KR20180092812A - Antimicrobial, antiparasite, antiviral and antioxidant compositions of fish comprising Rhus verniciflua lignum extract - Google Patents

Abstract

The present invention relates to antimicrobial, antiparasitic, antiviral and antioxidative compositions of fishes comprising a Rhus verniciflua xylem extract. By comprising the Rhus verniciflua xylem extract, the present invention relates to antimicrobial, antiparasitic, antiviral and antioxidative compositions of fishes comprising Rhus verniciflua xylem extract which can prevent or treat infections and infectious diseases caused by fish pathogenic bacteria, parasites or viruses, and prevent the death of fishes by inducing immune enhancement to increase the production amount of fish, and can minimize toxicities and rancidity caused by chemicals since the active substance is contained in a large amount in the extract.

Description

An antimicrobial, antiparasitic, antiviral and antioxidant composition of a fish comprising Rhus verniciflua extract,

The present invention relates to antimicrobial, antiparasitic, antiviral and antioxidative compositions of fish comprising Rhus verniciflua extract.

Fish farming is an innovative alternative to the depletion of fishery resources. Since the mid 1990s, the government has been actively promoting the main fish species such as flounder, rockfish, shrimp and red sea bream. Among them, , Which accounts for a large portion of the total fish production. However, the current rate of mulching in Korea is more than 20% per year, and the main cause of this is the incidence of intractable diseases, mixed infections (viruses, bacteria, parasites)

In particular, damage to fish pathogenic viruses, germs and parasites continues to occur, and the damage is significant. Thus, massive deaths have caused great problems in the aquaculture industry, but the development of appropriate medicines that can be used for the treatment is minimal.

Rhus verniciflua Stokes ( Rhus verniciflua Stokes) is a deciduous broad-leaved arboreous tree belonging to the Anacardiaceae family, and growing in Northeast Asia such as China and Japan.

The woody part of lacquer tree has been used as a health drink until now, and it is known that it is effective for the prevention of intestinal cancer and cancer. However, it has been reported that it has antibacterial, antiparasitic, antiviral and antioxidant activity against fish pathogenic bacteria, parasites and viruses Has not yet been reported.

Korean Patent Registration No. 10-918326

The present invention aims to provide a composition exhibiting antibacterial, antiparasitic, antiviral and antioxidative activities against fish pathogenic bacteria, parasites or viruses.

It is an object of the present invention to provide a composition capable of preventing or treating a disease caused by an infection caused by a fish pathogenic bacterium, a parasite or a virus.

It is an object of the present invention to provide a composition capable of inducing immune enhancement of fish to prevent mortality.

It is an object of the present invention to provide a composition capable of increasing the production amount of aquacultured fish by preventing the death of fish.

It is an object of the present invention to provide a composition capable of minimizing the toxic occurrence and chemical rancidity caused by chemicals.

1. An antimicrobial, antiparasitic, antiviral or antioxidant composition of fish comprising extracts of Rhus verniciflua woody.

2. The antimicrobial, antiparasitic, antiviral or antioxidant composition of fish according to 1 above, wherein the extract is water, methanol, ethanol or a mixed solvent thereof.

3. The antimicrobial, antiparasitic, antiviral or antioxidant composition of claim 1, wherein the extract is a water extract.

4. The antimicrobial, antiparasitic, antiviral or antioxidant composition of fish according to 1 above, wherein said extract is an aqueous solution of 10 to 70% by volume ethanol.

5. The antimicrobial, antiparasitic, antiviral or antioxidant composition of fish according to 1 above, wherein the extract is an organic solvent fraction of an extract of water, methanol, ethanol or a mixed solvent thereof.

6. The antimicrobial, antiparasitic, antiviral or antioxidant composition of fish according to 5 above, wherein the organic solvent is at least one selected from the group consisting of chloroform, ethyl acetate and hydrogenated butanol.

7. The antimicrobial, antiparasitic, antiviral, or antioxidant composition of fish according to item 1 above, wherein the extract is extracted at 80 to 110 ° C.

8. The antimicrobial, antiparasitic, antiviral or antioxidant composition of fish according to 1 above, wherein the extract is extracted with a solvent which is 10 to 40 times the weight of the rhizome wood part.

9. The antimicrobial, antiparasitic, antiviral or antioxidant composition of fish according to item 1 above, wherein the extract is extracted at 80 to 100 DEG C with an aqueous solution of 10 to 30% by volume of ethanol.

10. The antimicrobial, antiparasitic, antiviral or antioxidant composition of fish according to 1 above, wherein the extract is extracted at 50 to 70 vol.% Aqueous ethanol solution at 80 to 100 占 폚.

11. An antibacterial, antiparasitic, antiviral or antioxidant composition of fish, comprising 1 to 10% by weight of gallic acid, 10 to 40% by weight of fostin and 1 to 10% by weight of picetin.

12. A method for the prevention or treatment of bacterial, parasitic or viral infectious diseases of fish, comprising the step of administering to a fish a composition comprising a lacquerwood extract.

13. The method for preventing or treating a bacterial, parasitic or viral infection of fish according to item 12 above, wherein the administration is orally administered for 10 to 350 mg per 1 kg body weight of fish for 1 to 13 weeks.

14. A method for preventing or treating a bacterial, parasitic or viral infection of a fish, comprising the step of administering to a fish a composition comprising 1 to 10% by weight of gallic acid, 10 to 40% by weight of fuchsin and 1 to 10% .

15. The method for preventing or treating a bacterial, parasitic or viral infection of fish according to 14 above, wherein the administration is orally administered for 10 to 350 mg per 1 kg body weight of fish for 1 to 13 weeks.

The composition of the present invention can prevent or treat infections and infections caused by fish pathogenic bacteria, parasites or viruses.

The composition of the present invention can enhance the immunity of fish.

The composition of the present invention can prevent fish from dying.

The composition of the present invention can reduce the mortality of aquaculture fish and increase the production amount.

Since the composition of the present invention contains a large amount of a natural antioxidant active substance in the extract, it is possible to minimize the toxicity problem and rancidity of the food which may occur by using the chemical.

FIG. 1 is a graph showing the phenolic content, flavonoid content and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of each part of Rhus verniciflua.
Fig. 2 is a graph showing a preliminary experimental result of the antimicrobial activity (A) and the yield (B) of Rhus verniciflua Stokes extract (the line means a quadratic polynomial trend line).
FIG. 3 shows residual images of the antimicrobial activity regression model.
Figure 4 shows residual figures for the active regression model of Hanskutika.
Figure 5 shows residual figures for the yield regression model.
Figure 6 shows a predictive optimization picture of the antimicrobial activity and yield of the extract.
Figure 7 shows a predictive optimization picture of the activity and yield of the antiscucta extract of the extract.
Fig. 8 shows the reaction surface diagram showing the interaction effect between two independent variables, where A is the antimicrobial activity, B is the yield, C is the optimum activity and yield value predicted by the BBD (Box-Behnken design) .
Figure 9 shows the reaction surface figure showing the effect of the interaction between two independent variables, D representing the activity of the antiscuctic activity, E the yield and F the optimal activity and yield value predicted by BBD .
10 shows the composition of the 20 extract and the 60 extract in UV spectrum (254 nm).
FIG. 11 is a microscopic photograph showing antiviral activity against infectious hematopoietic necrosis virus (IHNV) of Rhus verniciflua extracts, wherein A is a normal control; B is a virus control; C was 3 ug / ml of hot water extract; D of 20.100.30 extract 3 mu g / ml; E was 3 ug / ml of 60.85.30 extract; F means 3 μM of ribavirin, and the scale bar is 100 μm.
Figure 12 shows the linearity, limit of detection (LOD) and limit of quantitation (LOQ) of gallic acid, fustin, and fisetin.
Figures 13 to 16 show the comparison of retention times of MRM data of standard compounds with multiple reaction monitoring (MRM) data of surface components in optimized rhizome woody extract (20 extract, 60 extract).
17 shows the cumulative mortality rate in olive flounder infected with E.tarda FJ1408 after oral administration of 20 vol% ethanol aqueous solution extract of lacquer wood (NC, simple control (Naive control); BC , Bacterial control (RPS, relative percent survival; *, P <0.05; **, P <0.01; ***, P <0.001).
Figure 18 shows the cumulative mortality rate in olive flounder infected with E.tarda FJ1408 after oral administration of 60 vol% aqueous ethanol extract of Rhus verniciflua var. Lucidum for 2 weeks (NC, naive control; BC , Bacterial control (RPS, relative percent survival; *, P <0.05; **, P <0.01; ***, P <0.001).
19 shows the cumulative mortality rate in olive flounder infected with E.tarda FJ1408 after oral administration of a 20 vol% ethanol aqueous solution extract of lacquer wood for 6 weeks (NC, naive control; BC , Bacterial control (RPS, relative percent survival; *, P <0.05; **, P <0.01; ***, P <0.001).
20 shows the cumulative mortality rate in olive flounder infected with E.tarda FJ1408 after oral administration of 60 vol% aqueous ethanol solution of Rhus verniciflua var. Lucidum for 6 weeks (NC, naive control; BC , Bacterial control; RPS, relative percent survival; *, P <0.05; **, P <0.01; ***, P <0.001).
Figure 21 shows the cumulative mortality rate in olive flounder infected with E.tarda FJ1408 after oral administration of 20 vol% ethanol aqueous extract of Rhus verniciflua var. Lucidum for 10 weeks (NC, naive control; BC , Bacterial control; RPS, relative percent survival; *, P <0.05; **, P <0.01; ***, P <0.001).
Figure 22 shows the cumulative mortality rate in olive flounder infected with E.tarda FJ1408 after oral administration of 60 vol% aqueous ethanol extract of Rhus verniciflua var. Lucidum for 10 weeks (NC, naive control; BC , Bacterial control; RPS, relative percent survival; *, P <0.05; **, P <0.01; ***, P <0.001).
Figure 23 shows clinical signs of flounder infected with E.tarda (FJ1408), where A is the external lesion of the diseased fish (abdominal distension and hernia); B is a congested intraoral and perineal lesion; C and D represent abscess and bacterial colonies of various sizes found in liver and kidney.
FIG. 24 shows the cumulative mortality rate in flounder infected with Sukutika puffer after oral administration of 20 vol.% Aqueous ethanol solution of Rhus verniciflua var.
25 shows the cumulative mortality rate in flounder infected with Sukutika puffer after oral administration of 20 vol% ethanol aqueous solution extract and 60 vol% ethanol aqueous solution extract of lacquer wood for 12 weeks, respectively.

The present invention relates to antimicrobial, antiparasitic, antiviral and antioxidative compositions of fishes containing Rhus verniciflua extract, and it can prevent or treat infections and infectious diseases caused by fish pathogenic bacteria, parasites or viruses by containing Rhus verniciflua extract , It can induce immune enhancement to prevent the death of fish, thereby increasing the production of fish. Also, it contains a large amount of natural antioxidant active substance in the extract, thereby minimizing the toxicity problem caused by the chemical and the rancid Antimicrobial, antiparasitic, antiviral and antioxidant compositions of fish comprising Rhus verniciflua extract.

Hereinafter, the present invention will be described in detail.

The "extract" means an extract obtained by extracting the rhus vernicifolia lacquer wood part, a diluted solution or concentrate of the extract, a dried product obtained by drying the extract, a dispersant or a purified product of the extract, .

"Fraction" means a result obtained by fractionating a specific component or a specific component group from a mixture containing various various components.

"Prevention" means any action that inhibits or slows the onset of infectious diseases caused by fish pathogenic bacteria, viruses or scuticociliatids by the composition, and "treatment" Or the act of infectious disease caused by Sukhitika infestation is improved or changed beneficially.

"Water" includes all kinds of water such as spring water, deep sea water, alkaline water, mineral water, natural carbonated water, ground water, mineral water, spring water, hexagonal water, ionized water, It also includes water at various temperatures.

In one aspect, the present invention provides an antimicrobial composition of fish. The fungus means a fish pathogenic bacterium.

The fish may be collectively referred to as all kinds of fish which can cause infectious diseases caused by fish pathogenic bacteria, viruses or scutica. Examples of the fish include, but are not limited to, flounder, rockfish, mullet, red sea bream, dolphin, sea bass, flounder, mackerel, lion fish, bluefin tuna, emotional fish and salmon. According to one embodiment of the present invention, the fish may be, but not limited to, flounder.

Fish pathogenic bacteria can be gram-positive or gram-negative. For example, Vibrio species (specifically, V. anguillarum, V. salmonicida, V. ordalii, V. alginoriticus, V V. choleraenon O1, V. fisheri, V. harvey, V. splendidus, V. ichthyoenteria, V. scophthalmi, V. rotiferianus, V. coralliilyticus, V. canolaea, V. pomeroyi, V. chagasii, V. fortis , V. hepatarius, V. neptunus, V. brasiliensis, V. xuii, V. agarivorans, V. lentus, etc. More specifically, V. anguillarum, V. salmonicida, V. ordalii, V. alginoriticus, V. vulnificus, V. choleraenonO1, V. harvey, V. splendidus, V. ichthyoenteri, V. scophthalmi, V. rotiferianus, etc.); Edwardsiella species (specifically, E. tarda et al.) That cause Ed ward disease; Tenacibaculum species (specifically, T. maritimum, etc.) that cause a sliding bacterium; Streptococcus species such as Streptococcus iniae, S. parauberis, S. difficilis, S. shiloi, S. mileri, S (E. coli), E. coli, and the like are examples of Gram- (such as Streptococcus iniae and S. parauberis), Lactococcus species (specifically L. garvieae, L. piscicum, etc.), Vagococcus species (specifically, V. salmoninarum, etc.) Enterococcus species (specifically, E. seriolicida, E. faecalis, etc.), Staphylococcus species (specifically, S. aureus etc.), etc .; Or Salmonella species which induce Salmonella disease (specifically, S. enterica, S. bongori), and the like.

In one embodiment of the present invention, it has been confirmed that the extract of Rhus verniciflua var. Lucidum and the fraction of the extract have excellent effects on the prevention or treatment of infection against fish pathogenic bacteria (Figs. 14 to 19). Thus, the composition of the present invention comprising extracts of Rhus verniciflua woody part or fractions of the extract is suitable for antibacterial or antifungal treatment against fish pathogenic bacteria.

The antimicrobial composition of the present invention for fish pathogenic bacteria includes Rhus verniciflua extract.

There is no particular limitation on the method for extracting rhizome woody part, and extraction can be carried out according to a method commonly used in the art. Examples of the extraction method include a hot water extraction method, an ultrasonic extraction method, a filtration method and a reflux extraction method. The extraction method may be performed alone or in combination with two or more methods, but is not limited thereto.

Rhus vermiculite extract can be obtained by extracting rhus verniciflua using any solvent known in the art. Examples of the solvent include water; C1 to C4 lower alcohols such as methanol, ethanol, propyl alcohol and butyl alcohol; Polyhydric alcohols such as glycerin, butylene glycol and propylene glycol; And hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Or a mixture thereof. Specifically, lower alcohols such as ethanol and methanol may be used alone or in combination of two or more.

According to one embodiment of the present invention, the Rhus verniciflua extract may be water, methanol, ethanol or a mixed solvent extract thereof, but is not limited thereto. According to a specific embodiment of the present invention, the Rhus verniciflua extract comprises water, 70 to 90% by volume aqueous methanol solution or 10 to 70% by volume (specifically 30 to 70% by volume, more particularly 55 to 65% Aqueous solution, but is not limited thereto. When extracted with water, methanol aqueous solution or ethanol aqueous solution within the above range, the antimicrobial activity against the pathogenic bacteria of the fish is maximized, and when the concentration is out of the above range, the antibacterial activity is decreased to lower the infection or therapeutic effect on the bacteria.

As a method for obtaining a lacquerwood woody part extract, a method of obtaining a lacquer wood lacquer part extract is characterized by 10 to 60 times, specifically 10 to 40 times, more specifically 25 to 35 times, more specifically 27 To 33 times the volume of the solvent, but is not limited thereto.

According to one embodiment of the present invention, the extract of Rhus verniciflua can be extracted with a solvent having a volume of 30 times the weight of Rhus verniciflua, but is not limited thereto.

The Rhus verniciflua extract may be extracted at 80 to 110 캜, specifically, 82 to 88 캜, more specifically 84 to 87 캜, but is not limited thereto.

Extracting the extract of Rhus verniciflua wood by weight ratio or temperature range within the above range maximizes the antimicrobial effect against fish pathogenic bacterium and maximizes the effect of preventing or treating the infectious disease caused by the bacterium. If the amount is outside the above range, the antimicrobial effect and the preventive or therapeutic effect on the infectious disease may be reduced.

According to one embodiment of the present invention, the extract of Rhus verniciflua can be extracted at 80 to 90 ° C in an aqueous solution of 50 to 70% by volume of ethanol, which is 10 to 40 times the weight of Rhus verniciflua, but is not limited thereto.

According to an embodiment of the present invention, the antimicrobial composition for a pathogenic bacterium of the present invention may include a fraction of the extract of Rhus verniciflua var. Woody part, and may specifically include an aqueous methanol solution of the Rhus vernicifolius wood part or an organic solvent fraction of the aqueous ethanol solution It is not limited.

The fractionation method for obtaining the fraction in the present invention is not particularly limited and may be carried out according to a method commonly used in the art. For example, the fraction can be obtained from the extract by treating the extract obtained by extracting rhizome lignin with a predetermined solvent, but is not limited thereto.

Examples of the organic solvent include ether, hexane, cyclohexane, chloroform, ethyl acetate, dichloromethane, hydrogenated butanol, benzene, dimethylsulfoxide and the like. Specifically, it may be at least one of chloroform, ethyl acetate and hydrogenated butanol But is not limited thereto.

In order to obtain an organic solvent fraction of Rhus verniciflua woody part extract, the crude extract of Rhus vernicifolia lacquer obtained above is suspended in distilled water, and an organic solvent of 1 to 100 times, specifically 1 to 5 times the volume of the suspension is added, The non-polar solvent soluble layer can be extracted and separated.

Further, a normal fractionation process may be further performed. Specifically, a crude extract of Rhus vernicifolia wood is suspended in water and successively extracted with an equal amount of n-hexane, chloroform and ethyl acetate to obtain a solvent extract of each solvent of Rhus wood. More specifically, , The same amount of n-hexane is mixed and then fractionated to obtain a n-hexane soluble fraction and a water-soluble fraction. The chloroform-soluble fraction and the water-soluble fraction can be obtained by adding chloroform to the water-soluble fraction, and ethyl acetate can be added to the water-soluble fraction to obtain an ethyl acetate-soluble fraction and a water-soluble fraction.

The composition of the present invention may further comprise a pharmaceutically or physiologically acceptable carrier.

"Carrier" means a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to the cell or mammal exposed to the dose and concentration employed. Examples of such carriers include buffers such as saline, Ringer's solution, buffered saline, phosphate, citrate and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than 10 residues) polypeptides, proteins such as serum albumin, Immunoglobulins; Other hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin, chelating agents such as EDTA, For example, mannitol or sorbitol, salt-forming counter ions such as sodium, and / or nonionic surfactants such as tweens, polyethylene glycol (PEG) and PLURONICS .

If necessary, the composition of the present invention may further contain other conventional additives such as antioxidants, buffers, bacteriostats, and the like. In addition, it can be formulated into injection formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders and lubricants, Specific antibody or other ligand can be used in combination with the carrier.

And further formulated by appropriate methods in the art. For example, it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions and syrups, and can be formulated in the form of external preparations, suppositories, and sterilized injection solutions. However, the composition of the present invention may be most preferably provided in the form of a bathing agent for the purpose of treating diseases of fish, but is not limited thereto.

The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to veterinary treatment and not causing side effects, The severity, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration, the rate of release, the duration of the treatment, factors including drugs used concurrently or concurrently, and other veterinary fields.

The present invention in another aspect provides an anti-parasitic composition of fish comprising Rhus verniciflua extract. The parasite means a fish pathogenic parasite.

The fish may be the fish described above.

The parasites may include various organisms such as protozoan parasites, protozoans, insects, mosquitoes, mosquitoes, vertebrates, and crustaceans, and may be specifically protozoan parasites.

(Eg, M. avidus), Philasterides species (specifically, P. dicentrarchi, etc.), Uronema sp. Strains causing S. scurvy as a protozoan parasite, (Specifically, U. marinum, U. nigricans, etc.), Tetrahymena species, Pseudocohnilemdus species (specifically, P. persalinus) and the like; Ichthyophthirius multifillis, Cryptocaryon irritans; Oodinium which induces an oridinium disease; Trichodina causing Trichoderma leprosy; Chilodonella, which causes kelodonella disease; Ambiphrya, which induces Ambi-free firearms; Apiosoma that causes apiomycosis; Epistylis causing Epstein's disease; Carchesium, which induces cachemium; And the like. However, the present invention is not limited thereto.

According to one embodiment of the present invention, the parasite is selected from the group consisting of the species Miamiensis (specifically, M. avidus, etc.), Philasterides species (specifically P. dicentrarchi etc.), Uronema (Eg, U. marinum, U. nigricans, etc.), Tetrahymena species, Pseudocohnilemdus species (specifically, P. persalinus), Ichthyophthirius multifillis, Cryptocaryon irritans, Oodinium, Trichodina, Chilodonella, and the like.

In one embodiment of the present invention, the extract of Rhus verniciflua and the fraction of the extract have excellent anti-parasitic effects (FIGS. 23 and 25). Thus, the composition of the present invention comprising the extract of Rhus verniciflua var. Rhizome and the fraction of the extract is suitable for use in the prevention or treatment of infectious diseases caused by an antiparasitic or parasitic organism.

The lacquer woody part extract can be obtained by extracting the lacquer wood part by using the above-mentioned solvent by the above-mentioned extraction method. According to one embodiment of the present invention, the Rhus verniciflua extract may be, but not limited to, water, an aqueous methanol solution or an aqueous ethanol solution. According to a specific embodiment of the present invention, the Rhus verniciflua extract comprises water, 70 to 90% by volume of methanol aqueous solution or 10 to 70% by volume of ethanol (specifically, 50 to 70% by volume of ethanol aqueous solution, 10 to 30% Aqueous ethanol solution, more specifically, 15 to 25% by volume aqueous ethanol solution). For example, Rhus verniciflua extract may be water, an aqueous 80 vol% methanol solution or an aqueous 20 vol% ethanol solution.

When ethanol is contained in the above range, the antimicrobial activity against the pathogenic bacteria of the fish is the most excellent. If the amount is outside the above range, there is a problem that the antibacterial activity is reduced and the infection or therapeutic effect of the fish pathogenic bacteria is lowered.

As a method for obtaining a lacquerwood woody part extract, a method of obtaining a lacquer wood lacquer part extract is characterized by 10 to 60 times, specifically 10 to 40 times, more specifically 25 to 35 times, more specifically 27 To 33 times the volume of the solvent, but is not limited thereto.

According to one embodiment of the present invention, the extract of Rhus verniciflua can be extracted with a solvent having a volume of 30 times the weight of Rhus verniciflua, but is not limited thereto.

The extract of Rhus verniciflua may be extracted at 80 to 110 ° C, specifically 90 to 105 ° C, more specifically 95 to 103 ° C, but is not limited thereto. For example, the extract may be extracted at 100 ° C.

According to one embodiment of the present invention, the extract of Rhus verniciflua can be extracted at 90 to 110 ° C in an aqueous solution of 10 to 30% by volume of ethanol, which is 10 to 40 times the weight of Rhus verniciflua, but is not limited thereto.

The composition of the present invention may comprise an organic solvent fraction of Rhus verniciflua extract.

The organic solvent may be the organic solvent described above.

The fraction can be obtained according to the above-mentioned fractionation method. For example, the crude extract of Rhus verniciflua is suspended in distilled water, and a non-polar solvent soluble layer is extracted and separated 1 to 10 times by adding a second organic solvent having a volume of 1 to 100 times, specifically 1 to 5 times, can do. Further, a normal fractionation process may be further performed.

The composition of the present invention may further include, but is not limited to, the aforementioned pharmaceutical or physiologically acceptable carrier.

In another aspect, the present invention provides an antiviral composition of fish comprising Rhus verniciflua extract. The virus means a fish pathogenic virus.

The fish may be the fish described above.

The fish pathogenic viruses may be Herpesvirus, Birnavirus, Rhabdovirus, Iridovirus, Nodavirus, and viruses, and specifically, Rabdovirus (Rhabdovirus).

Infectious pacreatic necrosis virus (IPNV) that causes infectious pancreatic necrosis with a virus or virus; But are not limited to, Avibirnavirus, Aquabirnavirus, Blosnavirus, Entomobirnavirus, and the like which cause burr and virus infection.

The viral hemorrhagic septicemia virus (VHSV), which causes viral exudative sepsis, is caused by the lapwort virus and the virus, the flounder rhabdovirus that causes the flounder rap virus virus, and the infectious hematopoietic necrosis Infectious hematopoietic necrosis virus (IHNV), spring viremia of carp causing carp spring virus disease, and the like, but are not limited thereto.

In this case, the viruses and viruses also cause viral diseases. The viruses include Megalocytivirus, Ranavirus, Iridovirus, Chloriridovirus, Lymphocystivirus, etc. But is not limited thereto.

Nodaviruses and viruses cause neuro-necrosis, and the viruses include, but are not limited to, alphanodavirus, betanodavirus, and the like.

According to one embodiment of the present invention, the virus is selected from the group consisting of viral hemorrhagic septicemia virus (VHSV), hirame rhabdovirus, infectious hematopoietic necrosis virus (IHNV), carp spring virus a spring viremia of carp, and the like.

In one embodiment of the present invention, the extract of Rhus verniciflua and the fraction of the extract showed excellent antiviral effect against fish pathogenic virus (FIG. 10). Accordingly, the composition of the present invention comprising the extract of Rhus verniciflua woody part and the fraction of the extract is suitable for the prevention or treatment of antiviral or viral infectious diseases against fish pathogenic viruses.

The Rhus verniciflua extract may be, but is not limited to, the above extraction method, extraction conditions, or solvent extraction.

According to one embodiment of the present invention, the Rhus verniciflua extract may be water, methanol, ethanol or a mixed solution thereof.

According to one embodiment of the present invention, the composition of the present invention may include an organic solvent fraction of the extract of Rhus verniciflua var. Lacquer, and specifically includes an organic solvent fraction of extract of Rhus verniciflua, methanol, ethanol, But is not limited thereto.

The fraction can be obtained by the above-mentioned fractionation method, and can be obtained by further performing a conventional fractionation process, but is not limited thereto.

The organic solvent may be, but not limited to, the organic solvents described above.

The composition of the present invention may further include, but is not limited to, the aforementioned pharmaceutical or physiologically acceptable carrier.

As shown in FIG. 1, the extract of Rhus verniciflua has an excellent antioxidative activity. Accordingly, the present invention provides an antioxidant composition containing Rhus verniciflua extract.

The antioxidant composition of the present invention can be used for various purposes such as immunization of fish, prevention of various diseases, prevention of oxidation of feed, and the like.

The Rhus verniciflua extract may be, but is not limited to, the above extraction method, extraction conditions, or solvent extraction.

According to one embodiment of the present invention, the Rhus verniciflua extract may be water, methanol, ethanol or a mixed solution thereof.

According to one embodiment of the present invention, the composition of the present invention may include an organic solvent fraction of the extract of Rhus verniciflua var. Lacquer, and specifically includes an organic solvent fraction of extract of Rhus verniciflua, methanol, ethanol, But is not limited thereto.

The fraction can be obtained by the above-mentioned fractionation method, and can be obtained by further performing a conventional fractionation process, but is not limited thereto.

The organic solvent may be, but not limited to, the organic solvents described above.

The composition of the present invention may further include, but is not limited to, the aforementioned pharmaceutical or physiologically acceptable carrier.

The antioxidant composition of the present invention can be included in health functional foods and medicines.

There is no limitation on the kind of the health functional food including the composition of the present invention. For example, there are no limitations on the kind of the health functional food including the meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, Soups, drinks, tea, drinks, alcoholic beverages, and vitamin complexes, and may include foods in a conventional sense.

The beverage composition of the health functional food including the composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention, but is not limited thereto. In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. The proportion of such additives is not critical, but may be selected from the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention, but is not limited thereto.

The pharmaceutical composition containing the composition of the present invention may be, for example, a powder, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup, and the like.

The pharmaceutical composition containing the composition of the present invention may be formulated and formulated using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like, but is not limited thereto.

Since the composition of the present invention can be used for the purpose of preventing or ameliorating a bacterial, parasitic or viral infection or infectious disease of fish, the present invention provides a feed additive comprising a Rhus verniciflua extract.

The lacquer wood extract has excellent antioxidant activity (Fig. 1), and can also prevent the oxidation of the feed.

The Rhus verniciflua extract may be, but is not limited to, the above extraction method, extraction conditions, or solvent extraction.

According to one embodiment of the present invention, the Rhus verniciflua extract may be water, methanol, ethanol or a mixed solution thereof.

According to one embodiment of the present invention, the feed additive of the present invention may include an organic solvent fraction of the extract of Rhus verniciflua var. Rhizome, and specifically includes an organic solvent fraction of extract of Rhus verniciflua, methanol, ethanol, But is not limited thereto.

The fraction can be obtained by the above-mentioned fractionation method, and can be obtained by further performing a conventional fractionation process, but is not limited thereto.

The organic solvent may be, but not limited to, the organic solvents described above.

The composition of the present invention may further include, but is not limited to, the aforementioned pharmaceutical or physiologically acceptable carrier.

The feed additives are collectively referred to as nutrient or substances added to the feed in trace amounts for specific purposes. The composition of the present invention may be included in the feed composition as the feed additive, and the feed is a generic term .

The feed composition of the present invention may further contain a binder, an emulsifying agent, a preservative, and the like added to prevent quality deterioration. The feed composition may contain an amino acid, a vitamin, an enzyme, a probiotics, a flavoring agent, A compound, a silicate, a buffer, a coloring agent, an extracting agent, an oligosaccharide, and the like, and may further include, but is not limited to, a feed mixture.

The compound feed commonly used in the feed composition for fish includes any one or more protein sources selected from fish meal, casein, bone meal, shrimp powder, or any one or more carbohydrate sources selected from sweet potato starch, potato starch, wheat starch, , Soybean oil, corn oil, pollack liver oil, squid liver oil, and the like, but the present invention is not limited thereto. Even when used in combination with the compound feed, the preventive or ameliorative effect of the disease occurs in the same way .

The present invention relates to the use of antimicrobial, anti-inflammatory, anti-inflammatory, anti-inflammatory, anti-inflammatory, anti-inflammatory, anti-inflammatory and antioxidant activities of fish including gallic acid, Parasite, antiviral or antioxidant composition.

Gallic acid has the chemical formula of 3,4,5-trihydroxybenzoic acid, and fustine is 2- (3,4-dihydroxyphenyl) -3,7-dihydroxy-2,3-dihydrochromen- 4-one, and picetine has the formula of 2- (3,4-dihydroxyphenyl) -3,7-dihydroxychromen-4-one.

The composition of the present invention comprises 1 to 10% by weight of gallic acid, 10 to 40% by weight of fostin and 1 to 10% by weight of picetin. When antioxidant, antiparasitic, antiviral or antioxidant activity of fish is maximized if it contains gallic acid, fucose, and phytoethin within the above range, and if it contains less than or more than the above range of gallic acid, Antiviral, or antioxidant activity is within the above range.

According to one embodiment of the present invention, the composition of the present invention may include 1 to 5 wt% of gallic acid, 15 to 25 wt% of fucin, and 1 to 5 wt% of picetin, but is not limited thereto.

The composition may further contain, in addition to the above components, the remaining components contained in the Rhus verniciflua extract. For example, it may further comprise gallate derivatives, flavonoid-based compounds (e.g., butin, sulfuretin, butein, etc.) % By weight, and the like.

In addition to the above components, all substances known in the art to be excellent in antibacterial, antiparasitic, antiviral or antioxidative activity may be included in the balance of the components other than the weight% of the components of the composition.

The present invention also provides a method for treating an infectious disease caused by a fish pathogenic bacterium, a parasitic virus or a virus, comprising a step of administering a composition containing Rhus verniciflua extract to a fish.

The fish may be the fish described above.

The Rhus verniciflua extract may be, but is not limited to, the above extraction method, extraction conditions, or solvent extraction.

According to one embodiment of the present invention, the Rhus verniciflua extract may be water, methanol, ethanol or a mixed solution thereof.

According to one embodiment of the present invention, the composition of the present invention may include an organic solvent fraction of the extract of Rhus verniciflua var. Lacquer, and specifically includes an organic solvent fraction of extract of Rhus verniciflua, methanol, ethanol, But is not limited thereto.

The fraction can be obtained by the above-mentioned fractionation method, and can be obtained by further performing a conventional fractionation process, but is not limited thereto.

The organic solvent may be, but not limited to, the organic solvents described above.

The composition of the present invention may further include, but is not limited to, the aforementioned pharmaceutical or physiologically acceptable carrier.

The method of the present invention may include any means by which the composition of the present invention is delivered directly or indirectly to the fish, such as by direct administration to the fish or in the farm. For example, the means may be, but is not limited to, direct injection, feeding, bathing or feeding (oral administration) to fish, and the like.

For example, the method of the present invention may be a method of preparing a drug solution at an effective concentration, bathing a fish having a lesion caused by infection with Sutica, and bathing the composition of the present invention, but the present invention is not limited thereto.

The dosage of the composition of the present invention may vary depending on the weight of the fish to be treated, the health condition, the administration time, and the severity of the disease.

According to one embodiment of the present invention, a composition comprising from 10 mg to 350 mg (specifically, 20 mg to 310 mg, more specifically, 30 mg to 300 mg) of Rhus verniciflua extract per gram of fish is applied for 1 to 13 weeks 2 weeks to 12 weeks).

When the composition is administered to fish in an amount and a period within the above range, the effect of preventing or treating a disease caused by the infection or infection with a fish pathogenic bacterium, a parasite or a virus can be maximized.

Since the extract of Rhus verniciflua is excellent in antimicrobial, antiparasitic or antiviral activity by containing glycine, fucose and picetin at a certain weight percentage, the present invention relates to a method for producing a plant extract comprising the step of administering to a fish a composition comprising gallic acid, A method for treating an infectious disease caused by a fish pathogenic bacterium, a parasite, or a virus.

Glycans, fuchsins, and picetins may be gallic acid, fucose and picetin as described above.

The composition of the present invention comprises 1 to 10% by weight of gallic acid, 10 to 40% by weight of fostin and 1 to 10% by weight of picetin. When antioxidant, antiparasitic, antiviral or antioxidant activity of fish is maximized if it contains gallic acid, fucose, and phytoethin within the above range, and if it contains less than or more than the above range of gallic acid, Antiviral, or antioxidant activity is within the above range.

According to one embodiment of the present invention, the composition of the present invention may include 1 to 5 wt% of gallic acid, 15 to 25 wt% of fucin, and 1 to 5 wt% of picetin, but is not limited thereto.

The composition may further contain, in addition to the above components, the remaining components contained in the Rhus verniciflua extract. For example, it may further comprise gallate derivatives, flavonoid-based compounds (e.g., butin, sulfuretin, butein, etc.) % By weight, and the like.

In addition to the above components, all substances known in the art to be excellent in antibacterial, antiparasitic, antiviral or antioxidative activity may be included in the balance of the components other than the weight% of the components of the composition.

Hereinafter, the present invention will be described in more detail with reference to the following examples. The following examples illustrate the invention and are not intended to limit the scope of the invention.

Example

In the following examples, 20 extract means 20 vol% ethanol aqueous solution extract of Rhus wood, 60 extract means 60 vol% ethanol aqueous solution extract of Rhus vernicifolia. The hydrothermal extract refers to an extract obtained by hot water extraction of lacquer wood.

Experimental Method

1. Selection test of lacquer tree

1.1. Manufacture of extracts from each part of Japanese lacquer tree

After the lacquer husks and woody parts were prepared, 10 g of the sample was extracted by extruding in 200 ml of methanol for 3 hours at room temperature. In order to investigate the activity of each fraction, 50 g of the sample was extracted with 600 ml of 80% methanol by ultrasonication. The extracts were collected and counted. 4 filter paper, and concentrated under reduced pressure. The residue was lyophilized for two days, and the sample was used as a test sample related to site selection after finishing the sample in a mortar.

1.2. Determination of phenolic compounds and flavonoids and the activation of DPPH radical scavenging by parts of Rhus verniciflua

Ten grams of each leaf of lacquer tree, woody part, and skin were extracted at room temperature in 200 ml methanol for 3 hours at room temperature. Each extract was dissolved in dimethyl sulfoxide and adjusted to a concentration of 5 mg / ml to measure phenolics, flavonoid contents and DPPH radical scavenging activity.

1.2.1. Phenolic measurement

The total content of phenolics was determined by the Folin-Ciocalteu assay. The standard calibration curve was used with galvanic acid, and the concentration of the standard was 50, 100, 200, 400 or 800 μg / ml. Take 4 μl of Galvanic by concentration, mix in 36 μl of H ₂ O, put in 96 well plate, add 4 μl of Folin-ciocalteu's phenol reagent, and mix for 5 minutes with a stirrer. Take 40 μl of 7% Na ₂ CO ₃ solution and add 4 μl of H ₂ O. After storing in a dark room for 90 minutes, absorbance is measured with a microplate reader at 750 nm wavelength to make a standard calibration curve. The standard calibration curve was called Gallic acid Equivalents (GAE) and expressed as mg (GAE) / mg (extract).

The phenolic content was measured by measuring the absorbance of leaves, woody part, and skin (5 mg / ml) of the lacquer tree in the same manner.

1.2.2. Determination of flavonoids

The total content of flavonoids was measured by an aluminum chloride colorimetric assay. Catechin was used as the standard calibration curve, and the concentration of the standard was 37.5, 75, 150, 300 or 600 μg / ml. Take 10 μl of catechin per concentration and mix in 40 μl of H ₂ O. Add 3 μl of 5% NaNO ₂ into the 96-well plate, and mix with a stirrer for 5 minutes. Take ₃ μl of 10% AlCl ₃ and mix with a stirrer for 6 minutes. Add 20 μl of 1 M NaOH and measure absorbance at 490 nm with a microplate reader to make a standard calibration curve. The standard calibration curve was called Catechin Equivalents (CE) and expressed as mg (CE) / mg (extract).

Leuconostoc spots, woody part, and skin (5mg / ml) were also measured for their flavonoid contents by the same method.

1.2.3. Measurement of DPPH radical scavenging activity

Add 2μl of leaf, woody part, and bark extract to a 96-well plate at a concentration of 2.5mg / ml, and add 50μl of methanol to each well. A methanol solution was added to DPPH to make a concentration of 0.3 mM, and 50 μl of the solution was added to each well. The mixture was allowed to stand for 10 minutes. The absorbance was measured at a wavelength of 550 nm with a microplate reader.

1.3. Search for antibacterial, antisuktica and antiviral activity

1.3.1. Disc diffusion assay (Antibacterial)

The antimicrobial activity of the samples was determined by the Disc diffusion assay. Samples were dissolved in absolute ethanol solution according to the composition of each extract solvent and adsorbed on a 6 mm diameter sterilized disc paper at a dose of 3 mg / disc or 2 mg / disc and dried on a clean bench until the solvent completely evaporated. E. tarda was suspended in phosphate buffered saline (PBS) at 10 ⁸ CFU / ml, and 100 μl was inoculated on BHI agar medium. The diameter of the inhibition zone including the disc was measured with an electronic caliper to the second decimal place in mm in the incubator for 24 hours at 25 ° C. As a positive control, a commercially available disc of oxytetracycline (30 μg) and amoxicillin (10 μg) was used.

1.3.2. Microdilution assay

The antimicrobial activity of the optimized extracts and major compounds in the extracts was determined by microdilution assay. Bacteria were inoculated into 10 ml of BHIB, cultured at 25 ° C for 24 hours according to a 0.5 Mcfarland standard, and the final bacterial count was adjusted to 1 × 10 ⁶ CFU / ml. The extracts and compounds were well dissolved in dimethyl sulfoxide (DMSO), and the final DMSO concentration was adjusted to 10% by the addition of BHIB and diluted 2-fold. Then, the same volume of the sample diluted solution and the bacterial solution was added to a 96-well plate to a final volume of 200 μl, and the plate was incubated in a 25 ° C. incubator for 24 hours. The p-INT was prepared at 0.2 mg / ml in PBS and injected 30 ㎕ per well. The reaction was allowed to proceed for 2 hours under dark conditions. The minimum inhibitory concentration (MIC) was determined for the unstained section. Oxytetracycline was used as a positive control.

1.3.3. Activity Hansukutika

DMSO is added to the lyophilized sample to make stock solution. Samples were diluted in DMEMO to a final concentration of 1% DMSO. Stepwise dilution in DMEM0 produces the desired concentration. Formalin was used as a positive control. 100 μl of a diluted sample and a formalin dilution solution were dispensed into a 96-well plate.

Sukutika culture medium cultured in fish coin cells for 4 days to 5 days was prepared. Since there is almost no cell suspension in the culture medium and the activity is the best, Sucutika sacrificed for 4 to 5 days was used. First, the number of scuticacas in the culture was confirmed, and then subjected to primary centrifugation under conditions such as 1000 rpm, 10 min, and 15 캜. After the primary centrifugation, the culture medium was discarded, and DMEM0 was added and centrifuged under the same conditions. DMEM0 was removed and fresh DMEM0 was added to float and diluted to the desired concentration. In this experiment, the number of Sucutica cells was adjusted so that ³ × 10 ³ cells / 10 μl per well could be contained.

Ultrathin samples were inoculated with Sucutica spp., And reacted in a 15 ° C incubator for 24 hours to determine minimal lethal concentration (MLC).

1.3.4. Antiviral activity

A) Coin cell culture

Fish coin cells for virus culture were prepared by adding CHSE-214 (Chinook Salmon Embryo-214) and FHN (Fathead minnow) cells to Eagle's minimum essential medium (MEM) with 100 IU / ml penicillin, / Ml of streptomycin and 10% fetal bovine serum (FBS) were added to each flask at a concentration of 1 × 10 ⁶ / ml and cultured at 20 ° C. for 20 hours. Respectively.

B) Virus culture

Viral hemorrhagic septicemia virus (VHSV, Wando 2005) and Infectious Hematopoietic Necrosis Virus (IHNV) were used in this study. For storage of the virus, 100 μl of the virus was inoculated into the FHM cell line in a 75 F flask and incubated at 15 ° C. for 10 days. After observing the cytopathic effect (CPE), the supernatant was dispensed into microtube And stored at -80 ° C.

C) Antiviral activity

The antiviral activity of the sample was measured using a CPE (cytopathic effect) reduction assay. Briefly, cells cultured on 96-well plates were inoculated with 4 sets of virus diluent and sample 1: 1, and incubated at 15 ° C for 1 hour. After 3 to 4 days, when virus denaturation (CPE) appeared uniformly in all wells in the virus control, the culture was stopped and CPE of each sample was observed under a microscope. The CPE of each sample was compared with the CPE of the virus control and scored. The concentration (EC ₅₀ ), which reduces the CPE by 50%, is plotted as a percentage reduction in CPE by concentration.

2. Optimization of extraction conditions

2.1. Preliminary experiments for setting extraction conditions

3g of lacquer wood samples were crushed with a mixer and filtered with a 1 mm sieve. The ethanol content (0, 10, 20, 40, 60 or 80% Respectively. The extract was filtered with Whatman No 4. filter paper, concentrated in a water bath at 40 ° C, and lyophilized for 24 hours. Based on the activity results, the level of independent variables for designing the experiment was determined.

2.2. Experimental Design

Box-Behnken design (BBD) of RSM was used to analyze the antimicrobial activity and yield of E. tarda. The independent variables in the extraction process were selected as ethanol content (X1), extraction temperature (X2), and sample to solvent ratio (X3) through preliminary experiments and the experiment was designed with three levels of coded -1, 0 and 1. Fifteen randomly designed experimental conditions were extracted and lyophilized to evaluate antimicrobial and antiscuctive activity. In order to predict the optimum conditions of activity and yield, we analyzed using MINITAB ^® release 14 program. The second regression equation for independent variables' dependent variables in response surface analysis is as follows.

 [Equation 1]

Y is the dependent variable in the model (antibacterial activity yield), X ₁ (ethanol content), X ₂ (extraction temperature), X ₃ (sample stage solvent ratio) is the independent variable, β ₀ is the intercept, β _{1, 2, 3} Β _{12, 13, and 23} are the coefficients for the interaction term, and β _{11, 22, and 33} are the coefficients for the quadratic term.

variable sign Coated level -One 0 One ethanol (%) X ₁ 20 40 60 Temperature (℃) X ₂ 70 85 100 Sample to solvent ratio X ₃ 30 40 50

2.3. Extraction method

Extraction was carried out according to the respective conditions for 15 experimental points designed in BBD by placing 3g of lacquer wood in a 250ml glass bottle. Extraction temperature was extracted from the set temperature chamber. The extraction time was 3 hours and 20 minutes, and the extraction time was 3 hours and 20 minutes. The extract was concentrated under reduced pressure and the solvent was completely blown. The remaining residue was lyophilized for 24 hours to obtain a sample. The antibacterial activity and the antiscuctica activity of the sample were examined, and the yield of the sample was also confirmed.

3. In vitro antibacterial, antiscuctive and antiviral activity of the optimized Rhus verniciflua extract

The in vitro antibacterial, antiscuctive and antiviral activities of the optimized extracts were compared with the hot water extracts to evaluate their activity.

4. Analysis of surface content of optimized Rhus verniciflua extract

Compounds were simultaneously quantified using LC-MS / MS to analyze the content of indicator components of the optimized extracts.

4.1. LC-MS / MS conditions

Analytical instruments were Agilent 1260series and Agilent 6460-triple quadrupole MS. The analytical column was Capcell pak C18 MG (4.6 x 250 mm, 5 μm) manufactured by Shiseido Co., Ltd. and was set to maintain 30 ° C using a column oven. The solvent was analyzed by HPLC grade methanol and water with 0.1% formic acid added at a flow rate of 0.8 mL / min using the slope condition (Rt) shown in Table 2 and the analysis time was 30 minutes. 5 μl of sample and standard solution were injected. Electro spray ion (ESI) negative mode was used as the ion generator. The device parameters were capillary voltage 4000volts, gas temperature 350 ℃, gas flow 10L / min, nebulizer pressure 45psi. Q1 and Q3 masses for gallic acid, fuestin and phicetin and the impact energy for each compound are shown in Table 2 below. The combination of time (minutes) and methanol (%) in solvent conditions was 0 min and 25%, 5 min and 35%, 15 min and 35%, 20 min and 100% or 30 min and 100%.

compound Rt (min) Q1 mass (m / z) Q3 mass (m / z) Impact energy (eV) Gallan 5.43 169 125.2 3 Fustin 15.76 287.4 269.4, 109.1 5, 20 Physetine 22.79 285.4 163.1, 135.2 10, 15

4.2. Standard solution preparation and calibration

A standard solution of 1 mg / ml was prepared with methanol to prepare a standard curve for the three compounds and then diluted to 30 ppm. Each diluted standard solution was equally diluted with 6 concentrations (10, 50, 100, 500, 1000 or 1500 ng / ml) in a mixture of 10 ppm mixed with three compounds. The chromatograms were analyzed by peak area for each concentration, and a standard curve was created. The linearity was confirmed by calculating the r ² value by regression analysis. LOD and LOQ were calculated according to ICH (International Conference on Harmonization) Q2B Guidelines.

4.3. Precision measurement

% RSD of the contents of three compounds of intraday (n = 3, 3 times / day) and interday (n = 9, consecutive 3 days) per concentration after 1, 3 and 5 ㎍ / (relative standard deviation).

4.4. Recovery rate measurement

After the active compounds were added to the optimum extracts at the concentrations shown in Table 3 below, the recovery was obtained by the difference in concentration of the active compounds in the optimized extract.

&Quot; (2) &quot;

20 extract 60 extract compound Spiked Amount (ng / ml, ppb) Gallan 15 15 30 30 60 60 Fustin 90 135 180 270 360 540 Physetine 15 15 30 30 60 60

4.5. Specificity measurement

The specificity was confirmed by comparing the MRM data and retention time in the separated and purified compound and the new herbal medicine extracts, and the MRM data of the peak with the same retention time were confirmed and the specificity was confirmed by comparing the parent ion and the product ion.

5. In vivo antibacterial, antiscuctive and antiviral activity of optimized Rhus verniciflua extract

5.1. Experimental language

The fishes were purchased from the flounder nursery located in Yeonggwang-gun, Jeollanam-do and purchased at Chonnam National Fisheries Research Institute in Yeosu-dong, Jeonnam Province for 3 months (mean weight: 13.43 ± 1.72g) 2.20 g) was used in this experiment.

5.2. Experimental fish breeding

In order to administer the optimized extracts, eight extractive feedstocks (120 liters per each) were fed orally with oral extracts of 30, 100, and 300 mg / kg bw for 2, 6, and 10 weeks, Was fed with the general diet (True Flounder No. 2, First Feed). The extracts were fed at the natural temperature (July to September, 22.5 ~ 28 ℃) during the feeding period and the shade was set up.

5.3. Mass production of optimized Rhus verniciflua extract

600g of lacquer wood samples were extracted. The extraction time was 10 hours in total, and the solvent was changed once every 3 hours and every 20 minutes to extract three times in total. The extract was filtered with a Whatman No. 4 filter, concentrated under reduced pressure, and the solvent was completely blown. After concentration under reduced pressure, the residue was freeze-dried for two days. The lyophilized sample was finely ground in a mortar and stored in a sealed container at 4 ° C refrigerator for use as an extractive adsorbent feed.

5.4. Optimized production and dosing of rusks supplemented with Rhus verniciflua extract

The freeze-dried Rhus verniciflua extract was dissolved in a 33% aqueous ethanol solution to prepare 30, 100 or 300 mg / kg bw. (300 mg / kg of body weight), and 2% of the body weight was orally administered once a day for 2 weeks, 6 weeks, and 10 weeks.

5.5. In vivo anthropometric study of the optimized Rhus verniciflua extract against E. tarda

The diets containing 30, 100 or 300 mg / kg of fish were harvested from the flounder at 2, 6, and 10 weeks of age, respectively, for 14 days in 20 liters of sea water. The rats were fed 0.5% of the body weight and divided twice a day. Infection was carried out by immersing 3 L of immersion liquid at 1x10 ⁶ and 5x10 ⁶ CFU / ml for 1 hour, and then adding 15 L of seawater to finally reach 20 L. After the infection, the extracts were adsorbed at 0.5% of the fish body weight until the animals were fed. During the experiment, sea water exchange was carried out 50% twice a day, and the water temperature was maintained at 22 ± 0.5 ℃. Daily observations were made to record cumulative mortality.

5.6. In vivo anthracnose infestation of the optimized Rhus verniciflua extract against Sututika spp.

2, and 12 weeks, respectively, for 15 days. The animals were fed with 30, 100, or 300 mg / kg of extracts at a dose of 30, 100 or 300 mg / 0.5%, and administered twice a day. The infections were injected intramuscularly at 5.24 × 10 ⁴ at week ² and 7.7 × 10 ⁵ cells / fish / 100 μl at week 12. During the experiment, sea water exchange was carried out 50% twice a day, and the water temperature was maintained at 22 ± 0.5 ℃. Daily observations were made to record cumulative mortality.

Experiment result

1. Phenolic acid, flavonoid content and DPPH radical scavenging activity

Total phenolics and flavonoid contents were four times higher in the woody parts than in the leaves and bark of Rhus verniciflua. DPPH radical scavenging activity was also two times higher than that of leaves and shells in woody parts. Therefore, it was confirmed that the contents of flavonoids and phenolic compounds were much higher than those of the lacquer wood parts, and that the antioxidative activity was also excellent due to these physiologically active substances (FIG. 1).

2. In vitro antimicrobial activity of antioxidant extracts of Lacquer

In all test strains, the extracts of Rhus verniciflua showed the highest activity. In addition, the extracts and fraction fractions of Rhus verniciflora showed very good activity compared to the peels.

Therefore, the results of the above experiment showed that the lacquer wood parts showed high levels of flavonoids and phenolic contents compared with the shells, and showed excellent antioxidant activity, antimicrobial activity and antiscuctic activity, and finally lacquer wood part was selected as the final sample.

Strain E. tarda KCTC12267 V. anguillarum KCTC2711 S. iniae KCTC3657 origin part 2 mg / disc (mm) 2 mg / disc (mm) 2 mg / disc (mm) xylem 11.74 12.70 10.46 Shell - 12.08 7.47 DMSO vehicle - - - 30 g of oxytetracycline 20.85 19.78 19.81 10 μg of amoxicillin 18.55 21.19 22.06 Number of bacteria (CFU / dish) 1.12x10 ⁷ 1.04x10 ⁷ 0.99x10 ⁷ -: No activity

Extract of lacquer tree or woody part or fraction thereof E. tarda
KCTC12267 V.anguillarum
KCTC2711 S.iniae
KCTC3657 M. avidus 2 mg / disc (cm) 2 mg / disc (cm) 2 mg / disc (cm) ppm ([mu] g / ml) Shell 80% methanol extract (T) 0.6 1.1 1.2 500 The n-hexane fraction (H) 0.0 1.9 2.1 500 The chloroform fraction (C) 0.0 1.7 1.8 250 The ethyl acetate fraction (EA) 1.1 0.0 0.0 500 The water saturated butanol fraction (Bu) 0.0 0.0 0.0 - The water fraction (W) 0.0 0.0 0.0 - xylem 80% methanol extract (T) 1.0 1.0 0.9 250 The n-hexane fraction (H) 0.0 0.0 0.0 1000 The chloroform fraction (C) 1.1 1.0 1.0 125 The ethyl acetate fraction (EA) 1.2 1.0 0.9 500 The water saturated butanol fraction (Bu) 0.0 0.0 0.0 500 The water fraction (W) 0.0 0.0 0.0 - Extract of hot water extract. 0.9 0.9 0.8 NT 30 g of oxetetracycline 2.2 2.0 2.0 formalin 10 μg of amoxicillin 2 2.4 2.4 31.25 Number of cells (CFU / dish) 1.76x10 ⁷ 1.77x10 ⁷ 1.71x10 ⁷ 3x10 ⁵ cells / ml -: No activity, NT: not tested

3. Antimicrobial, antiseptic activity and yield optimization of lacquer wood extract

3.1. Set the level of independent variables

The antimicrobial activity and the extraction yield of the extracts extracted with different ethanol contents were examined to establish the level of independent variables. Both ethanol and ethanol increased the activity and yield as the ethanol content increased. The antimicrobial activity showed the highest activity when the ethanol content was 40% and the highest value was obtained at the extraction yield of 60% 3). As a result of creating a trend line in each point, we confirmed that it is nonlinear. The minimum and maximum levels of the independent variables for the reaction surface analysis were determined to be 20, 60% ethanol, 70, 100 ℃, 30 and 50 times, respectively. .

3.2. Antibacterial, established Hansukutika active model

In order to obtain the optimum extraction and extraction conditions from Rhus verniciflua, the activity and yield of 15 experimental conditions obtained from BBD were obtained. The reaction surface regression analysis was performed based on the experimental results and the regression equation for each dependent variable Respectively. The predictive value ^a in the following table is generated from the mathematical model. Table 6 below shows the matrix in the BBD model.

try
Number of times Independent variable Dependent variable ethanol
% (X ₁ ) Temperature
° C (X ₂ ) Sample to solvent weight ratio
(X ₃₎ Suppression region
(mm, Y ₁₎ Minimum lethal concentration
(ppm, Y ₂ ) yield
(%, Y ₂ ) Actual value Predicted value ^a Actual value Predicted value Actual value Predicted value One 60 (+1) 70 (-1) 40 (0) 10.27 10.56 500 500 9.6 9.7 2 60 (+1) 100 (+1) 40 (0) 11.92 11.99 1000 938 9.3 9.3 3 60 (+1) 85 (0) 30 (-1) 12.09 12.00 500 531 9.9 9.9 4 40 (0) 70 (-1) 50 (+1) 11.56 11.54 500 469 9.0 9.0 5 40 (0) 85 (0) 40 (0) 12.15 12.04 500 500 9.2 9.2 6 60 (+1) 85 (0) 50 (+1) 11.91 11.64 500 531 9.8 9.7 7 20 (-1) 70 (-1) 40 (0) 10.51 10.44 500 562 7.8 7.8 8 20 (-1) 85 (0) 30 (-1) 11.20 11.47 250 219 8.5 8.6 9 20 (-1) 100 (+1) 40 (0) 11.80 11.51 250 250 9.2 9.1 10 20 (-1) 85 (0) 50 (+1) 11.47 11.56 250 219 9.1 9.1 11 40 (0) 100 (+1) 50 (+1) 11.52 11.72 500 531 9.0 9.1 12 40 (0) 100 (+1) 30 (-1) 12.89 12.91 500 531 9.3 9.3 13 40 (0) 85 (0) 40 (0) 12.04 12.04 500 500 9.0 9.2 14 40 (0) 85 (0) 40 (0) 11.92 12.04 500 500 9.3 9.2 15 40 (0) 70 (-1) 30 (-1) 10.81 10.61 500 469 8.5 8.4

A) Establishing an antimicrobial activity model

The antimicrobial activity model was established by the regression coefficient estimation and the significance test for the secondary polynomial model.

Terms Coefficient Standard error of coefficient T P a constant 12.0367 0.1739 69.222 0.000 ^* Linear X ₁ 0.1512 0.1065 1.420 0.215 X ₂ 0.6225 0.1065 5.846 0.002 ^* X ₃ -0.0662 0.1065 -0.622 0.561 circle

-0.4696 0.1567 -2.996 0.030 ^*

-0.4421 0.1567 -2.821 0.037 ^*

0.1004 0.1567 0.641 0.550 Interaction X ₁ X ₂ 0.0900 0.1506 0.598 0.576 X ₁ X ₂ -0.1125 0.1506 -0.747 0.489 X ₂ X ₃ -0.5300 0.1506 -3.520 0.017 ^* R ² = 93%, R ² (adj) = 80.5%
*: P < 0.05

,

, X₂ X₃ 항은 유의한 인자로 예측할 수 있다. 최종 적합 모델을 확인하기 위해 고차항부터 유의하지 않은 항을 차례로 제거하여 모형을 축소하였다. 단, 유의한 고차항에 포함된 주효과는 P값이 0.05이상이어도 계층적 모형을 유지하기 위해 최종 모델에 포함시켰다. 잔차 그림과 분산분석을 통해 모형의 유효성을 판단하였다. 잔차 그림을 통해 축소된 모형의 정규분포, 모든 적합치에서 분산의 동일성, 시간에 따른 임의성이 적합함을 확인하였으며, 분산분석에서 축소된 모델의 모든 항이 유의적임을 나타내었다. 모형의 적합성을 나타내는 적합성 결여(lack-of-fit) 값은 P > 0.05 임으로 축소된 모형이 적합함을 나타내었으며, 회귀모형의 반응변수에 대한 변동을 설명해주는 R² (adj)값은 84.6%로 확인되었다 (도 3). 축소된 최종적인 모형은 다음 식과 같다.Statistical significance of the model equations is evaluated by T- test, P <0.05 X ₂ eseo term significant commutative this model,

,

, And X ₂ X ₃ can be predicted with significant factors. In order to confirm the final fit model, the model was reduced by eliminating the high-order to the least-significant term in order. However, the main effects included in the significant higher order are included in the final model to maintain the hierarchical model even if the P value is 0.05 or higher. The validity of the model was assessed through residual figures and analysis of variance. It is confirmed that the normal distribution of the reduced model, the uniformity of the variance at all the fit values, and the randomness over time are appropriate through the residual images. All the terms in the reduced model are significant in the variance analysis. The R ² (adj) value, which accounts for the variance of the response variable of the regression model, is 84.6%, while the lack-of-fit value indicating the fit of the model is P> (Fig. 3). The final reduced model is as follows.

&Quot; (3) &quot;

Table 8 below shows the analysis deviations for the antimicrobial activity regression model.

sauce DF MS F P return 6 0.98886 13.79 0.001 Linear 3 1.10606 15.42 0.001 circle 2 0.74569 10.40 0.006 Interaction One 1.12360 15.67 0.004 Residual error 8 0.07172 - - Lack of conformity 6 0.09122 6.89 0.132 Pure error 2 0.01323 - - total 14 - - - R ² = 91.2% R ² (adj) = 84.6%
DF: degree of freedom; MS: Sum of square / DF

B) Hansukutika active model established

Hanskutika activity model was established by regression coefficient estimation and significance test for quadratic polynomial model.

Terms Coefficient Standard error of coefficient T P a constant 500.00 32.27 15.492 0.000 * Linear X ₁ 156.25 19.76 7.906 0.001 * X ₂ 31.25 19.76 1.581 0.175 X ₃ 0.00 19.76 0.000 1,000 circle

-31.25 29.09 -1.074 0.332

93.75 29.09 3.233 0.023 *

-93.75 29.09 -3.233 0.023 * Interaction X ₁ X ₂ 187.50 27.95 6.708 0.001 * X ₁ X ₂ -0.00 27.95 -0.000 1,000 X ₂ X ₃ 0.00 27.95 0.000 1,000 R ² = 96.4% R ² (adj) = 89.9%
*: P < 0.05

,

,

, X₁ X₂항은 유의한 인자로 예측할 수 있다. 최종 적합 모델을 확인하기 위해 고차항부터 유의하지 않은 항을 차례로 제거하여 모형을 축소하였다. 단, 유의한 고차항에 포함된 주효과는 P값이 0.05이상이어도 계층적 모형을 유지하기 위해 최종 모델에 포함시켰다. 잔차 그림과 분산분석을 통해 모형의 유효성을 판단하였다. 잔차 그림을 통해 축소된 모형의 정규분포, 모든 적합치에서 분산의 동일성, 시간에 따른 임의성이 적합함을 확인하였으며, 분산분석에서 축소된 모델의 모든 항이 유의적임을 나타내었다. 모형의 적합성을 나타내는 적합성 결여 값은 P > 0.05 임으로 축소된 모형이 적합함을 나타내었으며, 회귀모형의 반응변수에 대한 변동을 설명해주는 R² (adj)값은 89.9%로 확인되었다 (도 4). 축소된 최종적인 모형은 다음 식과 같다.The statistical significance of the model equations was evaluated by the T-test, and since the term P <0.05 was significant,

,

,

, And X ₁ X ₂ can be predicted with significant factors. In order to confirm the final fit model, the model was reduced by eliminating the high-order to the least-significant term in order. However, the main effects included in the significant higher order are included in the final model to maintain the hierarchical model even if the P value is 0.05 or higher. The validity of the model was assessed through residual figures and analysis of variance. It is confirmed that the normal distribution of the reduced model, the uniformity of the variance at all the fit values, and the randomness over time are appropriate through the residual images. All the terms in the reduced model are significant in the variance analysis. The R ² (adj) value, which describes the variance of the response variable of the regression model, was found to be 89.9% (Fig. 4) . The final reduced model is as follows.

&Quot; (4) &quot;

Table 10 below shows the analysis deviations for the Hanskutika active regression model.

sauce DF MS F P return 6 69017 28.71 0.000 Linear 3 67708 28.17 0.000 circle 2 35176 14.63 0.002 Interaction One 140625 58.50 0.000 Residual error 8 19231 - - Lack of conformity 6 19231 * * Pure error 2 0 - - total 14 - - - R ² = 91.2% R ² (adj) = 84.6%
DF: degree of freedom; MS: Sum of square / DF

C) Establishing a yield model

The yield model was established by regression coefficient estimation and significance test for the quadratic polynomial model.

Terms Coefficient Standard error of coefficient T P a constant 9.16667 0.07032 130.363 0.000 ^* Linear X ₁ 0.50000 0.04306 11.612 0.000 ^* X ₂ 0.23750 0.04306 5.516 0.003 ^* X ₃ 0.08750 0.04306 2.032 0.098 circle

0.09167 0.06338 1.446 0.208

-0.28333 0.06338 -4.470 0.007 ^*

0.06667 0.06338 1.052 0.341 Interaction X ₁ X ₂ -0.42500 0.06090 -6.979 0.001 ^* X ₁ X ₂ -0.17500 0.06090 -2.874 0.035 ^* X ₂ X ₃ -0.20000 0.06090 -3.284 0.022 ^* R ² = 98.1% R ² (adj) = 94.8%
*: P < 0.05

, X₁ X₂, X₁ X₂, X₂ X₃항은 유의한 인자로 예측할 수 있다. 최종 적합 모델을 확인하기 위해 고차항부터 유의하지 않은 항을 차례로 제거하여 모형을 축소하였다. 단, 유의한 고차항에 포함된 주효과는 P값이 0.05이상이어도 계층적 모형을 유지하기 위해 최종 모델에 포함시켰다. 잔차 그림과 분산분석을 통해 모형의 유효성을 판단하였다. 잔차 그림을 통해 축소된 모형의 정규분포, 모든 적합치에서 분산의 동일성, 시간에 따른 임의성이 적합함을 확인하였으며, 분산분석에서 축소된 모델의 모든 항이 유의적임을 나타내었다. 모형의 적합성을 나타내는 lack-of-fit 값은 P > 0.05 임으로 축소된 모형이 적합함을 나타내었으며, 회귀모형의 반응변수에 대한 변동을 설명해주는 R²(adj)값은 94.0%로 확인되었다 (도 5). 축소된 모형은 다음 식과 같다.The statistical significance of the model equations was evaluated by T-test, and since the term of P <0.05 was significant, X ₁ , X ₂ ,

, X ₁ X ₂ , X ₁ X ₂ , and X ₂ X ₃ can be predicted as significant factors. In order to confirm the final fit model, the model was reduced by eliminating the high-order to the least-significant term in order. However, the main effects included in the significant higher order are included in the final model to maintain the hierarchical model even if the P value is 0.05 or higher. The validity of the model was assessed through residual figures and analysis of variance. It is confirmed that the normal distribution of the reduced model, the uniformity of the variance at all the fit values, and the randomness over time are appropriate through the residual images. All the terms in the reduced model are significant in the variance analysis. The lack-of-fit value indicating fit of the model is P> 0.05, indicating that the reduced model is appropriate, and the R ² (adj) value that describes the variance of the response variable of the regression model is 94.0% 5). The reduced model is as follows.

&Quot; (5) &quot;

Table 12 below shows the analysis deviations for the antimicrobial activity regression model.

sauce DF MS F P return 7 0.54880 32.45 0.000 Linear 3 0.83750 49.52 0.000 circle One 0.32411 19.16 0.003 Interaction 3 0.33500 19.81 0.001 Residual error 7 0.01691 - - Lack of conformity 5 0.01435 0.61 0.714 Pure error 2 0.02333 - - total 14 - - - R ² = 97.0% R ² (adj) = 94.0%
DF: degree of freedom; MS: Sum of square / DF

3.3. Multiple Reaction Optimization

Through the final model of the antimicrobial activity and yield of Rhus verniciflua extract, the response surface analysis was carried out for two reaction parameters of more than 12mm antibacterial activity and 10% yield. The optimal reaction conditions were ethanol content 60%, extraction temperature 86.1645 ℃ , And the sample to solvent ratio was 30 times. The anticipated response of the dependent variable was 11.9254mm and the yield was 9.8438%. The total satisfaction (D) was 0.84733.

Through a final model of activity and yield of Hanskutika, reaction surface analysis was carried out for two reaction parameters of less than 200 ppm of Hanskutika activity and yield of 9%. The optimized reaction conditions were ethanol content 20%, extraction temperature 100 C, and a sample to solvent ratio of 30 times. The predicted response of the dependent variable was 173.08 ppm of antiscucta activity and 9.0625% of yield, and the overall satisfaction was 0.25.

For the extraction of antimicrobial and yield optima, it is difficult to set the extraction temperature to 86.1645 ℃ at the limit of the extraction process of this laboratory. Therefore, the extraction temperature was adjusted by adjusting the extraction temperature to 85 ℃. As a result of verifying the optimum reaction condition through multiple reaction optimization, it was confirmed that the final regression model explained the variation of the response variable with the antimicrobial activity of 12.09 mm and the yield of 10% (Fig. 6).

As a result of the validation experiment, the activity of Hanskutika was 225ppm and the yield was 9.3%. The final regression model explains the variation of the response variable. Likewise, (Fig. 7).

In order to investigate the interactions of the independent variables affecting the activity and yield of antimicrobial, antiscucta, the three-dimensional reaction surface diagram of the independent variables is shown in FIGS. 8 and 9 after the sample-to-solvent ratio is fixed at 30 times. The antimicrobial activity tended to increase as the extraction temperature increased. As the ethanol content increased at the extraction temperature of 100 ℃, the antimicrobial activity increased and the ethanol activity was highest at 40 ~ 50%. The activity of Huskutikka tended to increase as the ethanol content was lowered at 100 ℃ and it showed the maximum activity at 20% of ethanol content. In the case of the yield, it was confirmed that the extraction yield was higher as the extraction temperature was increased as well as the antibacterial activity. Also, the yield increased with increasing ethanol content, and the yield was found to be the maximum at an ethanol content of 60% and an extraction temperature of 80 to 90 ° C. a means a predicted value generated by a mathematical model, and B means not tested. Table 13 shows the comparison between the actual value and the predicted value determined in the optimization condition and the multiple reaction optimization.

reaction Optimization condition Complex fit
(D) Actual value Predicted value ^a Estimated Capacity (%) Ethanol% (X1) Temperature
(X2) Sample to solvent ratio
(X3) Antimicrobial activity (mm) 43 100 30 0.87939 NT ^b 12.88 - Activity of Hanskutika (ppm) 20 100 30 1.0000 225 173.08 76.92 Yield (%) 60 85 30 0.84474 10.00 9.84 101.63 Multiple Reaction Optimization 0.84733 - - - Antimicrobial activity (mm) 60 85 30 0.85083 12.09 11.93 101.34 Yield (%) 0.84384 10.00 9.84 101.63 Multiple Reaction Optimization 0.25000 - - - Activity of Hanskutika (ppm) 20 100 30 1.0000 225 173.08 76.92 Yield (%) 0.06250 9.30 9.06 102.62 Predicted value ^a : predicted value generated by mathematical model, NT ^b : not tested

In the following, 60.85.30 of the extracts were obtained by extracting 60 vol% ethanol aqueous solution of Rhus verniciflua var. Sp. At 85 ° C with 1: 30 ratio of lacquer wood to solvent, 20.100.30 of 20 vol% Ethanol aqueous solution extract at 100 ° C with a 1: 30 ratio of crushed lacquer to solvent.

4. In vitro antibacterial, antiscuctive and antiviral activity of optimized extracts

4.1. Antibacterial, active Hansukutika

The antimicrobial activities of the extracts of Rhus verniciflua and Rhus verniciflua were 1000 ㎍ / ml and 500 ㎍ / ml, respectively. The MIC of the optimized extracts was 250 ㎍ / ml each. The activity of Hansucutica was> 1000 ㎍ / ml for picetine and 250 ㎍ / ml for gallic acid. The MIC of the optimized extracts showed 225, 500 ㎍ / ml MLC at 20, 60 extracts, respectively. As a result of comparing the activities of natural products with hot water extracts, it was found that the antibacterial and antiseptic activities of the optimized extracts were superior to the hot water extracts and the extraction yield was also increased. In addition, it is found that the optimized extracts have higher activity than the antimicrobial activity of the main compounds that constitute the extract.

sample yield
(%) E. tarda FJ1408 E. tarda KCTC12267 V. anguillarum KCTC2711 S. iniae KCTC3657 M. avidus ppm ([mu] g / ml) ppm ([mu] g / ml) ppm
(占 퐂 / ml) ppm ([mu] g / ml) ppm ([mu] g / ml) Gallan - 500 > 2000 500 500 250 Fustin - 1000 1000 1000 1000 NT Physetine - 1000 1000 2000 1000 > 1000 Sulperitine - 1000 1000 NT NT NT 20 extract 9.0 250 250 1000 1000 225 60 extract 10.0 250 250 1000 1000 500 Hot water extract 7.5 500 NT NT NT 500 Oxytetracycline - 0.5 0.5 0.25 0.25 formalin 31.25 Number of bacteria (CFU / ml) - 7.5x10 ⁵ 6.1x10 ⁵ 7.7x10 ⁵ 8.4x10 ⁵ 3x10 ⁵ cells / ml -: No activity, NT: not tested

4.2. Antiviral activity

As shown in Figure 10, the EC ₅₀ for the viral hemorrhagic sepsis virus and the infectious hematopoietic necrosis virus of the optimized extracts 20 and 60 were 0.68 ± 0.01 and 0.83 ± 0.04 μg / ml, 0.83 ± 0.04 and 0.78 ± 0.12 μg / ml Respectively. In addition, the optimized extracts from the hot-water extracts showed excellent antiviral activity against the two pathogenic viruses. a means half-effect concentration.

extract EC ₅₀ ^a in FHM cells (占 퐂 / ml) VHSV IHNV Hot water extract 1.93 + 0.17 1.00 0.49 20.100.30 0.68 ± 0.01 0.67 + 0.03 60.85.30 0.83 + 0.04 0.78 + - 0.12

5. Analysis of the content of the indicator component among the optimized extracts (Method validation)

5.1. Linearity, LOD and LOQ output

The linearity (r ² ) of the three compounds showed a very high level of 0.9999 or more, and the respective LOD and LOQ values are shown in Table 16 below (FIG. 12).

compound Linearity (r ² ) LOD (ng / ml) LOQ (ng / ml) Gallan 0.99998 5.14 15.59 Fustin 0.99998 4.93 14.94 Physetine 0.99998 5.29 16.03

5.2. Precision measurement

As a result of the intra- and inter-day experiments for the precision measurement, RSD of less than 2% in all three compounds was confirmed, and it was confirmed that the precision of the present method was very high. Detailed results of each compound are shown in Table 17 below.

20 extract
(占 퐂 / ml) Accuracy: RSD (%) Intraday (n = 3) Interday (n = 9) Gallan Fustin Physetine Gallan Fustin Physetine One 0.80 0.26 0.94 1.88 0.72 0.71 3 1.53 0.39 0.54 1.39 0.53 0.57 5 0.47 1.86 0.32 0.77 1.11 0.25 60 extract
(占 퐂 / ml) Accuracy: RSD (%) Intraday (n = 3) Interday (n = 9) Gallan Fustin Physetine Gallan Fustin Physetine One 1.01 0.23 1.74 0.95 0.37 1.73 3 0.92 0.06 0.84 0.80 0.23 0.55 5 1.19 0.13 0.25 1.44 0.16 0.72

5.3. Measurement of accuracy

The recovery of the three compounds showed a high recovery of 92.09 to 105.16% and RSD was found to be less than 5.5%. Detailed results for each compound are shown in Tables 18 and 19 below.

compound 20 extract Original (ng / ml) Spiked (ng / ml) Detected (ng / ml) Recovery
(%) RSD (%) Gallan 29.65 15 44.34 99.31 0.41 30 54.93 92.09 1.78 60 86.08 96.02 2.09 Fustin 188.19 90 268.23 96.42 3.75 180 355.62 96.59 1.54 360 542.43 98.95 1.04 Physetine 31.97 15 47.67 101.49 5.31 30 57.91 93.45 5.44 60 89.72 97.55 1.98

compound 60 extract Original (ng / ml) Spiked (ng / ml) Detected (ng / ml) Recovery
(%) RSD (%) Gallan 18.33 15 30.81 92.44 4.34 30 44.74 92.57 5.03 60 75.68 96.62 1.58 Fustin 212.71 135 328.42 94.45 1.86 270 470.88 97.55 1.48 540 791.53 105.16 1.27 Physetine 27.31 15 40.34 95.34 3.53 30 53.98 94.19 2.43 60 84.53 96.82 1.17

5.4. Specificity

The MRM data of the surface components in the optimized extracts were compared with the retention times of MRM data of each standard compound. As a result, it was confirmed that the retention time of the reference material and the retention time of the indicator component in the extract were the same, and the ratio of the quantitative ion and the qualitative ion in the fragment pattern also coincided (FIGS. 13 to 16).

5.5. Analysis of the content of surface elements

Using the above analysis conditions, the contents of the indicator components in the optimized extracts were analyzed. The content of gallic acid in the optimized extracts of 1, 3 and 5 ㎍ / ml was 3.07 ± 0.06% in 20 extracts and 1.82 ± 0.05% in 60 extracts. The content of fostin was 19.77 ± 0.42% in 20 extracts, 60 extracts And 20.2 ± 0.58%, respectively. Finally, picetin was found to be 2.52 ± 0.02% in 20 extract and 2.12 ± 0.08% in 60 extract. The detailed results are shown in Tables 20 and 21 below.

20 extract
(占 퐂 / ml) Gallan Fustin Physetine %, Average
(mean) SD RSD
(%) %, Average
(mean) SD RSD
(%) %, Average
(mean) SD RSD
(%) One 3.04 0.06 1.88 20.09 0.14 0.72 2.52 0.02 0.71 3 3.13 0.04 1.39 19.98 0.11 0.53 2.52 0.01 0.57 5 3.04 0.02 0.77 19.23 0.21 1.11 2.51 0.01 0.25 Average 3.07 0.06 1.95 19.77 0.42 0.02 2.52 0.02 0.62

60 extract
(占 퐂 / ml) Gallan Fustin Physetine %, Average
(mean) SD RSD
(%) %, Average
(mean) SD RSD
(%) %, Average
(mean) SD RSD
(%) One 1.83 0.02 0.95 21.34 0.08 0.37 2.19 0.04 1.73 3 1.88 0.01 0.80 21.26 0.05 0.23 2.15 0.01 0.55 5 1.76 0.02 1.14 20.09 0.03 0.16 2.01 0.01 0.72 Average 1.82 0.05 2.96 20.90 0.58 2.78 2.12 0.08 3.74

6. In vivo efficacy of optimized Rhus verniciflua extract against E. tarda

6.1. Antibiotic activity at 2 weeks

5x10 ⁶ and 1x10 ⁶ CFU / ml, was immersed in the sphere of administration for two weeks with the test word A and 20 extract and an extract of 60 to day 30, 100 or 300 mg / kg in B infected with the control E.tarda FJ1408 , The relative survival rate was 7.1% and 100%, respectively, which was statistically significant (Figs. 17 and 18). Therefore, it has been proved that the optimized extract has very good anti - inflammatory effect on fish infectious diseases when administered to fish body.

6.2. Antimicrobial activity at 6 weeks

5, ¹⁰ and ⁶ × 10 ⁶ CFU / ml, respectively, and the test extracts A and B infected with E.tarda FJ1408 were treated with 30, 100 and 300 mg / kg of extracts of 20 and 60 for 6 weeks. Survival rate was at least 8.3% and maximal 83.3%, which was statistically significant (Figs. 19 and 20). Therefore, it has been proved that the optimized extract has very good anti - inflammatory effect on fish infectious diseases when administered to fish body.

6.3. Antimicrobial activity at 10 weeks

5x10 ⁶ and 1x10 immersed in ⁶ CFU / ml compared to the concentration of the sphere of control of administration for 10 weeks to 20, and 60 extract the control test A and B were infected with E.tarda FJ1408 to 30, 100, 300 mg / kg relative Survival rate was at least 54.5% and maximum 90.9%, which was statistically significant (Figs. 21 and 22). Therefore, it has been proved that the optimized extract has very good anti - inflammatory effect on fish infectious diseases when administered to fish body.

7. In vivo efficacy of optimized Rhus verniciflua extract against Sututika spp.

7.1. Activity of 2 weeks of oral Hucuksutika

The relative survival rates of the extracts treated with the optimized 20 extract at 10 and 100 mg / kg were 21.4% and 64.3%, respectively, confirming that the optimized extract had an excellent antiscucta activity (FIG. 24).

Control group 10 mg / kg 100 mg / kg death rate 93.3 73.3 33.3 RPS - 21.4 64.3

7.2. Activity of 12 weeks of treatment Hansukutika active

The optimum extracts of 20 and 60 extracts at 30, 100 and 300 mg / kg showed a relative survival rate of at least 18% and a maximum of 36%, respectively, as compared with the control. Thus, it was confirmed that the optimized extract had an excellent antiscucta activity (Fig. 25).

Claims (9)

An antimicrobial composition of fish comprising extracts of Rhus verniciflua woody part.
The antimicrobial composition of claim 1, wherein the extract is water, 70 to 90% by volume of methanol aqueous solution, 10 to 70% by volume of ethanol aqueous solution extract or mixed solvent extract thereof.
The antimicrobial composition according to claim 1, wherein the extract is a water extract.
The antimicrobial composition of fish according to claim 1, wherein the extract is extracted at 80-110.
The antimicrobial composition of fish according to claim 1, wherein the extract is extracted with a solvent which is 10 to 40 times the weight of the rhizome wood part.
The antimicrobial composition according to claim 1, wherein the extract is extracted at 80 to 90 ° C with an aqueous solution of 50 to 70% by volume of ethanol.
The antimicrobial composition of claim 1, wherein the extract is an organic solvent fraction of water, 70 to 90% by volume of methanol aqueous solution, 10 to 70% by volume of ethanol aqueous solution extract or mixed solvent extract thereof.
[Claim 7] The antimicrobial composition according to claim 7, wherein the organic solvent is at least one selected from the group consisting of chloroform, ethyl acetate and hydrogenated butanol.
The antimicrobial composition of claim 1, wherein the extract comprises 1 to 10% by weight of gallic acid, 10 to 40% by weight of fostin and 1 to 10% by weight of picetin.

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