KR101429112B1 - Anti-Viral Compositions against Viral Hemorrhagic Septisemia Virus - Google Patents

Abstract

The present invention relates to an antiviral composition against viral hemorrhagic sepsis virus, and is an antiviral composition against Viral Hemorrhagic Septisemia Virus (VHSV) containing curcumin as an active ingredient. According to the present invention, the antiviral composition of the present invention can provide a cell survival rate of about 5-fold for the viral hemorrhagic sepsis virus and understand the mechanism of infection and defense mechanism of the fish, It provides basic research that can be solved.

Description

[0001] Anti-Viral Compositions Against Viral Hemorrhagic Septisemia Virus [

The present invention is directed to an antiviral composition for viral hemorrhagic sepsis virus.

Flounder is the main fishery species of Korea, and the production of cultured flounder is about 47,000 tons as of 2008 and its production amount is 408.3 billion won, which is an important commodity item which accounts for 26.8% of the total production amount. However, various infectious diseases caused by viruses, germs or parasites that occur during the cultivation period cause about 40% of the stocks to be killed due to serious economic loss, and in particular, such as viral hemorrhagic septisemia (VHS) Viral diseases are reported to cause massive mortality in high-value-added fishes as well as tobacco, and the economic loss of farmed fish due to marine pollution and aquatic aquatic diseases is estimated to reach 300 billion won.

The possibility of influenza viruses in terrestrial animals (including humans) has been suggested, and the possibility of accepting fish pathogen viruses as a common pathogen in fisheries centering on the International Animal Health Organization (OIE) and the European Union's (EU) There is a need for review. Therefore, the issue of viral infectious diseases of aquaculture is not only necessary for producing safe aquaculture, but also for understanding the mechanism of infecting fishes and defenses, The need is increasing.

Studies on how viruses recognize clonality and induce apoptosis in relation to glucose metabolism and energy metabolism in host fish cells and studies to enhance antiviral effects by controlling factors that increase virus replication There is a trend.

Curcuma longa L. (tumeric) is a perennial plant belonging to the genus Zingiberaceae and has been widely used for edible purposes such as curry, tea and food additives. Curcumin, , Antiinflammation, antiviral, blood lipid lowering and anti-inflammatory action, and cholesterol inhibitory action have been reported. However, there is no report on the antiviral effect of curcumin on VHSV among fish disease-inducing viruses.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop an antiviral composition by investigating an infection mechanism and a defense mechanism against virus infectious diseases of aquaculture fish. As a result, the present invention was completed by identifying the antiviral effect of curcumin, a major component of Curcuma longa L., tumeric.

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

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

It is another object of the present invention to provide a composition for adding fish feed.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided an antiviral composition for Viral Hemorrhagic Septisemia Virus (VHSV) comprising curcumin as an active ingredient.

The present inventors have sought to develop an antiviral composition by investigating an infection mechanism and a defense mechanism against viral hemorrhagic sepsis, which is a viral infectious disease of aquaculture fish. As a result, the antiviral effect of Curcumin, a major component of Curcuma longa L., tumeric, against viral hemorrhagic sepsis virus was confirmed.

The curcumin used as an active ingredient in the present invention is a compound having a structure represented by the following formula

Formula 1

The active ingredient curcumin of the present invention includes not only isolated or synthesized but also a natural extract containing curcumin, preferably a herbal extract or a herbal fraction.

Cultured fish to which the composition of the present invention can be applied include flounder ( Paralichthys olivaceus ), defensive ( Seriola quinqueradiata ), red sea bream ( Pagrus Major , Takifugu rubripes), busiri (Seriola aureovittata , trout (e.g., Oncorhyncus nykiss), jaetbangeo (Seriola dumerili ), horse mackerel ( Pseudocaranx dentex , louse ( Epinephelus septemfasciatus ), bluefin tuna ( Thunnus thynnus , carp (e.g., Cyprinus carpio ), zebrafish ( Danino rerio ), catfish (e.g., Clarias gariepinus ), tilapia ( Oreochronis niloticus ), salmon ( Salmo Salar and Oryzias latipes ), but is not limited thereto. Most preferably it is a flounder.

As used herein, the term " comprising as an active ingredient " is meant to include an amount sufficient to achieve the efficacy or activity of curcumin. The present invention relates to curcuma longa L.), curcumin is used as an active ingredient. However, since there is no adverse effect on an animal even when it is administered in an excessive amount, the quantitative upper limit included in the composition of the present invention can be selected by a person skilled in the art within a suitable range.

Preferably, the curcumin inhibits the expression of PGC-1 alpha (Peroxisome proliferator-activated receptor Gamma Coactivator-1 alpha).

According to a preferred embodiment of the present invention, the composition of the present invention inhibits protein expression of PGC-1α, which is a VHSV transcriptional growth factor, and binds to viruses to decrease adherence to host cells, thereby inducing a decrease in infectivity, thereby increasing cell viability.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating viral hemorrhagic sepsis comprising curcumin as an active ingredient.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are those conventionally used in the field of manufacture and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. 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, etc. 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 pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, the composition can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, etc., .

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration route, animal receipt, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10000 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to another aspect of the present invention, there is provided a composition for preventing or treating a disease caused by a fish infectious viral hemorrhagic septicemia virus (VHSV) comprising curcumin as an active ingredient, .

The composition for fish feed addition of the present invention includes any composition conventionally used. For example, the composition for adding fish feed may be selected from the group consisting of marine protein (e.g. fish meal or krill mill), vegetable protein (e.g., soybean powder, wheat gluten, corn gluten, lupine powder, pea powder or sunflower seed powder) , At least one energy source selected from the group comprising at least one protein source selected from the group consisting of fish oil (e.g., squid liver oil) or vegetable oil (e.g., rapeseed oil, soybean oil, soybean oil) But are not limited to, mixtures.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides an antiviral composition for viral hemorrhagic sepsis virus.

(b) The antiviral composition of the present invention can provide about 5-fold cell survival rate for viral hemorrhagic sepsis virus.

(c) The present invention provides a basic research that can solve the problem of possibility as a future acceptance common pathogen by understanding infection mechanism and defense mechanism of fish.

FIG. 1 is a graph showing the activity of curcumin on the survival rate of virus-infected cells through inhibition of PGC-1 alpha.
FIG. 2 shows the result of inhibition of PGC-1α expression by curcumin by Western blotting.
3 is a graph showing the effect of increasing the survival rate of virus infected cells by curcumin treatment.
4 is a graph showing the effect of curcumin treatment to inhibit virus growth.
5 is a graph showing the effect of increasing the cell survival rate upon viral infection after neutralization treatment of curcumin with VHSV.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Identification of the role of PGC-1 alpha protein as an intracellular target substance for inhibiting the transcriptional proliferation of VHSV

The present invention relates to a method for producing a peroxisome proliferator-activated receptor (PPAR) γ (?) In a fish cell line, Fathead Minnow (FHM) cell (provided by a coin cell produced in a fish laboratory diagnosis laboratory of Chonnam National University) Coactivator -1α (PGC-1α) was overexpressed. Overexpression of PGC-1α in FHM cells was performed by transfection, in which DNA was directly introduced into cells to mutate the genetic traits of the cells. Reagents and recombinant PGC-1α DNA plasmids were transfected into FHM cells at a ratio of 2: 1 with reference to the transposon reagent FuGENE 6 (Roche, Cat. No. 1814 433) manual for over 48 hours. The viability of formazan crystals produced by dehydrogenase action of mitochondria present in living cells by inoculating PGC-1α over-expressing FHM cells with VHSV (Viral Hemorrhagic Septicemia Virus) was confirmed by spectroscopic method. As a result, PGC- Lt; RTI ID = 0.0 > 1α < / RTI > overexpression. This result is the first evidence that VHSV-induced apoptosis is induced through the PGC-1α-dependent pathway and that the intracellular target for inhibiting the effective proliferation of VHSV is the PGC-1α protein (FIG. 1) .

Example 2: Effect of PGC-1 alpha, a VHSV transcriptional growth factor of curcumin, as a protein expression inhibitor

To investigate the antiviral effect of curcumin (Sigma, USA) as a candidate substance to inhibit PGC-1α-dependent VHSV transcriptional proliferation, curcumin treatment and VHSV inoculation were performed on cells overexpressing the virus growth factor (PGC- Changes in cell viability were measured. Compared with the control without curcumin treatment in the same cells, the effect of inhibiting the expression of PGC-1α, a VHSV transcriptional growth factor, in curcumin treatment was confirmed by western blot and the cell survival rate was also found to increase 8.7 times (FIGS. 1 and 2). These results reaffirmed that PGC-1α is a VHSV transcriptional proliferative factor and proved to be a powerful compound capable of inhibiting the expression of PGC-1α, a VHSV transcriptional growth factor through curcumin.

Example  3: Curcumin  Increase of cell survival rate by virus infection by treatment

In order to investigate the concentration-dependent antiviral activity of curcumin, curcumin was treated with 0, 5, 10 and 50 μM of FHM cells, which are fish coin cells (provided by a coin cell produced by the Fisheries Biology Department of Fisheries Biology Department, Chonnam National University) And the cell viability was confirmed. As a result, it was confirmed that the cell survival rate of 4.3 times of VHSV infection was increased in a dose-dependent manner in the curcumin treatment group compared to the positive control group (untreated curcumin group) (FIG. 3).

Example  4: Curcumin  Effect of inhibiting virus transfer by treatment

In order to investigate whether the antiviral activity of curcumin inhibits direct viral transcription, FHM cells, a fish coin cell, were inoculated with VHSV in an experimental group treated with 50 μM, compared to a positive control group not treated with curcumin, PCR. The primers used for real-time PCR were quantitatively analyzed using QVHSV_3192F (5'-ACA AAC AAG GGC TTG GTC TC-3 ') and QVHSV_3261R (5'-CTG AGA CGC TGG TGA CTG AT-3').

As a result, it was confirmed that the experimental group treated with curcumin decreased the copy number by 12.6 times as compared with the positive control group (FIG. 4). These results demonstrate that curcumin inhibits VHSV transcription and thus has a higher cell survival rate than that of the positive control, demonstrating a strong antiviral effect against VHSV.

Example  5: With curcumin  Increase of cell survival rate by virus infection by neutralization treatment

To determine whether curcumin neutralizes the virus and increase the cell viability due to decreased infectivity by reducing the adherence to the host cells, the cell toxicity of curcumin after treatment with curcumin to the level of 160 μM was found to be considerably low (Fig. 5). In order to investigate the effect of curcumin on cell viability after neutralization treatment with VHSV at 0, 10, 20, 40, 80 and 160 μM concentration-dependent conditions, virus and curcumin were mixed for 2 hours each, The neutralized virus was inoculated into FHM cells, a fish coin cell (provided by a coin cell produced by a fishbone diagnosis laboratory in Chonnam National University), and the cell viability after infection was confirmed. As a result, it was confirmed that the cell survival rate up to 11-fold for VHSV infection was increased in a concentration-dependent manner in the curcumin treatment group compared to the positive control group (untreated curcumin group) (FIG. 5).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (8)

An antiviral composition for viral hemorrhagic septicemia virus (VHSV) which is a fish infectious virus containing curcumin as an active ingredient.
delete The fish according to claim 1, wherein the fish is one selected from the group consisting of flounder, defense, red snapper, loincloth, buckwheat, trout, cauliflower, horse mackerel, lion, tuna, carp, zebra fish, catfish, tilapia, salmon and sea lion ≪ / RTI >
4. The method of claim 3, wherein the fish is selected from the group consisting of flounder ( Paralichthys olivaceus . < / RTI >
2. The composition of claim 1, wherein said composition inhibits the expression of Peroxisome proliferator-activated receptor Gamma Coactivator-1 alpha (PGC-1 alpha).
The composition according to claim 1, wherein the curcumin is contained in a curd extract.
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