KR102558581B1 - Composition for preventing or treating PEDV comprising extract of Ulmus davidiana var. japonica supercritical extract residue and Chamaecyparis Obtusa emulsion complex - Google Patents

Abstract

The present invention relates to a composition for preventing or treating porcine epidemic diarrhea virus infection containing an extract of elm supercritical extract and a cypress oil-water complex as active ingredients.

Description

Composition for preventing or treating porcine epidemic diarrhea virus infection containing extract of elm supercritical extract and cypress oil water complex as active ingredients japonica supercritical extract residue and Chamaecyparis Obtusa emulsion complex}

The present invention relates to a composition for preventing or treating porcine epidemic diarrhea virus infection, and more particularly, to a composition for preventing or treating porcine epidemic diarrhea virus infection containing an extract of elm supercritical extract and a cypress oil water complex as active ingredients.

Porcine epidemic diarrhea virus (PEDV) is a virus that causes diarrhea in pigs and occurs throughout the year, causing pig epidemic diarrhea (PED), which causes enormous economic losses to pig farms. PEDV was first reported in Korea in 1992, and it usually sheds the virus in feces and occurs mainly in winter.

PED causes mortality due to diarrhea in piglets, and in the case of adult pigs, economic loss due to weight loss due to diarrhea and its role as a transmission source have become a problem. Although several types of vaccines are currently being introduced in Korea, PEDV is known to cause enormous economic losses to the domestic pig farming industry because the damage caused by PED is not decreasing. In order to prevent diarrhea caused by PEDV as described above, it is based on passive immunity in which antibodies formed by inoculating live vaccines to sows before parturition are transferred to piglets through colostrum. However, in the case of live vaccines, there is a high distrust in the preventive efficacy in outdoor farms, and the incidence of diarrhea in piglets is increasing every year due to non-vaccination. Accordingly, there is a need to develop new drugs for efficiently preventing the above diseases.

On the other hand, in order to use medicinal plants containing many active ingredients for various purposes, research and development are being actively conducted through equipment and systematic analysis methods. Since medicinal plants show marked differences in efficacy depending on the extraction method and conditions, selection of the extraction method and conditions of medicinal plants is an important part in raw material production of medicinal plants. Methods such as hot water extraction, ethanol extraction, ultrasonic extraction, pressurized extraction, and supercritical extraction are commonly known as extraction methods for medicinal plants, and in order to derive the desired effect of medicinal plants, it is necessary to set the conditions in the selected extraction method in detail as well as the selection of the above extraction method.

Conventionally, a composition for treating a disease caused by PEDV using a medicinal plant extract has been known. Korean Patent Registration No. 0839099 relates to a composition for the treatment and prevention of porcine epidemic diarrhea virus containing gall nut and moutan hull extract, and specifically discloses the PEDV infection inhibitory effect of gall nut hot water extract and mudan hull hot water extract. In addition, Korean Registered Patent No. 2160798 relates to a composition for preventing or treating porcine epidemic diarrhea containing a gosteen extract or a xanthone compound derived from a mangosteen extract as an active ingredient. are partitioning

Elm tree (Ulmus davidiana var. japonica) is a deciduous tree of the dicotyledonous nettle family Elmaceae. Six species grow wild in Korea, and there are about 20 species worldwide. In spring, the young leaves are used for food, and the bark is called Yupi, and especially the root bark is called Yugeunpi. Elm has been known to be effective for boils and swelling since ancient times, and recently there have been reports of antioxidant effects, antibacterial effects, anticancer effects, and antiviral effects. In addition, according to Donguibogam, the elm tree is effective for enteritis by getting rid of fever in the intestines and stomach and is effective in relieving insomnia. Conventionally, development of various compositions using the elm tree as described above has been made. Korean Patent Registration No. 1835543 relates to a composition for the prevention and treatment of infectious diseases caused by skin disease-causing bacteria containing an extract of elm, suggesting the antibacterial effect of a hot water extract of elm. In addition, Korean Patent Publication No. 2011-0049129 relates to an anticancer composition of elm extract, and suggests an anticancer (liver cancer, oral cancer, uterine cancer) effect of elm methanol extract. However, in the prior art, the PEDV infection inhibitory effect of the extract of elm supercritical extraction foil is not disclosed at all.

On the other hand, cypress (Chamaecyparis obtusa), also called cypress, is an evergreen arboreal tree of the gymnosperm coniferous Arborvitaceae family, and is widely cultivated as a plantation species in the southern regions of Korea. Phytoncide is known to contain sterilization and insecticidal components because it is secreted to resist pathogens, pests, molds, etc. as a self-defense material of plants produced in cypress trees. Phytoncide is composed of organic compounds such as phenolic compounds, alkaloids, and glycosides, and is known to have antibacterial, stress relieving, and sedative effects. Conventionally, development of various compositions using cypress trees as described above has been made. Korean Patent Publication No. 2016-0004878 relates to a method for producing cypress extract and a composition comprising the same, and discloses cypress oil, cypress extract, and stress hormone reducing effect, antibacterial and antifungal effect, and skin allergy improvement effect of cypress oil and cypress extract mixture. However, the prior art does not disclose the effect of inhibiting PEDV infection of cypress oil water at all.

Therefore, as a result of intensive research efforts to overcome the problems of the prior art, the present inventors confirmed that the active ingredient content in the extract of elm supercritical extract was significantly higher than the active ingredient content in the elm extract, and selected as an active ingredient for the composition for preventing or treating porcine epidemic diarrhea virus infection of the present invention. In the case of the extract of elm supercritical extract and cypress oil water complex, it was confirmed that porcine epidemic diarrhea virus infection can be prevented or treated, and the present invention was completed. It became.

KR 10-0839099 B1 KR 10-2160798 B1 KR 10-1835543 B1 KR 10-2011-0049129 A KR 10-2016-0004878 A

Accordingly, the main object of the present invention is to provide a composition for preventing or treating porcine epidemic diarrhea virus infection containing an extract of elm supercritical extract and a complex of cypress oil water as active ingredients.

Another object of the present invention is to provide a feed composition for preventing or improving porcine epidemic diarrhea virus infection containing an extract of elm supercritical extract and a cypress oil water complex as an active ingredient.

According to one aspect of the present invention, the present invention provides a composition for preventing or treating porcine epidemic diarrhea virus infection containing an extract of elm supercritical extract and a cypress oil water complex as active ingredients.

When preparing a complex preparation by mixing cypress oil itself with the extract of elm supercritical extract included as a composition for preventing or treating porcine epidemic diarrhea virus infection of the present invention, homogeneous mixing between cypress oil (oil) and plant extract (liquid) is impossible. In order to improve the problem of having to add chemical agents such as surfactants and alcohols, an emulsified water manufacturing device (Korean Patent No. 2057834) is used to prepare cypress oil water, which is the present invention was used to prepare a composite of Cypress oil water according to the present invention is produced by slowly mixing water or extraction water (cypress water) with cypress oil. More specifically, after homogenization by gradually mixing and stirring cypress oil with water (purified water) or extraction water (cypress water), a high-pressure pump is used to pass through an emulsifier under pressure, resulting in a stable cypress oil water in which cypress oil and water (purified water) or extraction water (cypress water) are not separated by complex mechanisms such as impact, compression, and vortex.

In the present invention, mixing with the extract of the elm supercritical extract using the cypress oil water prepared as described above, rather than the cypress oil, can easily obtain a homogeneous complex by mixing between liquids.

In the composition for preventing or treating porcine epidemic diarrhea virus infection of the present invention, the elm supercritical extraction foil is added to the pulverized elm tree in a supercritical extraction vessel, then carbon dioxide is flowed into the extraction vessel in a supercritical state at a temperature of 10 to 100 ° C. and an atmospheric pressure of 100 to 600 bar, and then 0.5 to 0.6 L of ethanol for 120 to 200 minutes for a total of 40 to 100 L It includes the extracted foil obtained after extraction by inputting. If the above conditions of temperature and atmospheric pressure are not satisfied, it is difficult to perform supercritical fluid extraction normally.

In the composition for preventing or treating porcine epidemic diarrhea virus infection of the present invention, the extract of elm supercritical extraction foil includes extracting elm supercritical extraction foil with water, lower ethanol having 1 to 4 carbon atoms or a mixed solvent thereof.

Based on the fact that medicinal plants show significant differences in efficacy depending on the extraction method and solvent, various extraction methods and extraction conditions were applied to the elm tree. As a result, the extract obtained after supercritical extraction of the elm tree was obtained after supercritical extraction of the elm tree rather than the elm tree extract.

According to an experimental example of the present invention, as a result of comparing the active ingredient content in the extract of elm supercritical extraction gourd and the elm extract, it was confirmed that the content of catechin glycoside, 7-O-β-D-apiofuranoside, which is an active ingredient, was about 10 times higher in the extract of elm supercritical extraction gourd (see Experimental Example 1 and Table 2). Through these results, the extract of elm supercritical extract was selected as a component of the composition for preventing or treating porcine epidemic diarrhea virus of the present invention.

Hereinafter, the method for preparing the extract of elm supercritical extract, which is an essential component of the composition for preventing or treating porcine epidemic diarrhea virus according to the present invention, will be described in detail step by step.

The extract of the elm supercritical extraction foil is obtained by (a) supercritical extraction of the elm tree to obtain a supercritical extraction foil; And (b) it is prepared through a manufacturing method comprising the step of obtaining an extract for the obtained elm supercritical extraction foil.

Specifically, in the step (a) of obtaining supercritical extraction foil by supercritical extraction of elm, the supercritical extract residue of elm refers to the residue left after extracting elm by a supercritical extraction method.

The elm tree used in the manufacture of the supercritical extraction foil of the present invention includes Ulmus plants, such as Ulmus davidiana, Ulmus davidiana var. japonica, Ulmus davidiana var. japonica for. (Ulmus laciniata) and one or more Elm genus plants selected from the group consisting of Ulmus macrocarpa, preferably Ulmus davidiana var. japonica, Ulmus macrocarpa or Ulmus davidiana.

The whole of the elm tree or parts of the elm tree selected from the group consisting of stems, roots, leaves, flowers, fruits, and seeds may be used, and preferably the branches, stems or roots of the elm tree may be used, and may be dried and / or pulverized before the supercritical extraction process.

In the present invention, the supercritical extraction method refers to a method of extracting fragrances, pigments, oils, or functional substances contained in natural products using a fluid in a supercritical state without denaturation. The fluid in a supercritical state means a fluid in a state in which gas and liquid are not distinguished beyond a certain high-temperature and high-pressure limit of a critical point. The supercritical fluid may be, for example, supercritical carbon dioxide. The supercritical carbon dioxide is carbon dioxide in a state exceeding the critical temperature (31° C.) and critical pressure (7.5 MPa), and is a fluid having both gas and liquid properties.

The supercritical extraction method in the present invention may be carried out, for example, by a pressure change method in which a mixture of a supercritical fluid and a solute is decompressed and expanded at the same temperature as the extraction temperature so that the solute is separated while the solvency of the supercritical fluid is reduced, or a temperature change method in which the supercritical fluid and the solute are separated by increasing the temperature of the supercritical fluid to decrease the solvency, but is not limited thereto. In addition, a co-solvent may be used together with the supercritical fluid, and the co-solvent includes a polar solvent commonly used in the art, preferably selected from the group consisting of alcohol, water, ethylene glycol, polyethylene glycol, propylene carbonate, formic acid, acetic acid, acetonitrile, chlorodifluoromethane, or mixtures thereof.

In the present invention, the vessel for supercritical extraction can be controlled in temperature and pressure, and is not particularly limited as long as it is a vessel configured to contact the extraction raw material and the supercritical fluid. In the present invention, the pressure in the supercritical extraction may be in the range of 100-600 bar, the temperature in the supercritical extraction may be in the range of 10-100 ° C, and the flow rate of the supercritical fluid and cosolvent in the supercritical extraction may be 10-100g / min. A person skilled in the art may appropriately adjust and use the pressure, temperature, flow rate of the fluid and cosolvent within the above ranges in supercritical extraction.

As described above, supercritical extraction foil, which is a residue generated after supercritical extraction of elm trees, is collected, and the collected supercritical extraction foil is used as a raw material for solvent extraction.

Next, (b) obtaining an extract of the obtained supercritical extraction foil of elm means obtaining an extract of supercritical extraction foil of elm through a solvent extraction method for the supercritical extraction foil.

The extract of the supercritical extraction foil of the elm tree of the present invention can be separated according to a conventional method known in the art for extracting an extract from a natural product, that is, using a conventional solvent under conditions of ordinary temperature and pressure. Specifically, a polar solvent or a non-polar solvent can be used. The polar solvent may be selected from the group consisting of water, alcohol (eg, methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol), acetic acid, DMFO (dimethylformamide) and DMSO (dimethyl sulfoxide), and non-polar solvents may include acetone, acetonitrile, ethyl acetate, methyl acetate, Fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, It may be selected from the group consisting of 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF (tetrahydrofuran), but is not limited thereto.

In the present invention, the solvent extraction process is completed by partial or complete removal of the extraction solvent, partial removal meaning concentration until an aqueous concentrate free of appreciable amounts of organic solvent is obtained, complete removal yielding a dry residue.

Hereinafter, a method for preparing cypress oil water, which is an essential component of the composition for preventing or treating porcine epidemic diarrhea virus according to the present invention, will be described in more detail step by step.

The cypress oil water is prepared by (a) heating and distilling one or more parts selected from leaves, stems, and fruits of cypress in a cauldron under normal pressure or reduced pressure; (b) collecting the steam generated in the heating and distillation steps and cooling and condensing it; (c) separating oil and water to recover oil and extraction water contained in the cooled or condensed extract; And (d) preparing through a manufacturing method comprising the step of preparing cypress oil water by mixing the separated oil with water (purified water) or extraction water (cypress water).

Specifically, at least one part selected from the leaves, stems and fruits of the (a) cypress tree is heated in a cauldron under normal pressure or reduced pressure, and the heating process in the distillation step is performed at a temperature range of 80 ° C to 105 ° C. When heated below 80 ° C, many components of low-molecular compounds with low boiling points among phytoncide components in cypress trees are contained, but low-molecular compound components are contained.

In addition, the distillation process means to heat a liquid to the boiling point and cool the evaporated material to make it into a liquid again.

In the present invention, a liquid obtained by collecting steam generated after heating by additionally adding water to cypress trees containing moisture can be prepared as an extract.

Next, in the step of (b) cooling and condensing, the steam generated through the heating and distillation process is collected at a temperature range of 80 ° C to 105 ° C, cooled and condensed.

Next, (c) the oil-water separation step using the difference in specific gravity between the oil and the extraction water (cypress water) in order to recover the oil and extraction water contained in the cooled or condensed extract is the oil floating on the condensed extract using an oil-water separator. The floating oil and the extraction water (cypress water) precipitated below can be physically separated, but is not limited thereto.

Next, (d) preparing cypress oil water by mixing the separated oil and water (purified water) or extraction water (cypress water) can be prepared using an emulsified water production device commonly used in the art, but is not limited thereto.

When preparing a complex preparation by mixing cypress oil itself with the extract of elm supercritical extract included as a composition for preventing or treating porcine epidemic diarrhea virus infection of the present invention, cypress oil (oil) and plant extract (liquid) Since homogeneous mixing between is impossible, cypress oil is prepared and used to improve the problem of having to add chemical agents such as surfactants and alcohols. Specifically, the method for producing cypress oil water is a method of greatly enhancing the antibacterial, antifungal, anticancer, and anti-inflammatory functions of cypress tree phytoncide. Cypress tree is placed in a cauldron, heated and distilled, and the resulting steam is cooled and condensed to produce cypress oil with high concentration of cypress tree phytoncide. Then, when the cypress oil is gradually put into water (purified water) or extraction water (cypress water) and stirred to homogenize, and then passed through an emulsifier by pressurization using a high pressure pump, cypress oil and water (purified water) or extraction water (cypress water) is used to produce stable cypress emulsified water that is not separated into oil and water.

In the composition for preventing or treating porcine epidemic diarrhea virus infection of the present invention, the cypress oil water is nano-dispersion method, ultrasonic method, impact method, compression method and swirl mechanism without chemical additives in order to fuse cypress oil with water (purified water) or extraction water (cypress water).

In the composition for preventing or treating porcine epidemic diarrhea virus infection of the present invention, the cypress oil water is obtained by oil-water separation of the cypress extract, and purified water or extract water is mixed in a weight ratio of 0.05 to 0.50%: 99.95 to 99.50%. When the content of the oil exceeds 0.5%, the antibacterial and antifungal properties of the cypress oil water increase, but it is undesirable from an economic point of view due to the increase in manufacturing cost.

In the composition for preventing or treating porcine epidemic diarrhea virus infection of the present invention, the extract of elm supercritical extraction gourd and cypress oil water complex is 0.001 to 0.1%: 99.999 It includes a mixture of cypress oil water and extract of elm supercritical extraction gourd in a weight ratio of 99.999 to 99.9%. When the content of the elm supercritical extraction foil exceeds 0.1%, the antioxidant, antibacterial, anticancer, and antiviral functions of the elm supercritical extraction foil are not desirable from an economic point of view because the effect of the elm supercritical extraction foil is not high compared to the content of the complex.

In the composition for preventing or treating porcine epidemic diarrhea virus infection of the present invention, the extract of the elm supercritical extract and the cypress oil water complex contain 10 to 60% by weight based on the total weight of the composition. When the composite is included at less than 10% by weight relative to the total weight of the composition, the effect of preventing or treating porcine epidemic diarrhea virus infection may be insignificant, and when it exceeds 60% by weight, the effect compared to the content of the composite is not high, which is not preferable from an economic point of view.

Since the extract of elm supercritical extraction gourd and the cypress oil water complex according to the present invention have little toxicity, they can be safely used even during long-term administration for preventive purposes.

The composition comprising the extract of elm supercritical extract and the cypress oil water complex according to the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions, and the pharmaceutical dosage form may be used in the form of a pharmaceutically acceptable salt thereof, and may also be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.

The composition comprising the extract of elm supercritical extraction foil and the cypress oil water complex according to the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions, respectively, according to conventional methods.

The preferred dosage of the composition comprising the extract of elm supercritical extract and cypress oil water complex according to the present invention varies depending on the condition and weight of the patient, the degree of disease, drug type, administration route and period, but can be appropriately selected by those skilled in the art. However, for a desirable effect, the composition containing the extract of the supercritical elm extract and the cypress oil water complex of the present invention is preferably administered at 0.0001 to 1,000 mg/kg. Administration may be administered once a day or divided into several doses, but the dosage is not intended to limit the scope of the present invention in any way.

The composition comprising the extract of elm supercritical extract and the cypress oil water complex according to the present invention can be administered through various routes. All modes of administration can be envisaged, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

According to another aspect of the present invention, the present invention provides a feed composition for preventing or improving porcine epidemic diarrhea virus infection containing an extract of elm supercritical extract and a cypress oil water complex as an active ingredient.

The feed composition for preventing or improving swine epidemic diarrhea virus infection according to the present invention may be prepared as a feed additive in the form of a powder, tablet, capsule, pill or liquid, and the extract of elm supercritical extract and cypress oil complex in the feed The amount of the composition may be included in 0.01 to 15% by weight relative to the total weight of the feed composition. When the composite is included at less than 0.01% by weight relative to the total weight of the composition, the effect of preventing or treating porcine epidemic diarrhea virus infection may be insignificant, and when it exceeds 15% by weight, the effect compared to the content of the composite is not high, which is not preferable from an economic point of view.

As described above, since the extract of elm supercritical extraction foil has a significantly higher content of active ingredients than the elm extract, the composition comprising the extract of elm supercritical extraction foil and the cypress oil water complex has a preventive or therapeutic effect of porcine epidemic diarrhea virus infection. It is excellent.

Figure 1 is a schematic diagram showing the process of extracting the ethanol extract (USCFR) of the supercritical extraction foil of the elm branch of the present invention.
Figure 2a is the result of HPLC analysis of catechin 7-O-β-D-apiofurannoside as a standard material, and Figure 2b is the result of HPLC quantitative analysis of catechin 7-O-β-D-apiofurannoside in the ethanol extract of elm supercritical extraction foil prepared in the preparation example of the present invention.
Figure 3 is the result of confirming the cytotoxicity according to Experimental Example 2.
Figure 4 is the result of confirming the antiviral activity according to Experimental Example 3.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are intended to illustrate the present invention only, the scope of the present invention is not to be construed as being limited by these examples.

Preparation Example: Preparation of ethanol extract of elm supercritical extraction foil

Elm tree (Ulmus) branches were purchased from Yangnyeong Market in Seoul, foreign substances were removed, washed, and then dried in the shade to be used as experimental materials. Supercritical extraction of elm branches was performed using supercritical fluid extraction production equipment (SCFE-P400, Ilsin Autoclave, Daejeon, Korea). Elm branches were purchased from Yangnyeong Market in Seoul, foreign substances were removed, washed, and then dried in the shade to be used as experimental materials. 100 kg of the dried sample was pulverized to pass through a 200 mesh pulverizing net, and the temperature of the pulverized elm branch was adjusted to 50° C. in the extraction tank to maintain the temperature. When the temperature is stabilized, the elm branch sample is put in, the CO ₂ gas is maintained at an equal pressure, and then the control valve is adjusted and injected until the experimental pressure condition of 400 bar is reached through a line using a high-pressure pump. After reaching the set pressure, extraction was performed by injecting a total of 60 to 80 L of ethanol (alcohol: edible alcohol) at a rate of 0.5 to 0.6 L per minute for 160 minutes at the bottom of the extraction tank, and the pressure and temperature set to remove the residual ethanol remaining in the sample. Extraction was completed by flowing CO ₂ using a high pressure pump for 60 to 120 minutes.

After supercritical extraction of elm branches as described above, 100 kg of the obtained elm supercritical extraction foil sample was dried in the shade, extracted once with 60% ethanol at room temperature, and filtered. The extract was concentrated under reduced pressure and lyophilized to obtain a final weight of 4.81 kg, and then used in the experiment. In the present invention, the supercritical extraction foil ethanol extract of elm branches was marked as "USCFR" (see Fig. 1).

Comparative Preparation Example: Preparation of elm ethanol extract

Elm tree (Ulmus) branches were purchased from Yangnyeong Market in Seoul, foreign substances were removed, washed, and then dried in the shade to be used as experimental materials. Elm branches prepared by drying in the shade were extracted once with 60% ethanol at room temperature and filtered. The extract was concentrated under reduced pressure and freeze-dried to obtain a final weight of 4.62 kg, and then used in the experiment. In the present invention, the ethanol extract of elm branches is indicated as "UR60E".

Experimental Example 1: HPLC analysis of alcohol extract of elm supercritical extraction foil

In order to analyze the content of catechin 7-O-β-D-apiofurannoside in the supercritical extraction foil ethanol extract of elm branches prepared in Preparation Example and the elm alcohol extract prepared in Comparative Preparation Example, a Waters 2695 separation module was used, and a Waters 2487 Dual λ Absorbance Detector was used as a detector. A SkyPak C18 column (250 X 4.6mm) was used as the column, and the column temperature was set at 25°C. The mobile phase was D.W. (A) and acetonitrile (B), and the analysis was performed using the Gradient program shown in Table 1.

Time (minutes) %A %B 0 95 5 5 90 10 7 85 15 30 75 25 31 95 5 35 95 5

The HPLC analysis result of catechin 7-O-β-D-apiofurannoside, which is a standard substance (active ingredient), is shown in FIG. 2a, and the HPLC quantitative analysis result of catechin 7-O-β-D-apiofurannoside in the ethanol extract of elm branch supercritical extraction foil (preparation example; USCFR) is shown in FIG. 2b, and the catechin 7-O-β-D- The content of apiofurannoside is shown in Table 2.

sample name RT(min.) Content (μg/ml) Catechin 7-O-β-D-Apiofurannoside 14.449±0.14 - Comparative Manufacturing Example 14.271±0.12 12.16±0.15 manufacturing example 14.482±0.16 131.45±0.18

As a result, as shown in Table 2, the content of catechin 7-O-β-D-apiofurannoside in the extract of the supercritical extraction foil of elm branches prepared in Preparation Example of the present invention was about 132 ug/ml, which was confirmed to be about 10 times higher than the content of catechin 7-O-β-D-apiofurannoside in the elm extract prepared in Comparative Preparation Example.

Through these results, the alcohol extract of supercritical elm branch supercritical extract with a significantly high content of active ingredients was selected as a component of the composition for preventing and treating porcine epidemic diarrhea virus infection.

Example: Preparation of alcohol extract of elm supercritical extraction gourd and cypress oil water complex

In order to prepare a composite of the alcohol extract of elm supercritical extraction foil and cypress oil water, cypress oil water was prepared.

Cypress oil water was prepared by warming cypress tree leaves in a cauldron at 80 ° C to 108 ° C, collecting steam generated by distillation, cooling and condensing cypress oil (0.05%), which was separated from oil and water, into a homogenization tank of an emulsifier filled with purified water (99.95%) in advance. .

99.98% by weight of cypress oil water prepared in Preparation Example was mixed with 0.02% by weight of alcohol extract of elm supercritical extraction foil and stirred to prepare an alcohol extract and cypress oil water complex of elm supercritical extraction foil.

Experimental Example 2: Confirmation of cytotoxicity (MTT assay)

MTT assay was used to determine the treatment concentration for vero cells of the composites prepared in Examples, and this quantification method was performed by modifying Mosmann's method. This assay uses the ability of mitochondria dehydrogenase in living cells to change the yellow water-soluble substrate MTT to dark blue water-insoluble formazan. The amount of formazan produced is proportional to the number of viable cells.

Specifically, by culturing the cells, put 180 μl of the cells in a 96 well plate with medium so that the number of cells per well is about 1×10 ⁵ , and incubate for 24 to 48 hours in an incubator (37° C., 5% CO ₂ ). Then, add 50 μl of MTT solution (0.5 mg/ml) and incubate again for 4 hours. Remove the supernatant, add 150 μl of DMSO to the formazan precipitate, leave it at room temperature for 15 minutes to dissolve formazan, and measure the absorbance with an ELISA reader at a wavelength of 570 nm to calculate cell viability. The results are shown in FIG. 3 by calculating % with respect to the control as in Equation 1.

[Equation 1]

As a result, as can be seen in Figure 3, no specific cytotoxicity was confirmed at all experimental concentrations of the composite (alcohol extract of elm supercritical extraction gourd and cypress oil water) prepared in Example.

Experimental Example 3: Measurement of inhibitory effect on porcine epidemic diarrhea virus

In order to confirm the inhibitory effect of the composite prepared in Example (the ethanol extract of elm supercritical extract and cypress oil water) against porcine epidemic diarrhea virus, the present inventors performed the following in vitro experiment using monkey kidney cell line Vero cells.

The method for measuring the virus growth inhibitory ability of the elm supercritical extract ethanol extract and the cypress oil water complex against porcine epidemic diarrhea virus (PEDV) was measured based on the method described in Korean Patent No. 0720151. The inhibitory effect was measured. Specifically, Vero cells were cultured in each well of a 96-well microplate until the bottom was completely covered with cells. After removing the existing culture medium and washing each well twice with phosphate buffer, a PEDV solution adjusted to a concentration of TCID50 was added to each well, the complex prepared in Example was dissolved in dimethylsulfoxide, and each solution was administered to each well at a concentration of 0.4 to 100 μg/ml and cultured for 48 hours. The shape of Vero cells was observed under a microscope to determine the antiviral activity. 4.

In addition, 70% acetone is added to each well, left at -20 ° C. for 1 hour, dried in a dryer, and 100 μl of 0.4% (w/v) SRB (srb) solution melted in 1% (v/v) acetic acid and dyeed for 30 minutes, and then SRB dyeing solution that is not binded to cells 1% (V) /V) washing 4 times with acetic acid and then dried again. At the bottom of each well, 100 μl of a 10 mM Tris solution (pH 10.5) of a dye bound to cells was added to each well to dissolve the dye, and then the absorbance was measured at 562 nm to compare the virus inhibitory effect with that of the control group. At this time, cells treated with only DMSO and cells treated with DMSO and PEDV were used as controls to confirm the inhibitory effect of the compound of the present invention against porcine epidemic diarrhea virus, and the results are shown in FIG. 5 .

As a result, as can be seen in Figure 5, 45.1 ± 3.21% at a concentration of 0.4 μg / ml of the complex, 69.6 ± 2.71% at a concentration of 0.8 μg / ml, 90.2 ± 5.78% at a concentration of 20 μg / ml, and 113.2 ± 0.36% at a concentration of 100 μg / ml. The ethanol extract and the cypress oil water complex have excellent antiviral efficacy.

All of the above experiments were repeated three times, and the average value and standard deviation were presented. In addition, using SPSS (Statistical Package for Social Science, ver. 18, IBM, Chicago IL. USA), significance was verified by Student's t-test on the average value of the experimental group and control group at P<0.05 level.

Claims (7)

A composition for preventing or treating porcine epidemic diarrhea virus infection containing an extract of elm supercritical extract and a complex of cypress oil water as active ingredients.
According to claim 1,
The elm supercritical extraction foil,
After adding the pulverized elm to a supercritical extraction vessel, carbon dioxide is flowed into the extraction vessel in a supercritical state at a temperature of 10 to 100 ° C. and an atmospheric pressure of 100 to 600 bar, and then 0.5 to 0.6 L of ethanol is added for 120 to 200 minutes. A composition for preventing or treating porcine epidemic diarrhea virus infection obtained after extraction by adding a total of 40 to 100 L.
According to claim 1,
The extract of the elm supercritical extraction foil is obtained by extracting the elm supercritical extraction foil with water, lower ethanol having 1 to 4 carbon atoms or a mixed solvent thereof, a composition for preventing or treating porcine epidemic diarrhea virus infection.
According to claim 1,
The cypress oil water is a composition for preventing or treating porcine epidemic diarrhea virus infection, which is a mixture of oil and purified water or extraction water obtained by separating the cypress extract from oil and water in a weight ratio of 0.05 to 0.50%: 99.95 to 99.50%.
According to claim 1,
The extract of elm supercritical extract and cypress oil complex is a mixture of extract of elm supercritical extract and cypress oil in a weight ratio of 0.001 to 0.1%: 99.999 to 99.9%, porcine epidemic diarrhea virus infection Prevention or treatment Composition.
According to claim 1,
The extract of the elm supercritical extract and the cypress oil water complex is a composition for preventing or treating porcine epidemic diarrhea virus infection, comprising 10 to 50% by weight relative to the total weight of the composition.
A feed composition for preventing or improving swine epidemic diarrhea virus infection containing an extract of elm supercritical extract and a complex of cypress oil water as active ingredients.