JPH0825893B2 - Antiviral agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a polysaccharide or protein polysaccharide collected from seaweeds, especially red algal plants, and an anti-bacterial composition containing the polysaccharide or protein polysaccharide (hereinafter abbreviated as “substance”) as an active ingredient. The present invention relates to viral agents, particularly antiretroviral agents, and inter alia anti-AIDS virus agents.

Recently AIDS (Acquired Immunodeficiency Syndrome)
Acquired immune deficiency syndrome, which is said to be a serious disease that causes death of patients, has been craving for effective therapeutic agents. 3'-azido-3'-deo, currently one of the nucleic acid derivatives as the only AIDS therapeutic agent
Only xy thymidine (AZT) is known. The cause of this disease is HIV (Human I), which is a type of retrovirus.
mmunodeficiency Virus) has been studied by adsorbing to human T ₄ lymphoid cells to start infection, and then by infecting other lymphoid cells one after another to destroy the immune system. The present inventors have already found many protein polysaccharides that act as BRM (Biological Response Modifier) on the immune system. As a result of extensive studies paying attention to the fact that the polysaccharide or protein polysaccharide that acts as a BRM has a large effect on the immune system, the substance extracted from a plant classified as a red alga in seagrass
Adsorption inhibitory activity of HIV to human-derived lymphoid cells, and HIV
The present invention was completed by discovering that it has an RTase (reverse transcriptase) inhibitory activity, which is an essential enzyme in the growth process of Escherichia coli. An example will be described in detail below.

Among the seaweeds, those which are classified as red algae and which are related to the present invention are as follows. That is, Yukio Yamada and Muneyoshi Segawa, "Primary Color Seaweed Encyclopedia" (nursery company) Showa
Issued June 1, 1969 and its "supplemented version" July 1, 1977
According to the daily publication, the seaweed Nori seaweed, which is classified in the primitive Rhodophyta, the eyes of the red seaweed, the eyes of the dark seaweed, the eyes of the delicious seaweed, the eyes of the sea urchin, the eyes of the dragonfly, the eyes of the hide-and-seek, and the seaweed glue Involved are seaweeds selected from eyes, dull eyes, and squid eyes. Particularly, the genus Amanori, the genus Astragalus, the genus Furinori, the genus Otori, and the genus Habutari are preferred. It was surprisingly found that the extracts from these seaweeds had HIV adsorption inhibitory activity and reverse transcriptase activity inhibition. Among the red algae, the present invention is the seaweed eyes, the dark green eyes, the dark eyes, the delicious eyes, the sea urchin eyes, the astringent eyes, the hide-and-seek eyes, the squirrel eyes, the dull eyes, The present invention relates to an antiviral agent containing this substance as an active ingredient, which is extracted from the dried product of seaweed selected from the order Astragalus or as it is (the drug substance).

The present invention is used for the algae, the red seaweed eyes, the red-eyed eyes, the dark-eyed eyes, the delicious eyes, the sea urchin eyes, the astringent eyes, the hide-and-seek eyes, the sewage eyes, the dull eyes, and the goose eyes. It is obtained by extracting a seaweed selected from the above with an aqueous solvent.

The aqueous solvent used in the extraction is water or a water-soluble organic solvent, a small amount of either acid, base or salt, for example 10%
It is composed of one kind or a combination of two or more kinds selected from an aqueous solution containing a certain amount or less. As the organic solvent, methanol, ethanol, isopropyl alcohol, etc. are mainly used. Examples of the acid include hydrochloric acid, sulfuric acid, acetic acid and the like. Examples of the base include ammonium, caustic soda, caustic potash, sodium carbonate and the like.

The extraction is carried out by using 5 times to 200 times the amount of the extraction liquid based on the raw material (dry basis), and usually treating at 4 ° C. to 120 ° C. for 20 minutes to 20 hours.

The purification treatment step means removing low molecular weight substances by applying one or more methods such as salting out, dialysis, ultrafiltration, reverse osmosis treatment, gel filtration, precipitation treatment with an organic solvent. It is a thing. From an engineering point of view, the ultrafiltration method, which is a membrane separation method by pressurization, and the reverse osmosis treatment method, are preferably used alone or in combination. If desired, these treatments may be carried out after the salting out step.

The salting-out agent used in the salting-out step is ammonium sulfate, sodium chloride, potassium chloride, barium carbonate, etc., but ammonium sulfate is most preferable. Further, as a post-treatment of the salting-out step, any one of dialysis, ultrafiltration, gel filtration, reverse osmosis treatment, etc., or a combination of two or more of these steps is required.

Dialysis is usually carried out using a semipermeable membrane such as a cellophane membrane or a collodion membrane. Gel filtration is performed using a column packed with dextran or polyacrylamide gel. Fillers sold under the names Sephadex and Biogel are usually used.

Both ultrafiltration and reverse osmosis are methods of fractionation using a membrane under pressure. The former is 0.5-5 Kg / cm ² , the latter is 20-
It is usually performed at 35 Kg / cm ² .

In the precipitation method using an organic solvent, it is common to use methanol, ethanol, isopropanol, acetone or the like. In addition to the above operation, an ion exchange treatment may be performed if necessary.

After completion of the above-mentioned purification operation, water is removed by spray drying, freeze-drying, etc., and then commercialized.

The treatment of this substance with an alkaline aqueous solution is more preferable because it surprisingly enhances the antiviral effect. The amount of this substance is 5 to 200 times 0.01N to 5N, preferably
0.1 to 2N alkaline aqueous solution, 40 ℃ to 250 ℃, preferably 60 ℃ to 200 ℃, most preferably 100 ℃ to 150 ℃
Treatment for 5 minutes to 2 hours, preferably 10 minutes to 1 hour. Next, the treatment liquid is neutralized and a purification treatment step is performed. The purification treatment step can be carried out by applying one or more methods such as salting out, dialysis, ultrafiltration, reverse osmosis treatment, gel filtration, precipitation treatment with an organic solvent, and the like. The conditions are the same as above. After this purification operation is completed, the product is commercialized after removing water by spray drying, freeze drying, or the like.

In addition, if this substance is further purified by chromatography using ion-exchange cellulose such as diethylaminoethyl (DEAE) -cellulose, the fraction eluted with the salt in the adsorbed substance, alkali, etc. has a strong antiviral effect. It is more preferable because a product can be obtained.

This substance shows positive or slight positive in α-naphthol sulfate reaction, indole sulfate reaction, anthuron sulfate reaction, phenol sulfate reaction and / or Lowry-Folin method, and ninhydrin reaction after hydrochloric acid hydrolysis. Elemental analysis results, carbon
It contains 20-55%, hydrogen 3-9%, and nitrogen less than 16% as main components.

 pH shows 6.0-7.5.

The sugar component of this substance contains at least glucose and xylose, and the protein component contains at least aspartic acid,
Contains glutamic acid and lysine.

Infrared absorption spectrum measured is 3600-3200cm ^-1
Absorption of a hydroxyl group in the vicinity and / or absorption derived from an amide group could be recognized in the vicinity of 1700 to 1600 cm ^-1 .

This substance is soluble in aqueous solvents and insoluble in organic solvents.
The water-based solvent is water or a water-based alcohol containing a water-soluble alcohol, acid, base or the like, and the organic solvent is chloroform, benzene, ether or the like.

The substance is white or brown and has a molecular weight of 10 ^{3 to} 3 × 10 ⁶ by gel filtration chromatography.

Rats (Gyuryu) 4 to 5 weeks old, weighing 100 to 150 g were orally administered to this substance at 1000 mg / Kg and observed for 7 days, but all of them were alive.

This substance is a safe substance with extremely low toxicity and almost no side effects.

It is generally known that a virus is replicated by being adsorbed to a target cell, injecting the nucleic acid of the virus into the cell, and further being integrated into the genome of the cell. In addition, particularly for retroviruses, a process is required in which RNA, which is a nucleic acid derived from a virus, is transcribed into DNA by the action of a reverse transcriptase, before being integrated into the cell genome. The present inventors
It was found to inhibit the adsorption of HIV to human-derived lymphoid cells and its subsequent infection, and to inhibit reverse transcriptase activity. That is, HIV is 50-1000 μg
After treatment with this substance at a concentration of / ml for 2 hours at 0 ° C, HIV was washed and added to MT-4 cells to be adsorbed and HI after 3 days of culture.
When the effect of this substance was examined by the method for measuring V antigen-positive cells, pretreatment with this substance almost eliminated HIV antigen-positive cells and confirmed a strong adsorption inhibitory effect of HIV on human-derived lymphoid cells. Was given. On the other hand, when the effect of this substance on the reverse transcriptase activity was measured using rat liver total messenger RNA as a template, this substance 500 μg /
A strong inhibition of reverse transcriptase activity was observed with the addition of ml.

These facts indicate that this substance has the effect of inhibiting the infection of viruses, conversely it inhibits the infection of retroviruses having reverse transcriptase, and in particular, that it is effective for AIDS caused by HIV infection. It is shown.

AZT, which is already used as an antiviral agent, has a side effect of inhibiting mitosis even in normal cells, but this substance has a very low acute toxicity and is a safe substance. It is useful as an antiviral agent because it has the effect of inhibiting viral infection. That is, viral infections, especially retroviral infections,
Especially effective for AIDS.

When used as an antiviral agent, this substance can be made into any dosage form. In addition, administration is also performed by each route. Furthermore, the drug of the present invention does not decrease in efficacy even in combination with the above-mentioned AZT which is used as an antiviral agent, and the combination with these other drugs can be used as an effective means.

In the case of oral administration, tablets, granules, powders, capsules and the like applied to the composition include binders, inclusion agents, excipients, lubricants, which are commonly used in their compositions.
May contain additives such as disintegrants, wetting agents,
When it is used as an oral liquid preparation, it may be in the form of an aqueous solution for internal use, a shaking mixture, a suspension, an emulsion, a syrup, or a dry product which is re-dissolved before use. Good. Further, such a liquid preparation may contain any of commonly used additives and preservatives. When injectable, the composition may contain additives such as stabilizers, buffers, preservatives, tonicity agents, and is provided in a unit-dose ampoule or a multi-dose container. . It should be noted that the composition may be in the form of an aqueous solution, suspension, solution, emulsion in an oily or aqueous vehicle, while the active ingredient is sterilized in a suitable vehicle, eg, pyrogen-free, before use. It may be a powder that is redissolved in water.

The antiviral agent of the present invention is orally or parenterally administered to humans and animals. Oral administration includes sublingual administration. Parenteral administration includes injection such as subcutaneous, intramuscular, intravenous injection,
Including drip etc. Since the dose of the antiviral agent of the present invention is affected by animals or humans, age, individual differences, medical conditions, etc., it may occur that an amount outside the following range is administered in some cases, but it is generally intended for humans. In this case, the oral dose of this substance is 1 kg of body weight, 0.1 to 1000 mg, preferably 1 to 100 mg per day in 1 to 3 divided doses.

 Examples will be shown below.

Example 1 100 g of dried pickled seaweed in the red algae, Asakusa nori (Amaranthus spp.), Put into a stainless steel tank with a capacity of 3 and added with 2000 ml of distilled water and stirred While maintaining the temperature at 90-95 ° C. After extracting for about 3 hours,
Cooled to room temperature.

The extraction slurry was separated into an extract and a residue by a centrifugal separator. Further, add 2000 ml of 0.4N-NaOH to the residue and add 90-95 ° C.
After extracting with 3 hours, cool to room temperature and p-p with 2N-HCl.
After adjusting H to 7.0, it was centrifuged to separate into an extract and a residue.

The extracts were combined, concentrated under reduced pressure, desalted by ultrafiltration, and then freeze-dried to obtain 30 g of a dried product (A).

Example 2 100 g of a dried product of agaricaceae, agaricaceae, agarella in the algae of red algae, is fragmented into a stainless steel tank having a capacity of 3, and 2000 ml of distilled water is added and the temperature is maintained with stirring. Was maintained at 90-95 ° C. After extracting for about 3 hours, it was cooled to room temperature.

The extraction slurry was separated into an extract and a residue by a centrifugal separator. Further, add 2000 ml of 0.4N-NaOH to the residue and add 90-95 ° C.
After extracting with 3 hours, cool to room temperature and p-p with 2N-HCl.
After adjusting H to 7.0, it was centrifuged to separate into an extract and a residue.

The extracts were combined, concentrated under reduced pressure, desalted by ultrafiltration, and then freeze-dried to obtain 25 g of a dried product (B).

Example 3 Hideout of red algae and 100 g of dried dried Hanafuri of the genus Furinaceae in the family Furinaceae are fragmented, put in a stainless steel tank of volume 3, and 2000 ml of distilled water is added to the mixture while stirring to adjust the temperature. Maintained at 90-95 ° C. After extracting for about 3 hours, it was cooled to room temperature.

The extraction slurry was separated into an extract and a residue by a centrifugal separator. Further, add 2000 ml of 0.4N-NaOH to the residue and add 90-95 ° C.
After extracting with 3 hours, cool to room temperature and p-p with 2N-HCl.
After adjusting H to 7.0, it was centrifuged to separate into an extract and a residue.

The extracts were combined, concentrated under reduced pressure, desalted by ultrafiltration, and then freeze-dried to obtain 27 g of a dried product (C).

Example 4 100 g of dried dried seaweed Nori seaweed Nori seaweed Nori seaweed in red algae 100 g was put into a stainless steel tank with a capacity of 3, 2000 ml of distilled water was added, and the temperature was 90- It was kept at 95 ° C. After extracting for about 3 hours, it was cooled to room temperature.

The extraction slurry was separated into an extract and a residue by a centrifugal separator. Further, add 2000 ml of 0.4N-NaOH to the residue and add 90-95 ° C.
After extracting with 3 hours, cool to room temperature and p-p with 2N-HCl.
After adjusting H to 7.0, it was centrifuged to separate into an extract and a residue.

The extracts were combined, concentrated under reduced pressure, desalted by ultrafiltration and then freeze-dried to obtain 23 g of a dried product (D).

Example 5 100 g of a dried product of Habutae paste of the genus Butaedori, which is a member of the genus Butaedori, is put into a stainless steel tank having a capacity of 3 and 2000 ml of distilled water is added. The temperature was maintained at 90-95 ° C with stirring. After extracting for about 3 hours, it was cooled to room temperature.

The extraction slurry was separated into an extract and a residue by a centrifugal separator. Further, add 2000 ml of 0.4N-NaOH to the residue and add 90-95 ° C.
After extracting with 3 hours, cool to room temperature and p-p with 2N-HCl.
After adjusting H to 7.0, it was centrifuged to separate into an extract and a residue.

The extracts were combined, concentrated under reduced pressure, desalted by ultrafiltration and then freeze-dried to obtain 22 g of a dried product (E).

Table 1 shows the physicochemical properties of substances newly extracted from various red algae. In this table, the phenol-sulfuric acid color reaction indicates the presence of saccharides and the Lowry-Folin method indicates the presence of peptide bonds. Regarding the molecular weight, the fractions which are present in average on the gel filtration method are described.

Example 6 With respect to the substances obtained in Examples 1 to 5, the degree of inhibition of reverse transcriptase specifically retained by retrovirus was measured by the following method.

All of this substance was obtained by dissolving 10 mg of lyophilized product in 10 ml of sterile distilled water (concentration: 1 mg / ml).

1 μ of 20 mM DTT (dithiothreitol: Sigma), 5 μ of 5 times concentration enzyme reaction solution (250 mM Tris-HC
l (pH8.3) -250mM KCl-40mM MgCl 2), 3d NT of 1μ
P solution (1 mM dATP-1 mM dGTP-1 mM dTTP: Sigma),
2μ of 100μg / ml oligo (dT) _12-18 (PL-biochemic
als), 1 μ of messenger RNA (from normal rat liver: 1 μg / μ), 0.5 μ of RNase Inhibitor (16 units
/ μ: Takara Shuzo Co., Ltd. and 1μ of [α- ³² P] dCTP (~ 80
0 Ci / mmol, 10 μCi / μ: Amersham Japan) 1.
Add to a 5 ml Eppendorf tube and place in a 37 ° C water bath.

After 5 minutes, 12.5μ of the substance prepared above at a concentration of 1mg / ml was added to the reaction tube, and 1μ of reverse transcriptase (7 units / μ: from Takara Shuzo, Rousassociated virus) was added, and the final reaction solution was added. The amount was adjusted to 25μ and the reaction was carried out at 37 ° C.

After 1 hour, 5 μ of the reaction solution was soaked in 2 cm × 2 cm DEAE paper (manufactured by Toyo Roshi Kaisha, Ltd.), air-dried, and 10 ml per filter paper was used.
Soak in 0.5M-Na ₂ HPO ₄ aqueous solution and shake while shaking the DNA on the filter paper.
The [α- ³² P] dCTP that was not used in the synthesis was washed (this operation was performed 5 times at intervals of 5 minutes).

Then, the DEAE paper was placed in a glass vial bottle containing 10 ml of liquid scintillation cocktail (Amersham Japan Co.) and the radioactivity (cpm) was counted for 1 minute with a scintillation counter (Aloka Co.).

The reverse transcriptase activity inhibition rate (%) was calculated by the following formula.

Co: Radioactivity without addition of this substance Cs: Radioactivity with addition of this substance Table 1 shows the reverse transcriptase (RTase) activity inhibition rate of each substance.
Shown in

Example 7 Inhibition of HIV (AIDS virus) adsorption on human lymphocytes by this substance was performed by the following method (all operations were performed under aseptic conditions).

1 ml of HIV suspension and 1 ml of this substance solution (800 μg / ml) were placed in a test tube and allowed to stand on ice. 2 hours later, 1 ml of the virus suspension was taken from the test tube and the human lymphocyte-derived cell line MT-4 was used.
[Jpn. J. Cancer Res. (Gann), 28 , 219-229 (1982)]
The virus was adsorbed to the cells at a multiplicity of infection (MOI) of approximately 2.
After centrifugation (2,000 rpm, 10 minutes), the supernatant was discarded and the MT-4 cells precipitated were added to RPMI1640 (Gibco Laborat) containing 20% FCS.
cells, NY) were suspended at a cell concentration of 2 × 10 ⁵ / ml.

The MT-4 cell suspension was dispensed into a 96-well plate by 100 μm and cultured under the conditions of 5% CO _{2 in} air and 37 ° C.
On the 3rd day of culture, HIV adsorbed cells and non-adsorbed cells were calculated by the indirect fluorescent antibody method.

 The HIV adsorption inhibition rate (%) was calculated by the following formula.

The results are shown in Table-1.

Example 8 Example 1 was applied to a No. 0 hard capsule using a pressure type automatic filling machine.
This substance was packed in an amount of 330 mg to prepare a capsule.

Front page continuation (72) Inventor Junji Kakuchi, 16-16, 31-6, Minamikarasuyama, Setagaya-ku, Tokyo (72) Inventor Kyofumi Sugita 2-10, Kasuga, Bunkyo-ku, Tokyo 13-402 (72) Inventor Takao Furuso Tokyo 1-6-13 Asahimachi, Miyakomachi City (72) Inventor Shigeaki Muto 2-23-2, 3-2, Higashihorikiri, Katsushika-ku, Tokyo (72) Inventor Chikao Yoshiku 2-19-46 East, Kunishi, Tokyo (72) Invention Masaaki Takahashi 1-5-33-314 Takanawa, Minato-ku, Tokyo

Claims (6)

[Claims] 1. An antiviral agent comprising a polysaccharide or protein polysaccharide produced from red algae in seaweeds as an active ingredient. [Claim 2] The red algae are chirimo eyes, red-eyed eyes, dark-eyed eyes, delicious-looking eyes, sea urchin eyes, aggression eyes, hide-and-seek eyes, squirrel eyes, and dull eyes. The antiviral agent according to claim 1, which is selected from the group consisting of Polygonum aeruginosa. 3. The antiviral agent according to claim 2, which is selected from the genus Amari, Genus Astragalus, Genus Nori, Genus Nori, and Habutae Nori. 4. An α-naphthol-sulfuric acid reaction, an indole-sulfuric acid reaction, anthuron-sulfuric acid reaction, a phenol-sulfuric acid reaction and / or a Lowry-Folin method, and a ninhydrin reaction after hydrolysis with hydrochloric acid are positive or slightly positive, and carbon 20 as an elemental analysis value. -55%, hydrogen 3-9%, nitrogen less than 16% as main components, pH is 6.0-7.5, contains at least glucose and xylose as sugar components, and contains at least aspartic acid, glutamic acid and lysine as protein components. It has an infrared absorption spectrum of a hydroxyl group near 3600 to 3200 cm ^-1 , and / or an infrared absorption spectrum of an amide group near 1700 to 1600 cm ^-1 , which is soluble in an aqueous solvent and organic. It is insoluble in solvent, white or brown, and the molecular weight is 10 ^{3 to} 3 × 1 by gel filtration chromatography.
The antiviral agent according to any one of claims 1 to 3, which is 0 ⁶ . 5. The antiviral agent according to claim 1, which is an antiretroviral agent. 6. The antiviral agent according to claim 1, which is an anti-AIDS virus agent.