KR19980053425A - Treatment of acquired immunodeficiency syndrome containing mixed water extracts of Hwangbaekpi and Matari plants - Google Patents

Abstract

The present invention relates to a treatment composition for acquired immunodeficiency syndrome containing a mixed water extract of Phellodendron amurense RUPRCHT cortex and Matari (Patrinia scabiosaefolia FISCH.) As an active ingredient.

The mixed water extract according to the present invention may exhibit excellent acquired immunodeficiency treatment effect because it has acquired effect of killing immunodeficiency virus (HIV), lymphocyte proliferation, and immune tissue regeneration. In addition, the treatment effect of the acquired immune deficiency of the mixed water extracts of Hwangbaekpi and Matari according to the present invention appears continuously and does not produce resistance to the drug has the advantage that the effect does not fall even if used for a long time, and there is almost no toxicity even in large doses It is believed to be a safe treatment for acquired immunodeficiency.

Description

Treatment of acquired immunodeficiency syndrome containing mixed water extracts of Hwangbaekpi and Matari plants

Figure 1 is a graph showing the results of measuring the cytotoxicity and anti-immune deficiency virus effect (anti-HIV effect) of Hwangbaekpi water extract according to Experimental Example 1.

Figure 2 is a graph showing the results of measuring the cytotoxicity and anti-acquired immunodeficiency virus effect of Matari water extract according to Experimental Example 1.

Figure 3 is a graph showing the results of measuring the cytotoxicity and anti-acquired immunodeficiency virus effect of the mixed water extracts of Hwangbaekpi and Matari plants according to the present invention according to Experimental Example 1.

4 is a graph showing the effect of the mixed water extracts of Hwangbaekpi and Matari plants of the present invention on the normal immune cell line.

FIG. 5 is a graph showing the effect of the mixed water extract of Hwangbaekpi and Matari of the present invention on T cell dependent antibody production in vivo.

6 is a graph showing the effect of the mixed water extracts of the Hwangbaekpi and Matari plants of the present invention on the proliferation of T cells.

Figure 7 is a graph showing the effect of the mixed water extract of Hwangbaekpi and Matari of the present invention on the proliferation of immune cells.

8 is a photograph showing the effect of the mixed water extract of Hwangbaekpi and Matari plants of the present invention on the liver, kidney and heart of the mouse (15 g / kg administration, HE staining, 8A Figure: liver x400, 8B Figure: kidney x200, 8C degrees: heart x200).

The present invention relates to a treatment composition for acquired immunodeficiency syndrome containing a mixed water extract of Hwangbaekpi (栢皮) and Matari plants (pack) as an active ingredient. More specifically, the present invention comprises a mixed water extract obtained by extracting a mixture of Phellodendron amurense RUPRCHT cortex and Matari (Patrinia scabiosaefolia FISCH.) As an active ingredient anti-immune deficiency virus effect (anti-HIV effect) And it relates to a pharmaceutical composition having a regenerative capacity for tissue cells damaged by the acquired immunodeficiency virus.

Acquired Immune Deficiency Syndrome (AIDS) has been the most problematic viral disease since it was first reported in 1981 and the number of patients doubled every six months. Recently, due to the development of science, the cause of HIV (Human Immunodeficiency Virus) infection and AIDS onset has been identified, but the effective treatment is rarely come out.

The pathogens of AIDS are immunodeficiency viruses and are classified as Lenti virus among retroviruses, and there are two types of type 1 and type 2 depending on the structural protein and gene structure. . Retroviruses are characterized by a reverse transcriptase with RNA (ribonucleic acid). In addition, HIV has high affinity for lymphocytes, especially helper T cells (T4) or macrophage, and when invading host cells, it reverses the transcriptase and transfers cDNA from RNA itself to double helix. After the DNA of helix is formed, it enters the DNA in the nucleus of the host cell as a provirus. When lymphocytes or macrophages, which are host cells, are activated by infection and inflammation, DNA replication of proviruses is rapidly increased, and newly formed HIV comes out of the cells. The result is a decrease in T4 cells, which results in immunodeficiency.

Infection with AIDS results in seroconversion within 2-8 weeks, and the infected person becomes HIV antibody positive. AIDS carriers with HIV that are not killed by the antibody are at risk as infectious agents but do not show any obvious symptoms. Some carriers of AIDS develop fever, diarrhea, systemic boredom, weakness, and swollen lymph gland swelling, leading to a persistent AIDS-related syndrome (ARC). In patients with AIDS-related syndrome or AIDS, HIV is parasitic to T4 lymphocytes in lymphocytes, impairing immune function. T4 lymphocytes promote the immune response, but T8 lymphocytes (Suppressor cells) primarily inhibit the function of T4 lymphocytes. In patients with AIDS, the T4 / T8 ratio is markedly reduced because T4 cells are reduced. In addition, a decrease in cellular immunity may include a negative tuberculin reaction, a lymphocytic reaction, a decreased phagocytosis of macrophages, and an increase in gamma globulin.

As already defined in WHO / CDC, AIDS is caused by a decrease in immunity. Opportunistic infection, malignant tumors and neuropathy appear, and opportunistic infections include pneumonia and fungal infection caused by carini protozoa. There are a lot of cases, and tuberculosis may recur. In addition, HIV enters the central nervous system through the cerebrovascular gate along with lymphocytes or macrophages, resulting in dementia, multiple neuropathy and peripheral neuropathy.

The path of infection of AIDS is as follows. AIDS is relatively difficult to transmit but is only infected by the body fluids of infected patients or by intimate contact with AIDS patients. For example, infected blood or blood products, sexual contact with an infected person, contaminated syringes, maternal infections, and the like.

The most frequent secondary diseases caused by AIDS infection are kaposis' sarcoma and Pneumocystis carini pneumonia. In addition, general progress drug syndrome, autoimmune thrombocytopenia, non-Hodgkin's lymphoma, advanced white matter brain disease, cheilitis, and anal tumors occur. In the case of AIDS infection, progressive immunodeficiency also destroys the host's defenses. For example, as the disease progresses, bacteria, viruses, fungi, and protozoa cause systemic systemic infections, resulting in death from sepsis or pneumonia pneumoniae pneumonia. About 50% of people with AIDS develop Kaposi's sarcoma (skin cancer), which continues to spread and gradually spreads to the whole body, involving lymph nodes and bowel relationships.

As the number of fatal AIDS infections increases rapidly, there is an urgent need for treatment. As a result, as anti-HIV drugs, chemicals that can inhibit the replication process of HIV are being developed and used. In addition, studies on adsorption inhibition (solubilizing CD4) to CD4 (helper cell), reverse transcriptase and protease inhibitors, and Tat (transcription factor) inhibitors are also in progress. 1) M.C. Hsu et al., Science, 254: 1799, 1991; M.C. Hsu et al., Abstract in the CP2 ICCAAC meeting (invited paper), 1992; And Tat inhibitor Ro5-3335 as described in S. Tan et al., 1st Royal Society of Chemistry International Symposium, Recent Advances in the Chemistry of Anti-Infective Agents, Churchill College, Cambridge England, July 5-8, 1992. The recovery of CD4 is thought to occur through the following mechanisms. HIV infection occurs by binding CD4 of lymphocytes to gp160 (glycoprotein) on HIV particles, but Ro5-3335 allows for recovery of CD4 by inhibiting post-transcription of this viral protein. Ro24-7429, a further development of this, is a drug that lowers the binding ability of benzodiazepines to receptors, and is expected to be a new type of AIDS inhibitor. 2) M. M. Kotler et al., Proc. Natl. Acad. Sci. USA, 85: 4185, 1988; M. L. Moore et al., Biochem. Biopys. Res. Common., 159: 420, 1989; And H. G. Krausslich et al., Proc. Natl. Acad. Sci. USA 86: 807, 1989, an inhibitor of HIV protease is an enzyme protein that is effective in the treatment of HIV infection and actively studies its tertiary structure, activity as a variant protein, and inhibitory function on other substrates. It is becoming. Currently developed protease inhibitors include Ro31-8959 and JG-365, which have similar structures at the N-terminus but different structures at the C-terminus. 3) In addition, reverse transcription inhibitors, adsorption inhibitors, cytokines, and vaccines are under clinical trials or under development. Currently used reverse transcription inhibitors such as AZT, ddI (2 ', 3'-dideoxyinosine) and ddC (2', 3'-dideoxycytidine) are generally symptoms of headache, vomiting, high fever and spots. This appears, there are side effects such as damage to the hematopoietic system, liver function, there is a problem such that resistance to the drug is formed during long-term treatment.

Therefore, the present inventors conducted a study to find a substance that minimizes the side effects of the anti-HIV therapeutic agent used in the past, has a proliferation inhibitory effect of HIV and has an immune enhancing function, and has a regenerative ability of damaged tissues caused by HIV. In particular, various natural medicinal plants were studied. As a result, it was confirmed that the mixed water extract extracted with water by mixing Hwangbaekpi and Matari plants among various natural plants can achieve the object as described above and completed the present invention.

Therefore, the first object of the present invention relates to a treatment composition for acquired immunodeficiency syndrome containing a mixed water extract of the baekbaekpi and matari plants.

A further object of the present invention is to provide a method for producing a mixed water extract, which is used as an active ingredient having an HIV anti-proliferative effect according to the present invention, from a mixture of baekbaekpi and matari.

Hereinafter, each object of the present invention will be described in detail.

The present invention is an AIDS therapeutic composition containing a mixed water extract of Hwangbaekpi and Matari as an active ingredient, in particular AIDS has a proliferation inhibitory effect and immune enhancing effect of the tissue cells damaged by HIV The present invention relates to an AIDS therapeutic composition having regenerative capacity and few side effects.

The mixed water extract of Hwangbaekpi and Matari, which is used as an active ingredient according to the present invention, is harmless to normal cells, as specifically demonstrated in the following experimental examples, and at the same time selectively inhibits the growth of the virus and is damaged by the virus. It has a regenerative capacity for tissue cells.

Hwangbaekpi used in the preparation of the extract of the present invention is a bark of a yellow wall or a yellow tree stem native to Korea, Japan, China, etc. A representative of the yellow-back tree is Phellodendron amurense RUPRECHT. His variants include Latifolia Nagai Kawamodo, Zapponicombe Cucumber, Pelodendron Insulin Nagai, Pelodendron Mullet Nagai, and Pelodendron Ricegent. Yellow and white skin contains 1.5% -4.5% of yellow or tan pigment and several alkaloids. The main component of alkaloids is berberine. It also contains palmatine, magnoflorine, guanidine, guateine, jateorrizine, phellodendrine, candicine and menisperine. High-density compounds are known to contain obakunone, obakulactone, and β-sitosterol. These ingredients have strong antibacterial action, blood pressure lowering action, central nervous system suppression effect, acetylcholine enhancement action and anti-inflammatory action. It has been used for bone disease and jaundice in herbal wood or pharmacy. It is also effective for typhoid fever and cholera, and it is effective for dry stomach and formal clothes. Their bark has been used in gastroenteritis, abdominal pain, jaundice, etc. However, there have been no reports that hwangbaekpi inhibits the growth of the virus or enhances or revives immune function.

Matari, another plant used in the manufacture of the extract of the present invention, is also known as a soak (敗 醬), Matari (Patrinia scabiosaefolia FISCH.), Dunggal (白花 敗 醬; Patrinia villosa JUSS.), Dolmatari (岩 敗 醬) ; Patrinia sibirica JUSS.). Matari is a perennial herbaceous plant native to Korea and in the temperate regions of the world. The herb is used as an anti-inflammatory agent in ophthalmology, Streptococcus pyogenes, edema, and fevers. . In addition, the hydrothermal extract of Matari has been reported to have antitumor effects (eg, uterine cancer, esophageal cancer, gastric cancer, intestinal cancer, lung cancer, etc.), and in vitro tests strongly inhibit the development of Staphylococcus aureus, Streptococcus, etc., It has regenerative ability of damaged liver cells, prevents its degeneration, improves circulation of portal vein and promotes the regeneration of hepatocytes. It also acts on the central nervous system to stabilize nerves, shows strong analgesic effect, and lowers blood pressure. And antidiuretic effects.

Matari's root contains trace alkaloids such as essential oils, various saponin components, carbohydrates and cumarin. The dermal layer of the root includes acetic acid, formic acid, valeric acid, and the like, and contains alkaloids such as catinine and valerianine. Valeric acid is known to have a very strong analgesic effect. Dried seeds contain 19.4-19.9% protein and 30-34.4% fat. However, there have been no reports showing that any extract of Matari has anti-proliferative effects on viruses, especially HIV.

In the present invention, the mixed water extract obtained by mixing Hwangbaekpi and Matari and extracting with water has a proliferation inhibitory effect of HIV virus, which is not known to Hwangpipi and Matari, and regeneration of tissue damaged by HIV, and also mixed water The extract has been experimentally confirmed to exhibit a significantly superior synergistic effect compared to the case of using the water extracts of Hwangbaekpi and Matari alone, and completed the present invention.

The mixed water extract of Hwangbaekpi and Matari used as an active ingredient in the present invention is obtained by the following method.

That is, the mixture of Hwangbaekpi and Matari plants was extracted with water, the extract was filtered, and the filtrate was saturated under high pressure to remove the coagulum precipitate. The filtrate was added with an organic solvent to remove the organic solvent-soluble substance. By separating the aqueous layer in lyophilized, a mixed water extract (RI-P) of sulfur white skin and matari desired in the present invention can be obtained in powder form.

At this time, Hwangbaekpi and Matari are used in a weight ratio of 1: 0.1-5, preferably 1: 1-2 by weight. In particular, in the present invention, it is preferable to combine the yellow and white skin and matari in a weight ratio of 1: 1.

In addition, the mixed water extract obtained above may be further purified as needed to separate only the water-soluble fraction thereof and used as an active ingredient in the present invention. That is, by dissolving the mixed water extract (RI-P) in water, purifying by physicochemical method, filtration and lyophilization of the separated filtrate to obtain the water-soluble fraction (RI-W) of the mixed water extract according to the present invention. It can also be used as an active ingredient of the composition.

The present invention also includes a method for producing a mixed water extract of baekbaekpi and matari as described above.

In the following, the mixed water extract should be understood to mean both the water extract obtained by extracting a mixture of Hwangbaekpi and Matari with water or its water-soluble fraction obtained by purifying it, unless otherwise specified.

Hereinafter, the method for preparing the mixed water extract of the present invention will be described in more detail.

According to the method of the present invention, the mixed water extract is pulverized a mixture of sulfur white skin and matari dried in the first step and extracted by heating under saturated vapor pressure (121 ° C., 15 pounds / in a ² ) using water such as constant or distilled water as a solvent. do.

The water used to extract the mixture of sulfur and skin in the first step is used in a ratio of 5-10 parts by weight, particularly preferably 7 parts by weight, based on 1 part by weight of the mixture.

Next, the extract is centrifuged in the second step to remove the precipitate and the extract is again saturated under high pressure, for example to boil under vapor pressure (121 ° C., 15 pounds / in a ² ) in an autoclave to coagulate the remaining protein. After filtration, the solution is filtered off by centrifugation.

In the third step, the separated filtrate is extracted with an organic solvent such as chloroform, hexane, dichloromethane, cyclohexane, preferably chloroform or hexane to remove impurities such as resin and fiber, and the aqueous layer is again subjected to talc or the like. After purification by lyophilization to give the desired mixed water extract (RI-P).

The mixed water extract (RI-P) thus obtained may be separated only by its water-soluble fraction through subsequent purification as follows, if necessary. That is, the mixed water extract (RI-P) is again dissolved in water such as constant or distilled water and left at a low temperature of about 1 to 5 ° C. for 18 to 24 hours, and then the precipitate is filtered off to remove the filtrate by a physicochemical method. For example, it refine | purifies using a talc at low temperature of 1-5 degreeC. The water-soluble fraction (RI-W) of the mixed water extract according to the present invention is obtained by lyophilizing the filtrate separating the purified aqueous solution again by filtration in a membrane filter apparatus at 1 to 5 ° C.

The mixed water extract of Hwangbaekpi and Matari obtained by the method of the present invention as described above is useful as an AIDS therapeutic composition by exhibiting the pharmacologically useful HIV antiproliferative effect and regeneration of tissues damaged by HIV as mentioned above. Can be used.

The mixed water extract of Hwangbaekpi and Matari or the water-soluble fraction thereof according to the present invention obtained by the method as described above can be used in the pharmaceutical field either alone or in combination with a pharmaceutically acceptable carrier, in a therapeutically effective amount. It may be formulated in the form of a suitable pharmaceutical composition by conventional methods and administered in a manner customary in the pharmaceutical art, preferably orally. In general, the compositions of the present invention may be formulated in the form of tablets, capsules, solutions, suspensions or syrups suitable for oral administration.

In addition, the dosage of the mixed water extract according to the present invention may vary depending on whether the extract is a mixed water extract or a water-soluble fraction thereof, or depending on the sex, age, weight, and desired effect of the patient to be administered. In the case of oral administration to an adult, a dose of 5 to 50 mg, preferably 10 to 30 mg per day, per kg of body weight can be administered. In addition, the extract according to the present invention may be combined or used in combination with other agents known to have a therapeutic effect on AIDS disease, if necessary.

Examples of AIDS therapeutic agents that can be used in combination or in combination in the compositions according to the invention include, for example, reverse transcription inhibitors AZT, ddI, ddC and the like and protease inhibitors.

The present invention is explained in more detail by the following examples and experimental examples, but these are provided merely to illustrate the present invention, and the present invention is not limited in any way by them.

Example 1 Preparation of Mixed Water Extract (RI-P)

100 g of powder ground by mixing dried Phellodendron amurense RUPRECHT and Matari (Patrinia scabiosaefolia FISCH.) In a weight ratio of 1: 1, was added thereto, and 3000 ml of distilled water was added thereto. After extraction for 40-60 minutes under / in a ² , the extract was aliquoted and the residue was removed. The extract was centrifuged to remove the precipitate, and the filtrate was concentrated and heated to 1500 ml in total and filtered. The filtrate was again saturated with steam at 121 ° C. under 15 pounds / in a ^{2 for} 15 minutes, and the precipitate containing the coagulated protein was removed by centrifugation and filtered. The filtrate was added to a separatory funnel, and chloroform 400ml was added to elute resin and fiber, and the chloroform layer was separated and removed. After the same operation was repeated twice, 200 ml of n-hexane was added to the aqueous layer again to elute remaining resins, fibers, and n-hexane soluble substances. The aqueous layer was separated, warmed to 60-80 ° C., and then stirred by adding 500 g of talc, followed by filtration under reduced pressure to remove talc, and the filtrate was gradually filtered again, and the filtrate was lyophilized to be powdered. By this method, about 15000 mg of mixed water extract (RI-P) was obtained from the mixture of Hwangbaekpi and Matari in a yield of about 15% (based on dry weight).

Example 2 Preparation of Water-Soluble Fraction (RI-W)

1000 mg of the mixed water extract RI-P obtained in Example 1 was aliquoted, dissolved in 500 ml of distilled water, and the resulting solution was left at low temperature of about 1 ° C. for one day, followed by stirring with 300 g of talc, under reduced pressure. Purification by filtration to remove talc. The purified filtrate was slowly passed through a Nucleopore apparatus at a temperature of about 5 ° C. to lyophilize the aqueous fraction of the filtrate passed through and powdered. This method yielded about 800 mg of an aqueous fraction (RI-W) according to the present invention in a yield of about 80% (dry weight basis).

Composition example

Composition 1: Tablet

250 mg of the freeze-dried powdered mixed water extract according to the present invention prepared in Example 1 was subjected to excipients 260 mg of lactose and 35 mg of Avicel (microcrystalline cellulose), 15 mg of sodium starch glyconate as a dissolution aid, and a binder. 80 mg of L-HPC (Low-hydroxypropylcellulose) for phosphorus was mixed and placed in a U-type mixer, and mixed for about 20 minutes. After the mixing was completed, 10 mg of magnesium stearate was further added as a lubricant and mixed for about 3 minutes, followed by tableting and film coating by a conventional method to prepare a tablet containing 250 mg of a mixed water extract per one party. It was.

Composition 2: Syrup

An appropriate amount of sucrose was dissolved in a certain amount of water, and 80 mg of paraoxymethylbenzoate and 16 mg of paraoxypropylbenzoate were added as a preservative, and the lyophilized powdered mixed water extract according to the present invention prepared in Example 1 was used. 4.5 g was added, maintained at 60 ° C., completely dissolved, cooled, and distilled water was added so that the total amount was 150 ml. A syrup containing 30 mg of mixed water extract was prepared per 1 ml.

Composition 3: Capsule

The capsules containing 300 mg of the mixed water extract 300 mg per capsule were mixed with 200 mg of lactose 200 mg of the freeze-dried powdered mixed water extract according to the present invention prepared in Example 1 as a carrier and filled into hard gelatin capsules. Prepared.

Experimental Example 1: Toxicity and Anti-HIV-1 Effect of Mixed Water Extracts on Cells

Toxicity and anti-HIV effects on the cells of the mixed water extracts of Hwangbaekpi and Matari according to the present invention were tested by the following method.

The anti-HIV effects of water extracts and mixed water extracts of Hwangbaekpi and Matari were measured using the T cell line CEM-SS infected with HIV-1. The CEM-SS T-leukemia cell line (CCI cell line cloned, NCI) used here has a high infectious effect against HIV, is 100% killed by viral infection, and multinuclear giant cells when infected. cell), so infection can be easily determined. The virus was isolated from AIDS patients in NCI and used a virus, HIV-1 Rf, cultured in H9 cell line.

In this experiment, the direct HIV killing effect of protecting the HIV-1 infected cells from the virus by the mixed water extract of the present invention was measured, and the cytotoxic effect was also measured. The viability of the cells was XTT (tetrazolium salt: 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl) -5-[(phenylamino) carbonyl] -2H-tetrazolium hydroxide) It was done using the method. XTT is degraded by mitochondrial enzymes in living cells to form water-soluble XTT formazan. Since the amount of water soluble XTT formazan produced is proportional to the number of viable cells, the OD (absorbance) value measured at 450 nm using an ELISA reader indicates cell viability. The specific experimental method is as follows.

CEM-SS T-leukemia cells were cultured in a 5% CO ₂ incubator at 37 ° C. using RPMI 1640 medium to which 10% FBS (fetal bovine serum) was added, and the mixed water extract of the present invention, the water extract of Matari and baekbaekpi Water extracts were dissolved in DMEM medium (containing 10% FBS) to which 0.1% DMSO (dimethylsulfoxide) was added, respectively, and the concentrations were serially diluted from 50 mg / ml by ½. CEM-SS cells were prepared to be 3 × 10 ⁵ cells for each well of a 96-well plate.

Group A is an antiviral effect experiment of drugs. After infection of the prepared CEM-SS cells with HIV-1 to a ratio of host cells and viruses at 10: 1 (MOI = 0.1), the drug diluted to each concentration was added. The total solution amount per well was 200 µl. Group B does not add drugs in the virus killing effect experiment. Group C adds a diluted drug to 3 × 10 ⁵ CEM-SS cells per well to measure the cytotoxicity of each of the mixed water extract, Matari water extract and Hwangbaekpi water extract of the present invention. Only cells were placed in each well to gain weight. All experimental groups were incubated for 6 days at 37 ° C., 5% CO ₂ incubator, treated with XTT, and measured by ELISA reader to obtain respective absorbance values.

The effects of the drugs were compared by obtaining IC ₅₀ and EC ₅₀ from the obtained absorbance values. The IC ₅₀ value represents the concentration at which 50% of the total cell number dies when the drug is added to the cells, from low to high concentrations, indicating a cytotoxic effect.

EC ₅₀ value is the anti-HIV effect of the drug at a concentration that can save 50% of all infected cells by protecting the virus from proliferation by inhibiting virus proliferation by the mixed water extract of the present invention. Indicates.

In this experiment, the mixed water extract of the present invention, the water extract of Matari and the extract of Hwangbaekpi were used at concentrations of 50, 25, 12.5, 6.3, 3.2, 1.6, 0.8, and 0.4 mg / ml, respectively. The results of measuring the cytotoxic effect of each drug and the cytoprotective effect by viral infection are shown in FIGS. 1 to 3. As shown in FIG. 1, the cytoprotective effect by viral infection was the greatest at 0.8 mg / ml of the extract of Hwangbaekpi, but it was difficult to evaluate the effective concentration due to its high cytotoxicity. Matari extract showed about 40% cytoprotective effect against virus infection at 0.8 mg / ml concentration, as shown in Figure 2, and cytotoxicity was low around 10%. On the other hand, in the case of the mixed water extract of the present invention, as shown in FIG. 3, the most effective concentration was 0.8 mg / ml, and the cytoprotective effect against viral infection was increased to 65% and cytotoxicity was about 10%. HIV effect was shown. That is, it can be seen that the synergistic effect is apparent when the mixed water extracts of Hwangbaekpi and Matari are used rather than each of them alone.

Experimental Example 2: Toxicity to Normal Cells of Mixed Water Extracts

In order to find out how the mixed water extract of Hwangbaekpi and Matari according to the present invention affects normal immune cells, in particular, it shows toxicity.

In this experiment, NIH-3T3 cell line distributed from National Cancer Institute, USA was used. All experiments were performed using cells passaged up to 10 times after receiving the culture. Cells were cultured in medium (R10) to which 10% FCS was added to RPMI1640 culture medium, and when performing SRB analysis, cells were cultured using RPMI1640 culture solution (R5) containing 5% FCS under conditions of 5% CO ₂ and 37 ° C. Incubated. Cell cultures containing 4 × 10 ⁴ cells / ml were dispensed into 96-well microplates and preincubated for about 24 hours under conditions of 5% CO ₂ , 37 ° C. to stabilize prior to drug administration.

Cytotoxicity was measured by administering the mixed water extract RI-P of Hwangbaekpi and Matari obtained in Example 1 at various concentrations between 0.03 μg / ml and 3 μg / ml (final concentration). After adding the mixed water extract, the cells were incubated for 48 hours under conditions of 5% CO ₂ , 37 ° C., and 100% relative humidity. Cytotoxicity was measured using SRB (Sulforhodamine B) assay. Monks, A et al., J. Natl. Cancer Inst. 83: 757-766, 1991. The measured results are shown in FIG. In FIG. 4, the X-axis (concentration) is represented by converting the concentration value into a log value.

As shown in Figure 4, the mixed water extract according to the present invention does not affect the normal cell line NIH-3T3 cells at all, no increase in cytotoxicity was observed even with increasing concentration.

Experimental Example 3 Effect of Mixed Water Extract on T Cell-Dependent Antibody Production in Vivo

Balb / c mice weighing 17-20 g ( ) 24 animals were divided into 6 groups of 4 animals each, and the first group was an untreated control group, and the second group was intraperitoneally implanted with SRBC (Sheep Red Blood Cell) 2.4x10 ⁸ cells, and the third and sixth groups were the second group. After treatment with SRBC as in the group, the mixed water extract RI-P of yellow and white skin obtained in Example 1 according to the present invention was added per 3 kg of body weight for 3 days (0 days, 1 day, 2 days) from the first day of transplantation. Oral doses of 10, 30 and 100 mg respectively. Four days after transplantation, the animals were sacrificed and the spleens were removed aseptically. The splenocytes were isolated, and the cells were liberated using a syringe plunger in a petri dish containing 3 ml of Earle's Balanced Salt Solution (EBSS). . The cell suspension obtained was transferred to 15 ml conical tube, left for 5 minutes, and 2 ml of the supernatant was taken and centrifuged at 1200 rpm for 10 minutes. The supernatant was discarded and the residue was again suspended in 2 ml of EBSS, diluted 30-fold with EBSS, and the splenocyte dilutions were used 100 μl each for one plaque forming cell assay. 100 μl of the splenocyte dilution thus obtained was washed with EBSS SRBC (25 μl, target cells), guinea pig complement (25 μl) diluted twice with EBSS, and agarose (0.85% in EBSS, 350 [Mu] l), poured into Petri dishes and solidified, and left in a CO ₂ incubator at 37 ° C for about 1 hour. The modified Jerne Plaque Assay (Hwan M. Kim, et al., J. Toxicological Sciences 21: 41-45, 1996) was then used to count plaque and cell numbers, resulting in 10 ⁶ cells. The number of sugar-producing cells is shown. The average value of the measured results is shown in Table 1 and FIG. 5.

TABLE 1 Effect of mixed water extract of the present invention on T-cell dependent antibody production response in vivo

As can be seen from these results, when the mixed water extract according to the present invention was administered, the number of antibody-forming cells increased proportionally with the concentration of the drug, so that the 100 mg / kg administration group produced the antibody as compared to the SRBC treatment group. An increase of 307% was shown in the number of cells. Therefore, it can be seen that the immuno-enhancing effect of the mixed water extract of Hwangbaekpi and Matari according to the present invention is very excellent.

Experimental Example 4 Effect of Mixed Water Extract on T Cell Activation Reaction

The effect on the T cell activation reaction of the mixed water extracts of Hwangbaekpi and Matari according to the present invention was measured by the mixed immune cell reaction method.

Major histocompatibility complex (MHC) class [2] antigens of different types of B6C3F1 (H-2k) and BDF1 (H-2d) mice with different spleens are aseptically removed and finely chopped to use mesh To release the splenocytes. Free splenocytes were adjusted to 2.5 × 10 ⁶ cells / ml, respectively, and 100 μl of B6C3F1 (H-2k) and BDF1 (H-2d) splenocytes were put into 96 well plates, respectively, to finally 200 μl.

Here, the mixed water extract RI-P obtained in Example 1 according to the present invention was treated at a concentration of 0.01 to 100 µl / ml, and then cultured for 3 days at 37 ° C. in a CO ₂ incubator. 1 μCi of [ ³ H] thymidine was added per well 18 hours before the end of the culture. After collecting cells using an automatic cell harvester, the degree of absorption of [ ³ H] thymidine was measured by a beta counter (Beta Counter, Beckman LSC) to measure the proliferation of immune cells. The measured results are shown in Tables 2 and 6 below.

[Table 2] Effect on the proliferation of T cells of the mixed water extract of the present invention

From the results described in Table 2 and FIG. 6, it can be seen that when the mixed water extract of the present invention is applied, the proliferation of T cells increases with the concentration thereof.

Experimental Example 5 Effect of Mixed Water Extract on Immune Cell Proliferation

After cervical distal bone, the abdomen was disinfected with 70% alcohol. In the sterile state, the abdomen was dissected, the spleen was removed, placed in a Petri dish containing buffer (EBSS), and the splenocytes were released using the plunge of the syringe. The suspension containing the splenocytes thus obtained was transferred to a tube, left for about 5-10 minutes, and the supernatant was collected and centrifuged (1200 rpm, 10 minutes). The supernatant was discarded and the precipitate was resuspended in RPMI 1640 culture [10% FCS (fetal calf serum), 2-mercaptoethanol (2-ME)], and then cell number and cell viability were measured. Separate the splenocytes at a concentration of 1 × 10 ⁶ cells / ml and dispense 200 μl per well of a 96-well plate and mix 0.01-100 μg of the mixed water extract (RI-P) obtained in Example 1 according to the present invention. After treatment at a concentration of / ㎖ was incubated for 3 days using a rocking plate (rocking plate) in an incubator filled with 5 psi mixed gas (7% O ₂ , 10% CO ₂ , 83% N ₂ ). After incubation, [ ³ H] -thymidine was added at a concentration of 5 μCi / ml and the degree of absorption was measured by beta counter to measure the proliferation of immune cells. The measured results are shown in Table 3 and FIG. 7.

TABLE 3 Influence on immune cell proliferation

As shown in Table 3 and the accompanying Figure 7, it can be seen that the application of the mixed water extract of the present invention increased the proliferation of immune cells in proportion to its concentration.

Experimental Example 6 Changes in the Immune Cell Composition of the Spleen, Thymus, and Bone Marrow by In Vivo Treatment of Mixed Water Extracts

After the oral administration of the mixed water extract RI-P1 of Hwangbaekpi and Matari prepared in Example 1 to mice at concentrations of 100 and 300 mg / kg for 3 days, the mice were sacrificed and the spleen, thymus and bone marrow were isolated. It was. Immune cells were separated from these organs by the same method as that of separating splenocytes in Experimental Example 2, followed by centrifugation. After discarding the supernatant, the cell precipitate was treated for 2 to 3 minutes using erythrocyte coagulation buffer (ACK buffer, 0.15M NH ₄ Cl, 0.01M KHCO ₃ , 0.1 mM Na ₂ EDTA, pH 7.2) for 1 ml per organ. . After washing the immune cells with medium, the cell concentration was adjusted to 10 ⁷ cells / ml, and then 100 μl per tube was dispensed. In order to measure B cells, phycoerythrin (PE) is bound to rat monoclonal antibody B220 against mouse B cell antigen, and Thy-1-PE (Caltag Laboratories) is used for T cells. For CD4 T cells CD4-FITC (Caltag Laboratories) and for CD8 T cells Ly2-FITC (Caltag Laboratories) were used. These antibodies were added to each tube and allowed to react on ice for about 40 minutes. After the reaction, the cells were washed with phosphate buffered saline (PBS), and the composition ratio of each immune cell was measured using FACScan (Becton Dickenson, San Jose, Calif.). The measured results are shown in Table 4 below.

[Table 4] Change in the percentage of immune cell composition by the mixed water extract of the present invention (unit%)

As a result of measuring 10,000 cells, the composition ratio of B cells, T cells, and CD4 T cells in the spleen was increased as shown in Table 4, and in the thymus, the composition ratio of T cells with Thy-1 antibody was increased. And CD4 T cells with CD4 antibody also increased. In the bone marrow, the proportion of B cells to which the B220 antibody was attached increased.

Experimental Example 7: Toxicity Test

LD ₅₀ (amount capable of killing 50% of experimental animals) is an important value that is an index indicating acute toxicity of drugs in order to confirm the safety of the mixed water extracts of P. alba and Matari according to the present invention. Obtained by the same method.

Normal ICR mouse ( , 19 ± 1g) divided into 30 groups of 6 dogs each into 5 groups of A to E, and to the group A to administer 3 g of the mixed water extracts of yellow and white skin and matari prepared in Example 1 according to the present invention per 1 kg of body weight. The doses were increased gradually and 6 g in group B, 9 g in group C, 12 g in group D, and 15 g in group E, respectively, followed by the Berens-Karber method [Reference: 高木 敬 次 郞In addition, LD ₅₀ value by oral (po) administration of the mixed water extract of the present invention was measured in accordance with the present invention. The results are shown in Table 5 below.

Table 5 Lethal dose by oral administration of the mixed water extract of the present invention (LD50)

z: ½ of the number of dead animals in two consecutive doses

** d: difference between two consecutive doses

As shown in the results shown in Table 5, even in the group that orally administered the mixed water extract of Hwangbaekpi and Matari according to the present invention in a high dose amount of 15 g per 1 kg of body weight, no animals died and oral administration of the mixed water extract LD ₅₀ value was 15 g / kg or more, and therefore, it was found that it was very safe for living bodies. In other words, it can be clearly seen that the mixed water extract of Hwangbaekpi and Matari according to the present invention can be safely administered with little toxicity.

In addition, autopsies and histopathological examinations were performed on the test animals used to measure the LD ₅₀ value in the following manner. At the end of the DL ₅₀ measurement test, all living animals were bled and killed by ether anesthesia. The autopsied organs were fixed in a 10% neutral formalin solution for at least 10 days for pathological examination, and then dehydrated and embedded in paraffin embedding (Fisher, Histomatic Tissue Processor, 166A, Shadon, UK). 5 micrometer sections were made with a microtome (AO Rotary Microtome, LEICA, Germany) and observed with hematoxylin and eosin staining.

All animals of each group were dissected and histopathologically examined under a microscope. That is, when oral administration of the mixed water extract of baekbaekpi and matari according to the present invention up to 15g per kg of mouse body weight, ideal findings by drug administration in liver tissues were not observed (Fig. 8A). In addition, no abnormalities were observed by drug administration in kidneys (Fig. 8B), and no abnormalities by drug administration were observed in cardiomyocytes of heart (Fig. 8C). Abnormalities due to drug administration were not observed in other major organs such as the gastrointestinal tract, pancreas, lung, spleen, adrenal gland, brain, testes, ovaries and bone marrow.

Therefore, the mixed water extract of Hwangbaekpi and Matari according to the present invention had no side effects due to acute toxicity to all organs even when 15 g per 1 kg of body weight, which is the maximum dose that can be administered to mice, and any organ damages. It was determined to be a safe drug that does not cause toxicity.

Claims (7)

A composition for treating acquired immunodeficiency syndrome, which contains a mixed water extract of baekbaekpi and matari plants. The method for treating acquired immunodeficiency of claim 1, further comprising a mixed water extract obtained by mixing the baekbaekpi and matari plants in a ratio of 1: 0.1 to 1: 5 by weight. According to claim 2, Acquired immune deficiency therapeutic composition comprising a mixed water extract extracted by mixing the baekbaekpi and matari plants in a ratio of 1: 1 to 1: 2 by weight. The method of claim 3, wherein the treatment composition for the treatment of acquired immunodeficiency containing a mixed water extract extracted by mixing the baekbaekpi and matari plants in a ratio of 1: 1 by weight. The method of claim 1, wherein the mixed water extract of Hwangbaekpi and Matari is a stagnant water-soluble fraction thereof. The therapeutic agent composition for treating acquired immunodeficiency of claim 1, further formulated in a pharmaceutical unit dosage form using a pharmaceutically acceptable carrier. The composition of claim 6, wherein the pharmaceutical unit dosage form is in the form of a tablet, capsule, solution, suspension, or syrup.