KR100204974B1 - An anti-viral agent - Google Patents

Abstract

The present invention relates to an antiviral agent consisting of asparagus linarias alkaline extract obtained by hot water extraction as an active ingredient, and inhibits retroviruses, bacteria, fungi and other microorganisms such as adult T-cell leukemia and AIDS virus. Antiviral agents, anticancer agents, antibacterial agents, and fungicides having no action, anticancer action, and protective action can be provided.

Description

Antiviral agents

The present invention relates to an antiviral agent comprising an alkali extract of Aspalathus linearis belonging to the legumes (Leguminosae family).

Numerous antiviral and anticancer agents have been known in the art.

However, antiviral agents mainly work only on bacteria and are extremely weak, even if they have an effect on viruses. Moreover, antibiotics usually have side effects that can sometimes be very harsh and serious.

Many diseases are caused by viruses such as influenza, hepatitis, measles, Japanese encephalitis, adult T-cell leukemia, and AIDS, but unfortunately, few drugs show clear efficacy against these conventional viral diseases. There was no drug effective enough against retroviruse to cause adult T-cell leukemia or AIDS without any side effects.

In addition, there is no known antiviral agent that can be taken in a daily diet and has a prophylactic effect without any side effects.

Therefore, the study of effective treatment of AIDS and AIDS-related complexes that do not have side effects is particularly difficult, and some drugs which have limited effects through long-term chemotherapy may have serious side effects.

Since anticancer drugs known to date not only destroy cancer cells but also damage normal cells, these anticancer drugs have shown severe side effects.

In addition, since these anticancer agents were difficult to use by oral administration, they have been commonly used by intravenous injection. There have been many problems with these anticancer agents.

Therefore, anti-HIV agents and anticancer agents do not have to have side effects and also have strong efficacy. As HIV (Human Immunodeficiency virus) agents, there is a need to invent a formulation which has no side effects and has the effect of inhibiting viral adsorption on cells, inhibiting virus replication, and activating immune activity.

Accordingly, an object of the present invention is to solve the above problems, to provide an antiviral agent that is effective against retroviruses, has a prophylactic effect without side effects, and can be orally administered.

The present inventors have diligently researched to solve the above problems, and as a result, alkali extracts from the leaves, stems, and / or roots of Asparagus linenaris have strong antiviral and anticancer effects, and the alkali extracts are applied to animals or humans. The present invention was developed by discovering that it is extremely safe and has no side effects.

The active ingredient of the present invention is derived from alkali extracts of leaves, stems and / or roots of Asparagus linenaris, more preferably by extracting the leaves, stems and / or roots from hot water, that is, hot water and then with an alkaline solution. Induced.

In the hot water extraction process, the amount of water should be 5 to 1000 times the weight of asparagus linaris, and should be extracted at 70 to 100 ° C. for 5 minutes to 10 hours. After extraction with hot water, the Asparagus linarias should be dried for 1 to 3 days under the sun or indoors.

To the hot water extracts of the leaves, stems and / or roots of Asparagus linarias, 0.05-5% alkaline solution (pH8-14) should be added and stirred at 10-60 ° C for 30 minutes to 10 hours.

Specific examples of the alkaline solution include sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium bicarbonate, sodium carbonate and ammonia, and extraction methods such as hot water extraction can be used. Separation of the extraction solution can be carried out by known methods such as filtration, decantation, centrifugation and the like.

In addition to water, ethanol, acetone, or the like may be used as an extraction solvent, and the solvent residue may be lyophilized or spray-dried by a conventional method.

The active ingredient of the extract can be mixed with a suitable carrier such as excipients, binders, diluents, etc., for example, granules, powders, hard capsules, soft capsules, ointments, syrups, suppositories, injections or For parenteral use or as it is mixed, it may be used alone or in a form such as a solution, powder, granule, tablet, emulsion, jelly, or the like, or mixed with other foods.

The dosage varies depending on the type and extent of the disease, but in the case of liquid form, 1 to 1000 mg / L solution should be administered at 2 mL to 500 mL / day, and when the extract is in powder form, 1 0.2 mg to 5 mg per day should be administered.

Alkaline extracts of Asparagus linarias were tested for acute toxicity in rats and mice. There were no deaths, no abnormal behavior was observed, blood biochemical tests and histopathological experience. No outliers were seen.

The present invention is an antiviral agent, an anticancer agent, an antibacterial agent and a bactericide. According to the present invention, various viruses such as influenza virus, herpes simplex virus (simple herpes virus), rotavirus and hepatitis virus, and adult T cell leukemia or Extracts that prevent infection of retroviruses such as HIV, inhibit replication, prevent the growth or proliferation of microorganisms such as bacteria and fungi, and improve and / or treat malignancies without causing any side effects. Can be obtained.

Below. The present invention is described in more detail through various preferred embodiments.

EXAMPLE

[Production Example 1]

The leaves and stems of Asparagus linarias were cut to a length of 5 mm, and rolling, enzyme fermentation and sunlight drying were carried out to obtain dried leaves. 3 g of these dried leaves were extracted in 100 ml of warm water at 85 ° C. for 3 hours, and then the leaves and stems were dried indoors. 50 ml of 1% sodium hydroxide was added to the obtained dried leaves, shaken vigorously at 45 ° C. for 3 hours, and then filtered through a sanitary cotton (0.5 cm thick) to obtain an extract containing various acidic polysaccharides. When the obtained extract was dried and powdered, this dry powder contained 16% reducing sugar and 20% neutral sugar.

[Production Example 2]

Instead of 1% sodium hydroxide as the alkaline solution, 2% ammonium hydroxide was used, and the dry powder of the extract was obtained in the same manner as in Preparation Example 1. This dry powder contained 14% of reducing sugar and 21% of neutral sugar.

[Manufacture example 3]

The dry powder of the extract was obtained in the same manner as in Preparation Example 1, using 1% sodium carbonate instead of 1% sodium hydroxide as the alkaline solution and one layer of gauze instead of sanitary cotton for filtration. This dry powder contained 18% reducing sugar and 29% neutral sugar.

[Production Example 4]

The leaves and stems of Asparagus linarias were cut to a length of 5 mm, and rolling, enzyme fermentation and sunlight drying were performed to obtain dried leaves. 50 g of 1% sodium hydroxide was added to 3 g of the dried leaves, which was vigorously shaken at 45 ° C. for 3 hours, and then filtered through a sanitary cotton (0.5 cm thick) to obtain an extract solution containing various acidic polysaccharides. When the obtained extraction solution was dried and made into a dry powder, this dry powder contained 22% reducing sugar and 37% neutral sugar.

Production Example 5

The leaves and stems of Asparagus linarias were cut to a length of 5 mm, and rolling, enzyme fermentation and sunlight drying were performed to obtain dried leaves. 3.5 g of the dried leaves obtained were extracted in 500 ml of warm water for 3 hours, and then the leaves with stems were dried indoors. The dried leaves were shaken vigorously for 3 hours at 45 ° C. in 50 ml of 1% potassium hydroxide and filtered through two layers of gauze to obtain an extract containing acidic polysaccharides. The obtained extraction solution was lyophilized, and the obtained dry powder contained 22% reducing sugar, 30% neutral sugar and 17% uronic acid.

[Manufacture example 6]

The leaves and stems of Asparagus linarias were cut to a length of 5 mm, and rolling, enzyme fermentation and sunlight drying were performed to obtain dried leaves. 3 g of the dried leaves obtained were extracted in 100 ml of 85 ° C. hot water for 3 hours, and then the leaves with the stems were dried indoors. The dried leaves were shaken vigorously for 3 hours at 45 ° C. in 50 ml of sodium hydroxide, and filtered through a sanitary cotton (0.5 cm thick) to obtain an extract. The extract was freeze-dried, and the dried powder obtained was used as a crude extract of Asparagus linarias. The crude extract was dissolved in distilled water and then ethanol was added, and the precipitated material was centrifuged at 300 rpm for 20 minutes between 25 to 75% ethanol concentration to obtain purified acidic polysaccharide. The dry powder of acidic polysaccharide contained 22.5% of uronic acid, 50.5% of neutral sugar, and 26.5% of reducing sugar.

[Manufacture example 7]

Dry powder of the extract (prepared extract) was obtained in the same manner as in Preparation Example 6. The crude extract was dissolved in distilled water, ethanol at 25 to 75% concentration was added, and the precipitated material was centrifuged at 3000 rpm for 20 minutes, and the precipitate obtained was dried and referred to as purified acidic polysaccharide. The dry powder of this acidic polysaccharide contained 22% of uronic acid, 49% of neutral sugar, and 25% of reducing sugar.

Experimental Example 1

The dry powder obtained in Preparation Example 1 was applied to cultured MDCK cells or MA104 cells. Immediately after application, MDCK was infected with influenza virus, MA104 was infected with herpes simplex virus, and spontaneous incubation with dry powder showed flaky formation. As a result, the influenza virus was inhibited completely (100%) by 0.1 mg / ml, and in the case of herpes virus flag formation, 90% or more was suppressed by 1 µg / ml of the dry powder (the extract).

Experimental Example 2

Microtiter plate with 96 wells containing MT-4 cells (2.5 × 10 ⁴ cells / well) with HIV-1 and immediately after infection with infected cells at various concentrations of the purified extract obtained in Preparation Example 1 Was incubated for 5 days in a 37C C ₂ incubator. The number of viable cells was measured by MTT method. Anti-HIV activity was expressed by the concentration (EC ₅₀ : 50% effective concentration) which shows 50% protection against the cell destruction by HIV by a sample. EC ₅₀ was 21 μg / ml or less.

Experimental Example 3

Immediately after MT-4 cells (2.5 × 10 ⁴ cells / well) were infected with HIV-1, the infected cells were pulsed onto a microtiter plate having 96 wells containing the purified extract obtained in Preparation Example 2 at various concentrations. And incubated in a CO ₂ incubator at 37 ° C. for 5 days. The number of viable cells was measured by MTT method. Anti-HIV activity was expressed by the concentration (EC ₅₀ : 50% effective concentration) which shows 50% protection against the cell destruction by HIV by a sample. EC ₅₀ was 48 µg / ml or less.

Experimental Example 4

Microtiter plate with 96 wells containing MT-4 cells (2.5 × 10 ⁴ cells / well) with HIV-1 and immediately after infection, with infected cells at various concentrations of the purified extract obtained in Preparation Example 3. Was incubated for 5 days in a 37C C ₂ incubator. The number of surviving trichotomy was measured by MTT method. Anti-HIY activity was expressed by the concentration (EC ₅₀ : 50% effective concentration) which shows 50% protection against the cell destruction by HIV by a sample. EC ₅₀ was 21 μg / ml or less.

Experimental Example 5

The extract obtained in Preparation Example 4 was applied to cultured MDCK cells or MA104 cells. MDCK cells were infected with influenza virus, MA104 cells were infected with herpes simplex virus, and then spontaneously cultured by dry powder, and flack formation was observed. Flag formation by influenza virus was almost 100% inhibited by 0.15 mg / ml, and in the case of herpes simplex virus, flag formation was inhibited by 90% or more by the extract 3 µg / ml.

Experimental Example 6

Microtiter plate with 96 wells containing MT-4 cells (2.5 × 10 ⁴ cells / well) with HIV-1 and immediately after infection with infected cells at various concentrations of the purified extract obtained in Preparation Example 6 Was incubated for 5 days in a 37C C ₂ incubator. The number of viable cells was measured by MTT method. The anti-HIV activity of the acidic polysaccharide was expressed as a concentration (EC ₅₀ : 50% effective concentration) showing 50% protection against cell-induced cell destruction caused by HIV. EC ₅₀ of the acidic polysaccharide was 15 µg / ml.

Experimental Example 7

NUGC-4 (1 × 10 ⁶ cells / ml) extracted from gastric cancer was subcutaneously injected into 20 rats.

From the next day, as an experimental group, 10 of these mice were injected daily with 1000 mg / ml of the extract obtained in Preparation Example 1 (i.p.).

Mice not administered the extract were used as controls. Cell growth was calculated for three weeks after administration of the extract, while the number of cells was significantly increased in the control group, whereas the increase in tumor cells was significantly suppressed in the experimental group.

Experimental Example 8

NUGC-4 (1 × 10 ⁶ cells / ml) extracted from gastric cancer was subcutaneously injected into 20 rats. From the next day, as an experimental group, 10 of these mice were injected daily with the 500 µg / ml of the extract obtained in Preparation Example 5 (ip). Mice not administered the extract were used as controls. When the growth of the cells was calculated for one month after administration of the extract, the number of cells in the control group increased markedly, the area of necrosis was somewhat present, and some animals died, but in the experimental group, the number of cells was significantly increased. No animals died under control.

Experimental Example 9

NUGC-4 (1 × 10 ⁶ cells / ml) extracted from gastric cancer was loaded into a microtiter plate containing 96 wells containing various concentrations of 75-subtraction obtained in Preparation Example 7, and CO ₂ at 37 ° C. The cells were incubated for 5 days in an incubator. The number of viable cells was measured by MTT method.

The anticancer activity of the acidic polysaccharide was expressed as a concentration (IC ₅₀ : 50% effective concentration) that inhibits the growth of cancer cells by 50%. The IC ₅₀ of the 75-fraction was 200 μg / ml, but the purified acidic polysaccharides (25-75% sedimentation fraction) did not inhibit tumor cell growth.

Claims (6)

An antiviral agent, comprising an alkali extract of Asparagus linarias as an active ingredient. The antiviral agent according to claim 1, wherein the alkaline extract extracted after hot water extraction of the leaves, stems, or roots of Asparagus linereas is used as an active ingredient. The antiviral agent according to claim 1 or 2, wherein the virus is human immunodeficiency virus (HIV). The method of claim 1 or 2, wherein the alkaline solution is sodium carbonate (NaCO ₃ ), sodium bicarbonate (NaHCO ₃ ), potassium hydroxide (KOH), sodium hydroxide (NaOH), ammonium hydroxide (NH ₄ OH), ammonia (NH ₃ Antiviral agent, characterized in that one of). A method for producing an antiviral agent, characterized in that the alkaline solution is further added to the hot water extract of the leaves, stems or roots of Asparagus linarias, and the extract is separated by filtration. The method of claim 5 wherein the alkaline solution is one of sodium carbonate (NaCO ₃ ), sodium bicarbonate (NaHCO ₃ ), potassium hydroxide (KOH), sodium hydroxide (NaOH), ammonium hydroxide (NH ₄ OH), ammonia (NH ₃ ) An antiviral agent, characterized in that.