WO2015065059A1

WO2015065059A1 - Composition for preventing or treating fish viral hemorrhagic septicemia comprising ecklonia cava extract

Info

Publication number: WO2015065059A1
Application number: PCT/KR2014/010285
Authority: WO
Inventors: 이우송; 류영배; 김영민; 노문철; 박수진; 정형재; 박지영; 이제희; 임봉수; 정성주
Original assignee: 한국생명공학연구원
Priority date: 2013-10-30
Filing date: 2014-10-30
Publication date: 2015-05-07
Also published as: KR101542882B1; KR20150049696A

Abstract

The present invention relates to: a pharmaceutical composition for preventing or treating VHS comprising an Ecklonia cava extract, a fraction thereof, a phlorotannic compound derived therefrom or a pharmaceutically acceptable salt thereof; a method for preventing or treating VHS using the pharmaceutical composition; a quasi-drug composition for preventing or treating VHS, a feed or a feed additive for preventing or ameliorating VHS comprising an Ecklonia cava extract, a fraction thereof, a phlorotannic compound derived therefrom or a pharmaceutically acceptable salt thereof; and a method of farming fish comprising a step of feeding the feed to fish. The Ecklonia cava extract, the fraction thereof or the phlorotannic compound derived therefrom of the present invention exhibits an effect of suppressing the activity of VHSV, and exhibits an effect of enhancing the prevention or treatment of fish VHS, with high safety using the same, and thus may be widely utilized in improving the survival rate of flatfish against infection of VHSV and in increasing the stability of the fishery industry due to an improvement of fishing productivity.

Description

Composition for preventing or treating fish viral hemorrhagic sepsis comprising Ecklonia cava extract

The present invention relates to a composition for the prevention or treatment of fish viral hemorrhagic sepsis comprising an Ecklonia cava extract, and more particularly, the present invention relates to a viral hemorrhagic septicemia (VHS) comprising Ecklonia cava extract or a fraction thereof. A prophylactic or therapeutic pharmaceutical composition, a prophylactic or therapeutic pharmaceutical composition of VHS comprising the fluorotannin-based compound derived from the Ecklonia cava extract or a fraction thereof or a pharmaceutically acceptable salt thereof as an active ingredient, by using the pharmaceutical composition Method for preventing or treating VHS, Ecklonia cava extract, Fractions thereof, phlotantanin compounds derived therefrom or quasi-drug compositions for the prevention or treatment of VHS, including pharmaceutically acceptable salts thereof, Feed for preventing or improving VHS Or feed additives and feeding the feed to fish It relates to a method of farming fish, including.

In general, the main causes of serious damage to the flounder industry are three or more complex infections, especially viral hemorrhagic septicemia virus (VHSV), the bacteria Edwardsiella sp., And streptococci. (Strepococcus sp.), Vibrio sp., Flexibacter sp., And the parasite Siamtica avidus. In particular, viral diseases, such as viral hemorrhagic septicemia (VHS), occur during the early stages of low temperature in the stocks of flounder, causing mass mortality, causing mass mortality not only in larvae but also in adult fish. It has been reported that the economic loss of farmed fish cost about 300 billion won.

The VHS was first reported in the flounder in 2001 in Korea, and has been reported in various marine fish since then, and the damage of the cultured flounder is a serious disease. In 2011, the major causes of mortality in the flounder farms in Korea were 12.5% of all mortality caused by VHSV. In addition, in Jeju Island, which produces about half of the flounder production in Korea, the highest rate of viral hemorrhagic sepsis was 30% among the samples for disease test from 2007 to 2010, and the damage is increasing. . In addition, the VHSV is defined as an OIE notifiable disease by the International Water Services Bureau (OIE), which is a quarantine target for the distribution of seafood, which is an important trade barrier for import and export of farmed fish including flounder. It is also a disease factor. VHSV has been reported for the first time as an important viral disease of rainbow trout. Recently, various fish, trout, rainbow trout, coho salmon, river trout, brown trout and steelhead trout, as well as salmon and fish, cod, herring, etc. It is also widely used in marine cultured fish such as fish and halibut in the natural water system, and is mainly found in Europe, North America, and Asia. In the naked eye of infected flounder, color blackening, whole body bleeding, abdominal distension and hernia due to ascites retention, gill discoloration, and red spot bleeding on the apical surface are observed. Onset water temperature ranges from 10 to 13 ° C, regardless of the size of the halibut, and death occurs. Currently, no treatment is available and care is taken not to spread to other areas. Therefore, there is an urgent need for an effective treatment method for viral hemorrhagic sepsis to reduce the economic loss of aquaculture fish caused by viral hemorrhagic sepsis and to smoothly export and distribute flounder.

VHSV is a negative single-stranded RNA virus, first reported in 1963 at the International Water Bureau's Fish Disease Symposium, bullet-shaped, 180 nm long, 60 nm in diameter, 11 kb in size, and a nucleocapsid protein. N), polymerase-associated phosphoprotein (P), matrix protein (M), surface glycoprotein (G), a unique non-virion protein (NV) ) And the rhabdovirus family, which consists of six proteins of virus polymerase (L). Genotypes are known to have four subtypes, type I to III isolated from Northern Europe, and type IV to North America. Based on the research on the structure and antigenic proteins of VHSV and these basic studies, researches on inactivated vaccines, recombinant protein vaccines, attenuated vaccines, DNA vaccines and the like have been conducted. However, while attenuated virus vaccines have been reported to be effective in experimental conditions, their use is not permitted because of the risk of pathogenic recovery of viruses and the risk of spreading into nature. In addition, there have been reports of rainbow trout and halibut in DNA vaccination experiments in which the vaccinated hemorrhagic sepsis virus G or N gene was injected into the muscles, a strong immune response and reduced mortality. Due to the problem, only one DNA vaccine has been approved to date, and there are few DNA vaccines in the commercialization stage. Therefore, research on this is being actively conducted, and a virus isolated from flounder which is commercially available in Korea. Vaccine development for the state is underway. However, the research on the virus itself is still insufficient, and researches to characterize the virus have become mainstream, and studies on agents capable of inhibiting the activity of the virus have not been conducted.

On the other hand, persimmons of perennial seaweed belonging to the brown seaweed kelp seaweed family are used as food for fish and shellfish when farming various fish and shellfish, but no farming method has been developed yet. Since the Ecklonia cava is known to have an anticancer effect, studies on its various pharmacological activities have been conducted. For example, Korean Patent Publication No. 2000-0065613 discloses a composition for preventing malodor and tooth decay, including Ecklonia cava extract, and Korean Patent Publication No. 2001-0098018 discloses a novel compound exhibiting antioxidant activity derived from Ecklonia cava extract. It is disclosed, Korean Patent Publication No. 2002-0015816 discloses a whitening cosmetic composition comprising an Ecklonia cava extract, Korean Patent Publication No. 2002-0085997 discloses a wrinkle improvement cosmetic composition comprising an Ecklonia cava extract, Korean Patent Publication No. 2011-0086474 discloses a composition for preventing or treating rotavirus infection comprising Ecklonia cava extract, and Korean Patent Publication No. 2011-0126413 discloses a cancer comprising a mixture of cynol and fucoidan derived from Ecklonia cava. A composition for preventing or treating is disclosed, and Korean Patent Publication No. 2012-0058695 discloses Ecklonia cava extract. Disclosed is a composition for preventing hair loss or promoting hair growth. As mentioned above, Ecklonia cava exhibits various pharmacological activities, and in particular, may be effective in preventing or treating viral infections, but only a few of them have been identified so far, and the effects of VHSV infections are not yet known.

As a result of intensive research to develop a method for preventing or treating VHS, the present inventors have shown that Ecklonia cava extract, a fraction thereof, or a phlorotannin-based compound isolated therefrom exhibits the activity of inhibiting VHSV. It confirmed and completed this invention.

One object of the present invention to provide a pharmaceutical composition for the prevention or treatment of viral hemorrhagic sepsis (VHS) comprising an Ecklonia cava extract or a fraction thereof as an active ingredient.

Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of VHS comprising a phlorotannin-based compound derived from the Ecklonia cava extract or a fraction thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

Still another object of the present invention is to provide a method for preventing or treating VHS using the pharmaceutical composition.

Still another object of the present invention is to provide an quasi-drug composition for the prevention or treatment of VHS comprising the Ecklonia cava extract, fractions thereof, phlorotannin-based compounds or pharmaceutically acceptable salts thereof as an active ingredient.

Still another object of the present invention is to provide a feed or feed additive for preventing or improving VHS comprising the Ecklonia cava extract, fractions thereof, phlorotannin-based compounds derived therefrom, or pharmaceutically acceptable salts thereof.

Still another object of the present invention is to provide a method of farming fish, comprising the step of feeding the feed to the fish.

Ecklonia cava extract, fractions thereof, or phlorotannin-based compounds derived therefrom exhibit an effect of inhibiting activity against VHSV, and thereby exhibit an effect of improving the prevention or treatment of fish VHS with high safety using it. In particular, it may be widely used to increase the stability of the fish by improving the survival rate and the productivity of the flounder for infection with VHSV.

1 is a graph showing the results of experiments to determine the concentration of the challenge test VHSV administration in order to verify the antiviral effect on VHSV of Ecklonia cava extract in vivo on the flounder.

Figure 2 is a graph showing the results of verifying the preventive effect on the VHS of Ecklonia cava extract in the olive flounder.

The present inventors came to pay attention to the Ecklonia cava, which is a kind of natural product, while conducting various studies to develop a method for preventing or treating VHS. The present inventors obtained the ethanol extract of Ecklonia cava, and separated and purified three compounds showing antiviral activity against VHSV. Subsequently, as a result of measuring the cytotoxicity and antiviral activity of the three purified compounds, it was confirmed that the antiviral activity was exhibited when the three compounds were treated to cells at a concentration that does not exhibit cytotoxicity. In addition, as a result of measuring the antiviral activity of the Ecklonia cava extract in the flounder, it was confirmed that the flounder pretreated with Ecklonia cava extract increased even if VHSV was infected.

As such, the Ecklonia cava extract, fractions thereof, or phlorotannin-based compounds derived therefrom exhibit an effect of preventing or treating VHS, and thus may be used as an active ingredient of a pharmaceutical composition or quasi-drug for preventing or treating VHS. It can be used as an active ingredient in food compositions, feed additives or feeds that have a preventive or improved effect of VHS.

As one embodiment of the present invention for achieving the above object, it provides a pharmaceutical composition for the prevention or treatment of viral hemorrhagic sepsis (VHS) comprising an Ecklonia cava extract or a fraction thereof as an active ingredient.

The term " Ecklonia cava " of the present invention refers to a kind of seaweed, whose length is usually 1 to 2 m, the stem is cylindrical, and the root is root-shaped. Morphically, the middle part is thick and young when it is young, but it is also hollow after it is grown. At the end of the stem is a flat middle leaf with a side leaf piece. The leaf is about 1 m long and brown, but when dried, it becomes black. Thick, leathery texture with pinnate small leaves on each side. Young plants appear in spring. In early years, stems are 5 to 10 cm long and about 5 mm in diameter. Middle leaves are 20 to 30 cm long and 4 cm wide. In the fall of the second year, the spores made in the middle leaf are released, and then the leaves fall off, only the stems hang, and the leaves hang in the middle of the old leaves. It is an important algae plant that makes up algae, and is a food source for abalone and conch. It is a major raw material for making alginic acid, iodine and potassium, and is sometimes collected and used for food. It is distributed in the south coast of Korea, Jeju Island, Ulleungdo Coast, and Japan.

In the present invention, the Ecklonia cava may be purchased and used commercially, or can be used directly collected.

The term "Ecklonia cava extract" of the present invention means an extract obtained by extracting the Ecklonia cava with water or an organic solvent.

In the present invention, the Ecklonia cava extract may be used as an active ingredient of the composition for the prevention, treatment or improvement of VHS, the Ecklonia cava extract may be an extract of Ecklonia cava using a polar solvent, a non-polar solvent or a mixed solvent thereof. have. The polar solvent may be water, anhydrous or hydrous alcohol having 1 to 6 carbon atoms (methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol, etc.) or Alcoholic compounds (ethylene glycol, propylene glycol, butylene glycol, etc.), acetic acid, dimethyl-formamide (DMFO), dimethyl sulfoxide (DMSO), and the like can be used alone or in combination. Non-polar solvents include hexane, uroalkane, Pentane, hexane, ethyl acetate, methyl acetate, butyl acetate, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1-chlorobutane, acetone, 1-chloropentane , o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, diethyl ether, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, 1,2-dichloro Ethane, annealed, di Ethylamine, ether, carbon tetrachloride, THF and the like can be used alone or in combination. The extract of the present invention is not particularly limited thereto, but preferably water, anhydrous or hydrous alcohol having 1 to 6 carbon atoms, acetone, ethyl acetate, chloroform, butyl acetate, 1,3-butylene glycol, hexane, diethyl ether May be obtained using a solvent alone or mixed, more preferably water, ethanol, butanol, ethyl acetate and the like alone or obtained using a mixed solvent, most preferably. Preferably ethanol.

Extraction using the solvent may be carried out according to a method known in the art, preferably cold immersion method to be immersed in the solvent, extraction at room temperature of 10 to 25 ℃, 40 to 100 ℃ It may be carried out by a method such as a heating extraction method of extraction by heating, reflux extraction method using a reflux cooler.

The term "fraction" of the present invention means the result obtained by the fractionation method of separating a specific component or a specific group from a mixture containing various components.

In the present invention, the fraction may be obtained by applying the Ecklonia cava extract to various fractionation methods, the fractionation method is not particularly limited to this, but the solvent fractionation method by treating a variety of solvents, constant molecular weight cut-off value The ultrafiltration fractionation method carried out by passing through the ultrafiltration membrane having, the chromatography fractionation method for performing a variety of chromatography (manufactured for separation according to the size, charge, hydrophobicity or affinity) and the like. In particular, the solvent used in the solvent fractionation method is not particularly limited, but the above-mentioned polar solvent or nonpolar solvent can be used, preferably a nonpolar solvent, most preferably hexane, ethyl acetate, etc. can be used. . For example, the solvent fractionation method may be performed by sequentially fractionating the upper leaf extract using a low solvent from a solvent having a high non-polar level, preferably the Ecklonia cava extract using hexane, ethyl acetate and water. Can be used to sequentially fractionate.

The term “viral hemorrhagic septicemia (VHS)” of the present invention refers to an infectious disease of fish caused by infection of viral hemorrhagic sepsis virus (VHSV), also called “Egtved diseases”. As bleeding occurs in the internal organs, such as internal organs, skin, muscles, external symptoms do not appear.

The term "viral hemorrhagic septicemia virus (VHSV)" of the present invention, also referred to as "Egtved virsu", refers to a kind of RNA virus containing 11 kb of negative sense single stranded RNA. The VHSVs are known to have different subspecies locally and may have various effects on each other, but do not have a particular effect on humans.

According to one embodiment of the present invention, 95% ethanol was added to the Ecklonia cava which can be easily collected in nature, and the Ecklonia cava ethanol extract was prepared by filtration, concentration and drying (Example 1). As a result of administering the prepared Ecklonia cava ethanol extract to the flounder infected with VHSV, it was confirmed that the survival rate is significantly increased (Figs. 1 and 2).

On the other hand, the pharmaceutical composition of the present invention may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture of the pharmaceutical composition. Specifically, the pharmaceutical composition may be formulated in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to conventional methods. Can be. In the present invention, carriers, excipients and diluents that may be included in the pharmaceutical composition 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, and the like in the above extracts and fractions thereof. , Sucrose or lactose, gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. 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, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester 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.

The content of the Ecklonia cava extract or fractions thereof contained in the pharmaceutical composition according to an embodiment of the present invention is not particularly limited thereto, but may be 0.0001 to 50% by weight, more preferably 0.01 to 5% by weight, based on the total weight of the final composition. It may be included in the content.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, the term "pharmaceutically effective amount" of the present invention is to treat or prevent the disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention Sufficient amount means an effective dose level means the severity of the disease, the activity of the drug, the age, weight, health, sex, sensitivity of the individual to the drug of the subject, the time of administration of the composition of the invention used, the route of administration and the rate of excretion The duration of treatment, factors including drugs used in combination or coincidental with the composition of the invention used, and other factors well known in the medical art can be determined. The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects.

The dosage of the pharmaceutical composition of the present invention may include, for example, the Ecklonia cava extract, a fraction thereof, the phlorotannin-based compound of any one of Formulas 1 to 3, or a pharmaceutically acceptable salt of the compound. The composition may be administered to fish at 1 to 200 mg / kg, more preferably 1 to 100 mg / kg, and the frequency of administration of the composition of the present invention is not particularly limited, but is administered once a day or Doses may be divided and administered several times.

As another embodiment of the present invention for achieving the above object, a pharmaceutical composition for the prophylaxis or treatment of VHS comprising a phlorotannin-based compound derived from Ecklonia cava extract or a fraction thereof or a pharmaceutically acceptable salt thereof as an active ingredient to provide.

At this time, the phlorotannin-based compound derived from the Ecklonia cava provided in the present invention may be used alone or in combination with eckol, fucodiphloroethol G or phlorofucoeckol A. Can be.

As used herein, the term "eckol" refers to a compound derived from Ecklonia cava and represented by the following Chemical Formula 1. The compound may be separated from natural sources such as Ecklonia cava, but is not limited thereto, and may be chemically synthesized by a method known in the art, or a commercially available material may be used.

Formula 1

As used herein, the term "fucodiphloroethol G" refers to a compound derived from Ecklonia cava and represented by the following formula (2). The compound may be separated from natural sources such as Ecklonia cava, but is not limited thereto, and may be chemically synthesized by a method known in the art, or a commercially available material may be used.

Formula 2

As used herein, the term "phlorofucoeckol A" refers to a compound derived from Ecklonia cava and represented by the following formula (3). The compound may be separated from natural sources such as Ecklonia cava, but is not limited thereto, and may be chemically synthesized by a method known in the art, or a commercially available material may be used.

Formula 3

As used herein, the term “pharmaceutically acceptable salt” refers to a salt in a form that can be used pharmaceutically among salts in which cations and anions are bound by an electrostatic attraction, and generally include metal salts and organic bases. Salts, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), aluminum salt, or the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N-dibenzylethylenediamine Salts and the like; Salts with inorganic acids can be salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like; Salts with organic acids can be salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; Salts with basic amino acids can be salts with arginine, lysine, ornithine and the like; Salts with acidic amino acids can be salts with aspartic acid, glutamic acid and the like.

According to one embodiment of the present invention, the ethanol extract of Ecklonia cava was fractionated using hexane, ethyl acetate, and distilled water, and the ethyl acetate extract was subjected to silica gel column chromatography using chloroform, methanol, and a mixed solvent thereof as a mobile phase. 15 fractions could be separated. The fifth fraction was separated into five fractions by silica gel column chromatography using chloroform, methanol, and a mixed solvent thereof as a mobile phase. Pure Compound 3 separated the chloroform, metaol and their mixed solvent (100: 0-7: 3, v / v) into mobile phase using MPLC instrument. Subsequently, Compounds 1 and 2 separated and purified the compounds of Formulas 1 to 3 by separating the water, methanol, and a mixed solvent thereof using a reverse phase column chromatography method in the fifth fraction into a mobile phase, respectively (Example 2). Next, the molecular weight and molecular formula of the separated compounds were analyzed using ¹ H NMR, ¹³ C NMR and 2D NMR spectroscopy data, melting point and infrared spectroscopy (IR) data through nuclear magnetic resonance (NMR) analysis. The structure was determined (Example 3). Through the determination of the molecular structure, compounds 1, 2 and 3 derived from the ethanol extract of Ecklonia cava are eckol, fucodiphloroethol G and phlorofucoeckol A, respectively. Confirmed. As a result of evaluating the VHSV inhibitory activity of the three phlorotannin-based compounds identified above, it was confirmed that excellent inhibitory activity against VHSV at concentrations without cytotoxicity (5.1, 0.7 and 1.8 μM, respectively) (Table 1) .

The dosage of the carrier, excipient or diluent and pharmaceutical composition which may be further included in the prophylactic or therapeutic pharmaceutical composition of VHS comprising the phlorotannin-based compound or a pharmaceutically acceptable salt thereof is the same as described above. In addition, the content of phlorotannin-based compounds or their pharmaceutically acceptable salts contained in the pharmaceutical composition is not particularly limited, but is 0.0001 to 10% by weight, more preferably 0.01 to 2% by weight based on the total weight of the final composition. It may be included in the content of.

As another embodiment of the present invention for achieving the above object, the present invention comprises the step of administering the pharmaceutical composition to a subject with or at risk of developing viral hemorrhagic sepsis (VHS), viral hemorrhagic Provided are methods for preventing or treating sepsis.

As described above, the Ecklonia cava extract provided in the present invention, fractions thereof, phlorotannin-based compounds derived therefrom or their pharmaceutically acceptable salts can inhibit the activity of VHSV. The pharmaceutical composition comprising the ingredient may be used for the prevention or treatment of VHS.

As used herein, the term "individual" includes, without limitation, fish, birds, mammals, etc., which may or may not develop VHS.

The term "administration" of the present invention refers to introducing the pharmaceutical composition of the present invention to a subject by any suitable method, and the route of administration may be administered through various routes, oral or parenteral, as long as the target tissue can be reached.

As used herein, the term "prevention" refers to any action that inhibits or delays the development of VHS by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action in which the symptoms of the disease are improved or beneficially altered by administration of the pharmaceutical composition according to the present invention.

In the method of treating VHS of the present invention, the route of administration of the pharmaceutical composition may be administered via any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonally, or rectally as desired. In addition, the composition may be administered by any device in which the active substance may migrate to the target cell.

As another embodiment for achieving the above object, the present invention provides the prevention or treatment of VHS comprising the Ecklonia cava extract, fractions thereof, phlorotannin-based compounds derived therefrom or pharmaceutically acceptable salts thereof as an active ingredient Provided is a quasi-drug composition for use.

The term "quasi drug" used in the present invention refers to articles that have a lesser action than drugs among those used for the purpose of diagnosing, treating, ameliorating, alleviating, treating or preventing diseases of humans or animals. According to this study, quasi-drugs are products that are used for the purpose of medicines, and are used for the treatment or prevention of diseases of humans and animals. These include sterilizing and insecticides to prevent infectious diseases. The kind or formulation of the quasi-drug composition of the present invention is not particularly limited, but may preferably be a disinfectant cleaner, a shower foam, a gagreen, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment, or a filter filler.

As another embodiment for achieving the above object, the present invention is for the prevention or improvement of VHS comprising Ecklonia cava extract, fractions thereof, phlorotannin-based compounds derived therefrom or pharmaceutically acceptable salts thereof as an active ingredient It provides a feed additive, a feed for the prevention or improvement of VHS containing the feed additive, and a method of farming fish comprising feeding the feed to the fish.

Ecklonia cava extract, fractions thereof, phlorotannin-based compounds derived from the same or their pharmaceutically acceptable salts can inhibit the activity of VHSV, so that they are prepared separately as feed additives and mixed in the feed It can be used in the manufacture of feed for the prevention or improvement of VHS, either by way of use or by addition directly in feed production. The prepared VHS preventive or improved feed may be used in aquaculture methods for feeding or preventing aquaculture fish, thereby preventing or improving VHS.

First, the feed additive provided by the present invention includes the Ecklonia cava extract, fractions thereof, phlorotannin-based compounds derived therefrom or their pharmaceutically acceptable salts as active ingredients, and further includes other additives as necessary. can do. The usable additives are not particularly limited as long as they can exhibit the effect of preventing or improving VHS provided by the present invention. Preferably, binders, emulsifiers and preservatives are added to prevent deterioration of feed quality; Amino acids, vitamins, enzymes, probiotics, flavoring agents, nonprotein nitrogen compounds, silicates, buffers, coloring agents, extractants, which are added for the purpose of increasing feed efficiency; Or a feed mixture or the like, alone or in combination, more preferably marine protein (fish meal or krill wheat), vegetable protein (bean powder, wheat gluten, corn gluten, lupine powder, pea or sunflower powder), Protein sources such as blood meal and bone meal; Energy sources such as fish oil (squid liver oil, etc.), vegetable oils (rapeseed oil, soybean oil, soybean oil, etc.), vitamin mixtures, and mineral mixtures may be used alone or in combination.

The form of the feed additive is not particularly limited, but may be preferably in the form of liquid or solid, more preferably in the form of a dry powder may be added during the preparation of the feed. At this time, the drying method used in the preparation of the dry powder is not particularly limited, but may be preferably used alone or in combination with a method such as ventilation drying, natural drying, spray drying, freeze drying.

Next, the term "feed" of the present invention means any natural or artificial diet, one meal, or the like or a component of the one meal for the animal to eat, ingest and digest.

Feed provided by the present invention is not particularly limited as long as it can exhibit the effect of preventing or improving the VHS provided by the present invention, preferably grains, fruits, food processing by-products, algae, fiber, pharmaceutical Busan Vegetable feeds such as logistics, oils, starches, gourds or grain by-products; Animal feeds such as proteins, minerals, fats and oils, minerals, fats and oils, single-cell proteins, zooplankton or foods; Seeds containing grains such as wheat, oats, and corn, and by-products obtained by refining grains, and by-products obtained from bran, beans, fluids, sesame seeds, linseed, coco, etc., including rice bran, bran and barley bran. Preparation of casein from residues such as residual starch, the main component of starch residue from phosphorus salt, sweet potato, potato, etc., meat meal, blood meal, milk powder, skim milk powder, milk cheese, and skim milk Thick foods including dry whey, yeast, chlorella, algae, etc., dried whey; Fill the silo with raw vegetables such as grasses, grasses and green grass, raw turnips, fodder beet, root vegetables such as turner, turnips, green grass crops and grains. Fertilizers such as silage, lactic acid fermentation, silage, hay, dried grass, straw of breeder, and leaves of legumes; Mineral feeds such as oyster shells and rock salt, urea feeds such as urea and its derivatives such as diureide isobutane, and natural ingredients are supplemented, or added to the feed mix in small amounts to increase the shelf life of the feed. Special feeds such as feed additives and dietary supplements, which are substances to be used, may be used alone or in combination.

Finally, the fish farming method provided in the present invention can be used for fish that can be VHS-induced VHS-induced fish, the fish that can be induced VHS is not particularly limited, but flounder ( Paralichthys olivaceus ), Defensive ( Seriola quinqueradiata ), red snapper ( Pagrus major ), self- propelled fish ( Takifugu rubripes ), bushy ( Seriola aureovittata ), trout ( Oncorhynchus masou ), rainbow trout ( Oncorhynchus mykiss ), spearfish ( Seriola dumerili ), horse mackerel ( Trachurus japonicus) ), Pseudocaranx dentex , Eelphe ( Epinephelus septemfasciatus ), Bluefin tuna ( Thunnus thynnus ), Koi ( Cyprinus carpio ), Zebrafish ( Danio rerio ), Catfish ( Silurus asotus ), Clarias gariepinus ( Clarias gariepinus) ), Tilapia ( Oreochronis niloticus ), salmon ( Oncorhynchus keta ), atlantic salmon ( Salmo salar ), larva ( Oryzias latipes ) and the like, more preferably flounder ( Paralic hthys olivaceus ).

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the following examples are merely to illustrate the present invention is not limited to the contents of the present invention.

실시예 1: 감태 추출물의 제조Example 1: Preparation of Ecklonia cava Extract

The Ecklonia cava used in the present invention is generally easily collected in nature and was used by pulverizing in the form of powder to obtain the extract of the present invention efficiently. Specifically, 20 l of 95% ethanol was added to 3 kg of the crushed Ecklonia cava, and extracted by leaving it for 7 days at room temperature. The resultant was filtered through a filter paper to obtain a liquid component, and the ethanol extract of Ecklonia cava was concentrated and dried (230). g) was prepared.

실시예 2: 감태 추출물의 VHSV에 대한 저해 효과Example 2: Inhibitory Effect of Ecklonia cava Extract on VHSV

First, as a result of measuring the EC ₅₀ of the Ecklonia cava ethanol extract prepared in Example 1, it was confirmed that 4.75 ㎍ / μl, in order to verify the antiviral effect of the Ecklonia cava extract against VHSV in vivo for the flounder The appropriate level of VHSV administration was determined (Figure 1).

1 is a graph showing the results of experiments to determine the concentration of challenge test VHSV administration in order to verify the antiviral effect on VHSV of Ecklonia cava extract in vivo in flounder. As shown in Figure 1, when the capacity to infect the cells VHSV TCID50 (when the titration of the virus), the results of the attack experiment at the concentration of 10E + 5.1, 10E + 6.1 or 10E + 7.1 TCID 50 / fish, When treated with a concentration of 10E + 7.1TCID50 / fish, mortality started from day 2 and confirmed that mortality of up to 80% was observed on day 11. Therefore, the proper dose of the VHSV challenge experiment was determined to be 10E + 7.1TCID50 / fish.

Next, the Ecklonia cava extract was added to the feed in a content of 0.3% and fed to the animals for 4 weeks, and then 15 animals were randomly selected for each experimental group, and the attack experiment was performed by artificial infection. The artificial challenge experiment was intraperitoneally injected with 100 μl of VHSV at a concentration of 10E + 7.1TCID50 / fish to all subjects, and they were raised together in a circular tank of about 1 ton scale. The return was carried out twice a day, the water temperature was maintained at about 14 ° C. using a cooler, and fasted during the breeding period. Then, relative mortality (Relative Percent Survival, RPS), including mortality after 4 weeks, was calculated in terms of (1-experimental mortality / control mortality) × 100), and the antiviral effect of Ecklonia cava extract on VHSV was analyzed (FIG. 2). At this time, the flounder cultured without feeding the Ecklonia cava extract was used as a control.

Figure 2 is a graph showing the results of verifying the preventive effect on the VHS of Ecklonia cava extract in the olive flounder. As shown in FIG. 2, the death of the cultured fish occurred between 2 and 3 weeks after the death started on the 7th day. The flounder in the control group had a cumulative mortality rate of about 53%, whereas the flounder pretreated with Ecklonia cava extract exhibited a cumulative mortality rate of about 20%. Therefore, the flounder which is provided in the present invention is a flounder that is anti-viral activity against VHSV. It can be seen that the effect provided to.

실시예 3: 감태 추출물로부터 분획물 및 화합물의 분리 및 정제Example 3: Separation and Purification of Fractions and Compounds from Ecklonia cava Extract

From the results of Example 2, it was confirmed that the Ecklonia cava extract exhibits the effect of providing antiviral activity to the olive flounder to the VHS, and to identify the active ingredient exhibiting the antiviral activity against the VHS.

Specifically, 2 liters of water was suspended in 230 g of the ethanol extract of Ecklonia cava obtained in Example 1, and suspended. This was placed in a separatory funnel, and fractionated sequentially using hexane, ethyl acetate and water to obtain a hexane fraction, an ethyl acetate fraction and a water fraction. The ethyl acetate fraction obtained above was concentrated and dried to obtain an ethyl acetate fraction, and 85 g of the ethyl acetate fraction was converted to chloroform, methanol and a mixed solvent thereof (100: 0 to 1: 3, v / v) as a mobile phase. Silica gel column chromatography [silica gel 700 g, 70 to 230 mesh] was performed to separate 15 fractions (Fraction 1 to 15). Among them, a fifth fraction from which a target compound is detected is selected, and the selected fifth fraction is used again as a mobile phase using chloroform, methanol, and a mixed solvent thereof (100: 0 to 1: 5, v / v) as a mobile phase. Chromatography (700 g of silica gel, 70 to 230 mesh) was performed to separate the five fractions. Pure Compound 3 separated the chloroform, metaol and their mixed solvents (100: 0 to 7: 3, v / v) into the mobile phase using an MPLC instrument. Subsequently, Compounds 1 and 2 separated water, methanol and their mixed solvents (100: 0 to 1: 4, v / v) using a reverse phase column chromatography in the fifth fraction, respectively, as mobile phases.

실시예 4: 화합물 1 내지 3의 구조 분석Example 4: Structural Analysis of Compounds 1 to 3

Molecular weight and molecular formula of the compounds 1 to 3 isolated in Example 3 were obtained through nuclear magnetic resonance (NMR) analysis (Bruker AM 300, 500) ¹ H NMR, ¹³ C NMR and 2D NMR spectroscopy data, and further As a result of determining their molecular structures using melting point and infrared spectroscopy (IR) data, the compounds 1 to 3 were identified as phlorotannin-based compounds.

Spectroscopic data of compound 1 (eckol).

Light brown powder-melting point (mp) 235 ° C., ESI-MS m / z = 371 [M−H], [calcd C ₁₈ H ₁₂ O ₉ , 372]; ¹ H NMR (400 MHz, Methanol-d4) d 6.15 (s, 1H, H-3), 5.96 (s, 2H, H-3, H-6), 6.03 (s, 3H, H-2 ', H -4 ', H-6'); ¹³ C NMR (100 MHz, Methanol-d4) d 161.9 (C-1 '), 160.3 (C-3', H-5 '), 154.6 (C-7), 147.3 (C-2), 147.2 (C -9), 144.3 (C-5a), 143.4 (C-4), 138.6 (C-10a), 125.7 (C-1), 124.9 (C-4a), 124.6 (C-9a), 99.9 (C- 8), 99.5 (C-3), 97.8 (C-4 '), 95.9 (C-6), 95.5 (C-4', C-6 ').

Spectroscopic data of compound 2 (fucodiphloroethol G).

White powder - ESI-MS m / z = 499 [M + H] +, [calcd C 24 H 16 O 12, 498]; ¹ H NMR (400 MHz, methanol-d4) d 6.13 (d, J = 2.44 Hz, 1H, H-4 "), 6.07 (s, 2H, H-3 '", H-5'"), 6.03 ( s, 1H, H-6 "), 6.03 (d, J = 0.98 Hz, 1H, H-5), 5.92 (s, 2H, H-30, H-5 '), 5.70 (d, J = 2.44 Hz , 1H, H-3); ¹³ C NMR (100 MHz, methanol-d4) d 159.6 (C-5 "), 159.5 (C-1"), 159.3 (C-3 "), 159.2 (C-4 '"), 158.1 (C-4 '), 157.6 (C-2), 156.5 (C-2'", C-6 '"), 153.9 (C-4), 152.3 (C-2', C-6 '), 152.1 (C-6 ), 124.9 (C-1), 124.4 (C-1 '), 102.1 (C-2 "), 101.9 (C-1'"), 98.1 (C-3), 97.6 (C-4 "), 96.8 (C-5 '"), 96.4 (C-3'", C-5 ', C-3'), 94.6 (C-5), 94.3 (C-6 ").

Spectroscopic data of compound 3 (phlorofucoeckol A).

Colorless solid-melting point (mp) 278 ° C., ESI-MS m / z = 601 [M−H], [calcd C ₃₀ H ₁₈ O ₁₄ , 602]; ¹ H NMR (400 MHz, methanol-d4) d 6.64 (s, 1H, H-13), 6.41 (s, 1H, H-9), 6.27 (s, 1H, H-3), 6.07 (d, 1H , J = 2.93 Hz, H-6), 5.99 (d, 2H, J = 1.95 Hz, H-2 ', H-6'), 5.95 (t, 1H, J = 1.95 Hz, H-4 '), 5.93 (t, 1H, J = 1.95 Hz, H-4 "), 5.90 (d, 2H, J = 1.95 Hz, H-2", H-6 "); ¹³ C NMR (100 MHz, methanol-d4) d 162.0 (C-1 ′, C-1 ″), 160.3 (C-3 ″, C-5 ″, C-3 ′, C-5 ′), 153.3 (C-12a), 151.8 (C-1 ′) ), 151.3 (C-11a), 146.1 (C-14), 144.1 (C-4), 138.5 (C-15a), 135.4 (C-5a), 128.3 (C-14a), 125.2 (C-4a) , 124.8 (C-1), 122.5 (C-11), 105.5 (C-7), 105.4 (C-6), 100.1 (C-9), 99.5 (C-3), 97.9 (C-4 ") , 97.8 (C-4 '), 96.3 (C-13), 95.5 (C-2', C-6 ', C-2 ", C-6").

실시예 5: 플로로탄닌계 화합물의 VHSV에 대한 저해 효과 및 세포독성 측정Example 5 Inhibition Effect of Cyclotannin Compounds on VHSV and Cytotoxicity

The cytopathic reduction effect and cytotoxicity of the phlorotannin-based compound obtained in Example 3 were measured.

First, in order to determine the inhibitory activity against VHSV, the cytopathic effect was measured as follows. 100 μl of FHM cells were dispensed into each well in a 96 well plate containing MEM10 medium, and 10 μl of VHSV (102.8) was injected into each well, and 4, 2, 1, 0.5, 0.25, or 0.125 μg was added to each well. Each compound was added to a concentration of / μl, an appropriate amount of MEM10 medium was added to a final volume of 200ul, and then reacted at 16 ° C for 1 hour. After the reaction was completed, four sets of each set of MEM10 medium removed from each well and sealed with a plastic paraffin film were prepared, and each of these sets was left at 16 ° C. for 10 to 14 days to survive. By counting the number of sets to calculate the survival rate, the cytopathic effect was measured (Table 1).

In addition, in order to measure cytotoxicity, EC ₅₀ (μM) values of the above compounds on FHM cells were measured (Table 1).

Table 1

Cytopathic Effect and EC <sub> 50 </ sub> (μM) Value of Phlorotannin Compounds

	Treatment concentration (μM)						EC ₅₀ (μM)
compound	4	2	One	0.5	0.25	0.125	EC ₅₀ (μM)
Compound 1	-	-	3/4	4/4	4/4	4/4	5.1
Compound 2	-	-	-	-	1/4	4/4	0.7
Compound 3	-	-	2/4	4/4	4/4	4/4	1.8

As shown in Table 1, each of the phlorotannin-based compounds isolated from the Ecklonia cava extract showed excellent inhibitory activity against VHSV at concentrations without cytotoxicity (5.1, 0.7, 1.8 μM, respectively).

Therefore, it was found that ecchol, fucodiphloroethol G, or phlorofucoeckol A, isolated from the Ecklonia cava extract, is an active ingredient exhibiting antiviral activity against VHSV.

Claims

Pharmaceutical composition for the prevention or treatment of viral hemorrhagic septicemia (VHS) comprising an Ecklonia cava extract or a fraction thereof as an active ingredient.
The method of claim 1,

The Ecklonia cava extract is composed of water, anhydrous or hydrous alcohol having 1 to 6 carbon atoms, acetone, ethyl acetate, chloroform, butyl acetate, 1,3-butylene glycol, hexane, diethyl ether, and combinations thereof. A composition obtained by extraction using a solvent selected from.
The method of claim 1,

The Ecklonia cava extract is obtained by extracting Ecklonia cava with ethanol.
The method of claim 1,

The fraction is a fraction obtained by applying the Ecklonia cava extract to a method selected from the group consisting of solvent fractionation, ultrafiltration fractionation, chromatography fractionation and combinations thereof.
A pharmaceutical composition for the prophylaxis or treatment of VHS, comprising a compound selected from the group consisting of ectopic extracts or fractions thereof, phlorotannin-based compounds, their pharmaceutically acceptable salts, and combinations thereof.
The method of claim 5,

Wherein the phlorotannin-based compound is Ecol (eckol) of formula (1), fucodiphloroethol G of the formula (2) or phlorofucoeckol A (phlorofucoeckol A) of the formula (3);

[Formula 1]

[Formula 2]

[Formula 3]

.
A method of preventing or treating viral hemorrhagic sepsis, comprising administering the pharmaceutical composition of any one of claims 1 to 6 to an individual with or at risk of developing viral hemorrhagic sepsis (VHS).
An quasi-drug composition for the prevention or treatment of VHS comprising the Ecklonia cava extract, fractions thereof, phlorotannin-based compounds derived therefrom, or pharmaceutically acceptable salts thereof as an active ingredient.
The method of claim 8,

The phlorotannin-based compound is eccol, fucodiphloroethol G or phlorofucoeckol A composition.
A feed additive for the prevention or improvement of VHS comprising an Ecklonia cava extract, a fraction thereof, a phlorotannin-based compound derived therefrom, or a pharmaceutically acceptable salt thereof as an active ingredient.
The method of claim 10,

The phlorotannin-based compound is ecchol, fucodiphloroethol G or phlorofucoeckol A.
A fish farming feed comprising the feed additive of claim 10.
A fish farming method comprising the step of feeding the fish of claim 12 to the fish.
The method of claim 13,

The fish are halibut ( Paralichthys olivaceus ), defensive ( Seriola quinqueradiata ), red snapper ( Pagrus major ), and swelling fish ( Takifugu rubripes ) that can develop VHS due to viral hemorrhagic septicemia virus (VHSV) infection. , busiri (Seriola aureovittata), trout (Oncorhynchus masou), rainbow trout (Oncorhynchus mykiss), jaetbangeo (Seriola dumerili), horse mackerel (Trachurus japonicus), black dot-line horse mackerel (Pseudocaranx dentex), grouper (Epinephelus septemfasciatus), bluefin tuna (Thunnus thynnus , carp ( Cyprinus carpio ), zebrafish ( Danio rerio ), catfish ( Silurus asotus ), Clarias gariepinus , Oreochronis niloticus , salmon ( Oncorhynchus keta ), Atlantic salmon ( Salmo salar ) And Oryzias latipes .
The method of claim 13,

The fish is a flounder ( Paralichthys olivaceus ).