheets

1. the preparation technology of Ai Ning extract

Prescription:

Radix Astragali 18g, Radix Ginseng 12g, Radix Angelicae Sinensis 12g, Fructus Lycii 10g, Fructus Schisandrae Chinensis 10g, Radix Ophiopogonis 9g, Radix Trichosanthis 9g, Poria 7g, Radix Glycyrrhizae 7g, Radix Bupleuri 3g, Rhizoma Cimicifugae 3g.

Preparation process:

Get the five tastes such as the above-mentioned Radix Astragali, Fructus Lycii, Radix Trichosanthis, Poria, Radix Glycyrrhizae, add the 357ml decocting to boil secondary, each 2 hours, collecting decoction filtered, and filtrate is concentrated into the clear paste that relative density is 0.9 (30～60 ℃), and is standby; Six-elements such as all the other Radix Ginsengs, Fructus Schisandrae Chinensis, Radix Angelicae Sinensis, Radix Ophiopogonis, Radix Bupleuri, Rhizoma Cimicifugae add 60% (m/m) ethanol of 245ml, the reflux secondary, each 2 hours, merge ethanol liquid, filter, decompression filtrate recycling ethanol also is concentrated into the clear paste that relative density is 1.1 (30～60 ℃), merge with above-mentioned clear paste, be concentrated into relative density 1.1 (30～60 ℃), spray drying gets dry extract 17.8g.

2. the preparation technology of Ai Ning sheet

Prescription:

Ai Ning extract 17.8g

Microcrystalline Cellulose 7.4g

Starch 2.9g

Pulvis Talci 0.5g

Micropowder silica gel 0.5g

Magnesium stearate 0.1g

Method for making:

In the prescription ratio, get dry extract, microcrystalline Cellulose, starch, Pulvis Talci and the micropowder silica gel of Ai Ning extract, cross 80 mesh sieves respectively, fully mixing, add ethanol system soft material, granulate, in 50 ℃～60 ℃ dryings 2 hours with 16 mesh sieves, after adding the magnesium stearate mixing, tabletting, promptly.

Embodiment 6 Ai Ning oral liquids

1. the preparation technology of Ai Ning extract

Prescription:

Radix Astragali 15g, Radix Ginseng 10g, Radix Angelicae Sinensis 10g, Fructus Lycii 12g, Fructus Schisandrae Chinensis 12g, Radix Ophiopogonis 8g, Radix Trichosanthis 9g, Poria 9g, Radix Glycyrrhizae 7g, Radix Bupleuri 5g, Rhizoma Cimicifugae 3g.

Get the five tastes such as the above-mentioned Radix Astragali, Fructus Lycii, Radix Trichosanthis, Poria, Radix Glycyrrhizae, add the 416ml decocting to boil secondary, each 2 hours, collecting decoction filtered, and filtrate is concentrated into the clear paste that relative density is 1.1 (30～60 ℃), and is standby; Six-elements such as all the other Radix Ginsengs, Fructus Schisandrae Chinensis, Radix Angelicae Sinensis, Radix Ophiopogonis, Radix Bupleuri, Rhizoma Cimicifugae add 60% (m/m) ethanol of 384ml, the reflux secondary, each 2 hours, merge ethanol liquid, filter, decompression filtrate recycling ethanol also is concentrated into the clear paste that relative density is 1.1 (30～60 ℃), merges with above-mentioned clear paste.Other gets sucrose 50g, and it is an amount of that glucose 8g adds water, and heating for dissolving adds sodium benzoate 0.2g, and the dissolving back filters, and mixing adds water, stir evenly, and embedding, promptly.

Below be the effect test embodiment of Chinese medicine composition of the present invention.

Experimental example 1 quality standard

Astragaloside content is measured in the embodiment 1-3 Ai Ning granule.

1) preparation of reference substance solution: it is an amount of that precision takes by weighing astragaloside reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 20mg/ props up), adds methanol and make the solution that every 1ml contains 0.5mg, promptly.

2) preparation of need testing solution: get the Ai Ning granule, porphyrize is got 2g, the accurate title, decide, and puts in the apparatus,Soxhlet's, adds methanol 40ml, merceration spends the night, and it is an amount of to add methanol again, heating and refluxing extraction 4 hours, extracting solution reclaims methanol and is concentrated into driedly, and residue adds water 10ml slight fever makes dissolving, with water saturated n-butyl alcohol jolting extraction 3 times, each 20ml merges n-butyl alcohol liquid, with ammonia solution washing 2 times, each 20ml, discard ammoniacal liquor, n-butyl alcohol liquid evaporate to dryness, residue add water 3-5ml makes dissolving, by D101 type macroporous adsorptive resins (internal diameter 1.5cm, long 12cm), with water 50ml eluting, discard water liquid, reuse 40% ethanol 30ml eluting, discard 40% ethanol elution, continue, collect eluent with 70% ethanol 50ml eluting, evaporate to dryness, with dissolve with methanol and be transferred in the 2ml measuring bottle, add methanol to scale, shake up, filter with microporous filter membrane (0.45 μ m), get subsequent filtrate promptly.

3) respectively accurate reference substance solution 2,4, the 8 μ l that draw of the formulation of standard curve inject chromatograph of liquid, are abscissa with the natural logrithm of reference substance sample size, are vertical coordinate with the natural logrithm of reference substance peak area, the drawing standard curve.Regression equation is Y=1.4828X+12.1376, r=0.9999.

4) measure the accurate reference substance solution 8 μ l that draw of branch, 12 μ l and need testing solution 10 μ l inject chromatograph of liquid, measure, promptly.

Above-mentioned determining instrument and condition are: U.S. HP1100 high performance liquid chromatograph, G1311A quaternary pump, G1313A automatic sampler, G1316A column oven, G1315A diode matrix detector HPCHEM chromatographic work station.Chromatographic column: ZORBAX RX-C ₁₈(4.6mm * 250mm), 5 μ m, wavelength 195～247nm; Flow velocity 1～3ml/min, 25～60 ℃ of column temperatures.

5) as a result in the Ai Ning granule Astragaloside content measurement result be: this product every bag (every bag 5 Ke Aining granule) contains the Radix Astragali by astragaloside (C ₄₁H ₆₈O ₁₄) meter, be no less than 0.85mg.

Experimental example 2 drug effects

(1) experiment material

1, medicine

Ai Ning granule (embodiment 1 preparation): brown ceramic powder, 0.1715g medicated powder is equivalent to the extract of 1g crude drug, and is water-soluble, prepares with cell culture fluid.

Zidovudine (Zidovudine, AZT) [anti-HIV-1 reverse transcriptase inhibitors]: available from Shanghai Disainuo Chemical Pharmaceutical Co., Ltd, lot number: 040201b.

Nevirapine (Nevirapine, NVP): available from the many doctorization Information Research Centers in pool, Nanjing, lot number: 0301001.Foscarnet sodium (PFA): Changzhou first pharmaceutical factory produces.

Saquinavir (Saquinavir, SQ): available from the Colorade USA medical center.

Methylimidazole.: ACROS company product.

ZHENQI FUZHENG KELI: Shenyuan Pharmaceutical Co., Ltd., Tonghua's product, lot number: 030411070.

Cyclosporin A: Sweden cyclosporin A company product, lot number: 478886.

2, animal

Animal Rhesus Macacus (Rhesus Macaques): male, body weight 6+1kg.Available from Kunming animal institute of the Chinese Academy of Medical Sciences.Confirm that through Serological testing no simian immunodeficiency virus (SIV), monkey reverse transcription D type virus (SRV), monkey T lymphocyte I type virus (STLV-I) infect.

Machin: male, body weight 4+1kg, 3-4 year, outward appearance health does not have the enlargement of superficial lymph knot through health check-up, detects SIV through serology, monkey reverse transcription D type virus (SRV) and monkey T lymphatic I type virus (STLV-I) negative antibody, tuberculin feminine gender, dysentery bacterium feminine gender.

Kunming mouse: male, the SPF level, body weight 18-20g is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute's Experimental Animal Center.

3, cell

(peripheral blood mononuclear cell PBMC) adopts routine techniques to separate acquisition, suspension culture in culture fluid from healthy fresh venous to human peripheral blood single nucleus cell.

The MT-4 cell: use RPMI Medium 1640 culture medium that contain 10% hyclone, 100IU/ml penicillin, 100 μ g/ml streptomycins and kanamycin and contain L-glutaminate, at 37 ℃, 5%CO ₂Cultivate in the incubator, went down to posterity once in per 3 days.

The chronically infected H9 cell of HIV-1IIIB, infectious disease system of U.S. Colorado university.

PBMC culture fluid composition: add in the 100ml RPMI Medium RPMI-1640: hyclone: 20ml, (phytohaemagglutinin: 300 μ g) interleukin-2: 1000U, antibiotics (each 10000U/ml of penicillin, streptomycin and kanamycin) 2ml.

4, virus

The laboratory HIV-1 IIIB Strain that goes down to posterity: U.S. Mount Sinai Medical Center, increase in cem cell.

Clinical separation AZT sensitive strain HIV-1018a and AZT persister HIV-1018c: infectious disease system of U.S. Colorado university.

HIV-1 intergrase system: Chinese microorganism of military medical sciences academy epidemiological study institute.

HIV (human immunodeficiency virus) 1 type reverse transcriptase P66/P51: Chinese microorganism of military medical sciences academy epidemiological study institute.

HIV (human immunodeficiency virus) 1 type protease: Chinese microorganism of military medical sciences academy epidemiological study institute.

Virus and modelling strain SIVmac251: U.S. Aaron Diamond acquired immune deficiency syndrome (AIDS) research center, Institute of Experimental Animals, Chinese Academy of Medical Sciences preserves.

SIVmac251 virus liquid: Aaron Diamond acquired immune deficiency syndrome (AIDS) research center, USA New York.

5, reagent

IMDM (Iscove ' s Modified Dulbecco ' s Medium, powder) culture medium and RPMI Medium1640 (powder) culture medium: U.S. GIBCO company product.

Hyclone: GIBCOBRL company product, lot number: 1025208;

86011) and streptomycin (lot number: 841121): North China Pharmaceutical Factory's product penicillin (lot number:;

Tetrazole indigo plant (Thiazolyl blue, MTT), citric acid: U.S. Sigma company product.

Triton X-100:KEBO AB STOCKHOLM company product.

N, N dimethyl formamide (N, N-Dimethyl Formamide): Beijing Chemical Plant's product.

HIV-1P24 antigen detecting agent box: U.S. ZeptoMetrix company product.

COVALINK NH ELISA Plate: Denmark Nunc company product.

EDC (1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide): U.S. Sigma chemical company product.

PNPP:Roche company product.

Positive control compound S Y: Chinese microorganism of military medical sciences academy epidemiological study institute.

Poly (A) is 15:Boehringer Mannheim GmbH company product (dT).

3H-dTTP:NEN company product.

DTT, bovine serum albumin: astronomical phenomena people company product.

The anti-Dig-NHS antibody of digoxigenin labeled-N-hydroxyl succinum ester, blocker and alkali phosphatase enzyme mark: Boehringer mannheim company product.

Synthetic peptide substrates: company is synthetic by the match Parkson.

Donor substrate and target substrate oligonucleotide: it is synthetic that worker bio-engineering corporation is given birth in Shanghai, uses the PAGE purification.

SIVP27 antigen ELISA detecting kit (RETRO-TEKTMSIV-1P27Antigen ELISA, CellularProducts Inc. product),

Mopterin ELISA test kit (ELItest Neopterin Germany produces), B2M ELISA method immunity elisa kit is provided by Immunotech Co.,

One anti-is mouse-anti people CD4 monoclonal antibody, two anti-sheep anti-mouse igg-FITC: Bang Ding company product.

Anti-mice CD3, CD4, CD8 and CD45 fluorescent labeling monoclonal antibody: produce available from U.S. e.B company.

6, instrument

EmaxTM microplate reader: be the LP 400 full-automatic microplate reader of U.S. Molecuar DevECes company product and France's production.

Flow cytometer: FACS420 Becton-Dickinson product.

Negative pressure super-clean bench: BHA48H BIOHAZARD company product, Britain.

Low-temperature and high-speed centrifuge: RS-20IV, TOMYSEIKO product, Japan.

CO ₂Incubator: CO-I TOKIWA company product, Japan.

PCR instrument: MODEL50/60, COYTEMPCYCLER company product, Britain.

(2) experiment in vitro

1, to the vitro inhibition effect of HIV-1 replicative enzyme.

(1) to the inhibitory action of HIV (human immunodeficiency virus) 1 type intergrase (HIV-1 IN).

Donor substrate bag by bread board, is added after washing plate: reaction buffer, variable concentrations medicinal liquid and demarcate the genetic engineering hiv integrase of concentration, 37 ℃ of reactions 1 hour.Add the target substrate, mix 37 ℃ of reactions.Wash plate.Add bovine serum albumin, room temperature reaction.Wash plate.The Avidin that adds alkali phosphatase enzyme mark, room temperature reaction.Wash plate.Add the chromogenic substrate colour developing.Add the reaction of 0.1N NaOH color development stopping.Microplate reader 405nm measures the OD value.Establish enzyme contrast and blank simultaneously, calculate IC ₅₀, test operation is referring to document: Guo Zhimin, the research of Chen Hong coral .HIV-1 intergrase linked immunosorbent adsorption test and inhibitor thereof. and China's experiment and clinical virology magazine .2002; 16 (2) 119-123.The results are shown in Table 1:

Table 1. Ai Ning granule suppresses the result of HIV-1 intergrase

(2) to the inhibitory action of HIV (human immunodeficiency virus) 1 type reverse transcriptase (HIV-1 RT).

With demarcate genetic engineering hiv reverse transcriptase (P66/51), the variable concentrations of concentration medicinal liquid, reaction buffer and ³After H-dTTP adds, mix 37 ℃ of reactions.0 ℃ of cessation reaction.Reactant liquor drips on filter paper, and cold trichloroacetic acid is washed 3 times.Dry after the ethanol dehydration.Liquid scintillation instrument is measured the cpm value.Establish the contrast of enzyme contrast and blank and positive drug simultaneously, calculate IC ₅₀, test operation is referring to document: Peng Zonggen, Chen Hongshan, Teng Li. and the active dynamic studies of the mice serum of HIV (human immunodeficiency virus) I type reverse transcriptase inhibitors. Chinese combination of Chinese and Western medicine magazine .2003; 23 (1): 514-517.The results are shown in Table 2.

Table 2. Ai Ning granule suppresses the result of HIV-1 reverse transcriptase

(3) to the inhibitory action of HIV (human immunodeficiency virus) 1 type protease (HIV-1PR).

Microwell plate adds reaction buffer and substrate, adds an amount of genetic engineering hiv protease and variable concentrations medicinal liquid, mixing, and 37 ℃ of reactions add the DMSO cessation reaction.Add sodium iodoacetate, mixing, 37 ℃ of reactions.Add Dig-NHS, mixing, 37 ℃ of reactions add glycine and interrupt reaction.Add in 96 orifice plates of marked by streptavidin, add reaction buffer simultaneously, mixing, room temperature reaction is attached to substrate on the ELISA Plate.Wash plate.Add the blocker room temperature reaction.The anti digoxin antibody Fab fragment that adds alkali phosphatase enzyme mark, room temperature reaction.Wash plate.Add substrate pNPP colour developing, microplate reader 405nm surveys the OD value.Establish enzyme contrast, blank and positive drug contrast simultaneously, calculate IC ₅₀, test operation is referring to document: Guo Zhimin, the research of Chen Hong coral .HIV-1 intergrase linked immunosorbent adsorption test and inhibitor thereof. and China's experiment and clinical virology magazine .2002; 16 (2) 119-123.The results are shown in Table 3.

Table 3. Ai Ning granule suppresses the result of HIV-1 protease

The anti-HIV-1 effect in cell culture of 2 Ai Ning granules

(1) toxicity of pair cell and inhibition HIV-1 effect in the MT-4 cell culture

The MTT staining is measured the toxicity (CC of medicine pair cell ₅₀And CC ₀)

MT-4 cell culture and MTT dyeing detect cytotoxicity: with 1 * 10 ⁵Cell/ml 100 μ l are inoculated in the 96 porocyte culture plates, add the Ai Ning granule and the positive drug AZT of 3 times of dilutions simultaneously respectively, or 2 times of dilute liquid medicines of NVP, and each dilution factor repeats 3 holes, establishes the cell matched group.Put 37 ℃, 5%CO ₂With cultivate in the saturated humidity incubator, every day the observation of cell pathological changes.The 4th day (96 hours) back is inhaled and is abandoned supernatant 100 μ l after the dosing, and every hole adds 10 μ l 5mg/ml MTT dyeing, 37 ℃, 5%CO ₂Continue in the saturated humidity incubator to cultivate 4 hours, every hole adds 100 μ l 50%DMF, 17%Triton X-100 destaining solution, and 37 ℃ are spent the night, and measuring wavelength on enzyme connection instrument is the OD value of 570nm, calculates the poisonous concentration (CC of medicine half ₅₀).

The MTT staining is measured the Ai Ning granule suppresses HIV in the MT-4 cell culture medium effective concentration.MT-4 cell 1 * 105/ml 50 μ l are inoculated in the 96 porocyte culture plates; the HIV-1 zoo virus strain IIIB culture fluid that adds 50 μ l 10-2; the variable concentrations Ai Ning granule or positive control drug AZT and the NVP medicinal liquid 100 μ l that add the following dilution of maximal non-toxic concentration simultaneously are at 37 ℃, 5%CO ₂With cultivate under the saturated humidity, the observation of cell pathological changes, in the 7th day with mtt assay dyeing, measure OD ₅₇₀Nm calculates medicine medium effective concentration (EC ₅₀), test operation is referring to document: Lu Changan, and Rabdosia rebia(W. W. Smith) Hara is to the HIV-1 experimental study of effect. Chinese TCM basis medical journal .1998,4 (4) 25-26.

The result is as follows:

Try to achieve three batches of experiment Ai Ning granules to MT-4 cell 50% toxic concentration CC with the Bliss method ₅₀=0.4683mg/ml.The results are shown in Table 4.

Table 4. Ai Ning granule is to 50% toxic concentration (three batches) of MT-4 cell

Protective effect to the HIV-1 infection cell

Calculate each dilution factor OD average and standard deviation respectively, calculate the protective rate of each dilution factor medicine with following formula again the HIV infection cell:

Reaching the dosage of 50% protection, is 50% valid density (EC ₅₀).About EC ₅₀Calculating, can be according to each dilution protective rate, utilize regular probit method (Bliss method) to calculate EC ₅₀Statistical procedure obtain.Virus infected cell is avoided taking place the protective rate of cytopathy (CPE), is exactly the suppression ratio that virus infected cell is taken place CPE, i.e. suppression ratio=protective rate.Medicine pair cell toxicity CC ₅₀The same EC of calculating ₅₀, be that HIV in the above-mentioned protective rate formula infects contrast OD value and answers substitution 0.

Three batches of result of the tests show that the particulate suppression ratio of Ai Ning is at 0.078-0.156mg/ml, and protective rate (suppression ratio) shows that greater than 50% it has certain inhibitory action to HIV-1, and suppression ratio is 54.7%～76.1%.Try to achieve the medium effective concentration EC that suppresses HIV-1 with the Bliss method ₅₀=0.0901mg/ml (three batches of experiment means), its selection index SI=CC ₅₀/ EC ₅₀=0.4683/0.0901=5.1976 (three batches of experiment means) (seeing Table 5,6,7).

Table 5. Ai Ning granule is to 50% toxic concentration (average) of MT-4 cell

The protective effect (three batches) of table 6. Ai Ning granule HIV-1 infection cell

Table 7. Ai Ning granule is to the protective effect (average) of HIV-1 infection cell

2 batches of experiments show, in the chronically infected H9 cell culture of HIV-1IIIB, and the TC that Ai Ning granule pair cell is cultivated ₅₀Be 964.46 ± 581.32 μ g/ml, EC ₅₀Be 641.42 ± 584.06 μ g/ml, SI is 1.85 ± 0.78.The TC that positive control drug AZT pair cell is cultivated ₅₀Be 156.49 ± 15.58 μ g/ml, EC ₅₀Be 39.51 μ g/ml, SI is 3.7.See Table 8.

Cytotoxicity and the inhibition HIV-1 P24 effect thereof of table 8 Ai Ning granule in HIV-1IIIB chronic infection H9 cell culture

The Ai Ning granule is to the toxicity T C of HIV-1IIIB chronic infection H9 cell culture ₅₀Be 964.46 ± 581.32 μ g/ml, in HIV-1IIIB chronic infection H9 cell culture, suppress the antigenic EC of HIV-1IIIB P24 ₅₀Be 641.42 ± 584.06 μ g/ml, SI is 1.85 ± 0.78.The result of positive control drug zidovudine (AZT) and relevant bibliographical information unanimity, illustrative experiment is credible.

(2) toxicity of inhibition antigenic effect of HIV-1-P24 and pair cell among the PBMC

The Ai Ning granule in the PBMC cell culture that HIV-1 infects to the antigenic inhibitory action of HIV-1-P24

Get healthy people's fresh venous, with FiColl separating medium separation of human peripheral blood lymphocytes (PBMC), with inoculum preparation 1.5 * 105 cells/ml suspension inoculation of containing PHA in culture bottle, 37 ℃ of 5%CO ₂Cultivated 72 hours in the incubator.Scrape collecting cell with cell, the viral liquid inductance transfect cell with AZT sensitivity (018a) and AZT drug resistance (018c) Strain 10-2 adsorbed 2 hours.With serum-free 1640 culture medium flush awaies viral adsorption not, the cell of infective virus is made into 1 * 10 with culture fluid ⁶/ ml.Plant 96 well culture plates, 100 μ l/ holes.Add the Ai Ning granule of the following 3 times of dilutions of non-toxic concn and the different dense medicinal liquid 100 μ l of positive control drug AZT of 2 times of dilutions simultaneously respectively.If cell contrast and virus infected cell matched group group.37 ℃ of 5%CO ₂Cultivate the 4th day (96 hours), the sucking-off supernatant ,-20 ℃ are frozen, HIV-1-P24 antigen titre to be measured.

The PBMC cell culture of above-mentioned HIV-1 virus treated adds the dyeing of MTT liquid after drawing supernatant, measure OD ₅₇₀The nm value is calculated CC ₅₀Test operation is referring to document: Zhang Xingquan, and Fan Jiang edits, and cell culture that Shen Lian .HIV infects and virusology detect. AIDS viral infection and acquired immune deficiency syndrome (AIDS). and the People's Health Publisher publishes 1999,295-304.

The ELISA method is measured HIV-1 P24 antigen

Frozen PBMC infects back dosing cell conditioned medium liquid, carries out dilution in 1: 100 after dissolving.Measure HIV-1 P24 titre by the operating procedure that examination HIV-1 P24 antigen-agent box provides, dosing group and virus control group are relatively calculated Ai Ning granule medium effective concentration (EC ₅₀).

Half toxic concentration (CC ₅₀) and medium effective concentration (EC ₅₀) and selection index (SI) computational methods

Medicine is pair cell or HIV-1P24 antigen inhibition percentage rate computational methods in cell culture

Suppress %=(experimental group OD-virus control group OD) * 100/ (normal control group OD-virus control group OD)

Reed ﹠amp; The Muench method is calculated the CC of medicine in cell culture ₅₀And EC ₅₀And the method for SI

Formula is as follows:

SI＝CC ₅₀/EC ₅₀

A, AZT sensitive strain HIV-1 018a and AZT persister HIV-1 018c infect after 2 hours the toxicity of pair cell in the PBMC cell culture

The result shows, two crowdes of poisonous concentration C C of experiment half ₅₀The responsive Strain 018a of AZT is respectively 1251.3 and 1190.8 μ g/ml, and average (1221.1 ± 42.78) μ g/ml is respectively 730.20 and 1232.2 μ g/ml to AZT multidrug resistant disease strain, average (981.20 ± 354.97) μ g/ml (seeing Table 9).

Table 9. Ai Ning granule is to the toxicity CC of HIV infection of PBMCs cell culture ₅₀(μ g/ml)

B, AZT sensitive strain HIV-1 018a and AZT persister HIV-1 018c infect in the back 2 hours PBMC cell culture the antigenic inhibitory action of P24

Result: Ai Ning granule EC ₅₀Be respectively 181.5 and 313.23 μ g/ml, average (247.37 ± 93.15) μ g/ml, SI is respectively: 5.2 and 6.2, average out to 5.7 ± 0.70.Two batches of experiments of positive control drug AZT EC ₅₀Not doing to the end, be respectively＜0.46 and＜0.01 μ g/ml, SI is respectively＞and 58.28 and＞100.0, average out to＞79.14 ± 29.50 (seeing Table 10).

Table 10. Ai Ning granule anti-HIV-1 018a (AZT sensitive strain) effect in the PBMC cell (dosing behind the absorption 2h)

Result: EC ₅₀Be respectively 499.70 and 134.53 μ g/ml, average (317.12 ± 258.21) μ g/ml, SI is respectively: 3.1 and 10.4, average out to 6.8 ± 5.11.To the 1st batch of experiment of positive drug AZT EC ₅₀Do not do to the end, the＜0.46,1st batch of experiment is 0.016 μ g/ml, average out to＜0.24 ± 0.31, and SI is respectively＞and 58.28 and＞62.5, average out to＞60.39 ± 2.98 (seeing Table 11).

Table 11 Ai Ning granule anti-HIV-1 o18c (AZT persister) effect in the PBMC cell (administration behind the absorption 2h)

3 in vitro tests conclusions

The Ai Ning granule has certain inhibitory action external to the HIV-1 intergrase, its IC ₅₀Be 153.29 ± 0.07 μ g/ml.To the 1000 μ g/ml unrestraint effects of HIV-1 reverse transcriptase, to the 00 μ g/ml unrestraint effect of HIV-1 protease 3.

Positive control medicine: HIV-1 intergrase SY, IC ₅₀For: 0.51 ± 0.06 μ g/ml; HIV-1 reverse transcriptase nevirapine (NVP), IC ₅₀For: 0.228 ± 0.035 μ g/ml; HIV-1 reverse transcriptase Saquinavir (SQ), IC ₅₀For: 8.122 ± 7.727nmol/L.All effective, consistent with document, illustrative experiment is credible.

According to the toxicity result of Ai Ning granule in cell culture hereinafter, the human T lymphocyte of promptly going down to posterity, MT-4 cell and human peripheral blood mononuclear cell's (PBMC) median toxic concentration (CC ₅₀) be respectively: 693.1 ± 432.1 μ g/ml, 200.9 ± 79.2 μ g/ml and 1221.1 ± 42.78 μ g/ml, it suppresses the IC of HIV-1 reverse transcriptase activity as can be known ₅₀Be 153.29 ± 0.07 μ g/ml, be actual inhibitory action, due to the non-cell toxicity.

The Ai Ning granule shows that to the MT-4 cytosis result that HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) I type (HIV-1IIIB) infects the Ai Ning granule is in the 0.078-0.156mg/ml dosage range, and the MT-4 cell that HIV-1 is infected has certain inhibitory action.Its average suppression ratio is 65.4%; The mean treatment index is 5.2.

The 2 batches of experiments of effect of anti-HIV in the chronically infected H9 cell culture of HIV-1 IIIB of Ai Ning granule show, in the chronically infected H9 cell culture of HIV-1IIIB, and the TC that Ai Ning granule pair cell is cultivated ₅₀Be 964.46 ± 581.32 μ g/ml, EC ₅₀Be 641.42 ± 584.06 μ g/ml, SI is 1.85 ± 0.78.The TC that positive control drug AZT pair cell is cultivated ₅₀Be 156.49 ± 15.58 μ g/ml, EC ₅₀Be 39.51 μ g/ml, SI is 3.7.

Cell MTT dyeing in the 4th day toxicity: the CC that the Ai Ning granule infects the responsive HIV-1 Strain of AZT in the human peripheral blood mononuclear cell cultivates ₅₀Be 1221.1 ± 42.78 μ g/ml, infect the 4th day supernatant of dosing in back 2 hours and measure P24 antigen EC ₅₀Be 247.37 ± 93.15 μ g/ml, selection index is 5.7 ± 0.7.

The cell that the Ai Ning granule infects AZT drug resistance HIV-1 Strain in the human peripheral blood mononuclear cell cultivates MTT dyeing in the 4th day toxicity: CC ₅₀Be 981.2 ± 354.97 μ g/ml, infect the 4th day supernatant of dosing in back 2 hours and measure P24 antigen EC ₅₀Be 317.12 ± 258.21 μ g/ml, selection index is 6.8 ± 5.11.

(3) experiment in the body

1, the Ai Ning granule is to the influence of immunosuppressed mice immunity function restructuring effect.

(1) set up the immunosuppressed mice animal model: Kunming mouse was fed three days at this institute secondary laboratory, injected 1 time next day of injecting cyclosporin A 0.5 milliliter (concentration is 1mg/ml) by 15 milligrams of consumptions of per kilogram of body weight to animal then, injected altogether 3 times.The 3rd injection back gastric infusion next day.

If normal control group, model control group, positive controls (ZHENQI FUZHENG KELI), 5 times of administration groups, 10 times of administration groups and 20 times of administration groups (the administration group is the Ai Ning groups of grains).20 of each treated animals.Administration every day 1 time, each 0.5ml (calculate by adult 59g crude drug amount/60 kg body weight, the dose,equivalent of mice medication is every 0.02g crude drug amount/only), successive administration 12 is big.

Administration is until the blood-letting of animal eye socket.Every Mus is used the 3K-EDTA anticoagulant then, gets the 0.1ml anticoagulation, by the reagent operation instruction carry out the T cell subsets detect [Guan Chongfen, Wang Jian, Xu Shuling etc. in grind the clinical and experimentation of II number treatment acquired immune deficiency syndrome (AIDS). Chinese combination of Chinese and Western medicine magazine 2003; 23 (7): 494-497].The testing result statistical analysis adopts the SPSS.11.0 statistical software to analyze.

(2) the results are shown in Table 11 '.The result shows, has successfully set up the immunosuppressed mice model with cyclosporine.Positive controls and model control group compare, difference P＜0.05.Approximate with dosage group result in the Ai Ning granule.The reconstruction that three Ai Ning granule administration groups all demonstrate immunosuppressant model mice immunologic function has facilitation, but the effect of low dosage, middle dosage is more obvious.

Table 11 '. the Ai Ning granule to the experimental result of immunosuppressed mice CD4+T cell (

N=20)

Compare with the normal control group ^*P＜0.01; Compare with model control group ^*P＜0.01; ^{* *}P＜0.05.

2, the Ai Ning granule is to immunodeficiency monkey disease poison (Simian immunodeficiency virus, SIV) the preventative-therapeutic influence of infection model in the body.

(1) modelling, grouping and administering mode.

The conventional anesthetized animal of ether, Strain SIVmac251, with every monkey 3MID/ml (100% low infective dose) through intravenous injection 1ml infection animal.With dissolved in distilled water, concentration is 1g/ml (crude drug) to the Ai Ning granule with preceding.Gavage 10ml/ time 1 time from infection slotting stomach tube every day on the same day.Azidothymidine AZT (Zidovudine, AZT) positive matched group, the 100mg/ grain, with before being dissolved among the normal saline 10ml, day gavages 1 time.The negative matched group of normal saline, medication is the same.8 weeks of successive administration, to withdraw weekly 1 time, the back continuation of 8 weeks observed for 4 weeks.Respectively took a blood sample 1 time in 2,4,8,12 weeks in the treatment back.Test operation is referring to document: A summary of the WHO Informal Consultation on Animal models forevaluation of drugs and vaccines for

(2) detect index.

1. virus is separated: the human T lymphocyte is CEMX 174, grows in the RPMI RPMI-1640.The infected monkey blood sampling, anticoagulant heparin, centrifugal 2000 rev/mins, 10 minutes, get blood plasma 200ul, do serial dilution with 50 times, respectively be equivalent in the blood plasma SIVmac251 titre and be 5,25,125,625,3125 and 15625TCID/ml, add 24 well culture plates (Lin 60 products), every hole and RBMI RPMI-1640 add to 1ml, add 2 * 10 again ⁵The CEM X174 cell suspension 0.5ml of cell/ml, at 37 ℃, 5%CO ₂Cultivate 4 weeks, routine observation cytopathy under the condition.

2. blood plasma SIV P27 antigen levels is measured: the ELISA method.Adopt SIV P27 antigen ELISA detecting kit, product description is pressed in operation.

3. monkey peripheral blood cd4 cell counting: adopt the flow cytometer method, one anti-ly is mouse-anti people CD4 monoclonal antibody, two anti-sheep anti-mouse igg-FITC.Isolating monkey PBMCs (PeriPheralblood mononuclear cells), behind the indirect method labelling, through 2% paraformaldehyde, 4 ℃ are fixedly spent the night, machine testing on next day.

4. serum mopterin level determination experimental technique: adopt enzymoimmunoassay, use ELItest Neopterin test kit, product description is pressed in operation.

5. serum ₂The mensuration that-microglobulin changes: adopt β ₂-MG ELISA test kit, by specification require to carry out test operation, and the drawing standard curve.Monkey serum is by different treatment time blood samplings, conventional separation of serum, cryogenic refrigerator-20 ℃ preservation.Experimental result uses the full-automatic microplate reader of France to sample OD pH-value determination pH, and by standard curve determination corresponding protein content.

6. pathologic finding: the experiment anesthesia that expires is put to death, and takes out internal organs, is fixed in 10% formalin, cuts into slices HE dyeing, microscopy.Test operation is referring to document: Guan Chongfen, Wang Jian, Xu Shuling etc. in grind II number the treatment acquired immune deficiency syndrome (AIDS) clinical and experimentation. Chinese combination of Chinese and Western medicine magazine 2003; 23 (7): 494-497.

(3) experimental result

1. the testing result of respectively organizing different phase plasma viral titre sees Table 12.

Table 12. is respectively organized the meansigma methods (log10/ml) of each stage plasma viral titre

Table 1 shows that with the 0.5log10/ml standard determination, administration group virus titer when 4 weeks descends, and virus is separated with AZT group result approximately during 12 weeks, and matched group virus is separated positive always.

2. the testing result of SIVp27 antigen levels sees Table 13.

Table 13. is respectively organized SIVp27 antigen levels relatively (pg/ml)

Table 13 demonstration obviously reduces when blood plasma SIV P27 antigen levels Ai Ning groups of grains is than 2 weeks when infecting for 4 weeks, and is close with the AZT group, and the negative control group variation is not obvious.

3. the testing result that infects each treated animal CD4 quantity of different times sees Table 14.

Table 14. infects the comparison (%) of each treated animal CD4 quantity of different times

Table 14 shows that cd4 cell percentage rate difference is little between preceding each treated animal of infection, infects back 4 all Ai Ning groups of grains bottom outs, level before approaching infection during 8 weeks, and AZT and negative control group are recovered slowly.

4. the antibody response experimental result of different group SIVmac sees Table 15.

The antibody response of the different group SIVmac of table 15. relatively

Table 15 shows that Ai Ning granule antibody response Zao and titre height occurs than model control group.

5. the result of variations of different time animal serum mopterin level sees Table 16.

Different time animal serum mopterin level variation before and after table 16. infects (nmol/L, x ± s)

Annotate: the mopterin level is 7.05 ± 3.01 before a, the infection, is the average that each treated animal adds up to;

Compare the t assay before b, the same infection: ^*P＜0.01, ^*P＞0.05.

6. animal serum β ₂-MG changes of contents the results are shown in Table 17.

Each treated animal serum before and after table 17. infects ₂-MG changes of contents (mg/L, X ± s)

The above results explanation, Ai Ning granule have the effect that suppresses infected monkey plasma viral mass formed by blood stasis and protection immune cell.

3, to of the influence of Ai Ning granule to the treatment of immunodeficiency monkey disease poison chronic infection model.

(1) modelling, grouping and administering mode

10 of machins are divided into Ai Ning groups of grains and model control group at random, and 5 every group, with SIVmac251 virus liquid, 5MID ₁₀₀(5 100% monkey infective doses) 1ml intravenous injection infection animal.Behind the taint with SIV 90 days, the Ai Ning groups of grains was that 0.5g/ml Ai Ning granule is inserted stomach tube and gavaged medicine with concentration, is equivalent to people's dosage every day 1g/kg (crude drug), every day 1 time, each 10ml, 8 weeks of successive administration.Model control group is given and is waited dosage water.Observed 2 months observation index project and sampled point after the drug withdrawal.Test operation is referring to document: Wu Xiaoxian, Zhang Fengxue, He Fuqiu etc. the foundation of simian immunodeficiency virus (SIV) chronic infection monkey model. and Traditional Chinese Medicine University Of Guangzhou's journal 2000; 17 (4): 355-357.

(2) inspection method

CD3, CD4 lymphocyte subgroup: the operating procedure by specification carries out, and measures CD3 ⁺CD4 ⁺Percentage of lymphocyte is calculated cd4 cell and cd8 cell % and CD4/CD8 ratio.

Plasma viral load: by sampled point requirement blood sampling, the EDTA anticoagulant after centrifuging and taking blood plasma is frozen, send the fifth-largest tumor of Paris, FRA and viruses molecule laboratory professor Lu Wei to locate to measure behind the zoogenetic infection SIV.Assay method is created by professor Lu Wei, and is published in Nature Medicine, and primer and probe prepare voluntarily, and uses the Roche LightCycler of company quantitative instrument to detect.

Lymph node biopsy: aseptic operation is got 1 side inguinal lymph nodes before the treatment, and treatment finishes to get 1 side inguinal lymph nodes in addition again after 8 weeks were observed in the back, is fixed in 10% neutral formalin liquid, paraffin embedding, H.E dyeing back om observation.The part lymph node section is made cell proliferation related antigen Ki-67 (clone B56, BD company) SABC inspection, (the anti-rabbit/mice IgG and the IgM of HRP labelling is ab2891) after the DAB colour developing for the Color Appearance System of employing Britain Abcam company, haematoxylin is slightly redyed, om observation.Test operation is referring to document: Lu Yaozeng, Wu Xiaoxian, Li Guoqiao etc. the pathological change of the acute and chronic infection lymph node of simian immunodeficiency virus. and Traditional Chinese Medicine University Of Guangzhou's journal 2003; 20 (3): 191.

(3) result

1. ordinary circumstance

The machin that experimental monkey groups adopts SIVmac251 to infect 90 days, the interior plasma viral load of monkey body this moment has all been reduced to set point (setpoint), i.e. plateau.Though owing to exist individual variation, each monkey to arrive the virus load level of this plateau and inequality, in long period of time after this, the virus load of each monkey all is maintained at a metastable level, the fluctuation range of its logarithm (log10) is general＜and 0.5.The ordinary circumstance of each monkey was fair after SIV infected.Ai Ning groups of grains treatment back ordinary circumstance is also still good, and body weight descends when treatment is all to 8 to some extent, generally keeps more stable after 8 weeks.Model control group two monkeys are intermittently suffered from diarrhoea always, may be relevant with body constitution and viral infection.Ai Ning groups of grains and model control group symptom and sign are not seen significant difference.

2. immunologic test result

Before the treatment of two groups of monkeys and treatment back is every detecting 1 T cell subsets CD4 and CDg dynamic levels 4 weeks, and the effect of observing medicine is analyzed the variation of CD4 and CD8 lymphocyte quantity and CD4/CD8 ratio respectively.

The result shows that the CD4 lymphocyte quantity that the Ai Ning groups of grains was treated after 4 weeks all rises than treatment is preceding, and 8 and 12 weeks maintained an equal level with treatment is preceding, and 16 weeks then descended.The corresponding point of model control group is promptly treated 4,8,12 weeks of back and is risen before the treatment.16 weeks also descended, but corresponding cd4 cell quantity treatment back each point is all than rise before the treatment (seeing Table 18,19,20).

The variation of table 18.CD4 (%) (X ± SD, n=5)

Compare before and after two groups of treatments of Ai Ning groups of grains and model control group ^*Compare after the drug withdrawal P＜0.01 ^*P＜0.05.

The variation of table 19.CD8 (%) (X ± SD, n=5)

Compare after two groups of treatment front and back of Ai Ning groups of grains and model control group, the drug withdrawal ^*P＞0.05.

The variation of table 20.CD4/CD8 ratio (X ± SD, n=5)

Compare before and after two groups of treatments of Ai Ning groups of grains and model control group ^*Compare after the drug withdrawal P＜0.05 ^*P＞0.05.

3. virusology check result

Before and after two groups of treatments the measurement result fluctuation range of each point plasma viral loads all＜0.5log10, do not have significant difference (seeing Table 21).

Plasma viral load (log10.copies/ml) before and after table 21. Ai Ning groups of grains and the model control group treatment

Compare before and after two groups of treatments of Ai Ning groups of grains and model control group ^*Compare after the drug withdrawal P＞0.05 ^*P＞0.05.

4. the result of lymph node biopsy histopathologic examination

The Ai Ning groups of grains: inguinal lymph nodes, folliculus, germinal center increases, and increases.There is a small amount of immune complex deposit in germinal center, and the centrocyte apoptosis is slight, and secondary cortical layer has more follicle hypertrophy, is hypertrophy type pathological changes.

Model control group: inguinal lymph nodes, lymph follicle dwindles, and folliculus quantity obviously reduces, and germinal center dwindles, and centrocyte is sparse, and immune complex deposit is arranged, and is regression type pathological change.

Ki-67 (monoclonal antibody) immunohistochemical detection result shows: the Ai Ning groups of grains all shows lymphocytosis, and the lymphocyte of model control group lymph node obviously reduces, and is the regression state.

Show: the Ai Ning granule has the lymphadenosis of promotion effect, or delays lymphoid tissue regression effect.

According to see Table 22 and table 23 with the evaluation of observing down monkey lymph node each several part variation before and after this test on show.

Table 22. simian acquired immunodeficiency syndrome model treatment back lymph node pathological change change list (N=5)

Utilization SPSS10.0 software carries out rank test, compares with model control group ^*P＜0.05 ^*P＜0.01.

Table 23. lymph node pathology changes to be estimated

4, Ai Ning particulate fraction symptom pharmacodynamic experiment research.

(1) experiment material

Animal: Kunming mouse;

Medicine: Ai Ning granule: 0.1715 gram medicated powder is equivalent to 1 gram crude drug;

ZHIKEJUHONG KOUFUYE, the Tongrentang Pharmaceutical Factory, Beijing TongrenTang Co., Ltd;

The Ginseng Bolus for tonifying spleen, the Tongrentang Pharmaceutical Factory, Beijing TongrenTang Co., Ltd;

SHENGMAI YIN, the Tongrentang Pharmaceutical Factory, Beijing TongrenTang Co., Ltd.

(2) experimental technique and result

1. the Ai Ning granule is to the antitussive effect of mice

Select 52 of Kunming mouses for use, male and female half and half, body weight 22 ± 2 grams, evenly be divided into 5 groups by body weight and sex, be respectively the heavy dose of group of administration 19.7g crude drug/kg body weight, middle dosage group 9.83g crude drug/kg body weight, small dose group 4.92g crude drug/kg body weight, during experiment Ai Ning granule medicated powder is configured to finite concentration, dosage is the 0.20ml/10g body weight.Positive controls gives ZHIKEJUHONG KOUFUYE, and dosage is the 0.1ml/10g body weight.The blank group gives the equivalent distilled water.Adopt the gastric infusion mode, successive administration 3 days, experiment administration on the same day placed animal in the beaker of 1000ml after 30 minutes, injected the cotton balls of 0.2ml ammonia in beaker, the cough number of times in the incubation period and 3 minutes of record mouse cough.The results are shown in Table 24.

Table 24. Ai Ning granule is to the antitussive effect of mice

Compare with the blank group: ^*P＜0.05, ^*P＜0.01.

By above result as seen, the Ai Ning granule can more significantly prolong the incubation period of mouse cough, obviously reduces the mouse cough number of times, points out this medical instrument that tangible antitussive effect is arranged.

2. the Ai Ning granule is to the influence of mice endurance

Select Kunming mouse for use, male and female half and half, body weight 20 ± 2 grams, evenly be divided into 5 groups by body weight and sex, be respectively the heavy dose of group of administration 19.7g crude drug/kg body weight, middle dosage group 9.83g crude drug/kg body weight, small dose group 4.92g crude drug/kg body weight, during experiment Ai Ning granule medicated powder is configured to finite concentration, dosage is the 0.20ml/10g body weight.Positive controls gives SHENGMAI YIN KOUFUYE, and dosage is the 0.1ml/10g body weight.The blank group gives the equivalent distilled water.Adopt the gastric infusion mode, successive administration 3 days, experiment administration on the same day was put into 25 ℃ water with animal after 30 minutes, and the swimming continuance time of record mice the results are shown in Table 25.

Table 25. Ai Ning granule is to the influence of mice swimming continuance time

Compare with the blank group: ^*P＜0.05, ^*P＜0.01.

By table 25 as seen, the big or middle dosage group of Ai Ning granule mice swimming continuance time obviously prolongs than the blank group, and T checks P＜0.01, and pointing out this medical instrument to have increases the muscle power effect preferably.

3. the Ai Ning granule is to the influence of mouse small intestine motion

Select Kunming mouse for use, male and female half and half, body weight 20 ± 2 grams, evenly be divided into 5 groups by body weight and sex, be respectively the heavy dose of group of administration 19.7g crude drug/kg body weight, middle dosage group 9.83g crude drug/kg body weight, small dose group 4.92g crude drug/kg body weight, during experiment Ai Ning granule medicated powder is configured to finite concentration, dosage is the 0.20ml/10g body weight; Positive controls gives 40% Ginseng Bolus for tonifying spleen, the 0.2ml/10g body weight; The blank group gives the equivalent distilled water.Adopt the gastric infusion mode, successive administration 3 days.The animal fasting be can't help water 24 hours before the experiment, experiment contained 5% arabic gum and 10% active carbon medicinal liquid once by same dosage the same day, after the administration 20 minutes, put to death animal, small intestinal total length and the active carbon forward travel distance at small intestinal is measured in dissection, the calculated activity charcoal the results are shown in Table 26 in the propelling ratio of small intestinal.

Table 26. Ai Ning granule is to the influence of mouse small intestine propulsion functions

Compare with the blank group: ^*P＜0.05, ^*P＜0.01.

All can significantly increase the advance distance of animal small intestinal by visible each the administration treated animal of table 26, prompting Ai Ning granule has invigorating the spleen and benefiting QI, strengthens the effect of gastrointestinal motility.

Conclusion: the experimental result of comprehensive above-mentioned main pharmacodynamics, prompting Ai Ning granule energy human body immunity improving function has certain inhibitory action to HIV (human immunodeficiency virus), is the medicable Chinese medicine preparation of treatment acquired immune deficiency syndrome (AIDS).

(4) clinical trial

1. clinical data

1.1 diagnostic criteria: mainly diagnose according to medical history, symptom and sign and laboratory Serological testing.

1.2 inclusion criteria: all patients all according to nineteen ninety-five China's AIDS diagnosis standard make a definite diagnosis, and meet inclusion criteria.

1.2.1 the age: 18-55 year.

1.2.2HIV the antibody conclusive evidence is positive.

1.2.3CD4 positive counting 100-500/mm ³

1.2.4 merge following opportunistic infection person (intestinal infection, skin infection, respiratory system infection).

1.2.5 signature Informed Consent Form person.

2. clinical observation

2.1 observation medicine: the Ai Ning granule, every day 3 times, each 1 bag, every bag 5 gram, 3 months is a course of treatment.

2.2 observational technique: before treatment, carry out virus load 3 the end of month respectively 1 the end of month, the immunologic function index (CD4, CD8, CD4/CD8 ratio, the CD45RA cell quantity detects), and fill in clinical observation table.

2.3 clinical observation and follow up a case by regular visits to content: observe weak, low grade fever in preceding 0 month of treatment and treatment back the 1st, 3 the end of month respectively, become thin, symptom and signs such as cough, indigestion and loss of appetite, diarrhoea, alopecia, prurigo, adopt the method representation of grade integration.

2.4 what virus load adopted is that the RT-PCR method is measured; The immunologic function index is used for detecting the T cell subsets, with cd4 cell absolute value and the CD45RA cell absolute value leading indicator as patient's immunologic function; Represented since the clinical symptoms sign, heavily remember 3 fens, middle note 2 minutes is gently remembered 1 fen, normally remembers 0 fen.

3. observed result

3.1 curative effect determinate standard: from virusology, immunology and clinical manifestation are judged.

Immunologic function:

Rise: the cd4 cell number rises＞30%;

Stable: cd4 cell is counted excursion ± 30%;

Descend: the cd4 cell number descends＞30%.

Virus load:

Descend: virus load is reduced to the level that detection do not go out (＜500 copies/ml); Or the above level of reduction 0.5log copy/ml;

Stable: virus load reduction＜0.5log copy;

Rise: the virus load ascensionist.

3.2 therapeutic outcome

Physical data: 14 routine HIV the infecteds, male's 9 examples, women's 5 examples, the age average 40 years old, is paid blood collecting and supplying and infects between 26-53.

The Ai Ning granule carries out trimestral treatment to 14 routine HIV the infecteds to be observed, and it the results are shown in Table 27～31.Its result shows: virus load, treat preceding 4.554 ± 0.975log, and treatment back 4.379 ± 1.082log, the 0.175log that on average descends, virus load 9 examples that descend wherein, obvious 5 examples (greater than 0.51Log) that descend, 3 routine stablizing, effective percentage is 64.28%; Immunologic function 3 examples that rise are stablized 5 examples, and effective percentage is 57.14%; Clinical symptoms makes moderate progress.Do not find obvious toxic-side effects.

The routine HIV the infected's changes in immune function of table 27.14 situation (routine number)

The routine HIV the infected's immunologic function of table 28.14 change detected situation

The routine HIV the infected's cardinal symptom of table 29.14 is improved situation

The routine HIV the infected's routine blood test of table 30.14, hepatic and renal function situation of change

The situation of change of table 31.14 routine HIV the infected's cd4 cell number and virus load

Claims (7)

1. treat the acquired immune deficiency syndrome (AIDS) Chinese medicine composition for one kind, it is characterized in that it is made by following raw medicaments in portion by weight: 3～8 parts of 15～20 parts of the Radixs Astragali, 7～12 parts of Radix Ginsengs, 8～13 parts of Radix Angelicae Sinensis, 9～14 parts of Fructus Lycii, 7～12 parts of Fructus Schisandrae Chinensis, 7～12 parts of Radix Ophiopogonis, 8～13 parts of Radix Trichosanthis, 7～12 parts in Poria, 7～12 parts in Radix Glycyrrhizae, 3～8 parts of Radix Bupleuri and Rhizoma Cimicifugaes.

2. Chinese medicine composition according to claim 1, it also comprises one or more pharmaceutically acceptable auxiliary additives.

3. Chinese medicine composition according to claim 2 is characterized in that, described auxiliary additive is lactose and dextrin.

4. Chinese medicine composition according to claim 3 is characterized in that, described Chinese medicine composition is a granule.

5. the preparation method of a Chinese medicine composition according to claim 1 is characterized in that, it comprises the following steps:

A) get the Radix Astragali, Fructus Lycii, Radix Trichosanthis, Poria and the Radix Glycyrrhizae 5 flavor medicines of described weight proportion, the decocting that adds 7～10 times of suitable crude drug amounts boils twice, and each 1～2.5 hour, collecting decoction, filter, relative density was 1～1.2 clear paste when filtrate was condensed into 30～60 ℃;

B) get Radix Ginseng, Radix Angelicae Sinensis, Fructus Schisandrae Chinensis, Radix Ophiopogonis, Radix Bupleuri and the Rhizoma Cimicifugae 6 flavor medicines of described weight proportion, percentage by weight 50～80% ethanol that add 5～8 times of suitable crude drug amounts, reflux or immersion secondary, each 1～2.5 hour, merge ethanol liquid, filter, decompression filtrate recycling ethanol and when being condensed into 30～60 ℃ relative density be 1～1.2 clear paste;

C) merge above-mentioned two clear paste, 30～60 ℃ are concentrated into relative density is 1～1.2 extractum, is the active component of medicine.

6. preparation method as claimed in claim 5 is characterized in that extractum spray drying or vacuum drying that it also obtains step c), gets dry extract.

7. as the application of each described Chinese medicine composition of claim 1～4 in the medicine of preparation AIDS virus resisting and/or raising immunologic function.