JPH0543477A - Human immunodeficiency virus proliferative inhibitor - Google Patents

Abstract

PURPOSE:To obtain an inhibitor free from side effects, preventing and treating acquired immunodeficiency syndrome, comprising an extract of SHIKON (root of Lithospermum erythrorhizon) with water as an active ingredient. CONSTITUTION:1 pt.wt. crude drug powder of SHIKON prepared by drying root of Lithospermum erythrorhizon of the family Borginaceae is extracted with 5-20 pts.wt. water (cold water, warm water or hot water) at 5-100 deg.C for several hours to several days, purified with a water-soluble alcohol such as methanol, dried and powdered to give an extract, which is mixed with a surfactant and an excipient to give the objective inhibitor. A dose as an AIDS preventive for male adult is 0.01-0.1g/kg/day and a dose as an AIDS remedy is 0.01-0.3 g/kg/day.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel human immunodeficiency virus (Human immunodefi-cienc).
y virus (hereinafter referred to as HIV) growth inhibitor. According to the HIV growth inhibitor of the present invention, the acquired immunodeficiency syndrome (Acquiredimmunodeficie)
The onset of ncy syndrome (hereinafter referred to as AIDS) can be prevented or AIDS can be treated.

[0002]

2. Description of the Related Art AIDS is an infectious disease caused by a virus and has a high mortality rate, so that the development of a therapeutic agent therefor is urgently needed. Several AIDS therapeutic agents and agents used for preventing AIDS development in HIV-infected persons who are the causative virus of AIDS have already been reported.
For example, azidothymidine, interferon, glycyrrhizin, dextran sulfate and the like can be mentioned. It has been reported that these have an HIV growth inhibitory effect. In particular, azidothymidine is actually used to treat AIDS patients, but it is highly toxic and AIDS in HIV-infected people is high.
There is a problem in using it for a long period of time to prevent the onset. Therefore, there is an urgent need to develop a drug having a lower toxicity and an anti-HIV effect. For this reason, the inhibitory effect on HIV growth has been investigated for synthetic chemical substances, natural substances or Chinese herbs. In particular, Kampo medicines are under study because they have few side effects due to administration. Although there are reports of clinical use of Shosaiko-to and Ninjin-to as Kampo medicines, their effectiveness has not been evaluated.

[0003]

Under the present circumstances, the present inventors have conducted a search using a large number of crude drugs in order to find a crude drug component having no side effect and an anti-HIV activity. That is, an object of the present invention is to find a galenical component which has a strong anti-HIV action and has no side effect even when administered, and provides an HIV growth inhibitor containing this as an active ingredient.

[0004]

In order to achieve the above object, the present invention uses the powder or water extract of the following 48 herbal medicine samples (water extraction sample 33, sample 15 as powder). An HIV growth suppression screening test was performed.
As a result, Sikon ( LithospermiRadi
It was found that only water extraction of x ) has a strong HIV growth inhibitory effect, and completed the present invention. That is, the present invention is based on Sikon ( Lithospermi Radix )
The present invention relates to an HIV inhibitor containing the water extract of the above as an active ingredient.

[0005] The following 48 crude drug samples were used as the crude drug samples subjected to the HIV growth suppression screen of the present inventors.
Crude drug used as a water extract; shikon, sugi, kokushi,
Bowie, Tangerine, Mokutenryo, Boufu, Saishin, Ziou, Bukuryo, Eucommia, Psycho, Bakuromontou, Touki, Thyroidis, Buttonpi, Maou, Tenmonto, Tanjin, Keihi, Epimedium, Sandpiper, Tojin, Kinginka, Oak, Kasshu, Sunyak, Sanyaku Gobaishi, sandalwood, inchinko, forsythia, solidago. Crude drug used in the experiment as a powder; succulent, sekiki, joteishi, banrankon, sow pea, yak chinin, gyokuchiku, range, jakujio, kanju, sohakuhi, fenugreek, baccat, bai, shazin.

As a result, HIV was extracted only from the water extract of Sikon.
It was found that it has an action of suppressing the growth of Shikon (purple root Lithospermi Radix ) is a member of the purple family Lithosperm ery.
throzon Sieboldet Zucca
rini) is a dried root and contains naphthoquinone derivatives such as shikonin, deoxyshikonin, anhydroshikonin, vomecitol, polysaccharides and the like, and is used as an antipyretic, antidote or external ointment.

The water extract used in the present invention is 5 parts by weight of water (cold water, hot water or hot water) to 1 part by weight of powdered Chinese herb medicine.
20 parts by weight is used, and the extract obtained by conducting extraction at 5 to 100 ° C. for several hours to several days is used. Even if a water-soluble solvent such as methanol or ethanol or a mixture thereof with water or a fat-soluble solvent such as hexane is used for this extraction, HI
The V growth inhibitory effect could not be obtained.

The obtained water extract may be powdered by subjecting it to a drying means such as freeze drying or spray drying.
Moreover, you may refine and use a water extract. Examples of the purification means include a method in which a water-soluble alcohol such as methanol is added to a water extract to remove insoluble matter such as a precipitate, and the residual liquid is dried to obtain a powder. In the present invention, such extract and dry powder are referred to as extract.

The extract thus obtained was subjected to the HIV growth inhibition screening test MT-4 described below.
When HIV growth inhibition test or giant cell formation inhibition test in cells was performed,
There was no growth inhibitory effect, and only this shikon extract showed an HIV growth inhibitory effect.

In the present invention, the shikon water extract can be administered as an oral preparation, a percutaneous absorption preparation, a nasal preparation or an injection preparation. Oral preparations are powders, granules, tablets, capsules prepared by mixing the shikon water extract with surfactants, excipients, lubricants, adjuvants, and other substances commonly known as formulation ingredients, and molding the mixture. It can be used as a drink, etc. In addition, a percutaneous absorption agent and a nasal agent can also be mixed with a generally known component for preparing these preparations to prepare a percutaneous absorption agent or a nasal agent by a usual method. Furthermore, the injection can be used as subcutaneous, intramuscular injection, intra-arterial injection, intravenous injection, etc. by dissolving the shikon extract in distilled water or physiological saline and filling the ampoule with this.

[0011] The dose, the purpose of use, symptoms of the patient, sex,
Although it depends on the age etc., when it is aimed at the prevention of AIDS, it is an adult male and a daily dry weight of 0.01-0.1 g /
Weight Kg, 0.01 ~ for the purpose of treatment
It is advisable to administer 0.3 g / Kg body weight in 2 to 3 divided doses per day. Sikon has long been used as a crude drug for a long time, and its toxicity, especially acute toxicity has not been reported until now, and acute toxicity does not exist. Further, Sicon has been conventionally used as an antidote, an antipyretic agent, or an external ointment,
In the present invention, a water extract of shikon is used, and as a result, a large dose is applied, and thus the application range is different from that of the conventional antidote and antipyretic and the ointment for external use.

[0012]

EXAMPLES Next, the present invention will be specifically described with reference to examples. In the examples of the present invention, crude drugs other than the shikon used in the test are also shown for comparison.

[Example 1] Preparation of water extract [0016] Silicone and other crude drug 3 as described above which were used in other experiments
Extraction was performed on the three samples by the following method. Other crude drugs were used as powder. (1) 200 ml of water was added to 25 g of cold-extracted crude drug powder, shaken, left at 5 ° C. for 2 days so as not to rot, strained with gauze, and then the filtrate was centrifuged, and then the supernatant was freeze-dried to obtain a sample. did. (2) Add 500 ml of water to 50 g of hot water extract crude drug powder, shake, heat for about 2 hours on low heat, strain with gauze,
The filtrate was concentrated under reduced pressure at 40 to 50 ° C. until it became approximately 100 ml. This was added to 10 volumes of methanol, the resulting precipitate was centrifuged, and methanol was distilled off under reduced pressure at 70 to 80 ° C. 50 ml of water was added to the residue, and the mixture was heated under reduced pressure for about 10 minutes to dissolve it, followed by centrifugation to collect a supernatant. A small amount of water was added to the residue, and the supernatants obtained by the same operation were combined and freeze-dried to obtain a sample. In addition, the hot water extract of crude drug Sikon was represented by A-1, and the cold water extract was represented by A-2.

Table 1 shows the extraction rate of the crude drug used.

[Table 1]

Example 2 HIV Proliferation Inhibition Test A. As the HIV growth inhibitory effect, the following (1) HIV growth inhibition screening test, (2) HIV growth inhibition test in MT-4 cells, and (3) Giant cell formation inhibition test were performed. (1) HIV Proliferation Inhibition Screening Test An experiment was conducted by the following method as a screening test for examining the anti-HIV proliferation suppression effect of a drug. Adult T-cell leukemia virus (HTLV-I, ATLV)
HIV to MT-4 cells, which is a cell line persistently infected with HIV.
It has been reported that HIV-4 rapidly proliferates and MT-4 cells die in 4 to 6 days when infected with E. coli.
Utilizing this phenomenon, the HIV growth inhibitory action of crude drugs was measured. HIV-1 (LAV-1 strain) 0.001 TCID
MT-4 cells were infected for 1 hour at a ratio of ₅₀ / cell (virus infectivity: 1 infectious HIV-1 particle was infected per 1000 cells) for 1 hour, and then the culture medium described below was used. Washed once. This HIV-1 infected MT-4
RPMI-104 containing a crude drug extract obtained by serially diluting cells
0 medium (10% fetal bovine serum, 100 U penicillin /
ml and streptomycin (including 100 μg / ml) at a concentration of 1 × 10 ⁵ cells / ml, and the plate was used for 5 days in a 96-well flat-bottomed culture plate at 200 μ / well.
Each well was cultured in an amount of 1 l. After culturing, HI by microscopic examination
CPE (Cylopa) of MT-4 cells by V-1 infection
The presence or absence of a thic effect; cytopathic effect) was observed.

(2) HIV Proliferation Inhibition Test in MT-4 Cells In the screening test, the following tests were carried out for substances that showed inhibition of CPE at concentrations that did not show cytotoxicity. HIV-1 (LAV-1 strain) 0.00
The cells were infected with 1 TICD ₅₀ / cell for 1 hour and then washed. The HIV-1 infected MT-4 cells and the non-infected MT-4 cells were suspended at various concentrations for 24 days for 5 days.
The culture was carried out in a flat-bottomed culture plate with holes in an amount of 1 ml per well. After culturing, the number of viable cells was counted by the trypan blue staining method.

(3) Giant cell formation inhibition test Non-infected cells bind around HIV-infected cells to form giant cells. By utilizing this phenomenon, HIV envelope protein of drug and Helper-T cell are used. HIV on the surface
The ability to block binding to the receptor (CD4 molecule) can be measured. Molt-4 cells and HIV-1
Molt-4 persistently infected with (HTLV-III strain)
The cells were mixed at a ratio of 1: 1 (cell concentration 5 × 10 ⁵ ce).
(11s / ml), and various concentrations of drugs were cultured in a 24-well flat bottom culture plate for 22 hours. The presence or absence of formed giant cells was observed microscopically.

B. Test Results (1) HIV Growth Suppression Screening Test When 32 kinds of crude drug extracts and 15 kinds of crude drug powders shown in Table 1 were subjected to an anti-HIV effect screening test, two kinds of A-1 and A-2 were obtained. , CPE was suppressed.

(2) HIV Proliferation Inhibition Test in MT-4 Cells Results of HIV proliferation inhibition test in MT-4 cells of A-1 and A-2 are shown in FIGS. 1 and 2. For A-1, in FIG. 1, when the herbal medicine extract is 0 μg / ml, HI
In V-1 non-infected cells, MT-4 cells proliferate up to 9 × 10 ⁵ cells / ml, but in MT-1 cells infected with HIV-1, the proliferation is suppressed by HIV-1 infection, The viable cell concentration was 7 × 10 ⁴ cells / ml. At a concentration of 250 μg / ml or more, suppression of growth of non-infected cells was shown. HI at 125 μg / ml
The concentration of V-1 infected MT-4 cells was 77% of that of uninfected MT-4 cells, and HIV-1 growth was suppressed. Even at 250 μg / ml or more, HIV-1 growth inhibition was remarkable, but some toxicity was observed. For A-2, as shown in FIG. 2, HIV-1 at 250 μg / ml was used.
It completely suppressed the growth of. Even at 125 μg / ml,
Almost 50% inhibition was observed. In this substance,
Toxicity appeared above 500 μg / ml.

(3) Giant cell formation inhibition test As shown in Table 2, both A-1 and A-2 were 250 μg / m 2.
1 with Molt-4 cells and HIV-1 infected Molt-4
It prevented giant cell formation observed when cells were mixed and cultured.

[0021]

[Table 2] Giant cell formation inhibition test ─────────────┬─────────────── Extract concentration (μg / ml) | A-1 A-2 ──────────────┼─────────────── 1000 │ − − 500 │ − − 250 │ ± − 125 │ + + 62. 5 │ ++ ++ 0 │ ++ ++ ─────────────┴─────────────────: Giant cell formation is not observed , +: Giant cell formation was observed.

C. Discussion MT-4 cells, which is a T-cell strain persistently infected with adult T-cell leukemia virus (ATLV-1), have a small number of HIV particles (1 HIV particle per 1000 cells).
Infection spreads rapidly and almost all cells die in 4 to 6 days. Utilizing this property, a method for determining the anti-HIV effect of a drug has been developed. This experimental system is based on the adsorption of HIV to cells,
It seems to be a simple method that can comprehensively judge the effect of suppressing the process of proliferation. To date some anti-HIV
Drugs with action have been discovered by this method. this time,
We have found that herbal extracts A-1 and A-2 suppress HIV-1 proliferation in MT-4 cells. A-1 which is hot water extraction is 125 μg / ml, A which is cold water extraction
-2 suppressed HIV-1 growth almost completely at 250 μg / ml. In addition, C which is a receptor for HIV and the cell side
In giant cell formation that seems to reflect the binding with D4 molecule, both A-1 and A-2 suppressed at 250 μg / ml. From this, it is highly possible that the HIV-suppressing action of A-1 and A-2 is caused by suppressing the binding of HIV to cells. In addition, comparing the effects of A-1 and A-2, in the CPE inhibition test in MT-4 cells, A-1 has a slightly higher inhibitory effect and toxicity.
The tendency was slightly higher than that of A-2. However, they are not so different, and it is considered that the essence of pharmacological action is not so different.

Glycyrrhizin (hereinafter referred to as GL) is known as an anti-HIV agent derived from crude drugs.
Comparing the concentrations showing the action with A-1 and A-2, the action was exhibited at GL of 500 μg / ml, whereas A-2 was 125 μg / ml and A-1 was 250 μg.
/ Ml, the effect was stronger than GL. Moreover, since A is unlikely to be a single substance and is considered to be a mixture of various substances, a substance having a stronger anti-HIV action may be identified by isolation and purification of crude drug A. Is high.

Although the mechanism of the anti-HIV action of the shikon water extract was not clear from the present experiment alone, it was found to be Molt-4 cells and HIV-infected Molt-4 cells.
It suppressed the giant cell formation that was observed when -4 cells were mixed, and it is considered that this may occur because of inhibition of HIV adsorption to cells. Since the main mechanism of anti-HIV action of GL is considered to be inhibition of protein kinase C, the mechanism of action of shikon water extract is GL.
It is considered different from.

[0025] In general, crude drugs have no strong side effects and are considered to be suitable for long-term administration. From the results of this example, it is considered that the toxicity of the water extract of Sikon is low, and considering that it has an immunostimulatory effect, not only the treatment of AIDS patients but also the development of AIDS in asymptomatic HIV infected persons (virus carriers). It can also be used for prevention.

Example 3 Formulation (1) 0.6 g of freeze-dried powder obtained in Example 1 (1)
Then, 0.2 g of corn starch and 0.2 g of lactose were added and kneaded to produce granules. The granules can be administered orally.

(2) 0.5 g of the freeze-dried powder obtained in Example 1 (2) was dissolved by adding 100 ml of sterilized physiological saline, and 5 ml was filled in an ampoule and sterilized in a sealed manner to obtain an intravenous injection. ..

[Brief description of drawings]

FIG. 1 shows the HIV growth inhibitory effect of Sikon hot water extract (A-1).

FIG. 2 shows the HIV growth inhibitory effect of shikon cold water extract (A-2).

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Katsuhiro Yamazaki 1-54-98, Otori Nishimachi, Sakai City, Osaka Prefecture No. 403, Highness Otori (72) Inventor, Shinichiro Abe, 3-17-35, Shinzonan, Toda City, Saitama Prefecture Japan Mining Industry Within the corporation

Claims (3)

[Claims] 1. Sikon ( Lithospermi Ra)
A human immunodeficiency virus growth inhibitor comprising a water extract of dix ) as an active ingredient. 2. The human immunodeficiency virus growth inhibitor according to claim 1, wherein the water extract is a cold water extract. 3. The human immunodeficiency virus growth inhibitor according to claim 1, wherein the water extract is a hot water extract, and the residue produced by removing the precipitate formed by adding methanol thereto.