KR20180128190A - Composition comprising extract of Garcinia Mangostana or xanthone compounds derived from for treating or preventing porcine epidemic diarrhea - Google Patents

Abstract

The present invention relates to a composition comprising a mangosteen extract or a xanthone-based compound separated from the mangosteen extract as an active ingredient for preventing or treating porcine epidemic diarrhea (PED). Inventors of the present invention identified that the mangosteen extract or α-mangostin, γ-mangostin, gartanin, deoxy gartanin, beta mangostin, 9-hydroxycalabaxanthone, and so on which are xanthone-based compounds derived from the mangosteen extract have anti-virus activity and a cytotoxicity effect by treating porcine epidemic diarrhea virus (PEDV) with the mangosteen extract or α-mangostin, γ-mangostin, gartanin, deoxy gartanin, beta mangostin, 9-hydroxycalabaxanthone, and so on. The present invention has been completed based on the identification above. PED is highly contagious and causes economic loss, such as deaths of piglets, and also leads to property losses in farms, and an economic impact from the consumer pork prices. Accordingly, when the mangosteen extract or α-mangostin, γ-mangostin, gartanin, deoxy gartanin, beta mangostin, 9-hydroxycalabaxanthone, and so on which are xanthone-based compounds derived from the mangosteen extract are used, a user can take action for disease caused by PEDV in an effective manner.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating porcine epidemic diarrhea comprising an extract of mangosteen or a mangosteen extract as an active ingredient and a composition for preventing or treating porcine epidemic diarrhea,

The present invention relates to a composition for preventing or treating porcine epidemic diarrhea (PED) comprising xanthone compound derived from mangosteen extract or mangosteen extract as an active ingredient, and more particularly to a composition for preventing or treating porcine epidemic diarrhea Mangostin, gamma-mangostin, gartanin, deoxygartanin, beta mangostin, mangosteen extracts or mangosteen extracts, which are derived from mangosteen extracts that are effective for the prevention or treatment of diarrhea. , And 9-hydroxycalabaxanthone.

Pandemic epidemic diarrhea (PED) is one of the most important viral diseases that cause enormous damage to the swine industry in Asian countries including Korea. Serious damage reports have been reported in North America and South America, including the United States and Canada, in Peru and Chile.

Pandemic seasonal diarrhea occurs specifically in piglets prior to winter weaning, and 50% -100% of piglets died due to dehydration with diarrhea, and seasonal characteristics occurred mainly in winter and early spring. Recently, however, the disease has also occurred in other seasons than the winter season, and the onset pattern is becoming more diverse. Since the first reports of the occurrence of porcine epidemic diarrhea virus (PEDV) in Europe in the late 1970s in Europe, it occurred in China and Japan in the 1980s and caused enormous damage to piglets. In Korea, . There have been many reports that the mutated pandemic strain of diarrhea virus (PEDV), which is highly pathogenic, is difficult to prevent by the vaccine, and the mutation of the virus has been reported from the US since the end of 2013, (Number of outbreaks: 231, number of infections: 46,540). However, primary pig farmers are avoiding farming due to concerns about the secondary economic loss of farms due to restrictions on livestock transport on the farms that have developed pandemic diarrhea.

The most obvious way to prevent damage from PEDV infections is to thoroughly disinfect and block livestock and livestock-related vehicles, and second, there is a method of prevention through appropriate vaccination. In Korea, PED is designated as a third type of infectious disease and currently live vaccines, inactivated vaccines and oral vaccines are commercialized and distributed to farm households. However, the vaccines were mainly produced in the early 2000s Pandemic influenza virus (PEDV), a newly emerging virus from the United States in 2013, is a genetically highly mutated virus that is said to be ineffective. In addition, live vaccines currently in circulation are being vaccinated through oral administration, which means that the vaccine diluent is soaked in the bread to feed the pigs, and the vaccine virus is effectively delivered to the small intestine due to digestion of gastric juices and digestive enzymes There is a lack of scientific evidence as to whether or not it can.

Accordingly, research has been conducted to develop a more effective composition for the prevention or treatment of swine diarrhea (Korean Patent No. 10-1170965).

DISCLOSURE Technical Problem Accordingly, the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a composition for preventing or treating diabetes mellitus, Pegylated diarrhea virus (PEDV), which is known to be involved in the pathogenesis of diarrhea, is known to be involved in the pathogenesis of diarrhea, such as alpha-mangostin, gamma-mangostin, gartanin, deoxygartanin, beta mangostin, and 9-hydroxycalabaxanthone. . As a result, it was confirmed that there was an antiviral activity and a cytotoxic effect, and on the basis thereof, the present invention was completed.

Accordingly, it is an object of the present invention to provide a composition for preventing or treating swine diseases caused by swine diarrhea virus, which comprises as an active ingredient, a minor tonic compound derived from mangosteen extract or mangosteen extract.

It is another object of the present invention to provide a pig feed additive comprising the composition.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating swine epidemic diarrhea comprising, as an active ingredient, a xanthone-based compound derived from mangosteen extract or mangosteen extract .

In one embodiment of the invention, the minor tonometric compound is selected from the group consisting of alpha-mangostin, gamma-mangostin, gartanin, deoxygartanin, beta mangostin, and 9-hydroxycalabaxanthone And at least one selected from the above.

In another embodiment of the invention, the mangosteen extract is selected from the group consisting of water, methanol, hexane, chloroform, ethyl acetate, n-butanol, C1 to C4 lower alcohols, n-hexane, acetone, butyl acetate, Is extracted with at least one solvent selected from the group consisting of ethylene glycol, propylene glycol, ethylene glycol, propylene glycol, ethylene glycol, propylene glycol,

The present invention also provides a pig feed additive comprising a pharmaceutical composition for preventing or treating swine epidemic diarrhea comprising, as an active ingredient, a xanthone-based compound derived from mangosteen extract or mangosteen extract.

In another embodiment of the present invention, the additive is a powder, a tablet, a capsule, a pill, or a solution.

The present invention also provides a method of preventing or treating swine epidemic diarrhea comprising administering to a subject a xanthone-based compound derived from mangosteen extract or mangosteen extract.

The present invention also provides the use of xanthone-based compounds derived from mangosteen extract or mangosteen extract for preventing or treating swine epidemic diarrhea.

The present invention relates to a pharmaceutical composition for preventing or treating swine epidemic diarrhea comprising, as an active ingredient, a xanthone-based compound derived from mangosteen extract or mangosteen extract. The present inventors have found that a mangosteen extract or a mangosteen extract Treatment of pig swine diarrhea virus (PEDV) with alpha-mangostin, gamma-mangostin, gartanin, deoxygartanin, beta mangostin and 9-hydroxycalabaxanthone As a result, it was confirmed that there was an antiviral activity and a cytotoxic effect, and based on this, the present invention was completed.

Pandemic Diarrhea (PED) is highly contagious and not only due to loss of property of the farm, which is the death of piglets, but also to the economic ripple effect of rising consumer price of pork, resulting in damage caused by PEDV However, the effectiveness of the vaccine has yet to be proven.

Accordingly, the present invention relates to a method for producing a compound of the present invention, which is a combination of a mangosteen extract or a mangosteen extract, which are α-mangostin, γ-mangostin, gartanin, deoxygartanin, beta mangostin, -hydroxycalabaxanthone is expected to be more effective against swine diarrhea.

Figure 1 is a schematic representation of an antiviral activity experimental design of alpha-mangostin, gamma-mangostin, and gartanin against porcine epidemic diarrhea virus (PEDV) CV777.
Figure 2 shows the results of testing antiviral activity of alpha-mangostin, gamma-mangostin, and gartanin against porcine epidemic diarrhea virus (PEDV) CV777 will be.
Fig. 3 shows the results of evaluating the cytotoxicity of alpha-mangostin, gamma-mangostin, and gartanin in Vero cells.

The present invention relates to a pharmaceutical composition containing xanthone-based compounds such as alpha-mangostin, gamma-mangostin, gartanin, deoxygartanin, beta mangostin, and the like, which are derived from mangosteen extract or mangosteen extract. 9-hydroxycalabaxanthone, and the like. The present inventors have found that a method of treating a piglet epidemic diarrhea virus (PEDV) with a zwitterionic compound derived from a mangosteen extract or a mangosteen extract, As a result, it was confirmed that there was an antiviral activity and a cytotoxic effect, and based on this, the present invention was completed.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating swine epidemic diarrhea comprising, as an active ingredient, a xanthone-based compound derived from mangosteen extract or mangosteen extract.

"Α-mangostin, γ-mangostin, and gartanin" of the present invention are major three-tonon compounds extracted from mangosteen hulls, and their antioxidative effects are known. In recent years, there has been a growing interest in synthetic compounds, and there is a growing interest in using relatively safe natural materials, so active research has been conducted on the effects of natural substances with useful physiological activities and low side effects.

The term " a disease caused by swine epidemic diarrhea virus (PEDV) "of the present invention includes porcine epidemic diarrhea (PED) and includes those occurring not only in swine but also in mammals including humans.

The minor tonometric compound may be at least one selected from the group consisting of alpha-mangostin, gamma-mangostin, gartanin, deoxygartanin, beta mangostin, and 9-hydroxycalabaxanthone , But is not limited thereto.

The above-mentioned mangosteen extracts can be used in combination with water, methanol, hexane, chloroform, ethyl acetate, n-butanol, C1 to C4 lower alcohols, n-hexane, acetone, butyl acetate, 1,3-butylene glycol, But the present invention is not limited to these solvents.

The porcine disease caused by the swine diarrhea virus may be porcine epidemic diarrhea (PED). However, the present invention is not limited to the use of a porcine epidemic virus diarrhea virus (PEDV)).

The present invention also provides a pig feed additive comprising a pharmaceutical composition for preventing or treating swine epidemic diarrhea comprising, as an active ingredient, a xanthone-based compound derived from mangosteen extract or mangosteen extract.

The additive may be a powder, a tablet, a capsule, a pill, or a liquid, but is not limited thereto.

In one embodiment of the present invention, SRB assay assays are performed to determine the anti-Pandemic anti-diarrhea virus (PEDV) anti-mangostin, gamma-mangostin, and gartanin anti- Virus activity was observed (see Example 2).

In another embodiment of the present invention, the cytotoxicity of alpha mangosteen, gamma mangosteen, and galatane in Vero cells was confirmed (see Example 3).

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

The content of the mangosteen extract is not particularly limited, but it is preferable that the content of the mangosteen extract is in the range of 0.1 to 50% by weight based on the total weight of the composition, so that the expected antiviral effect can be effectively exhibited in the therapeutic composition.

Since the mangosteen extract of the present invention has little toxicity and side effects, it can be safely used for prolonged administration even for prophylactic purposes.

The composition comprising the mangosteen extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the production of pharmaceutical compositions. The pharmaceutical dosage forms of the mangosteen extract 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 pharmacologically active compounds as well as in suitable aggregates.

The composition containing the mangosteen extract according to the present invention may be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions Can be used. Examples of carriers, excipients and diluents that can be included in the composition including the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propyleneglycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. The base of suppositories may be witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The preferred dosage of the mangosteen extract of the present invention may be appropriately selected by those skilled in the art, depending on the condition and body weight of the patient or conjunctiva, the degree of disease, the type of drug, the route of administration and the period of administration. However, for the desired effect, the mangosteen extract of the present invention is preferably administered at 0.0001 to 1,000 mg / kg. The dosage may be administered once a day or divided into several doses, but the dose is not limited in any way to the scope of the present invention.

The mangosteen 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 may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

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

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

Example  1. Experimental Preparation and Experimental Methods

One- 1. Mangosteen  Extract preparation method and mangosteen extract Janton  Method of Separation of Compound

The shaded mangosteen skin (1.3 kg) was sonicated three times with 8 L of 100% methanol, filtered and concentrated under reduced pressure to obtain 401 g of a methanol extract. A 164g methanol extract was suspended in distilled water and the solvent fraction was extracted with hexane, chloroform, ethyl acetate and n-butanol. After concentration under reduced pressure, the chloroform fraction 37.9 g). The mangosteen chloroform fraction (37.9 g) was purified by gradient silica gel (593 g) open column chromatography with hexane and ethyl acetate mixed solvent, and then divided into 49 fractions (GMC1 to GMC49).

Among them, GMC6 was separated _{GMC6-pk1 (3.9mg, t R} = 37.0min) using HPLC (Phenomenex Luna C18, ACN 98 %). GMC7 was purified _{GMC7-pk1 (4.1mg, t R} = 31.0min) using HPLC (Phenomenex Luna C18, ACN 98 %). GMC17 is a mobile phase of methanol and water as by performing a nanueotgo _{(MeOH: H 2 O, 80} :: 20to100 0) RP-MPLC 13 of minor fraction (GMC17-1 to GMC17-13), Substance GMC17-7 (175mg , 9-hydroxycalabaxanthone). GMC17-9 was purified by performing HPLC (Phenomenex Luna C18, ACN 90 %) GMC17-9-pk2 (3.8mg, t R = 30.2min, deoxygartanin).

GMC19 was purified _{GMC19-pk1 (2.8mg, t R} = 21.1min) and _{GMC19-pk2 (13.2mg, t R} = 26.4min, gartanin) using HPLC (Phenomenex Luna C18, ACN 80 %). GMC21 was divided into 12 small fractions (GMC21-1 to GMC21-12) by performing RP-MPLC with methanol and water as mobile phase (MeOH: H ₂ O, 80:20 to 100: 0) The single substance GMC21-7 (59.3 mg, beta mangostin) was. GMC26 (5.4 g), 27 (4.8 g) and 28 (2.2 g) were alpha-mangostin, a single substance. GMC35 (3.36 g) was subjected to gradient silica gel (120 g) MPLC with chloroform and acetone mixtures to obtain 23 fractions (GMC35-1 to GMC35-23). Among them, γ-mangostin (698 mg) was obtained by recrystallizing fraction 8 (GMC35-8, 890 mg) with methanol and distilled water.

1-2. Isolation of viruses Potency  Measure

PEDV (porcine epidemic diarrhea virus) CV777 virus purchased from ATCC using Vero cells was amplified and used. Each cell was cultured in a 5% CO ₂ incubator at 37 ° C in DMEM (Dulbecco's modified Eagle's medium) to maintain exponential growth of the cells. Cell culture dishes were used for 2-3 days After incubation, the cells were detached using 1 × Trypsin-EDTA, transferred to a 15 mL tube, and centrifuged at 1,000 rpm for 5 min. The separated cell pellet was resuspended in 1 × PBS (phosphate buffer saline), centrifuged, and the supernatant was removed to remove the cells. After repeated washing in the same manner, some of them are taken and 0.4% trypan blue is added. The stained cells are counted on a hemocyte counter, and fresh medium is added to a new culture dish to a concentration of 1 × 10 ⁴ cells / mL And then the test was carried out.

Each virus was diluted in 10-fold increments using a 96-well plate, and the activity was determined after Sulforhodamine B (SRB) assay. Each virus was diluted 10-fold in DMEM supplemented with 1% FBS, treated with 100 μL each of 96-well microplates in which A549 cells were cultured, and the plate was cultured until a proper level of cytopathic effect was observed . After incubation for 48 hours, 96-well microplates were washed with PBS, 100 μL of 70% cold acetone was added to each well and allowed to stand at -20 ° C. for 30 minutes.

Next, the acetone was removed, and then dried in a drier at 60 ° C for 30 minutes. 100 μL of a solution prepared by adding 0.4% (Weight / Volume; w / v) SRB to 1% acetic acid was added to each well And stored at room temperature for 30 minutes. Thereafter, the SRB solution was removed, washed 5 times with 1% acetic acid, and then dried in a dryer for 24 hours to be stained. To determine the absorbance of the stained plate, SRB should be dissolved. To this end, 100 μL of 10 mM Tris-base solution was added to each well and allowed to stand at room temperature for 30 minutes.

The absorbance of each well of the plate was measured using a multi-EIA reader (Molecular Device co.) At 562 nm. At the same time, the absorbance was measured at a wavelength of 620 nm and used as a control for the background color. The titers of the measured viruses were calculated as 50% Tissue Culture Infection Dose (TCID50) to determine the appropriate virus concentration.

1-3. SRB  Antiviral activity test by assay assay

As shown in FIG. 1, an antiviral activity experiment was planned, and an SRB assay (Sulforhodamine B (SRB) assay) analysis was performed to measure the antiviral activity in order to confirm the ability of the virus to suppress the cell lesion caused by the virus Respectively. SRB assay is an assay method to measure cell viability using Sulforhodamine, a protein staining reagent. It is more stable than MTT assay, more convenient than plaque assay, and currently used for screening anticancer drug.

First, A549 cells were prepared in each well of a 96-well culture plate in an amount of 2 × 10 ⁴ . Twenty-four hours later, cells were grown in 96-well culture plates at 90%. The culture solution was removed from each well and washed once with PBS. After that, PEDV CV777, which was confirmed by titration with 1% FBS, was diluted in DMEM at a concentration of TCID50 (tissue culture infection dose 50), and then mixed with 90 μL of a suitable concentration of alpha-mangostin, gamma mangosteen -mangostin, and 10 [mu] L of gartanin. Each compound was determined in four concentration ranges: 0.016 μg / mL, 0.08 μg / mL, 0.4 μg / mL, and 2 μg / mL. Three wells were used as the cell control without the antiviral agent and the other three wells were used as the cell control without virus and antiviral agent. The cells were cultured in a CO ₂ incubator at 37 ° C and 5% CO ₂ for 2 days, and the antiviral activity was measured.

1-4. Data analysis

Antiviral activity was expressed as a 50% inhibition concentration (IC ₅₀ ), for example, the concentration of the antiviral agent was determined to inhibit 50% of the cytopathic effects on the negative control. The measured results were converted to percentages of the IC ₅₀ values measured by the control and probit analysis.

1-5. SRB  Cytotoxicity test by assay assay

A549 cells cultured on a 96-well plate were treated with antiviral agents at different concentrations next to the virus-treated wells and cultured at 37 ° C for 2 days. For each antiviral agent, 3 wells were used as a cell control without antiviral agent. After incubation for 2 days in a CO ₂ incubator, cytotoxicity was assessed by SRB assay.

Example  2. PEDV  Identification of antiviral activity against CV777

Mangostin, gamma-mangostin, and gamma-mangostin against porcine epidemic diarrhea virus (PEDV) CV777 by performing SRB assay analysis according to the method of Example 1-3. After testing the antiviral activity of gartanin, the measured IC ₅₀ values were analyzed according to the methods of Examples 1-4.

As a result, as shown in FIG. 2, α-mangostin showed no antiviral activity at 0.016, 0.08, and 0.4 μg / mL among the concentrations used in the experiment, but showed 50% or more antiviral activity at a concentration of 2 μg / mL . In addition, gartanin showed no antiviral activity at concentrations of 0.016 μg / mL and 0.08 μg / mL, but showed antiviral activity of 50% or more at the concentrations of 0.4 and 2 μg / mL. On the other hand, γ-mangostin did not show any antiviral activity at the concentrations used in the experiment. The IC ₅₀ values for the 50% inhibition of viral activity were 0.84 μg / mL for α-mangostin and 0.27 μg / mL for gartanin, respectively.

Example  3. In Vero cells  Cytotoxicity Assessment

SRB assay analysis was performed according to the method of Example 1-5 to evaluate the cytotoxicity of alpha-mangostin, gamma-mangostin, and gartanin in Vero cells . The concentration of cytotoxicity was also determined to be 0.016 μg / mL, 0.08 μg / mL, 0.4 μg / mL and 2 μg / mL in the same manner as the concentration used in the antiviral activity measurement of Example 2 above.

As a result, as shown in Fig. 3, α-mangostin and gartanin did not show cytotoxicity up to 2 μg / mL, which is the highest concentration used in the experiment, but γ-mangostin showed about 50% cytotoxicity at a concentration of 2 μg / Respectively.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (5)

A pharmaceutical composition for preventing or treating swine epidemic diarrhea comprising, as an active ingredient, a xanthone-based compound derived from mangosteen extract or mangosteen extract.
The method according to claim 1,
The minor tonometric compound is at least one selected from the group consisting of alpha-mangostin, gamma-mangostin, gartanin, deoxygartanin, beta mangostin, and 9-hydroxycalabaxanthone Lt; / RTI >
The method according to claim 1,
The above-mentioned mangosteen extracts can be used in combination with water, methanol, hexane, chloroform, ethyl acetate, n-butanol, C1 to C4 lower alcohols, n-hexane, acetone, butyl acetate, 1,3-butylene glycol, ≪ / RTI > or a mixture of two or more solvents.
A pig feed additive comprising the composition of claim 1.
5. The method of claim 4,
Wherein the additive is a powder, a tablet, a capsule, a pill, or a liquid.

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