KR20110055266A - Composition for prevention or treating porcine epidemic diarrhea containing extract of lonicerae flos - Google Patents

Abstract

PURPOSE: A porcine epidemic diarrhea prevention or curing composition containing a lonicera japonica extract is provided to improve the anti-virus effect of the composition, and to effectively supply the massive amount of composition. CONSTITUTION: A porcine epidemic diarrhea prevention or curing composition contains a lonicera japonica extract as an active component. The composition is a feed additive in a form selected from the group consisting of a powder form, a tablet, a capsule, a pill, and a liquid. The lonicera japonica extract is obtained by extracting lonicera japonica with water, low class alcohol with 1~4carbons, or their mixture.

Description

Composition for prevention or treating Porcine Epidemic Diarrhea containing extract of Lonicerae Flos}

The present invention relates to a composition for the treatment and prevention of swine epidemic diarrheal disease, including the extract of sterling silver.

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, Avian infectious bronchitis virus, mouse hepatitis virus and Severe Acute Respiratory syndrome (SARS) virus in humans are among the corona viruses.

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 post motility, feed intake rate 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 gold and silver 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 a gold-silver extract 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 sterling silver is a herb that refers to the buds of the locust (Lonicera japonica Thunberg) or its variety, and has a unique smell, sweet taste, and cold nature. The gold and silver coins have been reported to have the effect of antibacterial, anti-inflammatory, antipyretic, leukocyte phagocytosis, central nervous system excitement, serum cholesterol lowering, ulcer prevention.

 In the present invention, gold and silver coins can be obtained by supercritical extraction in a conventional manner using carbon dioxide and water as a solvent. In addition, in the present invention, the gold and silver coins may be extracted by heating in a polar solvent such as ethanol and methanol or a mixed solution of them and purified water, and the gold and silver coins extract may be obtained by various methods such as concentration under reduced pressure. Preferably, hot water, methanol or the like may be used to obtain a sterling silver extract. More preferably, the sterling silver extract of the present invention may be extracted using a solvent selected from water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

Although the content of the sterling silver extract is not particularly limited, in order to effectively exhibit the antiviral effect expected in the therapeutic composition, it is preferably contained in 0.1 to 50% by weight relative to the total weight of the composition.

Since the sterling silver extract of the present invention has little toxicity and side effects, it can be used with confidence even for long-term administration for the purpose of prevention.

The composition comprising the sterling gold 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 sterling silver 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.

The composition containing the gold sterling extract according to the present invention, 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 a conventional method. Can be formulated and 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. . 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, etc. may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the sterling silver extract of the present invention depends 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, it is preferable to administer the gold coin extract of the present invention at 0.0001 to 1,000mg / kg. The administration may be administered once a day or may be divided several times, but the dosage does not limit the scope of the present invention in any aspect.

The gold coin extract of the present invention can be administered to various mammals such as mice, mice, livestock, humans. 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 feed additive for the prevention or improvement of diseases caused by swine epidemic diarrhea virus (PEDV) containing a sterling silver extract as an active ingredient. The feed additive of the present invention may be prepared in powder, tablet, capsule, pill or liquid form.

In addition, it may be added to the feed for the purpose of preventing the disease caused by the swine epidemic diarrhea virus. At this time, the amount of the extract in the feed may be added to 0.01 to 15% by weight of the total feed weight.

As described above, the present invention can effectively increase the antiviral effect on the epidemic diarrheal virus of pigs, and also the pig epidemic diarrheal disease in farms by including a gold-silver extract extract having a proliferation inhibitory effect on PEDV as an active ingredient There is an effect that can efficiently supply a large amount of the therapeutic composition.

Hereinafter, the present invention will be described in detail through examples. However, the following examples are only examples for describing the present invention, and the scope of the present invention is not limited thereto.

<Examples>

Example 1 Preparation of Gold Coin Extract

After weighing 63.43g of dried gold and silver coins, 1L of bottled water (white, Muhaksancheongsam Water, 800, Deokgyo-ri, Gyeongnam Sancheong-gun) was immersed for 1 hour, and then immersed for 3 hours at 100 ° C using Daewoong tangtang (DWP-5000M). 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 infection with CO ₂ , 37 ° C. incubator for 1 hour, 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 it 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 the sterling silver extract on the PEDV growth curve

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

As a result, as shown in FIG. 1, the PEDV titer increased with time in the control group (A), but the virus harvested from the cells to which the sterling gold extract was added was negative over time (B).

Next, in order to re-verify the effect of the sterling silver extract, the virus concentration was set to MOI = 0.01 over time, and the virus was harvested by titer-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 gold leaf extract (B). there was.

In addition, after inoculating the virus by concentration, the virus proliferation rate was measured by time, and the measuring method was performed by a plaque assay. As a result, as shown in Figure 3, it was confirmed that the virus titer of the experimental group (PED + 124) to which gold and silver is added is significantly less when compared to the control group (PED).

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 as the virus titer is lowered by the gold-silver coin extract, the shape or size of the plaque may be formed invisibly or invisibly due to the virus mutation. As a result, as shown in Figure 4, compared with the control group (A), even when added to the gold leaf extract (B) there was no significant change in the shape or size of the plaque (plaque). That is, there was no morphological change of the plaques, and the number of infectious viruses in the group added (B) was found to be reduced by 10 to 43 times (see Fig. 4). Therefore, it was confirmed that the gold silver extract has an inhibitory effect on PEDV.

Experimental Example 3: Evaluation of the effect of the sterling extract on CPE in PEDV cell culture

Dispense VERO-CELL into 1 × 10 ⁶ cells / flask in a 25 cm ² cell culture flask and add 5% CO after adding MOI (multiplicity of infection) = 0.01 when the virus solution (KPEDV-9) was monolayered. ₂ , 37 ℃ incubator (incubator) for 1 hour after the culture was removed and washed with PBS (phosphate buffered saline) and then added to the medium. Then, the extract was added gold and the CPE was evaluated over time.

As a result, Figure 5 is confirmed by magnification 200 times by the optical microscope whether CPE formation of cells at 48 hours and 72 hours after PEDV virus infection. According to (A) of FIG. 5, CPE formation was increased with time in the control group without mixing the sterling silver extract of the example according to the present invention, and finally, after 72 hours, all cells infected with PEDV virus were necrotic. I could confirm it. On the other hand, as shown in (B) of FIG. 5, in the cells to which the sterling silver extract of the embodiment according to the present invention was added, CPE was formed later and appeared weaker than the control group (A) 48 hours after virus inoculation. At 72 hours after the virus inoculation, CPE was seen, but the cells were still present.

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 represented by the following claims 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 interpreted.

Figure 1 shows the results of measuring the virus titer by quantitative-PCR by harvesting the virus by the time of inoculation at the virus concentration of MOI = 0.1, and then incubated with a virus concentration of MOI = 0.1 in order to confirm the anti-PEDV efficacy of the gold sterling extract will be.

Figure 2 shows the results of measuring the virus titer by quantitative-PCR by harvesting the virus by inoculation at the virus concentration of MOI = 0.01 and then adding the gold silver extract in order to confirm the anti-PEDV efficacy of the gold silver extract by growth curve will be.

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

Figure 4 shows the plaque assay (Plauqe assay) results, (A) is a control group containing only the PEDV, (B) shows the addition of the gold silver extract of the embodiment according to the present invention.

FIG. 5 is a photograph confirming the formation of CPE by the optical microscope in order to confirm the anti-PEDV efficacy of the sterling silver extract, showing a state after 48 hours and after 72 hours.

Claims (7)

Feed additives for the prevention or improvement of diseases caused by swine epidemic diarrhea virus (PEDV) containing gold silver extract as an active ingredient. The method of claim 1, Feed additives which are powders, tablets, capsules, pills or liquids. Composition for the prevention or treatment of diseases caused by swine epidemic diarrhea virus (PEDV) comprising a gold silver extract as an active ingredient. The method of claim 3, wherein The gold and silver coins are extracts soluble in a solvent selected from water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof. The method of claim 3, wherein The silver coin extract is a composition, characterized in that the content of 0.1 to 50% by weight. The method of claim 3, wherein The disease caused by the swine epidemic diarrhea virus is swine epidemic diarrheal disease (PED). The method of claim 3, wherein The disease is a composition, characterized in that the disease occurring in a mammal.

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