KR101140582B1 - Composition for prevention or treating Porcine Epidemic Diarrhea containing contract of Epimedium koreanum - Google Patents

Abstract

The present invention relates to a composition having antiviral activity, and more particularly, to a composition for preventing and treating porcine epidemic diarrhea (PED), which comprises an extract of Eumyang Kwak as an active ingredient.

Since the composition of the present invention has an excellent anti-virus against porcine epidemic diarrhea virus (PEDV), it can be used as a pharmaceutical composition and feed additive useful for the prevention and treatment of diseases caused by the virus infection. Can be.

Description

Composition for prevention or treating Porcine Epidemic Diarrhea containing contract of Epimedium koreanum}

The present invention relates to a composition for the treatment and prevention of swine epidemic diarrheal disease, which comprises the extract of Eum Yang Kwak.

Porcine Epidemic Disease (PED), along with Transmissible Gastroenteritidis (TGE), is a viral disease that causes great damage to pig farms in winter. Diarrhea can also be caused to foster and hog pigs, and high mortality rates can be a problem if they occur in young piglets. In particular, damage in Asia is prominent, and in Japan, 39,509 of the 56,256 heads of piglets in nine prefectures and 18 farms died in the winter of 1996, with 70% mortality. Corona virus includes calf coronavirus, swine TGE (Transmissible Gastroenteritidis) virus, hemagglutinating encephalomyelitis virus, feline infectious peritonitis virus, canine coronavirus, and bird coronavirus. Avian infectious bronchitis virus, mouse hepatitis virus and Severe Acute Respiratory syndrome (SARS) virus in humans belong to the corona virus.

PEDV's first report was Europe, first reported in the United Kingdom and Belgium in 1978, and has been occurring year-round regardless of season since Korea's first occurrence in 1993. In recent years, PEDs have been much higher than TGE, causing economic damage to hog farmers. In order to prevent such diarrhea, passive immunization has been mainly carried out so that antibodies formed by inoculating live vaccine into sows before delivery are delivered to mammalian pigs through colostrum. Domestic vaccine production companies also produce and sell live PED vaccines. Japanese foreign PED vaccines and PED-TGE mixed vaccines are imported.

However, in the case of live vaccines, in particular, the incidence of diarrhea in piglets is increasing every year due to the lack of vaccination due to the high distrust of preventive efficacy in outdoor farms for PEDV. Accordingly, there is an urgent need for the development of efficient formulations for effectively preventing or treating the diseases.

Therefore, the present inventors have conducted research to develop a natural-derived therapeutic agent that reduces diarrhea and small intestine damage caused by PEDV, the cause of swine pandemic diarrheal disease. The present invention was completed by identifying significant results in motility, feed intake, and the like.

An object of the present invention is to provide a composition for treating swine pandemic diarrheal disease that can effectively increase the anti-viral effect on swine pandemic diarrheal virus and efficiently supply large quantities to farms.

The present invention relates to a composition for preventing or treating swine pandemic diarrheal disease, characterized in that it comprises a medicinal herb extract as an active ingredient. More specifically, the present invention relates to a composition for the prevention or treatment of diseases caused by swine epidemic diarrhea virus (PEDV) comprising the extract of Eum Yang Kwak as an active ingredient. Diseases caused by PEDV of the present invention include swine noxious diarrheal disease (PED) and include those occurring in mammals, including humans.

The medicinal herb may be used as long as it is a medicinal herb that inhibits the propagation of viruses causing swine pandemic diarrheal disease. In the following examples, yin and yang were used.

The Yin Yang Kwak is the terrestrial part of Epimedium koreanum Nakai or other homologous root plants of the barberry family. This medicine is odorless, tastes spicy, sweet and warm in nature. Eum yanggae is used for erectile dysfunction, oil well, cold uterus, cold limb, skin paralysis, gut wasabi, forgetfulness, paraplegia, back and knee weakness, hypertension, polio. Pharmacological action promotes semen secretion, lowers blood pressure, increases coronary blood flow, lowers blood sugar, lowers cholesterol, boosts immune function, Jinhae, expectoration, Pyeongcheon, sedation, fungi, anti-inflammatory action, chicken femoral growth and protein polysaccharide activation Reported.

 In the present invention, the yin-yang box may be extracted by supercritical extraction by a conventional method using carbon dioxide and water as a solvent. In addition, the present invention may be extracted by heating the yinyanggak in a polar solvent such as ethanol and methanol or a mixed solution of them and purified water, it is possible to obtain the yinyangkuk extract by various methods such as concentration under reduced pressure. Preferably, hot water, methanol or the like may be used to obtain the Eum yang extract. More preferably, the yin yang extract of the present invention is soluble in a solvent selected from water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

Although the content of the herbal extract is not particularly limited, in order to effectively exhibit the antiviral effect expected in the therapeutic composition, it is preferable to contain 0.1 to 50% by weight based on the total weight of the composition.

Since the extract of the present invention has little toxicity and side effects, it can be used with confidence even during prolonged administration for the purpose of prevention.

The composition comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The pharmaceutical dosage forms of the extracts of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

Compositions comprising extracts according to the invention are formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be used. Carriers, excipients and diluents that may be included in the composition comprising the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, or the like in the compound. Or lactose, gelatin, or the like is mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propyleneglycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient or subject, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 1,000mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The extract of the present invention can be administered to mammals such as rats, mice, livestock, humans and the like in various routes. All modes of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In addition, the present invention relates to a health functional food for the prevention or improvement of diseases caused by swine epidemic diarrhea virus (PEDV) containing the extract of Eum Yang Kwak as an active ingredient. The functional food of the present invention may be prepared in tablet, capsule, pill or liquid form. Examples of the food to which the extract of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to food or beverages for the purpose of preventing the disease caused by the swine epidemic diarrhea virus. At this time, the amount of the extract in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml. have.

The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrzin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, such as flavoring agents, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually chosen in the range of 0 to 20 parts by weight per 100 parts by weight of the extract of the present invention.

As described above, the present invention can effectively increase the antiviral effect against the epidemic diarrheal virus in pigs, and by treating the epidemic diarrheal disease in farms by including a herb as an active ingredient having a proliferation inhibitory effect on PEDV as an active ingredient There is an effect that can efficiently supply a large amount of the composition for.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art to which the present invention pertains. Will be self-evident.

<Examples>

Example 1 Preparation of Yin-Yang Extract

After weighing 63.43g of dried Yin-Yang Kwak, 1L of bottled water (White, Muhaksan Cheong-Sam Water Co., Ltd., Deokgyo-ri, Gyeongnam Sancheong-gun, Deokgyo-ri) was immersed for 1 hour. After boiling to 100 mesh (mesh) to extract the solution (100 ml). The test material was diluted 20-fold and used for this experiment.

Example 2: Viruses and Cell Lines

The virus used in this example was KPEDV-9 strain, sm98 strain, which is a vaccine of PEDV, and the cells used for culture were vero-cell purchased from Seoul National University Cell Line Bank, and alpha minimum essential medium (alpha-MEM). Incubated at.

Example 3: Determination of Herbal Medicine Concentration

The water was reacted for one hour at 90 ℃ pre 5x10 ^ 4 / well of the 96well cell sewn to dispense a crude water extract 3.2, 1.6, 0.8 , 0.4, 0.2, 0.1 and 0.05 ㎎ / ml concentration of medicinal herbs (H ₂ Diluted in O) was added to the cell culture solution, and cultured for 96 hours to confirm whether the cells were necrotic. Subsequently, the culture medium of the plate wells in which cells were not necrotic was taken, and cytotoxicity was measured using a cytotoxicity assay kit.

Example 4: Virus Inoculation

Dispense VERO-CELL into 1 × 10 ⁶ cells / flask in a 25 cm ² cell culture flask and add 5% of virus fluid (KPEDV-9) to MOI (multiplicity of infection) = 0.01 when monolayer After incubation for 1 hour in a CO ₂ , 37 ° C. incubator, the culture medium was removed, washed with PBS (phosphate buffered saline), and fresh medium was added. At this time, the denatured effect of the cell aggregation is broken through the inverted microscope up to 3, 6, 9, 12, 24, 36, 48, 72 and 96 hours It was observed whether (cytopathic effects, CPE) appeared, and 120μl of the culture solution was harvested and stored at -80 ℃ over time.

Example 5: Preparation of One step Growth curve

Cells were prepared in 6-well plates and dispensed at a concentration of 1x10 ⁶ cell / well and monolayered at 5% CO ₂ , 37 ° C incubator. And incubated for 1 hour at presented earlier medicinal herb was added to each 100 ㎕ after inoculation was diluted with diluted while observing the CPE decimal virus culture who keep harvested over time up to 10 ^-6 37 ℃ incubator virus adsorption to the cells.

Agarplaque LE-agarose was made 1.5% in distilled water, dissolved in microwave, then left to stand in 45 ℃ water-bath and mixed with alpha-MEM to finally make 0.5% agarose. After completely removing the medium from the plate, the agarose gel prepared above was overlaid on the infected cells, and then incubated for 4-5 days at 37 ℃ incubator. After that, check whether the plaque was formed under a microscope, fix with 7% formalin, remove agarose, dye with crystal violet, and wash with water.

After drying, plaque-reading was performed. Proliferation curves were prepared to know the titer of the medicinal herbs added with the cultivation time.

Experimental Example

Experimental Example 1: Evaluation of the Effect of Eumyangkum Extract on PEDV Growth Curve

After inoculation according to the virus concentration (MOI = 0.1), the Chinese herbal medicine was added to the virus harvested by time and titer was determined by quantitative-PCR.

As a result, in the case of POSITIVE-CONTROL as shown in Figure 1 PEDV titer increased over time (A), the virus harvested from the cells added to the extract of Yin Yang-kyeok extract came out negative after 24 hours (B).

Next, in order to reaffirm the effect of the extract of Eumyangkum, the virus was harvested hourly with the virus concentration as MOI = 0.01, and titer was determined by quantitative-PCR. As a result, the same result as in FIG. 2 was obtained. That is, according to Figure 2, the PEDV virus titer was increased over time in the control group (A), but the PEDV virus titer was negative in 48 hours after the addition of the Eumyang extract (B). there was.

In addition, after inoculating the virus by concentration, the virus propagation rate was measured by time. The measurement method was performed by the plaque assay. As a result, it was confirmed that the virus titer of the experimental group (PED + Yin-Yum) to which Yin-Yang was added was significantly lower than that of control (PED) (see Table 1 and FIG. 3).

[Table 1] Virus growth rate by time (Virus unit: Log titer PFU / ML)

virus virus + Yin-Yang Extract added moi = 0.01 moi = 0.1 moi = 0.01 moi = 0.1 0h 4 4 4 4 18h 5.4x10 ^ 6 2.4x10 ^ 7 0 20 24h 3.4x10 ^ 7 1x10 ^ 7 0 30 48h 2x10 ^ 7 1.4x10 ^ 7 3.5x10 ^ 3 1x10 ^ 3

Experimental Example 2: Evaluation of the Effect on Plaque Shape or Size-Plaque Assay

Plaque assay evaluated the effect on the shape or size of the plaques. Plaque is formed by infecting a cell with an infectious virus. The result of a plaque assay is a method that can directly measure the number of virus particles. Plaque assay was carried out in the same manner as shown in Example 5 . Here, the reason for evaluating the shape or size of the plaque is that the virus titer is lowered by the yin and yang extract, so that the shape or size of the plaque may be small or invisible due to the virus mutation. As a result, as shown in Figure 4, when compared with the control (A) when added the extract of the yeast extract (B) there was no significant change in the shape or size of the plaque (plaque). In other words, there was no morphological change in the plaque, and the number of infectious viruses was found to be reduced in the Yin Yang-gak extract added group (see FIG. 4). As a result, the inhibitory effect on the PEDV of yinyanggak extract could be confirmed.

Experimental Example 3: Evaluation of the Effect of Yin Yang Kwak on CPE in PEDV Cell Culture

Incubate VERO-CELL with 1 × 10 ⁶ cells / flask in a 25 cm ² cell culture flask and add 5% CO ₂ , 37 ° C incubator after adding moi (multiplicity of infection) = 0.01 to the virus solution (KPEDV-9). After 1 hour of infection in the culture medium was removed and washed with PBS (phosphate buffered saline) and then added to the medium. After that, the extract of Yin-Yang and added to evaluate the CPE by time.

As a result, Figure 5 is confirmed by magnification 200 times by the optical microscope whether CPE formation of cells at 24 hours, 48 hours and 72 hours after infection. According to Figure 5 (A), CPE formation was increased with time in the control group without mixing the extract of the yeast extract of Example according to the present invention, after all the cells infected with PEDV virus necrosis after 72 hours Could confirm. On the other hand, as shown in Figure 5 (B), it was confirmed that in the cells to which the extract of the yeast extract of the embodiment according to the present invention does not show CPE formation over time.

Experimental Example 4: Antiviral Experiments on Sterile Pigs

A control group fed a diet containing 20 g of substitute oil and 0.6 g of Yin-Yang extract and 100 ml of water to sterile pigs and a control group of 20 g of 100 g of water and 100 ml of water to sterile pigs were added to pig feces over time. The concentration of virus present was measured. The virus concentration was measured by real time PCR.

The measurement results are as shown in FIG. 6. According to Figure 6, 24 hours after the inoculation did not detect the PED virus in the feces of the sterile pigs of the experimental group to which the extract of the umbilical cord extract was added, even when measured 48 hours and immediately after the autopsy, The concentration of the detected PED virus was lowered to 1/10 level, and it was confirmed that the herbal extracts had an effect of inhibiting the growth of the PED virus.

In addition, as shown in FIG. 7, the barrier of the control was thinner and the aqueous diarrhea was filled in the intestine (A-2), whereas the herbal medicine administration group contained the feces near the normal (A-1). ). Even in the feces state, the control group (B-2) is completely liquid at 24 hours after the virus inoculation, while the herbal medicine administration group (B-1) shows a solid shape to maintain the shape.

As described above, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. The scope of the present invention is shown by the claims to be described later rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts are included in the scope of the present invention. Should be.

1 shows the growth curve of the ANTI-PEDV efficacy of the extract of Yin-Yang Yang, showing the results of titration-PCR by harvesting the virus by time by adding the herbal medicine after inoculation according to the virus concentration (MOI = 0.1).

2 shows the growth curve of the ANTI-PEDV efficacy of the extract of Eum yangkum, showing the results of titration-PCR by harvesting the virus by the time of adding the herbal medicine after inoculation according to the virus concentration (MOI = 0.01).

Figure 3 is a graph showing the titer measured by the plaque assay (plauqe assay) the value of the virus rate measured by time after inoculating the virus by concentration.

Figure 4 shows the results of the plaque assay (Plauqe assay), A shows the PEDV, B shows the addition of the extract of the yeast.

Figure 5 is a picture confirming the ANTI-PEDV efficacy of Eumyangkum extract by CPE formation, showing the appearance after 24 hours, 48 hours and 72 hours.

Figure 6 shows a graph comparing the concentration of PEDV detected in feces of sterile pigs of the control group and the herbal medicine administration group.

Figure 7 shows the results of the ANTI-PEDV experiment performed on sterile pigs, the state of the bowel and bowel feces of sterile pigs.

Claims (8)

Composition for the prevention or treatment of diseases caused by swine epidemic diarrhea virus (PEDV) comprising the extract of Eum Yang Kwak as an active ingredient. delete The method of claim 1, The Yin Yang Kwak is an extract that is available in a solvent selected from water, lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof. The method of claim 1, The yinyang extract is a composition, characterized in that the content of 0.1 to 50% by weight The method of claim 1, The disease caused by the swine epidemic diarrhea virus is swine epidemic diarrheal disease (PED). The method of claim 1, The disease is a composition that occurs in a mammal, including humans. Health functional food for the prevention or improvement of diseases caused by swine epidemic diarrhea virus (PEDV) containing the extract of Eum Yang Kwak as an active ingredient. The method of claim 7, wherein A dietary supplement that is a tablet, capsule, pill, or liquid.

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