KR20060040407A - Anticoronavirus composition comprising genus zanthoxylum extract - Google Patents

Abstract

The present invention relates to an anti-coronavirus composition containing the herbaceous plant extract, more specifically, the organic solvent extracts of plants belonging to the genus Zanthoxylum (respiratory diseases, digestive diseases, liver disease, brain) in mammals The present invention relates to an antiviral composition comprising the extract by revealing that it has antiviral activity against coronavirus that causes diseases.

Perennial plant, Coronavirus, antiviral activity

Description

Anticoronavirus composition comprising genus Zucchinus extract {Anticoronavirus composition comprising genus Zanthoxylum extract}

The present invention relates to an anti-coronavirus composition containing the herbaceous plant extract, more specifically, the organic solvent extracts of plants belonging to the genus Zanthoxylum (respiratory diseases, digestive diseases, liver disease, brain) in mammals The present invention relates to an antiviral composition comprising the extract by revealing that it has antiviral activity against coronavirus that causes diseases.

Diseases caused by coronavirus infections vary widely. RNA viruses that cause respiratory, digestive, liver, and brain diseases in mammals and birds [Thomas M. Gallagher ™, and Michael J. Buchmeier MJ (2001) Coronavirus Spike Proteins in Viral Entry and Pathogenesis. Virology, 279 (2): 371-374.]. In particular, porcine epidemic diarrhea virus (PEDV), a virus belonging to the coronavirus, is a highly infectious viral disease that invades the gastrointestinal digestive system, causes dehydration and high fever due to vomiting and diarrhea, and has a high mortality rate. Duarte M, Laude H (1994) Sequence of the spike protein of the porcine epidemic diarrhoea virus. J Gen Virol., 75 (Pt 5): 1195-200.]. SARS is a type of coronavirus that causes respiratory diseases with high mortality. Although these viruses have a very high mortality rate, there are no treatments developed like other viral infections. Therefore, the vaccine development research to prevent the virus infection is ongoing, but the efficiency is still low [Alonso S, Sola I, Teifke JP, Reimann I, Izeta A, Balasch M, Plana-Duran J, Moormann RJ, Enjuanes L (2002) In vitro and in vivo expression of foreign genes by transmissible gastroenteritis coronavirus-derived minigenomes. J Gen Virol., 83 (Pt 3): 567-79.].

Plants have long been used as a treatment for many diseases [Seeff LB, Lindsay KL, Bacon BR, Kresina TF, Hoofnagle JH (2001) Complementary and alternative medicine in chronic liver disease. Hepatology, 34 (3): 595-603 .; Langmead L, Rampton DS (2001) Herbal treatment in gastrointestinal and liver disease--benefits and dangers. Aliment Pharmacol Ther., 15 (9): 1239-52.]. Viral diseases have also been reported to have antiviral activity in many plants [Kinghorn AD (1994) The discovery of drugs from higher plants. Biotechnology, 26: 81-108 .; Patrick L (1999) Hepatitis C: epidemiology and review of complementary / alternative medicine treatments. Altern Med Rev., 4 (4): 220-38.], Few studies have been conducted on plants with antiviral activity against coronaviruses [McCutcheon AR, Roberts TE, Gibbons E, Ellis SM, Babiuk LA, Hancock RE , Towers GH (1995) Antiviral screening of British Columbian medicinal plants. J Ethnopharmacol., 49 (2): 101-10.].

Cultivated plants have been traditionally used as flavoring foods in the Orient, and it has been reported to exhibit various pharmacological effects [Das NG et al. 2003. J Vector Borne Dis. 40 (1-2): 49-53; Hur JM et al. 2003. Biosci Biotechnol Biochem. 67 (5): 945-50; Thouvenel C et al. 2003. Phytother Res. 17 (6): 678-80; Rahman MT et al 2002. Fitoterapia. 73 (4): 340-2; de Moura NF et al. 2002. Planta Med. 68 (6): 534-8; Jo YS et al. 2002. Planta Med. 68 (1): 84-5; Arrieta J et al. 2001. Fitoterapia. 72 (3): 295-7; Bastos JK et al. 1999. Planta Med. 65 (6): 541-4; Kumar S and Muller K. 1999. Phytother Res. 13 (3): 214-7; Navarrete A and Hong E. 1996. Planta Med. 62 (3): 250-1]. Very little research has been carried out on the antiviral activity of creeper plants, and it has been reported that only fruit extracts from African creeper plants in Africa have antiviral activity against measles virus causing pertussis [Olila D 2002. Afr Health Sci. 2 (1): 2-10]. However, no herbaceous plant extract for anticoronavirus has been studied.

On the other hand, the present inventors have applied for a patent on the anti-coronavirus ability of the extract of the stem of the bark tree (Korean Patent Application No. 2003-29742).

Accordingly, the present inventors confirmed that the extract of the organic solvent of the plant of the herbaceous plant, which is totally different from the extract of the stem of the herbaceous tree, showed excellent anti-coronavirus ability, and the leaves, roots, heartwood, bark, etc. In other parts, the anticoronavirus ability was remarkably excellent, and the present invention was patented as a separate invention by revealing that the anticoronavirus ability was excellent not only in the bark tree but also in the genus plants including the same.

Accordingly, an object of the present invention is to provide an anti-coronavirus composition containing as a active ingredient chopped plant extract obtained by extracting genus Zanthoxylum with an organic solvent.

The present invention is characterized by an anti-coronavirus composition containing the chopsticks plant extract obtained by extracting genus Zanthoxylum with an organic solvent as an active ingredient.

Referring to the present invention in more detail as follows.

The present invention reveals that the organic solvent extracts of plants belonging to genus Zanthoxylum have antiviral activity against coronaviruses that cause respiratory diseases, digestive diseases, liver diseases, brain diseases, etc. in mammals. It relates to an antiviral composition comprising the extract.

Tissue pulverized materials such as fruits, stems, leaves, roots, heartwood and bark of Cultivated plants are placed in organic solvents, preferably methanol, ethanol, hexane, chloroform, etc., 5-30 times preferably 20 times the volume of each weight. Each tissue was ground to a mill, and then allowed to stand at 20 to 30 ° C., preferably at room temperature for 13 to 20 hours, preferably for 16 hours, and centrifuged at 6000 to 10000 rpm for 10 to 30 minutes to remove insoluble matters. After harvesting, the extract is dried with a reduced pressure extractor.

Particularly, in the present invention, the herbaceous plants are Zanthoxylum piperium , Zanthoxylum schinifolium , Zanthoxylum coreanum , Zanthoxylum planispinium , Zanthoxylum schinifolium var subinermis tree (Zanthoxylum schinifolium var microphyllum), Min pepper tree (Zanthoxylum schinifolium var inermis), zanthoxylum ailanthoides (Zanthoxylum ailanthoides), Min zanthoxylum ailanthoides (Z anthoxylumailanthoides var. inermis), Sommer ear tree (Z anthoxylum fauriei), teolcho linden (Zanthoxylumpiperitum var pubescens ) and the like.

The plant extract of the genus Chopi is anti-coronavirus (coronavirus) that is a virus causing respiratory diseases, digestive diseases, liver diseases, brain diseases, etc. in mammals or birds including humans or livestock such as pigs, dogs, cats, cows, etc. It was confirmed that it had viral activity.

Therefore, the anti-coronavirus composition of the present invention is expected to be usefully used as drugs and foods capable of preventing or treating infections such as porcine coronavirus, bovine coronavirus, dog coronavirus and SARS coronavirus.

Medical use of the super herbaceous plant extract according to the present invention in preparations orally administered such as formulations, such as tablets, capsules, troches, solutions, suspensions, etc., in combination with a conventional carrier in the pharmaceutical field; Injectable preparations in the form of injectable solutions or suspensions, or ready-to-use injectable dry powders which can be prepared and used as injectable distilled water at the time of injection; Or in various preparations such as ointments. Pharmaceutical preparations prepared using conventional carriers can be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally or topically.

In addition, the dosage of the vinegar extract is different depending on the weight of the mammal, the state of age, etc., but generally 20 to 100 mg / kg, preferably 40 to 60 mg / kg per day And, depending on the judgment of the doctor or pharmacist can be administered dividedly several times a day, preferably once or four times at regular time intervals.

In addition, the present invention includes a health food comprising the edible plant extract as an active ingredient.

The health food is a food prepared by adding the vinegar extract to food materials such as beverages, teas, spices, chewing gum, confectionery, or encapsulated, powdered, or suspension, and when ingested, has a specific health effect. It means coming, but unlike the general medicine has the advantage that there is no side effect that can occur when using the drug as a raw material for long-term use.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Reference Example 1 Preparation of Extract

Four crops of Zinoxylum piperium , Zanthoxylum schinifolium , Zanthoxylum coreanum and Zanthoxylum planispinium were harvested and dried in the shade at the Forest Research Institute. The bark tree used the root, heart, and leaf as the material, and the bark tree used the bark and leaf, and the bark tree used the leaf, the bark, and the root, and the flowering tree used the stem, the bark, the fruit, and the leaf. . Each material was dried and pulverized until the moisture content of the material was 10%. Methanol was added to each tissue mill in a volume of 20 times the weight of each weight and left for 16 hours at room temperature to obtain a methanol extract, which was centrifuged at 8000 rpm for 20 minutes to remove insoluble materials and harvested supernatant. Dry with an extractor.

Reference Example 2: Culture of Virus

The virus used to measure the antiviral activity of methanol extracts on the tissues of the four kinds of percutaneous plants obtained in Reference Example 1 was targeted to porcine epidemic diarrhea virus (PEDV), and PEDV was propagated in Vero cells. . Vero cells were cultured in a 37 ℃ incubator using MEM (minimal essential media) containing 10% fetal bovine serum.

Example 1: Virus Inhibition Assay

1) Virus Inhibition

In order to measure the virus proliferation inhibitory activity against PEDV of each methanol extract obtained in Reference Example 1, Vero cells were cultured in 96 well plates, and the existing culture solution was prepared when each cell was at the bottom of the well more than 90%. Removed and a new culture containing PEDV was administered to each well and each plant extract was administered to the wells by concentration (1, 10, 100, 1000 μg / ml media). Determination of the viral growth inhibitory activity of each extract was performed by SRB assay after infecting PEDV with Vero cells for 48 hours [Martin A, Martin C (1997) Comparison of 5 microplate colorimetric assay for in vitro cytoxicity testing and cell proliferation assay. Cytotechnology, 11: 49-54.]. 100 μl of 10% TCA (trichloroacetic acid) was added to each well, and then left at 4 ° C. for 1 hour and washed several times with distilled water. After drying at room temperature, 100 μl of 0.4% (w / v) sulforhodamine B (SRB) solution dissolved in 1% (v / v) acetic acid was added and stained for 30 minutes. The remaining SRB stain was removed and each well of 96 plates was washed several times with 1% (v / v) acetic acid and then dried again. 100 μl of 10 mM Tris solution (pH 10.5) was added to each well to sufficiently dissolve the dye bound to the cells, and the absorbance was measured at 560 nm. Each treatment group was labeled as virus-free (A), water-only extract (B), virus-only (C), and virus and water-soluble extracts (D). The cytotoxic activity (%) [Equation 1] of the extract and the virus growth inhibitory activity (%) [Equation 2] of the plant extracts were calculated.

2) cytotoxicity

The cytotoxic activity of the methanol extract of each of the plants of the genus Schizophyllum obtained in Reference Example 1 was analyzed. Methanol extracts of the tissues of the creeper plants showed different cytotoxicity against Vero cells used as virus culture cells. The results of measuring 50% cytotoxicity concentration (CC ₅₀ ) in each species of plants in S. pelvis are shown in Table 1 below. The bark root extract is 177 ㎍ / ml, the core extract is 726 ㎍ / ml, the leaf extract is 151 ㎍ / ml, the Japanese bark extract is 50 ㎍ / ml, the leaf extract is 68 ㎍ / ml, the bark extract is 100 More than ㎍ / ㎖, bark extract is 128 ㎍ / ml, root extract is 65 ㎍ / ml, hawthorn stem extract is 245 ㎍ / ml, bark extract is 66 ㎍ / ml, fruit extract is 170 ㎍ / ml, leaf The extract is 305 μg / ml.

3) Antiviral activity analysis for PEDV

The antiviral activity against PEDV of each tissue extract in the C. bark plants obtained in Reference Example 1 is shown in Table 1 below.

Positive controls include 10 mg / ml of glycyrrhizhin, quercetrin, catechin, coumarin, luteolin, and tannin, which are plant-derived compounds. Dissolved in dimethyl sulfoxide as much as possible to test 50% cell necrosis and 50% virus proliferation inhibitory to PEDV in the concentration range of 0.1, 1, 10, 100 ㎍ / ml and the results are shown in Table 1 below.

The plants of the genus Schisandra chinensis showed different antiviral activity in each tissue, but showed the effect of inhibiting the virus growth in the extracts of all four plants. Second linden the roots, the core material, leaf extract is 50 ㎍ / ㎖, 34.9 ㎍ / ㎖, 35 ㎍ / 50% by tissue in ㎖ viral proliferation inhibitory ability concentration (EC ₅₀₎ is significantly did not differ a CC ₅₀ value EC ₅₀ value When compared with the SI (selectivity index) value divided by the heartwood extract showed the most effective antiviral activity. Bark and leaf extracts were 6.4 ㎍ / ml and 3.7 ㎍ / ml, and their SI values were 18.6, higher than bark extracts. When comparing the SI values of the leaves, bark and root extracts, the SI values of root extracts were more than 50 times higher than those of leaves and bark extracts. The SI values of the stem, bark, fruit, and leaf extracts were 32.7, 11.4, 4.9, and 47.8, and the highest SI values of the leaf extracts were followed by the stem extracts. As such, it can be said that the herbaceous extract has a very high utility as an antiviral agent in medicine.

Example 2: Toxicity Test

Methanol extract obtained in Reference Example 1 was dissolved in dimethylsulfoxide (dimethylsulfoxide, DMSO), diluted with water, and then administered to the mice (10 mice per group) 100 mg / kg each, and observed for 7 days, but there was no death.

Preparation Example 1 Preparation of Powder and Capsule

10 mg of the methanol extract obtained in Reference Example 1 was mixed together with 14.8 mg of lactose, 3 mg of crystalline cellulose, and 0.2 mg of magnesium stearate. The mixture was filled into No. 5 gelatin capsules using a suitable apparatus.

The components of the powder and capsules are as follows.

10 mg of active ingredient

Lactose 14.8 mg

3 mg of crystalline cellulose

Magnesium Stearate 0.2mg

Preparation Example 2 Preparation of Injection Solution

Injection solution containing 10 mg of the methanol extract obtained in Reference Example 1 was prepared by the following method.

1 g of the methanol extract obtained in Reference Example 1, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

The components of the injection solution are as follows.

1 g of active ingredients

6 g sodium chloride

1 g of ascorbic acid

Distilled Water Determination

Formulation Example 3: Beverage Preparation

After dissolving 500 mg of the methanol extract obtained in Reference Example 1 in an appropriate amount of water, vitamin C as an auxiliary component, citric acid, sodium citrate, and oligosaccharides as an auxiliary component were added, and an appropriate amount of sodium benzoate was added as a preservative, and then water was added thereto. The total amount was made to 100 ml to prepare a beverage composition. At this time, taurine, myo-inositol, folic acid, pantothenic acid, etc. may be added alone or together.

As described above, the herbaceous plant extracts according to the present invention are confirmed to exhibit antiviral activity against coronaviruses that cause respiratory diseases, digestive diseases, liver diseases, brain diseases, etc. in mammals. It is expected to be a great help in the treatment.

Claims (6)

An anti-coronavirus composition comprising an extract of the genus Zanthoxylum extracted with an organic solvent as an active ingredient. The method of claim 1, wherein the plants of the genus Cortex are Zanthoxylum piperium , Zanthoxylum schinifolium , Zanthoxylum coreanum , Zanthoxylum planispinium , Zanthoxylum schinifolium var subinermis , Zanthoxylum schinifolium var microphyllum , Zanthoxylum schinifolium var inermis , Zanthoxylum ailanthoides , Z anthoxylumailanthoides var. Inermis , Z anthoxylum fauriei , and Zinthium (Z anthoxylum fauriei ) Zanthoxylum piperitum var.pubescens ). The anti-coronavirus composition according to claim 1, wherein the organic solvent is methanol, ethanol, hexane or chloroform. The anti-coronavirus composition according to any one of claims 1 to 3, wherein the corona virus is swine coronavirus, canine coronavirus, bovine coronavirus or SARS coronavirus. A health food comprising genus Zanthoxylum extract as an active ingredient. The method of claim 5, wherein the genus plants are Zanthoxylum piperium , Zanthoxylum schinifolium , Zanthoxylum coreanum , Zanthoxylum planispinium , Zanthoxylum schinifolium var subinermis , Zanthoxylum schinifolium var microphyllum , Zanthoxylum schinifolium var inermis , Zanthoxylum ailanthoides , Z anthoxylumailanthoides var. Inermis , Z anthoxylum fauriei , and Zinthium (Z anthoxylum fauriei ) Zanthoxylum piperitum var.pubescens ).