CN103585239B - A kind of preparation method of Boraginaceae extract and application thereof - Google Patents

Abstract

The invention relates to a preparation method and application of an extract of Serrata serrata, belonging to the field of medicine. The preparation method is to use Serenia serrata as raw material, extract with different concentrations of ethanol, and then purify it through two macroporous adsorption resins, including Serenia serrata extract A, Serra serrata extract B, Serra serrata extract C, Serpentine extract D. The Serrata serrata extract can be combined with pharmaceutically acceptable carriers or other suitable excipients, and prepared into a dosage form for oral administration according to conventional methods. The Serrata serrata extract of the invention can be used to prepare medicines for treating AIDS.

Description

A kind of preparation method and application of Serrata serrata extract

technical field

The invention belongs to the field of biomedicine and relates to a preparation method and application of an extract of Chinese medicinal materials, in particular to a preparation method and application of an extract of Serrata serrata.

Background technique

AIDS is a new viral infectious disease discovered in 1981, which was not recorded in traditional Chinese medicine. TCM differentiates AIDS and believes that AIDS belongs to the category of epidemics. The disease is caused by the invasion and attack of epidemic toxin and heat evil, and the weakness of righteousness. The pathogenesis is mainly that the defense is not solid, the yang is damaged, and the internal organs are involved, causing lung deficiency, spleen deficiency and kidney deficiency, and secondary infection. Heat damages the veins, qi and blood stagnation, and forms accumulations of lumps and tumors.

Differentiation of "syndrome" in traditional Chinese medicine: mainly to distinguish the growth and decline of evil and positive and the syndrome of deficiency and excess. The AIDS virus is an evil poison, and the evil poison enters and spreads and changes, and the dynamic change of fighting against the evil and righteousness of the body's righteousness is the development process of the disease. In the early stage of infection, evil and excess are the main factors, in the middle stage, "deficiency is caused by excess", and in the late stage, "deficiency is caused by excess". The replication of AIDS is variable, and both AIDS and its secondary infection can form "diseases" with different "types" and "periods". Traditional Chinese medicine has different diagnosis and treatment programs for AIDS, such as syndrome differentiation, classification and staging.

The treatment of AIDS with traditional Chinese medicine is still in the research stage. At present, there are no anti-HIV traditional Chinese medicines or traditional Chinese medicine monomer compounds that are recognized for their curative effect and approved for production. Anti-HIV botanical medicines and traditional Chinese medicine research are mainly divided into three ways. Some anti-HIV Chinese medicine botanical medicines and their components obtained from cigarettes have been used in clinical trials.

Since the discovery of HIV, HIV cell culture and in vitro experimental methods such as reverse transcriptase, protease and integrase have been used internationally to screen compounds extensively, and at the same time to screen a large number of plant and Chinese herbal medicine extracts and their components, and to study some Chinese medicines. Compound preparations have been found to be effective drugs for inhibiting HIV in vitro, and some can inhibit viremia in monkeys infected with Simian Immunodeficiency Virus (SIV). Some have been tested clinically, but the effect has not yet been determined.

Since 1987, my country has sent medical teams integrating traditional Chinese and Western medicine to treat AIDS patients in Africa. Before 2002, there was a lack of effective anti-HIV drugs in China. According to the theory of traditional Chinese medicine, traditional Chinese medicine and western medicine combined disease differentiation and syndrome differentiation for AIDS patients in clinical practice. , according to the performance of traditional Chinese medicine, select traditional Chinese medicine, prescription treatment. Through clinical practice, it is believed that some traditional Chinese medicine compound prescriptions can improve symptoms and alleviate the condition in the treatment of AIDS. In recent years, AIDS patients confirmed by experimental diagnosis have been treated with traditional Chinese medicine, and a control group has been set up. During the treatment period, blood viral load (HIV-RNA) and CD4 cell levels are regularly detected to evaluate the effect. It has been reported that it can improve symptoms and improve immunity, and most of them are still under clinical verification. Anti-HIV single herbal medicines or herbal medicines and Chinese medicine compound prescriptions that have entered clinical practice are as follows:

Glycyrrhizin: Glycyrrhizin (GL) is a glycyrrhizin saponin extracted from the traditional Chinese medicine Glycyrrhiza glabra L. It has broad-spectrum antiviral effects, inhibits HIV-1 reverse transcriptase in vitro, and inhibits HIV cell fusion in cell culture. It is also anti-influenza virus and SARS virus. In vivo experiments have proved that it can protect mice from pneumonia and lung lesions caused by influenza virus infection; protect mice from CCl ₄ toxic liver damage; induce gamma-interferon; and has hormone-like effects. It is commonly used in Japan to treat chronic hepatitis B by intravenous drip, and it has also been tried to treat AIDS patients. It can inhibit serum HIV-1-P24 antigen and increase CD4 cells. Professor Lu Weibo used Glycyrrhizin oral preparation Keaike to treat AIDS patients in Tanzania, Africa from 1988 to 1992, which can improve symptoms and slightly increase CD4 cells. Its effect has not been verified.

Trichosanthin protein: Trichosanthin (Trichosanthin, TCS, GLQ223, Q substance) is a traditional Chinese medicine Trichosanthes kirilowii (Trichosanthes kirilowii), which belongs to Cucurbitaceae (Cucurbitaceae). The active ingredient of anti-early pregnancy and induction of labor is trichosanthin, with a molecular weight of 27kD. Its primary, secondary and three-dimensional structures have been completely clarified. It is a phytotoxic protein that inhibits protein synthesis. It is a single-chain ribosome inactivating protein (Ribosome Inactivating Protein, RIP). When the United States screened anti-HIV-1 drugs, it was found that it inhibited HIV-1 cell fusion in lymphocyte cultures, and later found that HIV-1 P24 antigen and RNA in monocyte-macrophage cultures, HIV-1 in 3 monkeys Intravenous injection of (SIV) infected monkeys suppressed plasma HIV-1. The United States uses intravenous drip to treat AIDS patients in forest farms, which can inhibit HIV-1-infected T lymphocytes in peripheral blood and HIV-1-P24 antigen in mononuclear macrophages, and slightly increase the ratio of CD4/CD8. Clinical trials have been discontinued due to anaphylaxis death, neurotoxic insanity and nephrotoxicity. In recent years, another protein, TAP29, has been isolated from Trichosanthes pollen, with a molecular weight of 29 kD. It also has the effect of inhibiting HIV-1, but it is less toxic and has not been used in clinical trials. In addition, the bitter melon protein of MOmordica charantia and the pokeweed protein of Phytolacca americana have the effect of inhibiting HIV.

Hypericin: The plant Hypericum perforatum (St. John Wort, Hypericum perforatum) folk tradition uses its extract to treat depression. The active ingredient extracted from the flower is hypericin (hypericin), an aromatic polycyclic dimer Anthraquinone compounds, which are antidepressant drugs, have been fully synthesized. Hypericin has a variety of antiviral activities, inhibits HIV in cell culture, and its mechanism of action is to block the uncoating, assembly and release of the virus, has a virus-killing effect, and can pass through the blood-brain barrier. It can inhibit the reproduction of murine leukemia virus in cell culture. In vivo, it can inhibit the splenomegaly caused by murine leukemia virus infection in mice, and it can also inhibit the infection of duck blood virus DNA by duck hepatitis B virus, and has broad-spectrum antibacterial activity. It was used to treat AIDS patients in phase I/II clinical trial, but the trial was stopped due to photosensitivity.

D-allucin A: D-alderin A (+Calanolide A) is a natural compound isolated from the Malaysian tropical rainforest plant Calophyllum lanigerum reported in 1992. It is a mouse coumarin compound that inhibits HIV-1 reverse transcriptase, but has no HIV-2 is ineffective. Belonging to non-nucleoside HIV-1 reverse transcriptase inhibitors, it is effective for AZT-sensitive and drug-resistant HIV-1 clinically isolated in cell culture, and has a synergistic effect with AZT and NVP. It has been proved to have anti-HIV effect in the HIV hollow fiber mouse model, and the combined application with AZT can enhance the drug effect. It has been fully synthesized, and I-II clinical trials have shown that it has the therapeutic effect of inhibiting HIV-1 infection, has low toxicity, mild side effects, such as headache and loss of appetite, and is effective in combination with AZT. It is currently the only single plant ingredient that has entered phase II clinical trials. It is under observation and verification of efficacy, and has not yet been approved for production.

Xiao Chai Hu Tang: Xiao Chai Hu Tang (Bupleurum, Ginseng, Pinellia, Scutellaria, Licorice, Ginger, Jujube)

It is Zhang Zhongjing's Treatise on Febrile Diseases. Japan used it to treat hepatitis B and found that it inhibited HIV in cell culture. The United States has been clinically used to treat several AIDS patients, which can reduce the serum P24 antigen. We demonstrate that it inhibits HIV-1 reverse transcriptase and inhibits HIV-1 in cell culture. The effect of its seven components of traditional Chinese medicine was studied separately, and it was found that only the water extract of Scutellaria baicalensis and its component baicalin (baicalin) had the effect of inhibiting HIV-1, inhibiting HIV-1 reverse transcriptase in a cell-free system in vitro, and inhibiting HIV-1 reverse transcriptase in human T cells and Inhibition of HIV-1 cytopathy in cultured peripheral blood mononuclear cells, viral fluorescent antigen and P24 antigen, oral administration of murine leukemia virus infection in mice can inhibit splenomegaly and elevated blood leukocytes. Japanese Ono reported that baicalin (baicalien) inhibited HIV-1 reverse transcriptase.

Zhongyan No. I: Zhongyan No. I (the main drug is composed of Viola Didin, Trichosanthin, etc.) trade name: Ai

Ke Granules, with the principle of clearing away heat and detoxification, was formulated by the Chinese Academy of Traditional Chinese Medicine and was clinically used in Tanzania, Africa, in the early and middle stages of AIDS from 1992 to 1993. It is believed that it can improve symptoms and improve immunity. The laboratory has proved that the drug inhibits the activity of HIV reverse transcriptase in vitro, and the experimental treatment results in monkeys infected with HIV in vivo show that it can reduce the plasma virus titer, and can increase the number of CD4 cells, the ratio of CD4/CD8 and the value of T, B lymphocytes , Induce interferon and produce immune regulation function. It has been clinically tested in Africa and my country, but has not yet been approved for clinical verification.

Zhongyan No. 2: Zhongyan No. 2 (astragalus, ginseng, angelica, wolfberry and licorice, etc.) is a Chinese traditional medicine

Formation of the medical team of the research institute. In 1992, he treated AIDS patients in Tanzania, Africa. From 1998 to 1999, he treated 29 AIDS patients in African specialist outpatient clinics, 2/16 cases of HIV viral load decreased, and CD4 increased in 8/29 cases. From 2001 to 2002, he treated 15 AIDS patients in Beijing You'an Hospital, 3/15 cases had decreased viral load, and CD4 1/15 cases had increased CD4. It has the effect of improving symptoms. Experiments have shown that the drug can reduce the plasma virus titer, P27 antigen, increase the number of CD4 cells, and activate lymphocytes in monkeys infected with HIV.

Serenade is the dry aboveground part of Eritrichium rupestre (Pall.) Bunge, a plant of Boraginaceae. Harvest when flowers bloom in summer, remove roots and impurities, and dry in the shade. Included in 1998 "Ministry of Health of the People's Republic of China Drug Standard Mongolian Medicine Subvolume", it is cool in nature, bitter and sweet in taste. It has the functions of killing "sticky", clearing heat and detoxifying. It is used for warm flu, "Xie Ri" pulse syndrome, and "Xie Ri" fever. It is a commonly used drug in ethnic minority areas such as Tibet and Mongolia. However, the basic research on S. serrata is still very limited, which limits the subsequent promotion and application of this medicinal material. There are few reports on the chemical constituents and pharmacological effects of S. serrata in modern research.

According to domestic patent search results, there is no patent related to S. serrata.

In the above-mentioned documents and patents, there are no relevant reports on the preparation method and application of the Serrata serrata extract.

Contents of the invention

The purpose of the present invention is to provide a preparation method and application of Serrata serrata extract.

The present invention is achieved through the following technical solutions:

The raw material of Serrata serrata used in the present invention is the dry aerial part of Eritrichium rupestre (Pall.) Bunge, a plant of Boraginaceae.

A kind of preparation method and application thereof of Serrata extract of the present invention; The preparation method of described Serrata extract is:

(1) S. serrata, soak in water for 4-12 hours, use steam distillation to extract volatile oil, extraction time 1-4 hours, extraction times 1-3 times, add water 8-15 times the weight of S. serrata, collect Volatile oil, that is Serrata serrata extract A; filter the Serrata serrata water extract to obtain medicinal liquid A and medicinal residue A respectively;

(2) Use ethanol with a concentration of 50%-95% of the dregs A obtained in step (1) as a solvent, heat and reflux for extraction, the number of extractions is 1-3 times, each extraction time is 1-3 hours, and the amount of solvent used each time is 8-15 times the weight of S. serrata, filter, recover ethanol from the extract, concentrate, and dry to obtain S. serrata extract B;

(3) Concentrate the drug solution A obtained in step (1) to a relative density of d=1.10-1.20, filter it, pass the drug solution through a non-polar macroporous adsorption resin, and first elute with water, and the water eluent directly passes through the electrode macroporous resin; then elute the non-polar macroporous adsorption resin with ethanol solution with a concentration of 30%-70%, collect ethanol eluents with different concentrations, concentrate and dry, and then obtain Serrata serrata extract C; then use a concentration of 50% %-95% ethanol solution to elute the polar macroporous adsorption resin, collect ethanol eluents of different concentrations, concentrate and dry, and obtain Serrata serrata extract D;

(4) Serenia serrata extract A, Serenia serrata extract B, Serenia serrata extract C, and Serenia serrata extract D, one or two or three or four of which are mixed in a certain proportion, That is the Serrata serrata extract of the present invention.

A preparation method of Herba Serrata extract is:

(1) S. serrata, soak in water for 4-12 hours, use steam distillation to extract volatile oil, extraction time 1-4 hours, extraction times 1-3 times, add water 8-15 times the weight of S. serrata, collect Volatile oil, that is Serrata serrata extract A; filter the Serrata serrata water extract to obtain medicinal liquid A and medicinal residue A respectively;

(2) Use ethanol with a concentration of 50%-95% of the dregs A obtained in step (1) as a solvent, heat and reflux for extraction, the number of extractions is 1-3 times, each extraction time is 1-3 hours, and the amount of solvent used each time is 8-15 times the weight of S. serrata, filter, recover ethanol from the extract, concentrate, and dry to obtain S. serrata extract B;

(3) Concentrate the drug solution A obtained in step (1) to a relative density of d=1.10-1.20, filter it, pass the drug solution through a non-polar macroporous adsorption resin, and first elute with water, and the water eluent directly passes through the electrode macroporous resin; then elute the non-polar macroporous adsorption resin with ethanol solution with a concentration of 30%-70%, collect ethanol eluents with different concentrations, concentrate and dry, and then obtain Serrata serrata extract C; then use a concentration of 50% %-95% ethanol solution to elute the polar macroporous adsorption resin, collect ethanol eluents of different concentrations, concentrate and dry, and obtain Serrata serrata extract D;

(4) Mix the above-mentioned Serenia serrata extract A, Serra serrata extract B, Serra serrata extract C, and Serra serrata extract D to obtain the Serra serrata extract of the present invention.

The preparation method of a preferred Serrata serrata extract is:

(1) Serenia serrata, soak in water for 6 hours, extract the volatile oil by steam distillation, the extraction time is 2 hours, the extraction times are 2 times, the amount of water added is 12 times the weight of Serenia serrata, and the volatile oil is collected to obtain Serenia serrata extract substance A; the water extract of Serrata serrata was filtered to obtain medicinal solution A and medicinal residue A respectively;

(2) Use 60% ethanol as a solvent to extract the dregs A obtained in step (1), heat and reflux for extraction, the number of extractions is 2 times, each extraction time is 2 hours, and the amount of solvent used each time is 12 times the weight of S. serrata , filtered, the extract recovered ethanol, concentrated and dried to obtain Serrata serrata extract B;

(3) Concentrate the drug solution A obtained in step (1) to a relative density of d=1.12, filter it, pass the drug solution through D101 non-polar macroporous adsorption resin, and first elute with water, and the water eluent directly passes through DM130 Polar macroporous resin; then elute the D101 non-polar macroporous adsorption resin with 60% ethanol solution, collect the 60% ethanol eluate, concentrate and dry to obtain Serrata serrata extract C; % ethanol solution to elute the DM130 polar macroporous adsorption resin, collect the 75% concentration ethanol eluate, concentrate and dry, and obtain Serrata serrata extract D;

(4) Mix the above-mentioned Serenia serrata extract A, Serra serrata extract B, Serra serrata extract C, and Serra serrata extract D to obtain the Serra serrata extract of the present invention.

The preparation method of a kind of Serrata serrata extract of the present invention is characterized in that: the nonpolar macroporous adsorption resin adopted is D101 macroporous adsorption resin, AB-8 macroporous adsorption resin; The adsorption resin is DM130 macroporous adsorption resin and ADS-17 macroporous adsorption resin.

The preparation method and the application of a Serrata serrata extract of the present invention, the application is the application in the preparation of AIDS treatment drugs.

The medicine for treating AIDS composed of the Serra serrata extract and chemical medicine, traditional Chinese medicine or natural medicine.

The application of the Serra serrata extract A, the Serra serrata extract B, the Serra serrata extract C and the Serra serrata extract D of the present invention in the preparation of drugs for treating AIDS.

Serrata serrata extract A, Serra serrata extract B, Serra serrata extract C, and Serra serrata extract D of the present invention are AIDS-treating medicines composed of chemical medicines, traditional Chinese medicines or natural medicines.

The Serrata serrata extract of the present invention is prepared into pharmaceutical oral dosage forms such as tablets, granules, and capsules by adding various pharmaceutically acceptable auxiliary materials.

For the first time, the present invention explores and researches the extraction and preparation of the AIDS treatment extract from the dry aerial part of the plant Eritrichium rupestre (Pall.) Bunge of the Boraginaceae family. This experimental study shows that the Serrata serrata extract of the present invention can be used to prepare drugs for the treatment of AIDS. The Serrata serrata extract has little toxicity to C8166 cells and has significant inhibitory activity on HIV-1-induced syncytia formation in C8166 cells. It can be used in It is used in the preparation of drugs for the treatment of AIDS. Clinical treatment of AIDS patients has a remarkable effect.

Detailed ways

The preparation method and application of a Serrata serrata extract of the present invention will be further described below through specific experimental examples and examples, but not limited to the present invention.

Further details are given below through specific examples.

Embodiment 1: the preparation of Sara serrata extract

(1) 19kg of S. serrata, soaked in water for 6 hours, extracted the volatile oil by steam distillation, the extraction time was 2 hours, the extraction times were 2 times, the amount of water added was 12 times the weight of S. serrata, and the volatile oil was collected to obtain S. serrata extract A; filtering the Serrata serrata water extract to obtain medicinal liquid A and medicinal residue A respectively;

(2) Use 60% ethanol as a solvent to extract the dregs A obtained in step (1), heat and reflux for extraction, the number of extractions is 2 times, each extraction time is 2 hours, and the amount of solvent used each time is 12 times the weight of S. serrata , filtered, the extract recovered ethanol, concentrated and dried to obtain Serrata serrata extract B;

(3) Concentrate the drug solution A obtained in step (1) to a relative density of d=1.12, filter it, pass the drug solution through D101 non-polar macroporous adsorption resin, and first elute with water, and the water eluent directly passes through the DM130 electrode macroporous resin; then use 60% ethanol solution to elute D101 non-polar macroporous adsorption resin, collect 60% ethanol eluate, concentrate and dry to get Serrata serrata extract C; then use 75% concentration ethanol solution to elute DM130 polar macroporous adsorption resin, collect 75% ethanol eluate, concentrate and dry, and obtain Serrata serrata extract D;

(4) Mix the above-mentioned Serenia serrata extract A, Serra serrata extract B, Serra serrata extract C, and Serra serrata extract D to obtain the Serra serrata extract of the present invention.

Embodiment 2: the preparation of Sara serrata extract

(1) Serpentine 21kg, add water to soak for 4 hours, extract volatile oil by steam distillation, extraction time is 1 hour, extraction times 3 times, add water 8 times of the weight of Serpentine, collect volatile oil, and obtain Serpentine extract A; filtering the Serrata serrata water extract to obtain medicinal liquid A and medicinal residue A respectively;

(3) The dregs A obtained in step (1) were extracted with 50% ethanol as a solvent, heated and refluxed, and the number of extractions was once, and the extraction time was 3 hours each time, and the amount of solvent used each time was 8 times the weight of S. serrata , filtered, the extract recovered ethanol, concentrated and dried to obtain Serrata serrata extract B;

(2) Concentrate the drug solution A obtained in step (1) to a relative density of d=1.20, filter it, pass the drug solution through AB-8 non-polar macroporous adsorption resin, and first elute with water, and the water eluent passes through directly ADS-17 polar macroporous resin; then elute AB-8 non-polar macroporous adsorption resin with 30% ethanol solution, collect 30% ethanol eluate, concentrate and dry, and obtain Serrata serrata extract C Then elute the ADS-17 polar macroporous adsorption resin with 50% ethanol solution, collect the 50% ethanol eluate, concentrate and dry, and obtain Serrata serrata extract D;

(4) Mix the above-mentioned Serenia serrata extract A, Serra serrata extract B, Serra serrata extract C, and Serra serrata extract D to obtain the Serra serrata extract of the present invention.

Embodiment 3: the preparation of Sara serrata extract

(1) Serpentine 25kg, add water to soak for 12 hours, extract volatile oil by steam distillation, extraction time is 4 hours, extraction times 1 time, add water 15 times of the weight of Serpentine, collect volatile oil, and obtain Serpentine extract A; filtering the Serrata serrata water extract to obtain medicinal liquid A and medicinal residue A respectively;

(3) Use 95% ethanol as a solvent to extract the dregs A obtained in step (1), heat and reflux for extraction, the number of extractions is 3 times, each extraction time is 1 hour, and the amount of solvent used each time is 15 times the weight of Serrata serrata , filtered, the extract recovered ethanol, concentrated and dried to obtain Serrata serrata extract B;

(2) Concentrate the drug solution A obtained in step (1) to a relative density of d=1.10, filter it, pass the drug solution through D101 non-polar macroporous adsorption resin, and first elute with water, and the water eluent directly passes through the DM130 electrode macroporous resin; then use 70% ethanol solution to elute D101 non-polar macroporous adsorption resin, collect 70% ethanol eluate, concentrate and dry to obtain Serrata serrata extract C; then use 95% concentration ethanol solution to elute DM130 polar macroporous adsorption resin, collect 95% concentration ethanol eluate, concentrate and dry, and obtain Serrata serrata extract D;

(4) Mix the above-mentioned Serenia serrata extract A, Serra serrata extract B, Serra serrata extract C, and Serra serrata extract D to obtain the Serra serrata extract of the present invention.

Example 4: Preparation of Serrata Serrata Extract Tablets

Take 365g of Serrata serrata extract from Example 1, add 110g of starch, mix well, granulate, dry, sieve, add 15g of microcrystalline cellulose, 5g of magnesium stearate, mix well, press into 1000 tablets, and get tooth Marigold Extract Tablets.

Example 5: Preparation of Grass Serrata Extract Granules

Take 365 g of Serra serrata extract in Example 2, add 160 g of dextrin, mix evenly, granulate, dry, and granulate to obtain Serra serrata extract granules.

Embodiment 6: Preparation of Serrata Serrata Extract Capsules

Take 345 g of Serra serrata extract from Example 3, add 85 g of starch, mix evenly, granulate, granulate, and pack 1000 capsules to obtain Serra serrata extract capsules.

Experimental Example 1: A clinical observation trial on the treatment of AIDS with Serenola serrata extract capsules.

1. Qian Moumou, male, 43 years old, Changchun, Jilin Province, had a low-grade fever for one month, accompanied by sore throat, went to the hospital to check out AIDS, treated with western medicine for three months, did not improve, and developed itchy dermatitis, and later appeared bloodshot diarrhea. On June 10, 2010, I began to take the Serrata serrata extract capsules of the present invention, 5 capsules each time, 3 times a day, and the diarrhea stopped after three days, and the dermatitis disappeared after taking it for three months, and I can still work in the field so far. very good.

2. Sun Moumou, female, 32 years old, from Shenyang and Liaoning, had itchy tumors all over her body and sores on her perineum, and was diagnosed with AIDS in the hospital. After one month of western medicine treatment, it failed to take effect, but a small amount of bloody stool and dysentery occurred. Taking the Herba serrata extract capsules of the present invention, 5 capsules each time, 3 times a day, the diarrhea stopped in 4 days, and the perineal area was relieved after 4 months. The sore surface was cured, and at the same time, it was washed with the Serrata serrata extract of the present invention, once in 3 days, and washed nine times in total. So far, the body is good and can participate in normal work

Experimental example 2 In vitro anti-HIV-1 activity test of Serrata serrata extract

Samples to be tested: Drugs: Serenia serrata extract, Serra serrata extract A, Serra serrata extract B, Serra serrata extract C, Serra serrata extract D, all prepared according to the method of Example 1 of the present invention get. The positive control compound azidothymidine (AZT, 3'-Azido-3'-deoxythymidine) was purchased from Sigma. The samples to be tested were dissolved in dimethylsulfoxide (DMSO), and stored at 4°C after aliquoting; AZT was dissolved in RPMI-1640 complete medium, sterilized by 0.22um filter membrane, and stored at -20°C after aliquoting.

Reagents: HEPES (4-Hydroxyethylpyrrolethanethanesulfonic acid), MTT (3-(4,5-Dimethylpyrrole-2)-2,5-Diphenyltetrazolium sniffing salt), DMF ( N, N-dimethylformamide), penicillin (Penicillin), streptomycin sulfate (Streptomycin sulfate), glutamine (Glutamine) were all purchased from Sigma Company; 2-mercaptoethanol (2-ME,2 -Mercaptoethanol) is the product of Bio-Rad Company. RPMI-1640 and newborn calf serum are products of Gibco.

Medium: RPMI-1640 complete medium, containing 10% newborn calf serum, 2mM L-glutamine, 10mM HEPES, 50uM 2-mercaptoethanol, 100000IU penicillin, 100 μg/ml streptomycin.

Cells and viruses: human T lymphocyte cell line C8166, HIV-1 experimental strain HIV-1 _B. All were donated by British Medical Research Council, A-worker DS Reagent Project. All cells and viruses were cultured in RPM-1640 complete medium containing 10% calf serum. Prepare HIV-1 by conventional method _B , titrate and calculate the TCID ₅₀ of the virus. After aliquoting the virus stock solution, store it at -70°C. Cells and viruses were frozen and recovered according to conventional methods.

HIV-1 Infectivity Titration: HIV-1 _B is titrated according to Johnson & Byington (1990) method improvement, briefly described as follows: HIV-1 stock solution is diluted 4 times on a 96-well plate, 10 gradients, 6 replicate wells per gradient, and 6 wells of control wells are set at the same time . Add 50ul of C8166 cells (4×10 ⁵ /ml) to each well, and the final volume of each well is 200ul. Culture at 37°C in CO ₂ . On the third day, 100 μl of fresh RPMI-1640 complete medium was added, and on the seventh day, the cytopathic effect (Cytopathic Effect, CPE) induced by HIV-1 in each well was observed under an inverted microscope to check whether there was syncytium (Syncytium) in each well. Form OK. Calculate the TCID ₅₀ (50% Tissue Culture Infection Dose, half of the tissue culture infection dose) of the virus according to the Reed & Muench method.

Toxicity test on C8166 cells: 100 μl of 4×10 ⁵ /ml C8166 cell suspension was mixed with different drug solutions to be tested, and three replicate holes were set. At the same time, control wells without drug were set. Incubate at 37°C in _CO for 3 days, and detect cytotoxicity by MTT colorimetry. ELx800 microplate reader was used to measure the OD value, the measurement wavelength was 595nm, and the reference wavelength was 630nm. The CC ₅₀ value (50% Cytotoxic Concentration) was calculated, that is, the drug concentration at which 50% of the normal T lymphocyte line C8166 was toxic. The experimental results are shown in Table 1.

Table 1 The number of experiments on the toxic effect of Sara serrata extract on C8166 cells.

Test example 3 Sage serrata extract to HIV-1 _B cytopathic (CPE) inhibition test

Inoculate 50u1/well of 8×10 ⁵ /ml C8166 cells on a 96-well cell culture plate containing 100u1/well of gradient drug dilution, and then add 50u1 of HIV-1 _B Dilute the supernatant, 1300TCID ₅₀ /well. Set up 3 replicate holes. At the same time, a control well of normal cells without drugs was set. AZT is the positive drug control. Cultured at 37°C, 5% CO ₂ for 3 days, counted the formation of syncytia under an inverted microscope (100×). EC ₅₀ (50% Effective Concentration) is the drug concentration that inhibits syncytia formation by 50%. The experimental results are shown in Table 2.

Table 2 Serenola extract on HIV-1 Inhibition of _B -induced CPE in C8166 cells

The results showed that the extract of Sage serrata was effective against HIV-1 _B has a good inhibitory effect on inducing C8166 cell pathology.

The positive drug AZT (azidethymidine) was the first anti-HIV drug used clinically in 1987. It is used as a single or dual combination to treat HIV infection. Because of this treatment, obvious drug resistance usually occurs within 6 months, and The immune reconstruction of the body is not good, and there are side effects such as myelosuppression, leukopenia, total lymphocyte count decrease and megaloblastic anemia, so the comprehensive clinical effect is not ideal. The serrata serrata extract has little toxicity to C8166 cells, has significant inhibitory activity on the formation of syncytia induced by HIV-1 in C8166 cells, and can be used in the preparation of drugs for treating AIDS.

Claims (3)

1. the application of Boraginaceae extract in preparation treatment AIDS-treating medicine, the preparation method of described Boraginaceae extract is: Flos et Folium Eritrichii Rupestris, soak 4-12 hour, adopt extraction by steam distillation volatile oil, extraction time 1-4 hour, extraction time 1-3 time, amount of water is 8-15 times of Flos et Folium Eritrichii Rupestris weight, collect volatile oil, obtain Boraginaceae extract A, Boraginaceae extract A is Boraginaceae extract.

2. application according to claim 1, it is characterized in that the preparation method of described Boraginaceae extract is: Flos et Folium Eritrichii Rupestris, soak 6 hours, adopt extraction by steam distillation volatile oil, 2 hours extraction times, extraction time 2 times, amount of water is 14 times of Flos et Folium Eritrichii Rupestris weight, collect volatile oil, obtain Boraginaceae extract A, Boraginaceae extract A is Boraginaceae extract.

3. application according to claim 1, is characterized in that the treatment AIDS-treating medicine that described Boraginaceae extract and chemical drugs or Chinese medicine or natural drug form.