WO2003035091A1

WO2003035091A1 - Ai preparation and use thereof in preventing and treating infection with human immunodeficiency virus

Info

Publication number: WO2003035091A1
Application number: PCT/JP2002/009481
Authority: WO
Inventors: Osami Aki; Shinobu Matsuda; Naoki Yamamoto
Original assignee: Matsuda, Tasuku
Priority date: 2001-10-23
Filing date: 2002-09-17
Publication date: 2003-05-01
Also published as: JPWO2003035091A1; JP4121957B2

Abstract

It is intended to provide a novel plant-origin preparation which is efficacious in preventing and treating infection with human immunodeficiency virus (HIV). Namely, an “ai (polrgoum indigo) preparation” originating in “tadeai” (Polygonum tinctorium Lour.) which is a plant belonging to Polygonaceae and, in particular, a component having a remarkable anti-HIV effect which is contained in an aqueous extract of “sukumo” (fermented ai leaves) are provided. In the process where CD4 antigen binds to a chemokine receptor via a surface antigen gp120 and then invades into cells, the component contained in the aqueous extract of “sukumo” has an effect of inhibiting the binding of the antigen. This component also likely inhibits the replication and proliferation of HIV in the cells associating the infection. Moreover, the aqueous extract of “sukumo” has little toxicity and, therefore, is free from any risk of side effects even in prolonged administration for preventing or treating HIV infection. Therefore, it can be safely employed.

Description

Specification

Indigo preparation, and

Its use in preventing or treating human immunodeficiency virus infection

Name: Indigo preparation and its use in preventing or treating human immunodeficiency virus infection

Technical field

The present invention shows an anti-HIV effect that is effective for prevention of human immunodeficiency virus (HIV) infection and suppression of the development of acquired immunodeficiency syndrome (AIDS) caused by an increase in HIV-infected cells in the body of an infected person. Indigo preparation "or" indigo composition ". More specifically, the present invention relates to the indigenous plants of the Polygonum tinctrium Aiton (Polygonaceae J), the Ryukyu indigo (Strobilanthes flaccidifolium Nees), and the spurge Among the three species of Sanai (Mercuria Us leiocarpa Siebold), an indigo preparation prepared from the Polygonaceae plant Tateai, especially water from Sukumo derived from Tateino, using water as the extraction solvent. The present invention relates to the use of the extracted "sukumo" water extract or the anti-HIV active ingredient contained in the "sukumo" water extract in preventing or treating human immunodeficiency virus infection. Background technology

The number of people infected with the HIV (Human Immunodeficiency Virus: AIDS) virus has been reported to exceed 3,600,000 worldwide today. With the establishment of a general-purpose diagnostic kit for infection, it has been confirmed that these HIV-infected persons and patients are widely distributed not only in developed countries but also worldwide, It is a serious social threat in developing countries such as Southeast Asia and Eastern Europe. Research on prevention and treatment methods for this HIV infection is progressing rapidly, but not enough.

For example, the development of vaccines to prevent HIV Among the viruses, HIV is one of the special viruses, and its genomic mutations in clinically isolated mutants are extremely diverse. Therefore, development of an accurate vaccine that can protect the infection of these various mutants showing various genomic gene mutations in general has not yet been predicted.

On the other hand, regarding the therapeutic agent, the characteristic of the propagation process of this retrovirus, the HIV virus, is that when the virus is infected to host cells, the virus and genomic RNA are converted into type II, and the virus-derived reverse transcriptase is used in the host. Thus, a reverse transcriptase inhibitor that inhibits the process of replicating the corresponding DNA chain, that is, the reverse transcription process specific to the HIV virus, has been developed, and multiple species have been put to practical use. However, nucleotide reverse transcriptase inhibitors or non-nucleotide reverse transcriptase inhibitors used in this reverse transcriptase inhibitor have serious side effects, such as reduced production of leukocytes and erythrocytes, It has been shown that side effects such as inducing neuropathy and, in addition, the gene mutation of the virus itself in patients can relatively easily develop resistance to these reverse transcription-inhibiting drugs. .

In addition, virus-derived poly'proteins that are produced using the transcription and translation machinery of the host cell from viral genes that have been integrated into the genomic DNA of the host cell once through the reverse transcription process are: Through the cutting process by virus-derived HIV protease, it becomes various mature viral proteins. For example, inhibition of the cleavage process by the HIV protease inhibits the maturation of the reverse transcriptase derived from the above-mentioned virus, or the maturation of the outer coat protein of the virus. Production of viral molecules is suppressed. An HIV protease inhibitor targeting this HIV protease has also been developed, and several types have been put to practical use. However, HIV protease inhibitors have been reported to exhibit side effects such as psychiatric disorders, convulsions, and kidney stones.In addition, when used alone, resistance is quickly acquired due to the gene mutation of the virus itself, and the therapeutic effect is high. Is attenuated.

In order to alleviate the shortcomings such as emergence of resistance and side effects of these HIV drugs, an administration method called cocktail therapy was developed. Specifically, two types of HIV drugs The above is, for example, a method of simultaneously administering a reverse transcriptase inhibitor and an HIV protease inhibitor. Typically, one HIV protease inhibitor is administered in addition to two reverse transcriptase inhibitors, such as a combination of nucleotide reverse transcriptase inhibitors of different types of purine bases and pyrimidine bases. It is a method of simultaneously blocking different processes. In this cocktail therapy, the mechanism of acquiring resistance to each of the HIV drugs used is different, and the infections of resistant strains to each drug are mutually suppressed, thereby compensating for the above-mentioned disadvantages of monotherapy. be able to.

However, there is a real problem that even with this cocktail therapy, the emergence of drug-resistant mutant viruses is unavoidable sooner or later. In addition, treatment methods that use conventional HIV therapeutics have significant problems that can be a major obstacle to expanding their use. One is that, at this stage, drug prices are still high and treatment costs are high. The above HIV drugs suppress the increase of virus-infected cells in the body after HIV infection and exert a therapeutic effect to prevent the onset of AIDS symptoms. This means that the number of patients who can bear the cost of treatment is extremely small. Another problem is that the combined use of multiple drugs results in high overall doses and complicated dosing procedures, depending on the dosing regimen that is appropriate for each drug. As a result, it is often difficult for patients to continue to take a variety of medications regularly over a long period of time in accordance with established procedures. In general, complicated dosing procedures make it difficult to maintain adherence, i.e., aim for patients to actively participate in decisions on treatment and to carry out treatment according to their own decisions. However, the target treatment cannot be continued.

On the other hand, clinical application of this cocktail therapy has only just begun, and at this stage there is no record of long-term administration. In addition, long-term administration of many drugs may lead to a long-term increase in blood sugar levels, which may lead to diabetes, or an abnormal rise in total cholesterol levels, leading to hyperlipidemia. General, such as long lasting In the future, there is a concern that a serious, chronic illness could be predicted. Indeed, the number of deaths associated with AIDS appears to have declined temporarily in HIV patients who have been on medication for the first time since the cocktail therapy began. However, at present, these medications have yet to be confirmed as a sufficient and safe treatment for HIV infection. Disclosure of the invention

Although various attempts have been made to prevent and treat HIV infection, as described above, and to some extent achieved, many routes of transmission, for example, due to sexual contact, In human body fluids but due to the invasion of HIV. The route of transmission associated with this sexual contact is related to the human species preservation instinct of “sex,” and its prevention is not always easy. At this stage, only effective measures to prevent infection have been proposed, such as avoiding contact with body fluids of HIV-infected individuals by physical means.

On the other hand, current treatment for HIV infection requires taking many expensive drugs and continuing to administer them for a long period of time, and at the present stage, the effect is, for example, delayed AIDS onset. Although effective, it is still far from the stage of cure for HIV infection and, in addition, has many other problems besides its therapeutic effects.

In other words, the general principles currently being practiced in the treatment of HIV infection are: 1. Start strong anti-HIV therapy early in infection,

2: keep the amount of virus in plasma below the detection limit,

3: Interruption of treatment is not allowed,

4: To maintain adherence,

5 _: Anti-HIV drugs, etc.

These general principles apply to actual treatment of HIV infection as described above. Raises a very difficult problem.

INDUSTRIAL APPLICABILITY The present invention exerts a very clear effect in the prevention of HIV infection, the suppression of AIDS development after infection and the treatment of HIV infection, and has no adverse effects even when administered for a long period of time. The present invention provides an indigo preparation and its composition as a novel anti-HIV active ingredient that has a low resistance acquisition rate, is robust, can be obtained at low cost, and can be used for a long time.

The present inventors have reported that Polygonum tinctrium Aiton (Polygonum tinctrium Aiton), a Polygonaceae plant, and Ryukyu Ai (Strobilanthes flaccidifolium Nees), Poaceae plant, which have been used for medicinal purposes such as antipyretic and detoxifying for a long time. "Ai Ai"

(Mercurialis leiocarpa Siebold), and focused on their potential application in the prevention and treatment of HIV infection. As a result, among the three types of indigo, among the three indigo plants, indigo preparations using “Tateai” of the Polygonaceae plant as a raw material, in particular, a component that is remarkably resistant to components extracted with water from “Sukumo” prepared from “Tatei”. I found that it has HIV action. In addition, the indigo preparation, which is an aqueous extract of Sukumo, and the active ingredients contained therein are extremely low in toxic “I” life, and are suitable for the prevention of HIV infection, as well as for humans. Confirmed that it can be used with confidence.

That is, the first aspect of the present invention relates to an indigo preparation,

The indigo preparation according to the present invention is an indigo preparation derived from Polygonum tinctorium Aiton belonging to the Polygonaceae family,

An indigo preparation characterized by being an aqueous extract of "Sukumo" prepared from the leaves and stems of the indigo plant in the indigo production process.

In addition, the second aspect of the present invention relates to an active ingredient which provides an anti-HIV activity of an indigo preparation derived from Polygonum tinctorium Aiton belonging to the above-mentioned Polygonaceae, and thus the anti-HIV according to the present invention. The active ingredients are:

Contained in the water extract of "Sukumo", which is prepared in the process of producing indigo plants derived from the indigo plant belonging to the Polygonaceae family,

It has a molecular weight of more than 100,000 and is stable to heat, It is easily soluble in water and alkali, hardly soluble in acid,

It is a component having an anti-HIV action that is hardly soluble in organic solvents. In addition, the component having an anti-HIV action in the water extract of “Sugumo” contains a phenolic hydroxyl group that forms a blue precipitate due to trivalent iron ions and has a reducing property. On the other hand, a third aspect of the present invention relates to the use of the indigo preparation in the prevention or treatment of human immunodeficiency virus infection,

The first use of the indigo preparation according to the present invention is the use of the indigo preparation as one of the active ingredients in the production of a health food,

The health food is a use of the indigo preparation, characterized by comprising, as an active ingredient, the indigo preparation derived from the indigo plant belonging to the Polygonaceae plant according to the present invention. Also, the second use of the indigo preparation according to the present invention,

Use of an indigo preparation as one of the active ingredients in the manufacture of a pharmaceutical composition having an anti-HIV action,

The pharmaceutical composition according to the present invention is obtained by blending, as one of active ingredients having an anti-HIV activity, the indigo preparation derived from the above-mentioned fruit belonging to the Polygonaceae plant according to the present invention. Use of things. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the use of the HTLV-III B strain as HIV-1, infection of MT-4 cells derived from human and lymphocytes, and HIV in cells infected with the HTLV-III B virus. 1 shows the results of evaluating the inhibitory effect on the growth process by the "Sukumo solution" derived from Tateai added to the culture solution. The graph shown in FIG. 1, the HTLV III B strain virus dilution (0.0 0 1 / Ueru TCID _{5 0} of) one hour after infection manipulation of contacting, MT- 4 cells at various ratios "Sukumo After 5 days of culturing in a culture solution supplemented with the `` Solution '', the virus that multiplied following infection with HIV-1 in infected cells in a multiplicity of infection (MOI) = 0.01 infection state The HIV-1 virus eluted into the culture medium following the expression of the particles on the cell membrane surface The following shows the results of evaluating the concentration of the P24 antigen protein.

Fig. 2 shows a cell culture of MOLT-4 cells (MOLT-4ΊΠB) with persistent infection of HTLV-IIIB strain, using a “squid solution” derived from Tateai added to the culture. FIG. 9 shows the results of evaluating the effect of suppressing the concentration of the P-24 antigen protein derived from the HIV-1 virus contained in the supernatant.

Figure 3 shows that PBMCs, which produce T lymphocytes by stimulating PHA, were exposed to HTLV-IIIB strain virus solution for 2 hours (moi = 0.1). After culturing for 12 days in the presence of IL-12 in a culture solution supplemented with indigo-derived `` spider solution '', the effect of suppressing the concentration of HIV-1 virus-derived P24 antigen protein contained in the culture supernatant was reduced. The results of the evaluation are shown.

Fig. 4 shows that MOLT-4 cells were cultured for 24 hours in a culture solution supplemented with a squid solution derived from Tateai at various ratios and expressed CD4 antigen molecules on the cell surface. The result of evaluating the cell number ratio is shown.

Fig. 5 shows that four kinds of "Sukumo solution" derived from Tateai were prepared in a culture solution containing the "Sukumo solution" at a ratio of 20%, respectively, and cultured for 24 hours in the medium containing the "Sukumo solution". After treating the treated MOLT-4 cells with the HIV-1 virus, the cells are cultured in a new culture medium and cultured from the HIV-1 virus replicated and produced in the cells. The result of measuring the P24 antigen protein concentration in the supernatant is shown. BEST MODE FOR CARRYING OUT THE INVENTION

Polygonum tinctrium Lour (Polygonum tinctrium Lour) has been reported to have some medicinal properties in Kampo medicine. For example, fresh juice and decoction of indigo leaves, dried leaves, and seeds, when taken orally or externally, are effective against anti-inflammatory, detoxifying, antipyretic, hemostatic, insecticide, hemorrhoidal, tonsillitis, laryngitis, etc. Have been reported. Indigo also has the action of removing active oxygen from the body as a physiologically active substance.It has the properties of gallic acid, caffeic acid, kaemperol, It has been reported that it contains trybotanthrin, indimbin, and flavonoids having a platelet aggregation inhibitory action, which exhibit a cancer action and an antiallergic action.

In order to search for a new medically useful effect other than the above-mentioned already reported medicinal effects, the inventors of the present invention have described "Polygonaceae" which has been used for medicinal purposes such as antipyretic and detoxifying for a long time. (Polygonum tinctrium Aiton), Ryukyu indigo (Strobilanthes flaccidifolium Nees), a plant of the family Rhododendron, and the indigo plant (Mercurialis leiocarpa Siebold), a plant of the family Indaceae. Preparations ”We also investigated whether any useful pharmacological effects could be demonstrated in IHIV infection.

According to the study of the present inventors, the anti-HIV activity of the water extract of indigo, i.e., "sukumo", will be described in detail in the experimental examples below. “Sukumo” water extract contains HIV: the process by which human immunodeficiency virus enters healthy lymphocyte cells, specifically HIV

In the process of binding to the CD4 antigen molecule and the chemokine 'receptor expressed on the surface of T cells via (gP120) and invading the T cells, HIV has the effect of inhibiting the binding process.In addition, HIV causes mutations in infected cells, and repeats replication of virus particles and infection of other cells, resulting in the growth and infection of infected individuals. Although it is expanding, it has been found that it has the potential to prevent the process of spreading and multiplying the infection in the body of the infected person. Therefore, the water extract of Sukumo has a strong anti-HIV effect and is expected to play an important role in the prevention and treatment of HIV infection.

Hereinafter, the present invention will be described in more detail.

In the present invention, the “indigo preparation” is usually a Polygonum tinctrium Aiton (Polygonum tinctrium Aiton),

Rubik Ai (Strobilanthes flaccidifolium Nees) Plants and tissues of the Euphorbiaceae plant “Yamaai” (Mercurialis leiocarpa Siebold) include all processed products that have been subjected to physical or chemical treatment. In this case, the site used as a raw material and its preparation The processing method used for the process does not matter.

Therefore, the indigo fermented product called "Sukumo", which is made in the process of producing indigo dye, is harvested, cut and dried, and then fermented for 2 to 3 months with water. The fermented material looks like red-black humus, and is called "sukumo". This "sukumo" can be used as an indigo preparation in the present invention. The outline of the method for preparing the “indigo preparation” and the “indigo composition” used in the present invention is as follows.

`` Aio raw material ''

After immersing “Sukumo J” in purified water or deep sea water for 2 days, centrifuge and collect the soluble components contained in “Sukumo” as supernatant. The supernatant is filtered through a Millipore filter to remove bacteria. The filtrate after the sterilization treatment is referred to as a “steam solution” (square water extract).

`` Indigo heating material j

The above-mentioned “spring solution” is further heat-treated at 121 ° C. for 15 minutes to sterilize it. The heat-sterilized “spider solution” is particularly called “heat spider solution”. Immerse the indigo leaves in purified water or deep sea water, boil them for 20 minutes, and then cool. * Leave the soluble components. Next, the mixture is centrifuged, and the soluble component eluted by the heating and boiling treatment is collected as a supernatant. The supernatant obtained by filtering the supernatant with a Millipore filter 1 is referred to as a “heated indigo solution”.

Indigo drying "leaves, stems, roots, seeds, fruit powder"

Once harvested indigo leaves, stems and roots, the 'seed' fruit is dried once, crushed into fine powder, pressurized and heat sterilized. The dry fine powder sterilized by pressurizing and heating is referred to as indigo dried “leaf, stem, root, seed, fruit powder”.

"Indigo composition" Compounds derived from plants with anti-HIV activity have already been reported, but plants containing such compounds with anti-HIV activity, such as plants containing HIV, have already been shown to be somewhat effective in preventing and treating HIV infection. In the present invention, a composition in which a seed plant is mixed with, for example, the above-described “dried indigo leaf / stalk 'root' seed 'fruit” is referred to as “indigo composition”.

Specifically, the active ingredient exhibiting an anti-HIV activity derived from the Polygonaceae plant “Tateai” according to the present invention is usually obtained by extracting water from a fermented indigo product called “Sukumo” by water extraction. However, in some cases, it is possible to apply a method in which non-fermented plants are dried and pulverized and then collected by water extraction of the water-soluble components contained. Good. Hereinafter, the present invention will be described more specifically using specific examples.

In addition, in each of the following experimental examples, an example of a "sukumo solution" (sukumo water extract) prepared using the Polygonum tinctrium Aiton (Polygonum tinctrium Aiton) is shown.

Experimental example 1

Preparation of "heated blue-green solution"

50 g of freshly harvested indigo leaves are immersed in 1 liter of deep sea water, heated for 20 minutes, boiled, cooled to 10 ° C or less, and left for 2 days with occasional stirring. Next, the supernatant is collected from the immersed material, centrifuged at 10, OO Orpm for 30 minutes, and the supernatant is collected. Further, the solution is filtered through a 0.2 millimicron millipore filter to obtain about 900 ml of a filtrate. This filtrate is referred to as “heated blue raw solution”.

Experimental example 2

Preparation of "spider solution" and "heated spider solution"

Indigo leaves ・ Stem mixture is cut into appropriate size ・ After pulverized, fermented for 3 months to prepare “Sukumo”. Prepared "Sukumo": 50 g is immersed in 1 liter of deep sea water, centrifuged at 100,000 rpm for 30 minutes, and the supernatant is collected. You. The supernatant is filtered through a 0.2 millimicron Millipore filter to obtain a filtrate from which bacteria grown in the fermentation step have been removed. The sterilized filtrate is further heat-treated at 121 ° C. for 15 minutes to sterilize it.

The filtrate subjected to the sterilization treatment is referred to as a `` scoop solution '', and is usually used as the heat-sterilized `` spark solution ''. In particular, when distinguishing from the solution before the heat sterilization treatment, Heated spider solution ".

In addition, the freeze-drying process was performed on the above-mentioned “sparkling solution” and “heated spider solution”, and the extract dry weight was measured. As a result, the extract-containing concentration in the “sparkling solution” and “heated spider solution” was measured. Was 8 mgZm1.

Experimental example 3

Preparation of "Indigo dried leaves, stems, roots, seeds, fruit powder"

The harvested indigo leaves, stems, roots, seeds, and fruits are dried once, and the dried product is ground into a fine powder having a particle size of about 50 microns or less.Pressing is performed at 121 ° C for 15 minutes. And sterilize. The dried fine powder that has been sterilized by pressurization and heating is referred to as “blue-dried leaves, stems, roots, seeds, and fruit powder” depending on the location. The verification method used in the anti-HIV activity verification experiment described below is described below.

(1) Host cells

In the verification experiments described below, human lymphocyte-derived MT-4 cells (HTLV-I transformed T4-cell line) were used as host cells for infection with HIV.

MT—3 として as standard culture conditions for 4 cells. First, culture in a PRM I-1640 medium supplemented with 10% fetal serum, 100 μs 1 streptomycin and 100 U / ml penicillin G under the conditions of 5% CO ₂ was used.

(2) HIV-1 virus solution

HIV-1 virus solution is obtained from the culture supernatant of MOL T-14 cells (MOLT-4 / IIIB) that have been persistently infected with HTLV-IIIB as the source of HIV-1 used for infection. did. The amount of virus in such HIV-1 virus fluid is the indicator TCID ₅ . Was quantitatively evaluated in advance.

(3) Evaluation method of anti-HIV activity

The evaluation of the anti-HIV activity of the test substance was based on the cytopathogenic effect of HIV-1 in MT-4 cells as an index, and the inhibitory effect was evaluated.

The cytopathogenic effect of HIV-1 on MT-4 cells was measured according to the method described in the literature: Harada, Koyanagi & Yamamoto, Science, Vol. 229 p.563-566 (1985).

MT-4 cells were contacted with a solution containing HIV-1 (0.001 / well TC ID ₅₀ ) for 1 hour to infect the cells, and then unadsorbed virus was washed and removed. Then, the MT- 4 cells subjected to the infection process, RPMI-were resuspended at a concentration of ^{1. 5 X 1 0 5 eel 1} / m 1 in 1640. The cells were cultured for 5 days on a 96-well culture plate with 200 μl of 1-well cell suspension containing various concentrations of the test substance. As a control, a culture in which HIV-1 infected cells (positive control) or uninfected cells (negative control) were similarly cultured without adding a test substance to the medium was used. After cultivation for 5 days, the CPE (cytopathic effect: giant cell formation) induced by HIV-1 was observed using an optical microscope, and the CPE was completely inhibited based on the observation results of the various concentration-added groups. The concentration of the test substance to be added . It was decided as.

In addition, as an evaluation of the cytotoxicity of the test substance itself, a cell proliferation test in a medium to which the test substance was added at various concentrations was separately performed to determine a concentration at which the survival rate of MT-4 cells was reduced. The survival rate of the cultured cells was evaluated using MT based on the reduction reaction of viable cells against 3- (4,5-dimethythithiazoi-2-yl-2,5-diphenylentetrazolium bromide (MTT). The method is performed according to the T-Atsey method (J. Virol. Methods 20 (4), p. 309-21 (1988)).

Experiment 4

According to the preparation method described in Experimental Example 2, We examined its anti-HIV activity.

First, the addition ratio of the "sparkling solution" to the medium is 100% for the "sparkling solution" itself, and 0% for the medium without the "sparkling solution", and the addition ratio is determined by the volume ratio. Was defined and used in the following experiments. Prior to the anti-HIV activity evaluation experiment, it was examined in advance whether or not the “spider solution” itself had cytotoxicity. 'In other words, MT-4 cells and MT-4 cells infected with the HIV-1 virus were cultured in a culture medium supplemented with various proportions of the "spider solution", and the MTT assay was performed. Assess its survival. As a result, it was confirmed that MTT Assay in MT-4 cells infected with HIV-1 virus showed no cytotoxicity even when the addition ratio of “Sukumotsu solution” was 20% at moi = 0.01 (multiplicity of infection). Was. Next, the anti-HIV activity was verified in the range of the addition ratio of the “spin solution”, which was confirmed not to exhibit the cytotoxicity. The evaluation of the suppression of the effect of HIV-1 on the cytopathogenic effect of MT-4 cells by the addition of a "steam solution" to the medium was evaluated.

After infecting MT-4 cells with HIV-1 solution (supernatant of MOLT-4 / IIIB cells) and infecting (MOI (multiplicity of infection) = 0.01), washing the HIV virus The cells were cultured for 5 days in the culture medium to which the "sparkling solution" was added at the ratio of. After that, following the proliferation of HIV-1 in infected cells, the morphological changes of cells (CPE (cytopathic effect): giant cell formation) associated with the expression of the propagated virus particles on the cell membrane surface are optically examined. Observed with a microscope. Table 1 shows the results of evaluating the presence or absence of CPE (cytopathic effect). Even with the addition ratio of 1.25% of the "scoop solution", no CPE (cytopathic effect) was found, and it was shown that cell fusion was completely inhibited. table 1

Inhibition of giant cell formation in MT-4 cells infected with HT LV-III B strain MT-4 I n f e c t ion (III B, mo i = 0.01)

At the same time, after culturing for 5 days as described above, the concentration of P24 antigen protein present in the culture supernatant was measured. Figure 1 shows the measurement results. Corresponding to the inhibitory effect of the above-mentioned CPE, the P24 antigen, which corresponds to the release of HIV-1 virus produced in infected cells to the culture supernatant even at an addition ratio of 1.25% of the `` spider solution '', It is very clear that the increase in protein concentration is suppressed.

Experimental example 5

P24 antigen test: MOLT-4 / IIIB cells were cultured in a culture solution containing “Sukumo's solution” at various addition ratios, and 4 days later, the p24 antigen protein in the culture supernatant was cultured. The substance concentration was measured. Figure 2 shows the measurement results.

When cultivating MOLT-4 cells (MOLT-4 / IIIB) that have been persistently infected with HTLV-IIIB, they produce high concentrations of the HIV-1 virus in the culture broth while undergoing mitotic growth. Thus, maintenance of a persistently infected cell group is performed. After culturing these MOLT-4 IIB cells at various ratios in a culture solution supplemented with the `` spider solution '' for 4 days, the culture supernatant, which corresponds to the concentration of the HIV-1 virus contained in the culture solution, is used. P24 antigen protein concentration was measured. Under the conditions of 0.6%, 1.25%, and 2.5% ai, the concentration of P24 antigen protein in the culture supernatant was higher than that of the control (without the addition of the "sparkling solution"). No significant change is observed. Under the condition of the addition ratio of “Sukumotsu Solution” ai 20%, the concentration of P24 antigen protein in the culture supernatant was clearly reduced as compared with the control, and the reduction ratio was about 57%. Was. Experiment 6

After collecting heparin, the isolated peripheral blood mononuclear cells (PBMC: Peripheral blood monocytes) were stimulated with phytohemagglutinin (PHA) for 3 days to confirm that lymphocytes were proliferating. After confirming the growth, the HIV-1 virus (culture supernatant of MOLT-4 iB cells) was adsorbed to the PMBC for 2 hours, followed by infection treatment (mo i = 0.1). After that, the infected PMBC was cultured for 12 days in the presence of T-cell growth factor IL-2 in a culture medium supplemented with various proportions of "Sukumotsu", and then the P24 antigen protein in the culture supernatant was Measure the concentration 1

FIG. 3 shows the measurement results of the P24 antigen protein concentration in the culture supernatant. The concentration of P24 antigen protein in the culture supernatant decreases along with the addition ratio ai of the "sparkle solution" added to the culture solution. At 24% ai, P24 antigen was hardly detected in the culture supernatant, and almost 100% was suppressed. In particular, under the condition that the ratio of addition of the “scoop solution” a i was 20%, no P24 antigen was observed in the culture supernatant after culturing for 12 days.

Experimental example 7

Glycoprotein gp120 present on the surface of the HIV virus coat has the ability to form a complex with the CD4 surface antigen molecule, a leukocyte differentiation antigen of human T lymphocytes (receptor function). In addition, the CD4 molecule, which forms a complex with HIV gp120, plays a role as a receptor in the process of HIV infection of T cells. On the other hand, the “spider solution” added to the culture solution reduced the surface expression of the CD4 antigen molecule, a receptor for HIV-1, in MOLT-4 cells in a concentration (addition ratio) -dependent manner. Figure 4 shows the results of estimating the ratio of the number of cells expressing CD4 antigen molecules on the cell surface by culturing MOLT-4 cells for 24 hours in culture medium supplemented with various proportions of the Sukumotsu solution. Is shown. Relative evaluation based on the ratio of the number of cells expressing and expressing the CD4 antigen in the culture of unpolished sorghum (100%) When the ai 2.5% solution was added to ai 2.5%, 92%, ai 5%, 42%, ai 10%, 26%, and ai 20%, 10% The surface expression of the CD4 antigen molecule is suppressed in a concentration (addition ratio) -dependent manner.

Furthermore, after treating for 24 hours in the culture solution to which the above “sparkle solution” is added, the plate is washed and a new “sparkle solution” is not added! / When culturing MOLT-4 cells in the culture medium, it is also confirmed that the surface expression ratio of CD4 antigen increases again 6 hours after the start of culture.

Experiment 8

After 24 hours of cultivation in a culture solution supplemented with the “spider solution”, the MOLT-4 cells treated with the “spider solution” are transferred to HIV-1 virus (MOLT-4 / IIIB cells). (Culture supernatant). Then, the cells were cultured in a new culture medium, and the concentration of the P24 antigen protein in the culture medium derived from the P24 antigen protein produced in the cultured cells was observed. In this experiment, a culture solution supplemented with “smooth solution” at a ratio of 20% was used.

Specifically, four types of "sparkle solution" were prepared, and each "splatter solution" was prepared in a culture solution supplemented with 20% of the "splatter solution", and cultured for 24 hours in a medium containing the "splatter solution". Treated MOLT-4 cells were infected with HIV-1 virus. Then, the cells were cultured in a new culture medium, and the concentration of P24 antigen protein in the culture supernatant derived from the HIV-1 virus replicated and produced in the cells was measured. FIG. 5 shows the measurement results of the P24 antigen protein concentration. Relative evaluation using the P24 antigen protein concentration as a reference (100%), which is measured in a positive control (positive 0 ntro 1) in which the pre-culture treatment is performed using a culture solution without the “spider solution”, The concentration of the P24 antigen protein measured in the group added with the four kinds of “spider solution” was within several% at the maximum. Therefore, in the above-mentioned four “smoking solution” supplemented groups, the P-1 antigen in the culture broth caused by the HIV-1 virus infection and the production of the virus-derived P24 antigen protein after the infection. The increase in the protein concentration is almost 100% suppressed.

Experiment 9

Several further experiments were performed to determine the active ingredients involved in the anti-HIV activity of the “spider solution”.

First, the presence or absence of anti-HIV activity was examined for components already known to be contained in indigo. For example, indicans (glycosides of indoxyl), such as eyes (Persicariatinctria), indigo, the main component of natural indigo dyes produced by fermenting plants containing this indican, and derivatives indigoca rmi ne (5, 5) '-Indigotine' diso-lenoic acid disodium salt) does not have the above-mentioned anti-HIV activity at all, specifically, the above-mentioned inhibitory effect of HIV-1 infection or the HIV virus particle table after infection. The effect of suppressing the outflow was confirmed by a test system such as the use of the above-mentioned MTT assay method and a ce11-fusion (Syncytium formulation) assay fe in MOLT-4, MOLT-4 / ΠΙ coculture.

It was found that the active ingredient involved in the anti-HIV activity indicated by the “spider solution” was a component having the following characteristic properties.

A water-soluble component having a molecular weight of 10,000 or more, fractionated by an ultrafiltration membrane;

When heat treatment was performed during the preparation of Sukumo, at least immersion in water, heat boiling at 100 ° C for 1 hour, and heating in the fermentation process for a total of three months in preparing Sukumo (60 minutes) The anti-HIV activity is not lost by thermal treatment (eg, 70 ° C), and furthermore, heat sterilization at 121 ° C for 15 minutes for the “Smoking solution” is performed, and 100 ° C, 1 Anti-HIV activity is not lost by heat treatment for a long time or drying treatment by evaporation of water solvent;

The "spider solution" produces a precipitate upon treatment with 0.5N HC1, and the precipitate has anti-HIV activity, while the supernatant has no activity; The "spider solution" is unchanged by the treatment with 0.5N NaOH, and the solution retains anti-HIV activity;

It is sparingly soluble in organic solvents and, when ethanol is added to the "spider solution", it forms a precipitate, which has anti-HIV activity but no activity in the supernatant;

The “spider solution” is a blue precipitate formed by the addition of an aqueous solution of .l% FeCl ₃ , 1% potassium ferricyanide (potassium hexacyanoferrate (III)) containing trivalent iron ions, Indicates the presence of a reducing, phenolic OH group.

For example, "Sukumo solution" was evaporated to dryness processing, the dry product obtained, an organic solvent (methanol, pyridine, Asetonitoriru) can not be re-dissolved in, 0. IN NaHC 0 ₃ aqueous solution, 0. 5N N a OH It can be redissolved in aqueous solution or water. Further, the “heated spider solution” described in Experimental Example 2 described above was subjected to a heat sterilization treatment at 121 ° C for 15 minutes in the manufacturing process, but was further heated at 100 ° C for 1 hour. Even with the treatment, the anti-HIV activity remained to a considerable extent. In addition, even if the “calo-hot scoop solution” was treated with a 0.5N NaOH aqueous solution, no precipitate or the like was formed, and the anti-HIV activity was maintained. On the other hand, when the “heated spider solution” was treated with a 0.5N HCl aqueous solution, a precipitate was formed. The supernatant was separated, neutralized by dialysis, and its anti-HIV activity was evaluated. No anti-HIV activity was found. It separated該沈lees are in water or an organic solvent without remelting, 0. 1 N NaHC0 ₃ aqueous solution or the addition of 0. 5N N a OH water溶裤and redissolved. The re-dissolved solution of the precipitate was returned to neutral by dialysis, and its anti-HIV activity was evaluated. As a result, anti-HIV activity was found.

The "heated spider solution" is converted into a water-soluble component having a molecular weight of less than 10,000 and a water-soluble component having a molecular weight of 10,000 or more remaining on the ultrafiltration membrane by an ultrafiltration membrane having a molecular weight of 10,000. No anti-HIV activity was found in the filtrate of a water-soluble component having a molecular weight of less than 10,000 that permeated the ultrafiltration membrane. Ultrafiltration membrane in a liquid which is redissolved molecular weight 10, 000 or more soluble water-soluble components remain on only anti HI active '! ¹ production was found. Evaluation of the anti-HIV activity when the above-mentioned various treatments were performed was performed using the ce11-fusion (Syncyti urn formation) assay method using MOLT-4, MOL T-4 / III B cocuture. The addition ratio (IC ₅₀ ) giving% inhibition was measured. Incidentally, separately, the sum of component that is part of in each test solution, measured as dry weight, the I c _5. Represents the weight of the dry matter (dry matter weight / m1 unit) added per unit volume of the medium. Table 2 below shows the results of the evaluation of anti-HIV activity.

Table 2

In addition, a mixture of ethanol containing 5% sodium acetate / 2.5M acetic acid was mixed with the heated spider solution at a ratio of 1 vol of the scoop solution to 3 vol of the ethanol mixture, and the solution was allowed to stand at 4 ° C. Upon precipitation, a precipitate formed. The ethanol precipitate was separated by filtration, dried in a vacuum desiccator, and redissolved by adding water to the dried product. When the anti-HIV activity of this ethanol precipitation solution was evaluated, the anti-HIV activity was found.

Similarly, the methanol-extracted residue and water extract obtained by extracting the water-soluble components remaining in the residue by reflux extraction with methanol after reflux extraction for 4 hours with methanol are also the same. When anti-HIV activity was evaluated, anti-HIV activity was found. In other words, it is thought that the components extracted in water after removing methanol-soluble and water-soluble components by methanol extraction in advance correspond to the active components contained in the “heated spider solution”. .

The evaluation of the anti-HIV activity was based on the measurement of the addition ratio (EC ₅ ) that inhibited cell degeneration by 50% by using the MTT assay method. Separately, the total component contained in each test aqueous solution was measured as a dry weight, and the EC ₅ was determined. Represents the weight of the dry matter (dry matter weight / ml unit) added per unit volume of the medium. Also measured simultaneously, the addition ratio to inhibit cell growth by 50% (50% cytotoxic个生concentration:. CC ₅₎ for were also expressed in (dry matter weight Zm l units). Table 3 below shows the evaluation results of anti-HIV activity. Table 3

Therefore, the following procedure can be used to purify the active ingredients involved in the anti-HIV activity exhibited by the “squid solution”.

(i) First, the water-soluble components having a molecular weight of less than 10,000 are removed by fractionation using an ultrafiltration membrane, and the water-soluble components having a molecular weight of 10,000 or more remaining on the ultrafiltration membrane are separated into water. Redissolve in

(ii) To this re-dissolved aqueous solution, for example, an ethanol mixture containing 5% sodium acetate Z2.5 M acetic acid is mixed at a ratio of 1 volume of the re-dissolved aqueous solution: 3 volumes of the ethanol mixed solution, and After standing at 4 ° C., the active ingredient is reprecipitated and separated and recovered. Separation • The collected re-precipitate of the active ingredient is separated by filtration, dried in a vacuum desiccator, and stored.

When the active ingredient thus purified and dried is redissolved in water, its anti-HIV activity per unit dry weight is compared with the anti-HIV activity per unit dry weight in the original "drip solution". It will be much higher.

Karoete the Etanoru precipitation was redissolved further 0. 5N HC 1 subjected to treatment with an aqueous solution, the resulting precipitate separated 'recovered, 0. IN NaHC_〇 ₃ aqueous solution or, 0. 5N N a OH solution Further purification can be carried out by adding and re-dissolving the mixture. Industrial applicability

The indigo preparation according to the present invention, in particular, the water extract of "Sukumo" derived from Polygonum tinctorium Aiton belonging to the Polygonaceae family has anti-HIV virus activity, and particularly, non-infected cells of HIV virus. To stop the invasion process of HIV, to prevent the propagation of the HIV virus in cells after infection, And has almost completely suppressed the different processes involved in the spread of infections in the body, and has multiple types of anti-HIV virus that have different mechanisms of action, resulting in low resistance. And its effectiveness in prevention and treatment is dramatic. In addition, the indigo preparation according to the present invention, in particular, the water extract of Sukumo, which is derived from Tatsuai, has extremely low side effects, high safety, and can be provided at low cost. It is considered to be well tolerated for periodical administration. In addition, the indigo preparation according to the present invention, in particular, the "Sukumo" extract derived from Tatsuai, itself has a plurality of types of anti-HIV virus activities with different mechanisms of action. Even when used in combination with other drugs, it is possible to reduce the number of drugs administered to patients and consequently delay the onset of side effects and the acquisition of resistance of these concomitant drugs. Thus, it seems that it can have a positive effect on current drug treatment. Including these advantages, the indigo preparation according to the present invention, in particular, the water extract of "Sukumo" derived from the indigo plant, is particularly useful in drug treatment aimed at preventing infection and preventing the onset of AIDS. Useful as an anti-HIV drug.

Claims

The scope of the claims

1. An indigo preparation derived from Polygonum tinctorium Aiton belonging to the Polygonaceae family,

2. Included in the water extract of "Sukumo" prepared in the indigo production process derived from Polygonum tinctorium Aiton belonging to the Polygonaceae plant.

A component having a molecular weight of 10,000 or more,

Stable to heat,

It is easily soluble in water and alkali, hardly soluble in acid,

A component having an anti-HIV action that is hardly soluble in organic solvents.

3. The use of an indigo preparation as one of the active ingredients in the manufacture of health foods,

Use of the indigo preparation, which is characterized in that the health food comprises the indigo preparation according to claim 1 as one of the active ingredients.

4. Use of an indigo preparation as one of the active ingredients in the manufacture of a pharmaceutical composition having an anti-HIV effect,

Use of the indigo preparation, wherein the pharmaceutical composition comprises the indigo preparation according to claim 1 as one of active ingredients having an anti-HIV action.