KR102534348B1 - Composition for antivirus comprising Glycine max extract as effective component - Google Patents

Abstract

The present invention relates to an antiviral composition containing a Glycine max extract as an active component. The Glycine max extract of the present invention, more specifically, the Glycine max extract with the genetic name GL 2626 /96 or Pu Ru Ragi, has an excellent effect of inhibiting the infection of influenza A virus (A/PR8/ 34), thereby being able to be usefully used as a treatment for viral infectious diseases and as a material for antiviral health functional foods.

Description

Antiviral composition containing soybean extract as an active ingredient {Composition for antivirus comprising Glycine max extract as effective component}

The present invention relates to an antiviral composition containing a soybean extract, specifically a soybean extract of genetic origin GL 2626/96 or Pu Ru Ragi as an active ingredient.

Soybean ( Glycine max ) is also called 'soybean' and is widely cultivated as an edible crop. The stem is 60-100 cm high, stands upright, and there are also vine varieties. The root has many rhizomes (root nodules). The leaves are alternate and have 3 small leaves, and the small leaves are egg-shaped or elliptical with flat edges. Flowers bloom in July-August in purplish red or white, and run in a raceme on a short peduncle from the axil. The calyx is bell-shaped and the tip is split into 5 pieces. The petals are butterfly-shaped, and there are 10 stamens. Fruits are legumes, line-shaped flat ovals, and contain 1 to 4 seeds. Seeds vary greatly in size, such as round shape, flat and round shape, depending on the variety. The color of seeds is yellow, black, brown, green, etc., and there are many. Soybeans have evolved from wild soybeans (vine beans) into cultivated crops. It is generally accepted that beans originate from Manchuria and the northern part of the Korean Peninsula.

On the other hand, genetic resources is a term that encompasses currently cultivated species, wild species, and primitive species that have useful or potentially useful traits. These genetic resources are stored as seeds or tissues and are used for plant breeding, conservation, or research purposes.

As the diversity of plant genetic resources rapidly decreases due to recent rapid environmental changes, the importance of securing genetic resources is gradually increasing. We are making efforts to conserve a large number of plant genetic resources by securing genetic resource centers in developed countries. Plant genetic resources are expected to play a pivotal role in sustainable agriculture, food nutrition and food safety in the future. It is predicted that it will be possible to secure excellent plant genetic resources as the genotype/phenotype analysis of various plants has become easier with the recent development of molecular biology technology. Through the Green Revolution in the 1960s, the possibility of standardization of genetic diversity of food resources emerged, and various efforts to secure genetic diversity were carried out nationally. Various complementary measures are proposed and implemented. In Korea, the National Institute of Agricultural Sciences of the Rural Development Administration is operating a seed bank (http://genebank.rda.go.kr).

Korean Patent No. 1099967 discloses an isoflavone derivative of techtorgenin, its preparation, and an antiviral medicine containing it as an active ingredient, and Korean Patent No. 0872910 discloses soybean yogurt fermented with kimchi lactic acid bacteria as an active ingredient Antiviral compositions for avian flu, influenza and acute respiratory infectious disease viruses containing SARS have been disclosed, but so far, no antiviral composition containing the soybean extract of the present invention as an active ingredient has been disclosed.

The present invention has been derived from the above needs, and the present invention provides an antiviral composition containing soybean extract as an active ingredient, and the genetic origin of the seed bank according to the present invention is GL 2626 / 96 or Pu Ru Ragi. The present invention was completed by confirming that the soybean extract had a remarkable inhibitory effect on influenza A virus (A/PR8/34) infection compared to other soybean sources.

In order to achieve the above object, the present invention provides an antiviral pharmaceutical composition for viral infection containing as an active ingredient a soybean extract whose genetic origin is GL 2626 / 96 (IT228347) or Pu Ru Ragi (IT196925).

In addition, the present invention provides an antiviral health functional food composition containing as an active ingredient a soybean extract whose genetic source name is GL 2626/96 (IT228347) or Pu Ru Ragi (IT196925).

In addition, the present invention provides an antiviral feed composition containing as an active ingredient a soybean extract whose genetic origin is GL 2626 / 96 (IT228347) or Pu Ru Ragi (IT196925).

In addition, the present invention is a method for enhancing antiviral activity against influenza virus infection in animals, characterized in that the soybean extract having the genetic source name GL 2626 / 96 (IT228347) or Pu Ru Ragi (IT196925) is administered to animals other than humans provides

The present invention relates to an antiviral composition containing soybean extract as an active ingredient, and the soybean extract whose genetic source name is GL 2626 / 96 or Pu Ru Ragi of the seed bank according to the present invention is resistant to influenza A virus in preparation for other soybean resources ( A/PR8/34) The infection inhibitory effect is remarkable.

Figure 1 is the result of confirming the virus infection inhibitory effect on 12 kinds of soybean resources containing the soybean resource GL 2626 / 96 or Pu Ru Ragi extract of the present invention. Virus control is a group treated with virus without treatment with soybean extract. *, ** means that the virus infection rate of GL 2626 /96 or Pu Ru Ragi extract was statistically significantly reduced compared to Control, * is p <0.05, and ** is p <0.01.
Figure 2 is a result confirming the inhibitory effect on the decrease in survival rate according to the influenza A virus mouse infection of the soybean resource GL 2626 / 96 extract of the present invention. Negative is a group not infected with virus, and Virus control is a group infected with virus without administration of soybean extract.
Figure 3 is a result confirming the inhibitory effect on weight loss according to influenza A virus mouse infection of the soybean resource GL 2626 / 96 extract of the present invention. Negative is a group not infected with virus, and Virus control is a group infected with virus without administration of soybean extract.

The present invention relates to an antiviral pharmaceutical composition containing, as an active ingredient, a soybean extract whose genetic source name is GL 2626/96 (IT228347) or Pu Ru Ragi (IT196925) of a seed bank.

The soybean with the genetic resource name GL 2626 /96 (IT228347) or Pu Ru Ragi (IT196925) is sold as a resource stored in the Seed Bank (http://genebank.rda.go.kr) operated by the National Academy of Agricultural Sciences of the Rural Development Administration. This is a possible genetic resource.

The soybean extract may be prepared by a method comprising the following steps, but is not limited thereto:

(1) extracting by adding an extraction solvent to dried beans;

(2) filtering the extract of step (1); and

(3) preparing an extract by drying the filtered extract of step (2).

In the step (1), the extraction solvent is preferably selected from water, C ₁ ~ C ₄ lower alcohol or a mixture thereof, more preferably ethanol, but is not limited thereto. In the preparation method, all conventional methods known in the art such as filtration, hot water extraction, immersion extraction, reflux cooling extraction and ultrasonic extraction may be used as the extraction method. The extraction solvent is preferably extracted by adding 1 to 20 times the weight of soybeans. The extraction temperature is preferably 30 ~ 100 ℃, but is not limited thereto. In addition, the extraction time is preferably 1 to 6 hours, but is not limited thereto. In the step (3), drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, more preferably freeze drying, but not limited thereto.

The virus is preferably an influenza virus, but is not limited thereto, and infection with the influenza virus is characterized by causing any one disease selected from influenza, new flu, cold, sore throat, bronchitis, pneumonia and bird flu .

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent in addition to the above active ingredients, but is not limited thereto.

The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is one commonly used in preparation, and may include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, lactose, water Cross, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate ates, talc, magnesium stearate and mineral oil, and the like, but are not limited thereto. In addition to the above components, antioxidants, buffers, bacteriostatic agents, diluents, surfactants, binders, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, or preservatives may be further included.

A suitable dosage of the pharmaceutical composition of the present invention will be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity. can

The administration route of the pharmaceutical composition of the present invention may be administered through any generally accepted route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but depending on the purpose, intramuscular administration, eye drop administration, intraperitoneal administration, intravenous administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, It may be administered through a route such as intrarectal administration.

In addition, the present invention relates to an antiviral health functional food composition containing a soybean extract having a genetic source name of GL 2626/96 (IT228347) or Pu Ru Ragi (IT196925) as an active ingredient.

The health functional food composition is preferably prepared in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto. The health functional food composition of the present invention may be prepared by adding the active ingredient as it is or mixing it with other foods or food ingredients, and may be appropriately prepared according to a conventional method. Examples of foods to which the soybean extract can be added include caramel, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea , It may be in the form of any one selected from drinks, alcoholic beverages and vitamin complexes, and includes all health functional foods in the conventional sense. That is, there is no particular limitation on the type of food. The health functional food composition includes various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners , pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like. It may also contain fruit flesh for the preparation of natural fruit juices and vegetable beverages. The above components may be used independently or in combination. In addition, the health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional components, and the natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, dextrin, and cyclosaccharides. polysaccharides such as dextrin, sugar alcohols such as xylitol, sorbitol, and erythritol. The ratio of the natural carbohydrates is not very important, but is preferably 0.01 to 0.04 g, more preferably 0.02 to 0.03 g, with respect to 100 g of the composition of the present invention, but is not limited thereto. As the sweetener, natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame may be used.

In addition, the present invention relates to an antiviral feed composition containing a soybean extract having a genetic source name of GL 2626/96 (IT228347) or Pu Ru Ragi (IT196925) as an active ingredient.

The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act. In the present invention, the term 'feed' may refer to any natural or artificial diet, one meal, etc., or a component of the one meal meal, suitable for or suitable for consumption by animals. The type of feed is not particularly limited, and feeds commonly used in the art may be used. Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal or grain by-products; Animal feed such as proteins, inorganic materials, oils, mineral oils, oils, single cell proteins, zooplankton, or food may be mentioned. These may be used alone or in combination of two or more.

In addition, the present invention is a method for enhancing antiviral activity against influenza virus infection in animals, characterized in that the soybean extract having the genetic source name GL 2626 / 96 (IT228347) or Pu Ru Ragi (IT196925) is administered to animals other than humans It is about. In the enhancement of antiviral activity against influenza virus infection in animals, the dose, route of administration, timing of administration, etc. It is clear to those skilled in the art that it can be appropriately selected from conventional techniques known in the field.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for explaining the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

Example 1. Preparation of soybean extract

15 times 70% (v/v) ethanol was added to dried soybean (seed) whose genetic source name was GL 2626 /96 or Pu Ru Ragi in the seed bank, and ultrasonically extracted for 2 hours, and qualitative filter paper NO.1 (11㎛ ), concentrated and freeze-dried. As a comparative example, extracts were prepared from each of the dried seeds of 12 soybean sources (see Table 1) by the above method. In cell experiments, it was used after dissolving with dimethyl sulfoxide, and in animal experiments, it was used after dissolving in sterile distilled water.

Example 2. Confirmation of virus infection inhibitory effect in immune cells by soybean extract

Raw 264.7 cells were cultured with 200, 400 or 800 μg/ml of GL 2626/96 or Pu Ru Ragi extract in DMEM medium supplemented with 10% Fetal bovine serum (FBS) and 1% penicillin/streptomycin. concentration for 18 hours. After culturing, the cells were infected with influenza A virus (A/PR8/34-GFP, 10 MOI) in DMEM medium supplemented with 1% penicillin/streptomycin. After 2 hours of infection, the infection solution (supernatant) was removed, washed with PBS (phosphate buffer saline), and DMEM supplemented with 10% FBS and 1% penicillin/streptomycin was added and cultured for 24 hours. Cells were collected, fixed with 4% paraformaldehyde, washed with PBS, and GFP of the virus was quantified by flow cytometry. The cell infection rate for the virus was expressed as an infection rate (%) compared to a group not treated with soybean extract (Virus control).

As a result, as shown in FIG. 1 , it was confirmed that influenza A virus infection was inhibited when GL 2626/96 or Pu Ru Ragi extract was treated. In contrast, extracts from 12 other soybean sources showed little efficacy in inhibiting influenza A virus infection.

Example 3. Soyasaponin and isoflavone content analysis of soybean resource extract

To measure the contents of soya saponin and isoflavones in soybean resource extracts, the content analysis was performed using a Dionex UltiMate 3000 system (Sunnyvale, CA, USA) and Thermo's quadrupole-orbitrap mass spectrometer (Thermo Q-Exactive, Bremen, Germany) equipped with an electrospray ionization (ESI) source were used, and the content analysis in soybean resource extracts was searched for in parallel. It was carried out by parallel reaction monitoring (PRM). Xcalibur (version 3.0, Milford, MA, USA) was used for collection and qualitative analysis of all data, and TraceFinder 3.2.512 (Thermo, Bremen, Germany) was used for quantitative analysis.

The contents of soya saponin and isoflavones for each soybean resource are disclosed in Tables 1 and 2, and the composition of soya saponin and isoflavones varies depending on the soybean resource. It was found that the flavone content was not high. Therefore, it was determined that the remarkable anti-influenza A virus efficacy of GL 2626/96 or Pu Ru Ragi bean extract of the present invention was not caused by soyasaponin and isoflavone.

Soya saponin content of soybean resource extract (mg/10g extract) genetic source name IT number Soyasaponin Aa Soyasaponin Ab Soyasaponin B Soyasaponin I Soyasaponin II GL 2626/96 228347 345.984 18.594 3.046 45.419 n.d Pu Ru Ragi 196925 n.d 137.886 3.587 47.494 n.d Sachiyutaka 238169 n.d 199.446 5.653 61.745 n.d Suwon103 24470 218.425 18.488 2.588 33.73 n.d Heugcheong 229367 n.d 100.568 4.212 36.766 n.d Bosuk Kong 213209 n.d 248.956 6.357 68.348 n.d Sinrok 252262 n.d 191.34 7.693 52.475 n.d Dongsan133 154786 2.946 308.599 6.349 73.359 n.d Sugae #43 (A) 229363 n.d 94.039 4.751 56.781 n.d Suwongaetong #2 229364 381.204 20.621 8.485 64.941 n.d CS 380-14 115585 458.795 18.549 3.554 36.882 n.d Yu tae 228397 n.d 195.634 8.725 59.178 n.d CS 629-10 115768 n.d 97.385 1.999 28.287 n.d CS544-2 219625 262.478 18.613 3.94 44.007 n.d

n.d, not detected (limit of quantification of Soyasaponin II is 3.6 μg/10 g extract).

Isoflavone content of soybean resource extract (mg/10g extract) genetic source name IT number Daidzin Daidzein Glycitin Glycitein Genistin Genistein GL 2626/96 228347 11.773 0.235 4.914 0.052 32.877 0.265 Pu Ru Ragi 196925 7.267 0.283 2.852 0.063 20.828 0.362 Sachiyutaka 238169 25.111 0.209 11.135 0.038 76.342 0.113 Suwon103 24470 12.294 0.098 3.872 0.015 36.894 0.072 Heugcheong 229367 8.484 0.143 4.672 0.045 19.731 0.118 Bosuk Kong 213209 11.166 0.27 10.057 0.097 32.01 0.158 Sinrok 252262 9.182 0.107 7.718 0.058 28.247 0.096 Dongsan133 154786 7.573 0.467 3.342 0.053 23.247 0.277 Sugae #43 (A) 229363 5.878 0.206 4.754 0.043 26.513 0.188 Suwongaetong #2 229364 9.869 0.124 4.412 0.029 32.47 0.134 CS 380-14 115585 13.421 0.085 6.25 0.026 28.91 0.063 Yu tae 228397 4.89 0.021 3.66 0.008 20.853 0.039 CS 629-10 115768 12.194 0.089 5.408 0.02 39.196 0.086 CS544-2 219625 12.574 0.089 7.885 0.041 33.263 0.099

Example 4. Confirmation of antiviral efficacy in virus-infected animal models by soybean extract

6-week-old BALB/c female mice were divided into 4 groups with 10 mice randomly assigned to each group. Soybean resource GL 2626/96 or Sinrok extract dissolved in sterile distilled water was orally administered once daily at a concentration of 300mg/kg for 2 weeks, and the virus-free group (Negative) and the virus-infected group without soybean extract administration ( Virus control) was administered with sterile distilled water.

Body weight and survival rate were measured daily for 9 days after intranasal infection with influenza A virus. A weight loss of more than 25% of the starting body weight before infection was considered death from an ethical point of view.

As a result, as shown in FIG. 2, the soybean source GL 2626 / 96 extract administration group was able to significantly reduce deaths due to viral infection compared to the virus control group or the Sinrok extract administration group.

In addition, as shown in Figure 3, it was confirmed that the soybean source GL 2626 / 96 extract administration group suppressed the weight loss due to viral infection compared to the Virus control group or the Sinrok extract administration group.

Claims (9)

An antiviral pharmaceutical composition for infection with influenza A (H1N1) virus containing soybean ethanol extract having genetic origin name GL 2626/96 (IT228347) or Pu Ru Ragi (IT196925) as an active ingredient. delete delete The antiviral pharmaceutical composition according to claim 1, wherein infection with the influenza A (H1N1) virus causes any one disease selected from influenza, swine flu, cold, sore throat, bronchitis, pneumonia and avian flu. The antiviral pharmaceutical composition according to claim 1, further comprising a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient. A health functional food composition for antiviral against infection of influenza A (H1N1) virus containing soybean ethanol extract having genetic origin name GL 2626/96 (IT228347) or Pu Ru Ragi (IT196925) as an active ingredient. The antiviral health functional food composition according to claim 6, wherein the composition is prepared in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage. Antiviral feed composition for infection of influenza A (H1N1) virus containing soybean ethanol extract of genetic origin name GL 2626 / 96 (IT228347) or Pu Ru Ragi (IT196925) as an active ingredient. Enhancement of antiviral activity against influenza A (H1N1) virus infection in animals characterized by administering soybean ethanol extract with genetic source name GL 2626 /96 (IT228347) or Pu Ru Ragi (IT196925) to non-human animals method.

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