KR19990085819A - Antiviral drugs and functional foods with therapeutic and preventive functions of the cold - Google Patents

Abstract

The present invention relates to antiviral medicines and functional foods for preventing and treating colds, which comprises extracts of nevus, cinnabar, gilt eum, peony, and astragalus.

According to the present invention, medicines and functional foods excellent in the prevention and / or therapeutic effect of influenza virus A type and having little cytotoxicity can be obtained.

Description

Antiviral drugs and functional foods with therapeutic and preventive functions of the cold

The present invention relates to an antiviral agent and a functional food which are excellent in the treatment and prevention of a cold because they have a very excellent inhibitory activity against influenza virus A type.

Amantadine hydrochloride, which is currently chemically synthesized and used as an influenza virus A-type inhibitor, exhibits anti-influenza virus activity, but has a disadvantage that it is harmful to human body because it has cytotoxicity against erythrocytes.

On the other hand, the herbal medicine extract is used to treat influenza. For example, napposers have effects such as asthma, gingival herb, fever, and ginger, making the cheeks cool and sweating. Therefore, by using the herbal extract combination, the herbal extract preparation .

The inventors of the present invention selected 100 kinds of herbal medicine raw materials in order to find out a new herbal preparation which has excellent inhibitory activity against influenza virus A and does not show cytotoxicity compared with the conventional herbal and oriental herbal medicine, and the first influenza virus A In vitro activity inhibition and cytotoxicity tests were carried out on the antimicrobials, and then the antimicrobial agents were tested for their secondary activity and toxicity. As a result, the present inventors have found an extraction agent having not only excellent effect of inhibiting influenza virus A form, but also little cytotoxicity, thereby completing the present invention.

Therefore, it is an object of the present invention to provide a novel extract drug or functional food which is excellent in the prevention and / or therapeutic effect of influenza virus A type and has little cytotoxicity.

FIG. 1 is a photograph showing the results of a test for inhibiting erythrocyte aggregation of an extractant and a negative control according to the present invention.

According to the present invention, there is provided an antiviral agent for preventing and treating a cold, which comprises an extract of Angelica keiskei, Angelica keiskei koidz.

Raphani Semen is a seed of Raphanus sativus Linne and Cruciferae. It is a herbicide that has the effect of purifying the energy, melting the fence and eliminating the enemy. It has been used for indications such as follicles, asthma, seawater, asthma, diarrhea and dysentery.

Meliae Fructs is a fruit of Melilla azedarach Linne var. Japonica Makino, and is recognized as an efficacy such as 气 气 止痛, 殺 虫 药 药, , Colic, saliva, and duodenum.

Lonicerae Flos is a bud of Lonicera japonica Thunberg. It has been recognized for its efficacy in relieving fever and poisoning, and has been used for indications such as warts, fever, and fever.

Peony Root is the root of Paeonia albiflora (Pallas var. Trichocarpa Bunge) and its related plants (Ranunculaceae). It shows the action of softening the liver, protecting and converging blood. It is used for indications such as menstrual relief, chest, side, abdominal pain, and hyperhidrosis.

Cnidium Rhizome is the roots of the Cnidium officinale Makino, which are left intact or in hot water. I have been using it for a sick indication because I have a tendency to manage tummy blood and wind in one room, and have trouble with menstruation, headache, chest and side.

The inventors of the present invention have found that an extraction agent obtained by combining these five herbal medicines exhibits excellent inhibitory activity against influenza virus type A as well as little cytotoxicity.

The extraction agent according to the present invention is prepared by extracting a mixed drug of nevada, cinnabar, gilt europaea, peony and cinnabar using an appropriate extraction solvent, followed by filtration, separation, concentration, and lyophilization.

As the extraction solvent, hot water, methanol and the like can be used.

It is preferable to use a domestic herbal medicine as a raw material shortening agent.

When hot water is used as the extraction solvent, 10-40 L of distilled water per 1 kg of the raw material is added to the mixed raw material consisting of napposer, celadon, gold, silver, peony and celeste, boiled for 2-4 hours, primary filtered using a gauze, Centrifugation for 10-20 minutes, secondary filtration, concentration with a concentrator, and freeze-drying.

When methanol is used as the extraction solvent, 5-20 L of methanol per 1 kg of the raw material is added to the raw material, and the mixture is immersed in a 50-70 ° C water bath for 15-20 hours, centrifuged at 8000 xg for 10-20 minutes, Filtered, concentrated with a concentrator, and lyophilized to prepare a final extraction agent.

The extractant produced by the present invention exhibits excellent influenza virus A type inhibitory activity and has almost no toxicity, so that it can be used as a medicament for treating cold.

In case of using as a medicine, it is preferable to administer the drug extract obtained by using 60 g in equal ratio of napposers, cinnabar, gilt euphonium, peony, and astragalus dividedly three times a day.

In addition, functional foods for prevention of cold can be prepared by using extracts of napper, cinnabar, ginger, peony, and astringent mixed prescription raw materials according to the present invention.

For example, when producing a functional beverage, purified water is added to the raw material in an amount of 5-10 liters per 1 kg of the raw material, and then the mixture is heated at a high temperature (100-120 ° C), a high pressure (1-2 kg / m < ² >) for 2-3 hours, and the filtrate is further treated with the same amount of water for 2-3 hours under the same conditions, followed by filtration. The obtained filtrate may be well mixed with an additive commonly added to the beverage, such as glucose, maltose, saccharose, oligosaccharide, or natural honey, and sterilized, followed by commercial beverage production.

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

Example 1

1300 ml of distilled water was added to a total of 60 g of each of 12 g of napposer, cinnabar, gold euhae, peony, and cinnabar, and the mixture was boiled for 2 hours and 30 minutes, followed by primary filtration using a gauze. Then, the extracted solution was centrifuged at 8000xg for 15 minutes, filtered, concentrated by a concentrator, and lyophilized to obtain about 6g of a drug.

Example 2

500g of 80% - methanol was added to 60g in total of 12g each of Nepheline, Cheonjunja, Geumgwha, Peony, and Taeungguk, and they were extracted by immersing in 60 ℃ water bath for 18 hours. The extracted methanol solution was centrifuged at 8000xg for 15 minutes, filtered, concentrated with a concentrator and lyophilized to obtain about 2g of the drug.

Example 3

Nabokja, cloth ryeonja, honeysuckle, peony and Cnidium into each 200g of purified water by the total 6ℓ 1kg, and for 2 hours, then filtered under a master 120 ℃, a pressure of 1.5kg / m ^2, the same condition by adding more water to the same volume of filtration residue Lt; / RTI > for 3 hours and then filtered. The resulting filtrate was mixed well with 50 g of natural honey per 1 kg of the raw material while stirring well, and sterilized by passing it through an instant sterilizer (110-120 ° C). The sterilized beverage was poured into a 120 ml can dispenser in an amount of 120 ml, and the mixture was cooled and packed to prepare a functional beverage.

Test Example 1

In order to compare antiviral activity and cytotoxicity against each of the extracts of endophytic agents used in the present invention, the extractant according to the present invention, amantadine hydrochloride, which is a conventional antiviral agent, and Korean medicinal herb extracts marketed at home and abroad, Haemagglutination Inhibition Test (HIT) and Haemagglutination Assay.

Each of the starch extracts was extracted, separated, concentrated, and lyophilized according to the method of Example 1 (extracted using the same amount of a single agent as the total amount of the mixed drug used in preparing the drug extract according to the present invention ).

Compared with Chinese herbal extracts, there are two kinds of herbal extracts: SsanghwangTM ™ (Donghwa Pharm: K-1), Galgangtang ™ (Samyoung Pharm: K-2) (J-1), Gagonal ™ (Shin-Etsu Chemical Co., Ltd .: J-2), which is commercially available in Japan, and Anticancer Insecticide ™ (Sacheon Chemical Co., (C-1), Pang-ram Root (C-2) and Cheongmyeong Cheol-hyeong (C-3) were used.

Virus preparation

In this study, influenza virus [A / Taiwan / 1/86 (H ₁ N ₁ )] was distributed from the National Institutes of Health respiratory virus department.

Virus growth

Specific pathogen-free embryonated hen's eggs were incubated for 10 days at 37 ° C in a pellet to propagate the virus, and fertilized eggs, except contaminated eggs and oocytes, were used. 0.1 ml of virus (0.08HA unit) was injected into the allantoic fluid of fertilized eggs and pores were blocked with paraffin wax. After the inoculation, the virus was grown at 37 ° C for 3 days, then left at 4 ° C for 18 hours, and the urinary juice was obtained in an amount of 10 ml per egg. Enzymes containing virus were centrifuged at 4 ° C, 8000xg for 10 minutes, and culture supernatant was used.

Virus titer measurement

In order to measure the activity of the virus proliferated in the urinary juice, the erythrocyte agglutination test was performed. The viral culture stock solution isolated from the prolonged urine was diluted 2-fold with PBS and dispensed 0.05 ml into 96-well plates. 0.05 ml of 0.5% RBC solution diluted with PBS was added to each well and shaken. The reaction was then allowed to proceed at 25 캜 for 60 minutes, and the agglutination titers were measured in comparison with the negative control.

As a result of the proliferation of viruses in three 10-day-old SPF fertilized eggs and measuring the activity through erythrocyte agglutination, the concentration of virus in the A SPF fertilized egg was 4HA unit / 0.025 ml, and the B SPF fertilized egg , The titers of the viruses grown in the C SPF fertilized eggs were 256 HA unites / 0.025 ml. In the dual C SPF fertilized eggs, the virus propagated was used for haemagglutination assay and haemagglutination inhibition test.

Preparation of erythrocyte solution

Blood was collected from the subcutaneous vein of chicken and collected in Alsever solution to prevent blood clotting. This was centrifuged at 1800 xg for 5 minutes to collect only the red blood cells, suspended in phosphate buffered saline (PBS, pH 7.4), stored at 4 캜 for 5 days, and subjected to hemagglutination assay and Haemagglutination Inhibition Test : HIT).

Evaluation of anti-influenza virus activity: erythrocyte agglutination inhibition test

Haemagglutination inhibition test (HIT) was performed to measure anti-influenza virus activity of prescription of herbal medicine. The stock solution (100 mg / ml) was diluted 2-fold in each step, and 0.025 ml was added to each well. The virus solution measured in the red cell coagulation titer was diluted with 4 haemagglutination units (HA units), 0.025 ml was added to each well, and the mixture was reacted at room temperature for 30 minutes. Next, 0.05 ml of 0.5% RBC solution was dispensed into each well, gently shaken, and reacted at 25 캜 for 60 minutes. After the reaction, the positive control group and the negative control group were compared, and the effect of the drug extracted by the present invention to inhibit the adsorption of virus and RBC was searched.

Table 1 shows the effects of amantadine hydrochloride, which is a conventional antiviral agent, and Korean herbal medicine extracts, which are commercially available at home and abroad, and Table 2 shows the effects of each of the shortening agents and the extracting agents of the present invention.

In the above and following tables, Con. Is the dilution concentration of each extract, ++, +,? And - indicate strong positive, positive, weak positive and negative responses, respectively, and NC is the negative control.

As can be seen from the above results, amantadine hydrochloride showed antiviral activity only at 12.5 mg / ml, and Korean herbal medicine K-1, which is marketed in Korea, showed 3.13 mg / ml-6.25 mg / ml, 50 mg / At the concentration of 100 mg / ml, weak antiviral activity was exhibited at a concentration of 12.5 mg / ml-25 mg / ml. K-2 showed a weak antiviral activity at a concentration of 12.5 mg / ml and 100 mg / Activity exhibited strong antiviral activity at 25 mg / ml-50 mg / ml, and K-3 showed strong antiviral activity at 25 mg / ml-100 mg / ml at 12.5 mg / , And K-4 had a weak antiviral activity between 25 mg / ml and 100 mg / ml. J-1, a herbal medicine extract commercialized in Japan, has a strong viral activity between 6.25 mg / ml and 100 mg / ml and has a relatively weak antiviral activity between 1.56 mg / ml and 3.13 mg / ml , J-2 had a strong antiviral activity between 6.25 mg / ml and 100 mg / ml, and a relatively weak antiviral activity at 3.13 mg / ml. C-1, a Chinese herbal medicine, has weak antiviral activity at a concentration of 6.25 mg / ml-50 mg / ml and has a strong antiviral activity at a concentration of 100 mg / ml. C- / Ml, and C-3 had weak antiviral activity in the concentration range of 6.25 mg / ml-25 mg / ml and a strong antiviral activity in the concentration range of 50 mg / ml-100 mg / Respectively.

Inhibitory effect of a single agent and a mixed extraction agent on influenza A virus type ConNo -1 (100 mg) -2 (50) -3 (25) -4 (12.5) -5 (6.25) -6 (3.13) -7 (1.56) -8 (780 [mu] g) -9 (390) -10 (195) -11 (97.5) -12N Napkin ++ ++ ++ ++ ++ ++ + - - - - Tianjin ++ ++ ++ ++ ++ ++ ++ + - - - Gold silver - ++ ++ + - - - - - - - Peony - - - - + - - - - - - Celestial + + + + + + - - - - - Example 1 ++ ++ ++ ++ ++ ++ ++ ++ - - - -

The potent antiviral activity was shown in the wide sample concentration range of 3.13 mg / ml-100 mg / ml and 1.56 mg / ml-100 mg / ml, respectively, The antiviral activity was much stronger than that of herbal extract.

The ginseng extracted with hot water showed antiviral activity in the concentration range of 12.5 mg / ㎖-50 mg / ㎖, and the concentration of peony was 6.25 ㎎ / ㎖ at the concentration range of 3.13 ㎎ / ㎖ -100 ㎎ / ㎖, Showed weak antiviral activity.

The extractant of Example 1 prepared according to the present invention exhibited much stronger viral activity over a wider concentration range than commercial herbal extract preparations available at home and abroad. FIG. 1 is a photograph showing the experimental results of inhibition of erythrocyte aggregation of an extractant according to the present invention.

Cytotoxicity test for erythrocytes: erythrocyte agglutination test

The stock solution (100 mg / ml) was diluted 2-fold in each step, and 0.025 ml was added to each well. After 0.025 ml of PBS (pH 7.4) was added to each well, 0.05 ml of 0.5% diluted RBC solution was added to each well. The mixture was gently shaken and reacted at 25 ° C for 60 minutes. After the reaction, the cytotoxicity of amantadine hydrochloride, commercial herbal medicine extract, various finely divided product extract and pharmaceutical extract according to the present invention to red blood cells (RBC) was examined in comparison with the negative control group.

Amantadine · HCl showed cytotoxicity in the concentration range of 25 mg / ㎖-200 mg / ㎖, and herbal cold extracts sold in Korea and Japan showed no cytotoxicity to RBC. Therefore, herbal cold extracts The toxicity to the cells was much less.

The cytotoxicity of red cells to each of the starch 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 [mu] g) -9 (390) -10 (195) -11 (97.5) -12N Napkin - - - - - - - - - - - - Tianjin - - - - - - - - - - - - Gold silver + - - - - - - - - - - - Peony + + + + + - - - - - - - Celestial - - - - - - - - - - - - Example 1 - - - - - - - - - - - -

Nebulizers and tennis players showing strong viral activity in a wide concentration range showed no cytotoxicity against RBCs, whereas gingivalis showed mild cytotoxicity at a concentration of 100 mg / ml, and peanuts showed 6.25 mg / ml -100 mg / ml And showed cytotoxicity in a wide concentration range. Cynomolgus showed weak viral activity in a wide concentration range and did not show cytotoxicity at all. The extractant according to the present invention showed no toxicity in the cytotoxicity test on erythrocytes.

In vivo Evaluation of antiviral activity using fertilized eggs

a. Virus growth in embryos

The embryos were incubated for 10-11 days in the paddle, 100 μl of 0.5 HA influenza virus was inoculated into the urine of the embryos and sealed with paraffin, incubated at 34 ° C for 2 days, and left at 4 ° C for 18 hours or more . After that, the urine was collected using a syringe. This procedure was repeated several times to produce influenza virus adapted to embryos and concentrated using an ultracentrifuge at 25000 rpm, 4 ° C for 2 hours. The concentrated virus was used to obtain EID ₅₀ .

b. Determining the EID ₅₀ value

EID ₅₀ values can be obtained by erythrocyte agglutination test after infecting the enriched influenza virus into the urine of the fertilized egg. The enriched virus was diluted 10 times in PBS by incubating the embryos cultured at 37 ° C for 10 to 11 days. 100 μl of the virus was inoculated into the prolonged urine of the embryos according to the respective concentrations, and the inoculation site was sealed with paraffin. After incubation for 2 days at 34 ° C, the cultured embryos were allowed to stand at 4 ° C for more than 18 hours. The embryo urine was removed by syringe and the erythrocyte agglutination test was conducted to confirm the virus proliferation in the enriched urine. ₅₀ was obtained.

In this experiment, the first erythrocyte agglutination test was carried out to proliferate the virus and 20EID ₅₀ was measured. For the pharmaceutically active evaluated by 2 X 10 ⁶ EID ₅₀ or higher should be so concentrated using an ultracentrifuge to proliferate the virus in embryos as possible, was obtained the EID ₅₀ values using the fertilized egg back, 2 X 10 ²² EID ₅₀ appear.

c. Evaluation of drug activity

0.1 ml and 0.2 ml of the extractant of Example 1 according to the present invention (10 μg / ml, 100 μg / ml, diluted with 1000 μg / ml) were mixed with virus (2 × 10 ⁶ EID ₅₀ ) After the inoculation of the urine of the urine, the hole is blocked with paraffin wax. After the inoculation, the virus was grown at 37 ° C for 2 days and then allowed to stand at 4 ° C for 18 hours to obtain urine. The virus-containing enterotoxin was centrifuged at 4 ° C for 10 minutes at 8000xg, and the supernatant was assayed for erythrocyte agglutination activity [McLaren, LC: Haemagglutination Inhibition and Hematology in Clinical Virology Manual (Specter S, Lancz , G, eds) pp 243-249, Elsevier science publishing, New York, 1992].

(NP-97-1) and NPW-7 (NP-97-2) in the fertilized egg using 2 × 10 ²² EID ₅₀ values, (Table 5).

The anti-influenza virus activity in the embryo of the extractant according to the present invention con ([mu] g) No. -1 (1000) -2 (100) -3 (10) Example 1 + + +

The extraction agent according to the present invention has a concentration of about 8 times that of amantadine hydrochloride, which is a conventional chemical synthesizer, and is about 4 times as much as the concentration of influenza virus A in Korea, Japan, and China Prophylactic and inhibitory action, and no cytotoxicity.

According to the present invention, it is possible to obtain a pharmaceutical agent which is excellent in the prevention and treatment effect of influenza virus type A, has little cytotoxicity unlike a chemical synthesis drug, and a functional food using the same.

Claims (5)

An antiviral drug for prevention and treatment of a cold, which comprises an extract of Angelica keiskei koidz. The method according to claim 1, Wherein the extract is obtained by adding 10 to 40 L of distilled water per 1 kg of raw material of napposer, cinnabar, gingkojie, peony, and mantis, followed by heating and extracting. The method according to claim 1, Wherein the extract is obtained by adding 5-20 L of methanol per 1 kg of the raw material of napposer, cinnabar, gilt eum, phoenix, and mantis, followed by sedimentation extraction in a water bath at 50-70 캜. Functional food for prevention of cold, including napposer, cinnabar, golden earthenware, peony, and astringent. 5. The method of claim 4, Wherein the functional food is a beverage, The beverage is prepared by adding 5-10 L of purified water per 1 kg of raw material of napkin, cinnabar, cinnabar, peony, and cinnabar and separating the primary filtrate by heating for 2-3 hours under high temperature and high pressure; Separating the filtration residue with an equal amount of purified water for 2-3 hours under the same conditions and separating the obtained secondary filtrate; And adding a beverage additive to the mixture of the primary and secondary filtrate and sterilizing the mixture.