JPH0825892B2 - Antiviral agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a polysaccharide or protein polysaccharide collected from seaweeds, especially brown algae, and an antiviral agent containing the polysaccharide or protein polysaccharide (hereinafter abbreviated as “substance”) as an active ingredient. , Especially antiretroviral agents, and especially anti-AIDS viral agents.

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 intensive studies focusing on 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 plants classified as brown algae among seaweeds
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 enzyme essential for the growth process of Escherichia coli. An example will be described in detail below.

Seaweed is roughly classified into brown algae, red algae, and green algae.
Although it can be divided into two, the ones involved in the present invention belong to brown algae. In other words, brown algae are written by Yukio Yamada and Sokichi Segawa in "Primary Color Seaweed Encyclopedia" (nursing company) issued on June 1, 1972 and its "enlarged version" issued on July 1, 1977, Shiomi. Thick eyes, black eyes, whip eyes, red eyes, long-eyed eyes, eager eyes, rush eyes, frightening eyes, squid eyes, hump eyes,
The genus Mozuku, the genus Tororokonbu, the genus Konbu, the genus Wakame, and the genus Hijiki are preferred, although they can be further divided into the genus Hiba. It was surprisingly found that the extracts from these seaweeds have HIV adsorption inhibitory activity and reverse transcriptase inhibitory activity. Among the brown algae, the present invention relates to the green-eye, the black-eye, the whip-eye, the red-eye, the long-eyed eye, the eye-catching eye, the light-eye eye, the red-eye eye, the eye-eye. The present invention relates to an antiviral agent containing the present substance as an active ingredient, which is extracted from the dried product of seaweed selected from the order of Kokume, Kumome, Himabatama or as it is (the drug substance).

This substance is used in the brown algae plant
Whip eyes, Amajigusa eyes, Nagamatsu eyes, Kayashi eyes,
It can be obtained by extracting a seaweed selected from Urushigusa, Hadamadoki, Uiimomo, Konbu, and Hiba-mata 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 and the adsorbed substance is eluted with salt, alkali, etc., a strong antiviral effect can be obtained. Therefore, it is more preferable.

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 glucuronic acid, and the protein component contains at least aspartic acid, glutamic acid, and glycine.

Infrared absorption spectrum measured is 3600-3200cm ^-1
Absorption of a hydroxyl group and / or absorption derived from an amide group could be observed near 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 an effect of inhibiting viral infection, in particular, inhibiting retroviral infection with reverse transcriptase, and is particularly effective against AIDS caused by HIV infection. It is a thing.

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 by exhibiting 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 a viral drug, and 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 a dried product of mozuku, which is selected from the group belonging to the moth genus of the algae, moth, and moth, among the brown algae, is shredded into a stainless steel tank having a capacity of 3, and 2000 ml of water is added and stirred. The temperature was kept between 90 ° C and 95 ° C. After extracting for about 3 hours,
Cooled to room temperature.

The extraction slurry is separated into an extract and a residue by a centrifuge. 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 to 400 ml with a vacuum concentration device, desalted with an ultrafiltration device, and then freeze-dried to obtain 21 g of a dried product (A).

Example 2 100 g of a dried product of Tororokonbu, which is selected from the member of the family Kombuchin and the genus Rorokonbu of brown algae, is fragmented and placed in a stainless steel tank having a capacity of 3, and 2000 ml of water is added to the temperature while stirring. Was maintained at 90 ° C to 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 to 400 ml by a vacuum concentration device, desalted using an ultrafiltration device, and then dried by freeze-drying to obtain 23 g of a dried product (B).

Example 3 100 g of dried dried kelp of Konbu family selected from the genus Konbu of the family Konbu family of brown algae was fragmented and placed in a stainless steel tank having a capacity of 3, and 2000 ml of water was added and the temperature was adjusted to 90 while stirring. Keep 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 to 400 ml with a vacuum concentration device, desalted with an ultrafiltration device, and then freeze-dried to obtain 20 g of a dried product (C).

Example 4 100 g of dried product of seaweed selected from the genus Wakame in the family Kombu family of brown algae is shredded into a stainless steel tank having a capacity of 3, and 2000 ml of water is added and the temperature is kept at 90 while stirring. Keep 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 to 400 ml with a vacuum concentration device, desalted with an ultrafiltration device, and then freeze-dried to obtain 18 g of a dried product (D).

Example 5 100 g of a dried product of hijiki selected from the hijimata in the brown algae, Hondawara family, hijiki genus, was fragmented, put in a stainless steel tank of volume 3, and 2000 ml of water was added and the temperature was raised with stirring. Maintained at 90-95 ° C. After extracting for about 3 hours,
Cooled to room temperature.

The hijiki extract slurry was separated into an extract and a residue by a centrifuge.

The extract was concentrated to 400 ml by a vacuum concentration device and dried by freeze-drying to obtain 20 g of dried product (E-1).

Example 6 100 g of dried hijiki was fragmented, put in a stainless steel tank having a capacity of 3, 2000 ml of water was added, and the temperature was maintained at 90 to 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 to 400 ml by a vacuum concentration device, desalted using an ultrafiltration device, and then dried by freeze-drying to obtain 25 g of a dried product (E-2).

Example 7 20 g of the hot extract of Hijiki obtained in Example 5 was treated with 1N-NaOH.
It was dissolved in an aqueous solution and heat-treated at 120 ° C. for 20 minutes.
Then, after cooling to room temperature, the treatment liquid was adjusted to pH with 2N-HCl.
Was adjusted to 7.0, desalted using an ultrafiltration device, and dried by freeze-drying to give 19 g of the alkali-treated product (E
-3) was obtained.

Table 1 shows the physicochemical properties of substances newly extracted from various brown 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 8 With respect to the substances obtained in Examples 1 to 7, 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 messenger RNA (derived from normal rat liver: 1 μg / μ), 0.5 μ RNase Inhitor (16 unit /
μ: Takara Shuzo Co., Ltd. and 1μ of [α- ³² P] dCTP (up to 800)
Ci / mmol, 10 μCi / μ: Amersham Japan Co.) 1.5
In addition to an Eppendorf tube of ml capacity, it was placed 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

As representatives of brown algae, mozuku extract (A), grated yam extract (B), kumbu extract (C), wakame extract (D), hijiki extract (E-1, E-2, E- Select 3),
The reverse transcriptase activity inhibition rate of each substance is shown in Table 1.

Example 9 Inhibition of HIV (AIDS virus) adsorption on human lymphocytes by this substance was carried out 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 and 37 ° C. Culture 3
On the day, 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 10 Example 1 was applied to No. 0 hard capsules using a pressure type automatic filling machine.
This substance was packed in an amount of 330 mg to prepare a capsule.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kunitaka Hirose 3-26-1, Hyakunin-cho, Shinjuku-ku, Tokyo (72) Inventor Junji Kakuchi 6-16-31-406 (72) Inventor, Minamikarasuyama, Setagaya-ku, Tokyo Sugita Kyobun, Bunkyo-ku, Tokyo, Kasuga 2 10-13, 402 (72) Inventor Takao Furuso 1-6-13 Asahimachi, Machida-shi, Tokyo (72) Inventor Chikao Yoshikumi 2-19-46, Higashi, Kunitachi, Tokyo ( 72) Inventor Masaaki Takahashi 1-5-33-314 Takanawa, Minato-ku, Tokyo

Claims (7)

[Claims] 1. An antiviral agent comprising a polysaccharide or protein polysaccharide produced from brown algae in seaweeds as an active ingredient. 2. The brown algae are those of the green eyes, the black eyes,
Whip also eyes, Ajijigusa eyes, Nagamatsu eye, eyelid eyes,
The antiviral agent according to claim 1, which is selected from Urushigusa eyes, Hadamadoki eyes, Ukihamo eyes, Kumbu eyes, and Hibama eyes. 3. In the eyes of the brown algae, all the seaweeds belonging to the families of Nagamatsu and Mozuku, Kumbu-kombu, Hibamata-Hondawara are selected in the eye.
The antiviral agent according to the item. 4. The antiviral agent according to claim 3, which is selected from the genus Mozuku, the genus Tororokonbu, the genus Konbu, the genus Wakame, and the genus Hijiki. 5. 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 ninhydroline reaction after hydrolysis with hydrochloric acid are positive or slightly positive, and the elemental analysis value is carbon 20. -55%, hydrogen 3-9%, nitrogen less than 16% as main components, pH 6.0-7.5, sugar components at least glucose and glucuronic acid, protein components at least aspartic acid, glutamic acid, glycine Containing an infrared absorption spectrum of a hydroxyl group near 3600 to 3200 cm ^-1 , and an infrared absorption spectrum of an amide group near 1700 to 1600 cm ^-1 , which is soluble in an aqueous solvent and an organic solvent. It is insoluble in water and has a white or brown molecular weight of 10 ^{3 to} 3 × 10 ⁶ by gel filtration chromatography.
The antiviral agent according to any one of claims 1 to 4, which is 6. The antiviral agent according to claim 1, which is an antiretroviral agent. 7. The antiviral agent according to claim 1, which is an anti-AIDS virus agent.