KR20000022593A - Anti-viral pharmaceutical and food for treatment and prevention of influenza infection - Google Patents

Abstract

PURPOSE: An anti-virus medicament and a functional food containing extract of chaenomelis fructus, an unripe fruit of citrum junos sieb., a zizyphi fructus and magnolia bark are provided, which has no cellular toxicity but the excellent efficacious of treatment and prevention against influenza virus A. CONSTITUTION: An extract is prepared by extracting, filtering, isolating, concentrating and freeze drying of the mixed medicaments of chaenomelis fructus, an unripe fruit of citrum junos sieb., a zizyphi fructus and magnolia bark using extracting solvent. The extracting solvent can be distilled water or methanols: 10-40 liter of distilled water is added to 1 kg of mixed materials of chaenomelis fructus, an unripe fruit of citrum junos sieb., a zizyphi fructus and magnolia bark, then the solution is boiled for 2-4 hrs, filtered by using gause, centrifuged at 8000xg for 10-20 min, filtered again, concentrated and freeze dried; or 5-20 liter of methanol is added to 1 kg of mixed materials of the chaenomelis fructus, an unripe fruit of citrum junos sieb., a zizyphi fructus and magnolia bark, then the solution is deposited in the chamber for 15-20 hrs to be extracted, centrifuged at 8000xg for 10-20 min, filtered again, concentrated and freeze dried.

Description

ANTI-VIRAL PHARMACEUTICAL AND FOOD FOR TREATMENT AND PREVENTION OF INFLUENZA INFECTION}

The present invention has a very good inhibitory activity against influenza virus type A, and relates to antiviral drugs and functional foods excellent in the treatment and prevention of colds.

Dextran, which is currently chemically synthesized and used as an influenza virus type A inhibitor, exhibits anti-influenza virus activity, but has cytotoxicity against erythrocytes, which is detrimental to humans.

Meanwhile, herbal extracts are used to treat influenza. For example, moths are effective in asthma, gold and silver coins lower the heat, and ginger tends to soften the inside and cool the sweat, such as herbal extract formulations using extracts of these combinations Produces.

The inventors of the present invention selected primary herbal influenza virus A to find new herbal preparations that have superior inhibitory activity against influenza virus type A and do not exhibit cytotoxicity, as compared to the conventional two-way and herbal cold medicines. After the in vitro activity inhibition and cytotoxicity test on the type, the extracts of the newly combined complex drug were prepared based on the Military Sinjwasa and Chiljeong theory, and the second activity and toxicity test was performed. As a result, it was found that an extracting agent which is very excellent in influenza virus type A inhibitory effect and has little cytotoxicity, thus completing the present invention.

Therefore, an object of the present invention is to provide a novel extracting agent or functional food which is excellent in the prophylactic and / or therapeutic effect of influenza virus type A and little cytotoxicity.

In order to achieve the above object, according to the present invention, there is provided an antiviral agent for the prevention and treatment of colds comprising a Chinese quince, unripe citron, jujube and pepper extract.

Chaenomelis Fructus is the ripe fruit of the Chinese quince tree and is an herbal sweetener that is effective in removing moisture, softening tendons and lowering energy. It has been used to be effective for indications such as ovulation, vomiting, diarrhea, dysentery, each, thirst.

Citrus junos sieb. Fruits are water fruit, round in shape, rugged and wrinkled on the outside. When it ripens, it becomes yellow, the fruit peel is peeled off well, it is fragrant, and the flesh is sour. Recently, it has been reported to affect immune function and anticancer activity and to have anti-inflammatory effect.

Jujube (Zizyphi Fructus) protects the rain, helps the chicks, releases the juice and evens out various weaknesses. In particular, it is known to melt the heart and lungs, stop coughing, disperse the gut, and protect the intestines. It has been used for non-hearing diarrhea, palpitations, and dry mouth and tongue.

Magnolia Bark is the bark of stems and branches of Magnolia officinalis Rehder et Wilson and Magnolia obovata Thunberg. It is recognized that the effect of warming up, lowering energy, and controlling humidity, wall, and enemy in one shot is recognized. When the stomach and chest are full and painful, it has been used for indications such as vomiting, diarrhea and dysentery.

The present inventors have found the surprising fact that the extraction agent obtained by combining and extracting these four herbal medicines shows not only excellent inhibitory activity against influenza virus type A but also little cytotoxicity.

The extraction agent according to the present invention is prepared by extracting a mixed drug of Chinese quince, unripe citron, jujube and thick gourd using an appropriate extraction solvent, followed by filtration, separation, concentration and freeze drying.

Hot water, methanol, etc. can be used as an extraction solvent.

It is preferable to use domestic herbal medicines, and in the case of citron, it is better to use unripe ones to have excellent antiviral effect.

When using hot water as the extraction solvent, add 10-40 liters of distilled water per kg of raw materials to a mixed raw material consisting of Chinese quince, unripe citron, jujube and thick pepper and boil for 2-4 hours, and then filtered with gauze and filtered at 8000xg. After centrifugation for 20 minutes, the resultant was filtered and concentrated in a concentrator, followed by freeze drying.

When methanol is used as an extraction solvent, 5-20 liters of methanol per 1 kg of raw material is added to the mixed raw material, which is extracted by dipping for 15 to 20 hours in a 50-70 ° C. water bath, followed by centrifugation at 8000xg for 10-20 minutes, and then the secondary Filtration was concentrated with a concentrator and then lyophilized to prepare the final extraction medicament.

The extraction agent prepared according to the present invention exhibits excellent influenza virus type A inhibitory activity and little toxicity, and thus can be used as a medicine for treating colds.

When used as a medicament, it is preferable to divide oral administration of the pharmaceutical extract obtained by using a total of 60 g of quince, unripe citron, jujube and pepper in equal proportions, three times a day.

In addition, it is possible to prepare a functional food for cold prevention using the extract of the Chinese quince, less ripe citron, jujube and thick gourd formulations according to the present invention.

For example, in the manufacture of functional beverages, purified water is added to the Chinese quince, unripe citron, jujube and pepper in an amount of 5-10 liters per kilogram of raw material, followed by high temperature (100-120 ° C.) and high pressure (1-2 kg / After filtering for 2 hours under m ² ), add the same amount of water to the filter residue, and filter for 2 hours again under the same conditions. While stirring the obtained filtrate well, additives commonly added to beverages, for example, glucose, maltose, saccharose, oligosaccharides, natural honey, etc. can be mixed well, sterilized and then commercialized according to the conventional beverage preparation method.

Next, the Example of this invention is described. However, these examples are provided to facilitate the understanding of the present invention, and the scope of the present invention is not limited to these examples.

Example 1

15 g of Chinese quince, unripe citron, jujube and thick gourd were put in a total of 60 g and 1200 ml of distilled water was boiled for 3 hours, followed by primary filtration using gauze. Then, the extracted solution was centrifuged at 8000xg for 15 minutes, filtered, concentrated in a concentrator and lyophilized to obtain about 5g of the drug.

Example 2

15 g of Chinese quince, unripe citron, jujube and white gourd were put in a total of 60 g of 500 ml of 80% -methanol, and then extracted by dipping in a 60 ° C. water bath for 18 hours. The extracted methanol solution was centrifuged at 8000xg for 15 minutes, filtered, concentrated in a concentrator and lyophilized to obtain about 1.8g of drug.

Example 3

Add 6 liters of purified water to 1 kg of Chinese quince, unripe citron, jujube and pepper, and add 1 liter of purified water for 2 hours under 120 ℃, 1.5kg / m ² pressure, filter, and add the same amount of water to the filter residue under the same conditions. Filtered again after 3 hours decoction. While stirring the obtained filtrate well, 50 g of natural honey was added per 1 kg of raw material, mixed well, and sterilized by passing through a sterilizer (110-120 ° C.). The sterilized beverage was injected into 120ml cans 120ml each, and then cooled and packaged to prepare a functional beverage.

Test Example 1

In order to compare the antiviral activity and cytotoxicity against each of the extracts of sweetener used in the present invention, the extracting agent according to the present invention, the dextran which is a conventional antiviral agent, and the Chinese herbal extracts which are sold at home and abroad, Haemagglutination Inhibition Test (HIT) and Haemagglutination Assay.

Each sweetener extract was obtained by extracting, separating, concentrating and freeze drying according to the method of Example 1 (extracted by using the same amount of sweetener as the total amount of the mixed drug used in the preparation of the pharmaceutical extract according to the present invention). ).

Comparative Chinese medicine extracts include Ssanghwatang ™ (Donghwa Pharm: K-1), Gal Geuntang ™ (Samyoung Pharm .: K-2), Ssanggamtang ™ (K-3), Ginseng Samultang ™ (Haedong Pharm. -4), 葛根 湯 液 カ ネ ボ ウ ™ (J-1) and ガ ゴ ナ-ル ™ (J & D: J-2), which are commercially available in Japan, were used.

Virus preparation

In this test, influenza virus [A / Taiwan / 1/86 (H ₁ N ₁ )] was distributed from the National Institute of Respiratory Viruses.

Virus propagation

In order to propagate the virus, SPF fertilized eggs (Specific pathogen free embryonated hen's eggs) were hatched for 10 days at 37 ℃ incubation period, and fertilized eggs except fertilized eggs and unfertilized eggs were used. 0.1 ml of the virus (0.08HA unit) was inoculated into the fertilized egg's allantoic fluid and the pores were closed with paraffin wax. After inoculation, the virus was propagated at 37 ° C. for 3 days, and then allowed to stand at 4 ° C. for 18 hours. The urine solution containing the virus was centrifuged at 8000xg for 10 minutes at 4 ° C., and the culture supernatant was used.

Virus titer

Hemagglutination experiments were performed to determine the titer of virus propagated in the urine. Virus culture stocks separated from the intestinal fluid were diluted 2-fold with PBS and dispensed in 0.05-well plates in 96-well plates. PBS was used to shake the red blood cell (RBC) solution diluted to 0.5% in each well by 0.05 ml, and the reaction was carried out at 25 ° C. for 60 minutes, and the aggregation titer was measured in comparison with the negative control group.

The titer of the virus propagated from three 10-day-old SPF fertilized eggs after proliferation by erythrocyte agglutination reaction showed that the titer of virus propagated in A SPF fertilized egg was 4HA unites / 0.025ml and B SPF fertilized egg. The virus propagated at showed no titer, and the titer of virus propagated at C SPF fertilized eggs was 256HA units / 0.025ml. Viruses propagated in C SPF fertilized eggs were used for haemagglutination assay and haemagglutination inhibition test.

Erythrocyte Solution Preparation

Blood was collected from the chicken's parotid vein and collected in Alsever's solution to prevent blood clotting. Centrifugation at 1800xg for 5 minutes to collect only red blood cells, suspended in phosphate buffered saline (PBS, pH7.4), and stored for 5 days at 4 ℃ haemagglutination assay and haemagglutination inhibition test (Haemagglutination Inhibition Test) : HIT).

Anti-influenza virus activity assessment: red blood cell aggregation inhibition test

Haemagglutination Inhibition Test (HIT) was performed to measure anti-influenza virus activity. Each stock sample (100 mg / ml) was diluted 2-fold step by step and dispensed in each well by 0.025 ml. The virus solution measured in the hemagglutination titer test was diluted with 4 haemagglutination units (HA units), mixed well with 0.025 ml of each well, and reacted at room temperature for 30 minutes. Subsequently, 0.05 ml of the RBC solution diluted to 0.5% was dispensed into each well, shaken gently, and reacted at 25 ° C. for 60 minutes. After the reaction, the positive control group and the negative control group were compared, and the drug extracted by the present invention was searched for the effect of inhibiting the process of adsorption of the virus and RBC.

Table 1 shows the effects of dextran, which is a conventional antiviral agent, and Chinese herbal extracts commercially available at home and abroad, and Table 2 shows the effects of the extracts of the present invention.

Inhibitory Effects of Dextran and Herbal Extracts on the Market for Influenza Virus A Conno -1 (200 mg) -2 (100) -3 (50) -4 (25) -5 (12.5) -6 (6.25) -7 (3.13) -8 (1.56) -9 (780 µg) -10 (390) -11 (195) -12 (97.5) Dextran △ △ ++ ++ + - - - - - - - Conno -1 (100 mg) -2 (50) -3 (25) -4 (12.5) -5 (6.25) -6 (3.13) -7 (1.56) -8 (780 µg) -9 (390) -10 (195) -11 (97.5) -12 (48.7) K-1 + + ++ ++ + + - - - - - - K-2 + ++ ++ + - - - - - - - - K-3 ++ ++ ++ + - - - - - - - - K-4 + + + - - - - - - - - - J-1 ++ ++ ++ ++ ++ + + - - - - - J-2 ++ ++ ++ ++ ++ + - - - - - -

In the above and the following table, Con. Is the dilution concentration of each extract, ++, +, Δ and-represent strong positive reaction, positive reaction, weak positive reaction and negative reaction, respectively, and NC is negative control.

As can be seen from the above results, dextran has very weak antiviral activity at 100 mg / ml-200 mg / ml and strong antiviral activity at 25 mg / ml-50 mg / ml. Showed weak antiviral activity. K-1, a Korean herbal extract manufactured in Korea, exhibits weak antiviral activity at concentrations of 3.13 mg / ml-6.25 mg / ml and 50 mg / ml-100 mg / ml. It showed strong antiviral activity at, K-2 showed weak antiviral activity at concentrations of 12.5 mg / ml and 100 mg / ml, and strong antiviral activity at 25 mg / ml-50 mg / ml. 3 shows strong antiviral activity between 25 mg / ml-100 mg / ml and weak antiviral activity at 12.5 mg / ml, and K-4 shows weak antiviral activity between 25 mg / ml-100 mg / ml. It was found to have. J-1, a Chinese herbal extract formulation marketed in Japan, has a strong viral activity between 6.25 mg / ml-100 mg / ml and a relatively weak antiviral activity between 1.56 mg / ml-3.13 mg / ml. , J-2, showed strong antiviral activity between 6.25 mg / ml-100 mg / ml and relatively weak antiviral activity at 3.13 mg / ml.

Inhibitory Effects of Single and Mixed Extract Agents on Influenza Virus Type A Conno -1 (100 mg) -2 (50) -3 (25) -4 (12.5) -5 (6.25) -6 (3.13) -7 (1.56) -8 (780 µg) -9 (390) -10 (195) -11 (97.5) -12N Quince ++ ++ ++ ++ ++ ++ ++ ++ ++ + - Unripe citron ++ ++ ++ ++ ++ + + + + + + Ripe citron + + + + - - - - - - - Jujube + ++ ++ + + + - - - - - Thick - - - - - - - ++ + - - Example 1 ++ ++ ++ ++ ++ ++ ++ ++ ++ + - -

Hot water extracted Chinese quince and unripe citron showed strong antiviral activity in a wide sample concentration range of 390 µg / ml-100 mg / ml and 6.25 mg / ml-100 mg / ml, respectively.

Hot water extracted jujube showed strong antiviral activity in concentration range of 25mg / ml-50mg / ml, weak antiviral activity at concentrations of 3.13mg / ml-12.5mg / ml and 100mg / ml. Was found to have antiviral activity in the concentration range of 390 µg / ml-780 µg / ml, and ripe citron showed relatively weak antiviral activity in the concentration range of 12.5 mg / ml-100 mg / ml.

The extracting agent of Example 1 prepared according to the present invention showed strong antiviral activity in the concentration range of 0.0195 mg / ml-100 mg / ml, and showed much stronger viral activity than dextran, which is a domestic and international herbal medicine extract preparation. Indicated.

Cytotoxicity Test on Erythrocytes: Erythrocyte Agglutination Test

Each stock sample (100 mg / ml) was diluted 2-fold step by step and dispensed in each well by 0.025 ml. In each well, 0.025 ml of PBS (pH 7.4) was added and mixed well. After diluting 0.5 ml of RBC solution in each well, 0.05 ml of each well was shaken gently, and the mixture was reacted at 25 ° C. for 60 minutes. After the reaction, the cytotoxicity of dextran, commercial Chinese herbal extracts, various sweetener extracts, and pharmaceutical extracts according to the present invention on red blood cells (RBCs) was compared with the negative controls.

Cytotoxicity of Dextran and Commercial Chinese Herbal Extracts against Red Blood Cells Conno -1 (200 mg) -2 (100) -3 (50) -4 (25) -5 (12.5) -6 (6.25) -7 (3.13) -8 (1.58) -9 (780 µg) -10 (390) -11 (195) -12 (97.5) Dextran + + + + - - - - - - - - Conno -1 (100 mg) -2 (50) -3 (25) -4 (12.5) -5 (6.25) -6 (3.13) -7 (1.56) -8 (780 µg) -9 (390) -10 (195) -11 (97.5) -12 (48.7) K-1 - - - - - - - - - - - - K-2 - - - - - - - - - - - - K-3 - - - - - - - - - - - - K-4 - - - - - - - - - - - - J-1 - - - - - - - - - - - - J-2 - - - - - - - - - - - -

Dextran showed cytotoxicity in the concentration range of 25mg / ml-200mg / ml, and herbal cold extracts sold in Korea and Japan showed no cytotoxicity to RBC. It was shown to be less toxic to cells.

Cytotoxicity of Erythrocytes by Various Single and Mixed Extracts Conno -1 (100 mg) -2 (50) -3 (25) -4 (12.5) -5 (6.25) -6 (3.13) -7 (1.56) -8 (780 µg) -9 (390) -10 (195) -11 (97.5) -12N Quince △ △ - - - - - - - - - - Unripe citron - - - - - - - - - - - - Ripe citron - - - - - - - - - - - - Jujube - - - - - - - - - - - - Thick + + + + + + + - - - - - Example 1 - - - - - - - - - - - -

The unripe citron and jujube showing strong viral activity in a wide concentration range showed no cytotoxicity against RBC, whereas the Chinese quince showed mild cytotoxicity in the concentration range of 50 mg / ml-100 mg / ml, while the hunch was 1.56 Cytotoxicity was shown in the mg / ml-100 mg / ml concentration interval. The extraction agent according to the present invention showed no toxicity in the cytotoxicity test for red blood cells.

Evaluation of antiviral activity using fertilized eggs in vivo

a. Virus propagation in fertilized eggs

After incubating the fertilized egg for 10 to 11 days in the incubation period, 100 μl of influenza virus at 0.5 HA concentration was inoculated into the fertilizer of fertilized egg, sealed with paraffin, incubated at 34 ° C. for 2 days, and left at 4 ° C. for 18 hours or more. . Thereafter, the intestinal fluid was collected using a syringe. This process was repeated several times to produce influenza virus adapted to fertilized eggs and concentrated using an ultracentrifuge for 25000rpm, 4 ℃, 2 hours. The concentrated virus was used to obtain EID ₅₀ .

b. Determination of the EID ₅₀ Value

Concentrated influenza virus can be infected in the fertilizer's urinary fluid and then subjected to erythrocyte agglutination test to obtain an EID ₅₀ value. Viruses concentrated in fertilized eggs cultured at 37 ° C. for 10 to 11 days were diluted 10-fold in PBS to inoculate 100 μl of the virus according to each concentration into the fertilizer's urinary fluid, and the inoculation sites were sealed with paraffin. After incubation at 34 ° C. for 2 days, the fertilized eggs were left at 4 ° C. for 18 hours or longer, and the fertilized egg urine was extracted with a syringe, and red blood cell aggregation test was performed to confirm the virus proliferation. The value of ₅₀ was calculated | required.

In the present experiment, the virus was propagated and subjected to the first hemagglutination test. As a result, 20EID ₅₀ was measured. In order to evaluate the activity of the drug, it should be more than 2 X 10 ⁶ EID _50. Therefore, the virus was propagated to the maximum and concentrated using an ultracentrifuge. The EID ₅₀ value was again determined using the fertilized egg, and the result was 2 X 10 ²² EID ₅₀ . .

c. Drug activity evaluation

11 days of fertilized egg by mixing 0.1 ml and 0.2 ml of the extraction agent (10 μg / ml, 100 μg / ml, 1000 μg / ml diluted drug) of Example 1 according to the present invention with virus (2 × 10 ⁶ EID ₅₀ ) After inoculation into the urinary tract, the hole is closed with paraffin wax. After inoculation, the virus was propagated at 37 ° C. for 2 days, and then left at 4 ° C. for 18 hours to obtain a gut. After centrifugation of virus-containing urinary fluid at 4 ° C. and 8000 × g for 10 minutes, the culture supernatant was evaluated for drug activity by hemagglutination [McLaren, LC: Haemagglutination Inhibition and Hemadsorption in Clinical Virology Manual (Specter S, Lancz) , G, eds) pp 243-249, Elsevier science publishing, New York, 1992].

As a result of evaluating drug activity in the fertilized eggs using the 2 X 10 ²² EID ₅₀ value, anti-influenza virus activity was also strong in the fertilized egg which is an animal experimental model (Table 5 and Figure 3). In Fig. 3, A is a dextran 10,000 µg / ml administration group, B is a dextran 1,000 µg / ml administration group, C is a dextran 100 µg / ml administration group, D is a dextran 10 µg / ml administration group, and E is 1 µg of dextran. / Ml administration group, F is positive control group, G is negative control group, H is the extraction drug of Example 1 10,000 ㎍ / ㎖ administration group, I is the extraction drug of Example 1 1,000 ㎍ / ㎖ administration group, J is the extraction agent of Example 1 100 µg / ml administration group, K is 10 µg / ml administration group of the extraction agent of Example 1, L is 1 µg / ml administration group of the extraction agent of Example 1, M is the positive control group and N is the negative control group.

Anti-Influenza Virus Activity in Fertilized Eggs of Extracted Drugs According to the Present Invention No.con (μg) Number of fertilized eggs that tested positive 10000 1000 100 10 One Dextran 6/6 0/6 1/6 0/6 1/6 Example 1 6/6 6/6 4/6 4/6 2/6

The extraction agent according to the present invention is about 32 times the concentration of dextran, which is a conventional chemical synthesis agent, and about 16 times the concentration of influenza virus type A compared to Korean herbal medicine cold medicine preparations of Korea and Japan. It was found to be inhibitory and not cytotoxic.

According to the present invention, a prophylactic and therapeutic effect of influenza virus type A is excellent, and unlike chemical synthetic drugs, drugs with little cytotoxicity and functional foods using the same can be obtained.

Claims (5)

An antiviral agent for the prevention and treatment of colds, comprising a Chinese quince, less ripe citron, jujube, and a pepper extract. The method of claim 1, The antiviral drug for preventing and treating colds, characterized in that the extract is obtained by adding 10-40 L of distilled water per 1 kg of raw materials of Chinese quince, unripe citron, jujube and thick gourd. The method of claim 1, The antiviral agent for the prevention and treatment of colds, characterized in that the extract is obtained by adding 5-20 liters of methanol per kilogram of raw materials of Chinese quince, unripe citron, jujube and peppermint and immersion extraction in a water bath at 50-70 ℃. Functional food for the prevention of colds, including Chinese quince, unripe citron, jujube and pepper extract. The method of claim 4, wherein The functional food is a beverage, The beverage is added to 5-10 L of purified water per kg of raw materials of Chinese quince, less ripe citron, jujube and thick gourd, and separating the primary filtrate for 2-3 hours under high temperature and high pressure; Separating the secondary filtrate obtained by diluting the filtered residue with the same amount of purified water for 2-3 hours under the same conditions; Functional food for the prevention of cold, characterized in that it is prepared by the step of adding a beverage additive to the mixed solution of the primary and secondary filtrate and sterilization.