JP2016529243A - Antiviral composition of fish containing quercetin as an active ingredient - Google Patents

Abstract

Disclosed is an antiviral composition for a viral hemorrhagic septic virus in fish, comprising quercetin as an active ingredient. Prevention or treatment of diseases caused by viral hemorrhagic septic viruses that have a large effect on the mortality of cultured fish by intraperitoneal injection, oral administration, or aquarium administration of the composition according to the present invention. Will ultimately contribute greatly to cost savings and increased added value in the fish farming industry. [Selection] Figure 5

Description

  The present invention relates to an antiviral composition against fish viral hemorrhagic septic virus (hereinafter referred to as “VHSV”) containing quercetin as an active ingredient, and more particularly, an appropriate amount of quercetin is obtained using various methods. The present invention relates to a composition for preventing infection of fish with viral hemorrhagic sepsis or treating infected fish by administration to fish.

  Viral hemorrhagic septicemia (VHS) was distributed mainly in the European region, but was isolated from various marine fish species after being discovered from coho salmon and chinook salmon migrating to the western Pacific coast of the United States in 1988. It was. Recently, viral hemorrhagic septicemia was not only detected from naturally occurring marine fish species in Japan and Korea, but it was reported to cause fatal diseases in cultured flounder during the low water temperature period (Non-patent Document 1). Examples of fish species that have been confirmed to be infected include flounder, rainbow trout, Atlantic salmon, brown trout, coho salmon, chinook salmon, and cod. In South Korea, it has been detected not only in seawater-cultured fish but also in naturally occurring fish in the Tokai and South Seas. Such hemorrhagic sepsis virus is an important pathogen of European trout and Korean and Japanese flounder.

  VHSV causes economic loss by causing drowning not only for small fish but also for fish with a large shipping time. For example, flounder is the main aquaculture fish species in Korea, the production amount is about 47,000 tons based on 2008, and the production value is 408.3 billion won, which is the total aquaculture production value It is an important aquaculture item accounting for 26.8%. However, various infectious diseases caused by viruses, bacteria, parasites, and the like cause about 40% of deaths and serious economic losses. In particular, viral diseases such as VHS cause massive moribundity not only in fry, but also in commercial products with high commercial value. However, the annual loss of aquatic marine product diseases and morbidity due to marine pollution is about 3 It is reported that it will reach 100 billion won.

  VHSV is also inactivated by ultraviolet treatment and easily inactivated by disinfectants such as chlorine, hypochlorous acid, and iodophor. Therefore, it is known that it is effective to disinfect instruments and water tanks in aquaculture. ing. However, like other viral diseases, a chemotherapeutic agent for treating VHS has not been developed yet (Non-patent Document 2).

  Patent Document 1 discloses a method and a system for preventing infection with Japanese flounder by VHSV by adjusting the water temperature for flounder culture to be maintained at 18-22 ° C. However, this method has a problem that the heating cost for maintaining the water temperature in winter is too high.

  Patent Document 2 discloses an antiviral composition against VHSV containing curcumin as an active ingredient. However, since the above-described conventional technology has confirmed antiviral activity at the cellular level, it has not been confirmed whether or not a desired effect can be obtained from an actual cultured individual, and even in the field. Even if effective, there is a drawback that it is difficult to obtain a large amount of curcumin.

  Attenuated virus vaccines are at risk of virus virulence repair and spread to nature. Moreover, in a DNA vaccine experiment in which a plasmid in which a hemorrhagic septic virus G or N gene was implanted was injected intramuscularly, it was reported that a strong immune reaction was induced and mortality was also reduced in rainbow trout and flounder (non-patented). Reference 3) has not yet reached a practical level.

  Quercetin is one of the flavonoids, and is known to be abundant in red wine, onions, kiwi, green tea, apples, berries, Chinese cabbage (cabbage, broccoli, cauliflower, turnip, etc.). Quercetin is known to have various physiological activities such as removal of heavy metals, reduction of blood cholesterol, prevention of adult diseases such as hypertension and diabetes, and antioxidant and antiviral activities. There are only a few papers on the impact on reducing the incidence of coronary artery disease, stroke, hypertension, cancer, etc. In particular, there are no reports on the antiviral effects and studies of quercetin on VHSV. The chemical structure of quercetin is as follows.

  The inventors of the present invention have made extensive efforts to develop an antiviral composition by investigating the infection mechanism and the defense mechanism of virus infectious diseases in cultured fish. As a result, quercetin was found to exhibit an excellent antiviral effect against viral hemorrhagic sepsis, and the present invention was completed.

Korean Published Patent No. 10-2009-0077303 Korean Published Patent No. 10-2013-0058104

Takano et al. Bulletin of the European Association of Fish Pathologies, 186-192, 2000; Issiki et. , Al. Diseases of Aquatic Organisms, 87-99, 2001 National Fisheries Chemistry, http: // fdcc. nfrdi. re. kr / 02 / 01_click. jsp? DSS_CD = IV009 Byon et al. , Vaccine, 921-930, 2005

  It is an object of the present invention to provide an antiviral composition for fish.

  Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases caused by fish infectious viruses.

  Still another object of the present invention is to provide a fish feed composition and feed for the prevention or treatment of diseases caused by fish infectious viruses.

  Still another object of the present invention is to provide a method for preventing infection with fish-infectious viruses in fish, which comprises feeding fish with an antiviral composition.

  The present invention made to achieve the above-mentioned object is characterized by being an antiviral composition against VHSV of fish containing quercetin as an active ingredient.

  “As an active ingredient” means containing an amount sufficient to achieve the efficacy or activity of quercetin. As will be apparent from the following examples, toxic doses of quercetin contained in the composition of the present invention are within an appropriate range for those skilled in the art, since toxic doses do not appear in animal cells even if quercetin is administered in excess. Can be selected.

  The quercetin in the present invention is synthesized, purely isolated, extracted from a plant body or fractionated, but as much as possible a natural extract containing quercetin, preferably Contains onion extract or onion fraction.

  The composition of the present invention is applied to any fish species that is a host of hemorrhagic septic virus. For example, flounder, yellowtail, red sea bream, tiger puffer, flatfish, trout, amberjack, horse mackerel, main tuna, carp, zebrafish, catfish, tilapia, salmon, medaka or shrimp that are naturally produced or cultivated. In the following examples, actual biological tests were conducted on Japanese flounder, but quercetin inhibits hemorrhagic septic virus replication and increases the survival rate of cells infected with hemorrhagic septic virus. When other examples in which the above is confirmed are also examined, it goes without saying that the composition of the present invention is applied to any fish species that can host hemorrhagic septic viruses.

  In other words, the present invention is a pharmaceutical composition for preventing or treating viral hemorrhagic sepsis containing quercetin as an active ingredient.

  The pharmaceutical composition of the present invention can comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is one that is usually used at the time of preparation. Lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, fine crystallinity Cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like are included, but are not limited thereto. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). The dosage form of the pharmaceutical composition according to the invention is a solution, suspension or emulsified liquid in oil or aqueous medium, or is in the form of an extract, powder, granule, tablet or capsule and is dispersed. An agent or stabilizer may further be included.

  The antiviral composition (or prophylactic or therapeutic pharmaceutical composition) according to the present invention is administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection , Intraperitoneal injection, transdermal administration, etc., preferably oral administration. In the following examples, the peritoneal injection method was applied in order to obtain early experimental results, but the present invention is not limited to this.

  The preferred dosage of the pharmaceutical composition of the present invention is determined by factors such as formulation method, mode of administration, animal age, body weight, sex, pathological condition, administration time, route of administration, excretion rate and reaction sensitivity. And usually a skilled physician can easily determine and prescribe the effective dose for the desired treatment or prevention. For example, it is preferable that the composition of the present invention is administered by intraperitoneal injection or oral administration once every 1 to 3 days so that the active ingredient content is 1 mg per 1 kg of the living body.

  According to another aspect of the present invention, the present invention provides a fish feed for preventing or ameliorating fish infectious VHSV disease or a composition for adding fish feed, comprising quercetin as an active ingredient.

  The fish feed or fish feed composition according to the present invention can be produced together with various commonly used components. For example, any selected from the group consisting of marine protein (eg, fish meal or krill meal), vegetable protein (eg, bean powder, wheat gluten, corn gluten, gauze bean powder, bean powder or sunflower seed powder), blood meal and bone meal One or more protein sources, one or more energy sources selected from the group consisting of fish oil (eg, squid liver oil) or vegetable oil (eg, rapeseed oil, bean oil and soybean oil), a vitamin mixture and an inorganic mixture Can be included.

  According to a preliminary experiment, when a normal fish feed containing 0.0025% quercetin was administered, it showed excellent antiviral activity in Japanese flounder. For this reason, it is preferable to contain 0.0005 to 0.005% of quercetin in the feed for fish farming. If the content of quercetin is lower than this, almost no preventive and therapeutic effect appears, and if the content of quercetin is higher than this, the effectiveness is lower than the added amount.

  On the other hand, quercetin is chemically and physically very stable in an aqueous solution. For this reason, the preventive or therapeutic effect of VHSV disease is obtained by the method of directly administering the antiviral composition according to the present invention to the farm.

  The specific dose and administration interval can be variously changed according to the type of fish to be cultured, the culture density, the water temperature of the farm, etc., but every other day with reference to other farm additives It is preferable to administer quercetin to a concentration of 0.1 to 10 ppm.

  As described above, the composition according to the present invention has an effect of suppressing replication of VHSV, so that the survival rate of the host fish against VHSV is ultimately increased.

  For this reason, the intraperitoneal injection, the oral administration, and the aquarium administration of the composition according to the present invention enables prevention or treatment of diseases caused by VHSV that greatly affect the mortality rate of cultured fish.

  This greatly contributes to cost reduction and added value increase in the fish farming industry.

It is a graph which shows the result of having compared and confirmed the antiviral activity with respect to the virus infection of a quercetin and a quercetin glycoside. It is a graph which shows the result of having confirmed the cytotoxicity in the fish cell (Fat head Minnow; FHM cell) which processed quercetin concentration-dependently. It is a graph which shows the result of having confirmed the survival rate of the fish cell (FHM cell) by VHSV infection after processing a quercetin concentration-dependently. It is a graph which shows the result of having confirmed the VHSV replication inhibitory ability in a cell culture solution by a quercetin process. It is a graph which shows the experimental result in the living body which compared the survival rate with respect to VHSV infection in the Japanese flounder by intraperitoneal administration of quercetin. It is a graph which shows the experimental result in the living body which compared the survival rate with respect to VHSV infection in the flounder by oral administration of quercetin.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are merely examples for easily explaining the contents and scope of the technical idea of the present invention, and the technical scope of the present invention is not limited or changed by these examples. Needless to say, various modifications and changes can be made within the scope of the technical idea of the present invention based on these examples.

Example 1: Comparison of antiviral activity of quercetin and quercetin glycosides It was confirmed that quercetin and similar compounds were excellent in antiviral activity of quercetin.

FHM cells (ATCC CRL-2872, Manassa, VA), a fish cell line derived from carp showing the highest susceptibility to VHSV infection, were cultured in 96 well plates at 1.0 × 10 ⁵ cells / 100 μL in each well for 24 hours. . After removing all Libobitz L-15 medium containing 10% fetal bovine serum (FBS), quercetin (Sigma, Q4951) and 7 quercetin glycosides (quercetin-3-β-D-glucoside, quercetin) -3-O-α-L-arabinopyranoside, kaempferol-3-O-β-D-glucopyranoside, quercetin-3-O-β-D-glucopyranoside, quercetin-3-glucuronide, rutin, quercetin-3 -O-β-D-galactoside) was each treated with 100 μM for 90 minutes. The residual fluid was then removed and fish cells were infected with VHSV at a 0.2 MOI level for 2 hours. After 2 hours, the residual liquid was removed, sympathized with fresh medium, and cultured for 42 hours. Each well was treated with 10 μL of the Cell Counting Kit-8 reagent and cultured for 8 hours, and then the absorbance value at 450 nm was measured using a microplate reader to compare the cell viability. As a result, it was confirmed that quercetin was superior in antiviral activity against VHSV compared to other quercetin glycosides (FIG. 1).

Example 2: Confirmation of the toxicity of quercetin to fish cells The presence or absence of toxicity of quercetin to fish cells was confirmed.

Fish cells were cultured in a 96-well plate at 1.0 × 10 ⁵ cells / 100 μL in each well for 24 hours, and quercetin was treated at 25-400 μM for 24 hours. Next, the medium was replaced with a new medium, 10 μL of Cell Counting Kit-8 reagent was treated in each well and cultured for 8 hours, and then the absorbance value at 450 nm was measured using a microplate reader to determine the cell viability. Compared. As a result, it was confirmed that quercetin was not toxic to fish cells when compared with the negative control group not treated with quercetin (FIG. 2).

Example 3: Antiviral activity of quercetin An attempt was made to confirm an increase in cell viability against VHSV infection upon pretreatment with quercetin.

Fish cells were cultured in a 96-well plate at 1.0 × 10 ⁵ cells / 100 μL in each well for 24 hours, and quercetin was treated at 25-400 μM for 24 hours. The medium was then removed and VHSV was infected at a concentration of 0.2 MOI for 2 hours. Subsequently, it was replaced with a new medium and cultured for 42 hours. Each well of Cell Counting Kit-8 Reagent was treated at 10 μL and cultured for 8 hours, and then the absorbance value at 450 nm was measured using a microplate reader to compare the cell viability with increasing concentration. As a result, it was confirmed that as the concentration increased, the cell viability increased by 40% compared to the control group not treated with quercetin (FIG. 3).

Example 4: Confirmation of VHSV gene replication control of quercetin The presence or absence of VHSV replication control during the pretreatment of quercetin was confirmed.

Fish cells were cultured in a 60 mm dish at 3.0 × 10 ⁶ cells / 3 mL for 24 hours and treated with 100 μM quercetin for 90 minutes. The medium was then removed and VHSV was infected at a concentration of 0.2 MOI for 2 hours. Subsequently, it was replaced with a new medium and cultured for 42 hours.

  The supernatants of the positive control group not treated with quercetin and the experimental group treated with 100 μM were analyzed using the real-time polymerase chain reaction (PCR) method. The VHSV genes to be confirmed using the real-time polymerase chain reaction (PCR) method are NV-genes, NV_F (sequence 1, 5′-TTG TCC TTC GCG AGA TGA TCG-3 ′) and NV_R (sequence 2, 5 '-TTT CTG ACC GAT CGA GGT CAC TG-3') primer was used for absolute quantitative analysis.

  As a result, it was confirmed that the experimental group treated with quercetin showed a replication reduction rate of about 42% compared to the positive control group (FIG. 4). This shows that quercetin has the effect of suppressing VHSV replication and increasing cell viability compared to the positive control group, and confirms that quercetin is an antiviral agent against VHSV.

Example 5: Confirmation of the effect of prolonging the survival rate of Japanese flounder by intraperitoneal administration of quercetin The antiviral change of flounder administered intraperitoneally with quercetin against VHSV was examined.

About 14 flounder of about 100 g were divided into a flowing water tank and acclimatized, and then subjected to the experiment. The water temperature was kept at 12 ° C. during the experiment. Quercetin was allowed to be injected intramuscularly in 0.1 mL at a concentration of 0.1 mg / fish (ie, 1 mg per 1 kg of living body), and then the experimental group in which quercetin was injected intramuscularly and the positive control group that was not administered were grouped. . Twenty-four hours after the intramuscular injection of quercetin, VHSV 10 ^6.8 TCID ₅₀ / mL was intramuscularly injected into the experimental group and the positive control group. Three days after the infection with VHSV, quercetin was injected intramuscularly once for the experiment.

  I stopped feeding the feed and examined the mortality rate. When compared with the positive control group, which was quercetin-untreated group after 2 weeks, the effect of prolonging the survival rate of Japanese flounder appeared in the experimental group treated with quercetin three days later after treatment with quercetin 0.1 mg / fish ( FIG. 5).

Example 6: Confirmation of the effect of prolonging the survival rate of flounder by oral administration of quercetin The antiviral change of flounder administered orally with quercetin against VHSV was examined.

  About 10 flounder fish of about 100 g were set as one experimental group and a control group, and were raised in a circulating filtration system for 2 days. Feeding was stopped from the experiment start date, and the experimental group was administered quercetin at a concentration of 10 mg / 0.1 mL and then administered 0.1 mL each using an oral sonde. The control group was orally administered only PBS containing a transmitter (1% polyoxyethylene sorbitan monooleate (Tween-20) and 0.5% dimethyl sulfoxide (DMSO) instead of quercetin.

Twelve hours after the oral administration of quercetin, 0.1 mL of VHSV 10 ^6.8 TCID ₅₀ / mL was injected intramuscularly into the experimental group and the positive control group. The experiment was conducted by further oral administration of quercetin once every other day for 6 days after infection with VHSV. During the experiment, the water temperature was kept at 12 ° C. to examine the mortality rate. When the mortality inhibitory effect by oral administration of quercetin was compared after 8 days, there was no difference in the survival rate of flounder in the positive control group which was an untreated group and the experimental group which was treated with quercetin 10 mg / fish. The reason why the inhibitory effect on the mortality rate is different between the intraperitoneal administration and the oral administration seems to be that the flounder that reacts more sensitively to the environmental conditions was subjected to considerable stress by artificial oral administration (FIG. 6).

Claims (7)

  An antiviral composition for a viral hemorrhagic septic virus in fish containing quercetin as an active ingredient.   The antiviral composition according to claim 1, wherein the quercetin is synthesized, purely isolated, extracted from a plant, or fractionated. The fish
Claim 1 or claim 2 characterized by being a flounder, yellowtail, red sea bream, tiger pufferfish, flounder, trout, amberjack, horse mackerel, mahata, this tuna, carp, zebrafish, catfish, tilapia, salmon, medaka or shrimp An antiviral composition as described.   The parenteral or oral administration is performed by any one method selected from the group consisting of intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, and transdermal administration. 2. The antiviral composition according to 2.   The antiviral composition according to claim 1 or 2, wherein the composition is a composition for adding fish feed for prevention or treatment of diseases caused by viral hemorrhagic sepsis virus.   Fish culture feed characterized by containing quercetin 0.0005 to 0.005%.   A method for the prevention and treatment of viral hemorrhagic septic infection of fish, characterized in that quercetin is added to a fish culture aquarium at a concentration of 0.1 to 10 ppm every 1 to 3 days.