KR20240023573A - Antimicrobial composition against fish bacterial disease comprising Cudrania tricuspidata Bureau extract or fraction as an active ingredient - Google Patents

Abstract

The present invention relates to an antibacterial composition against bacterial diseases in fish containing an extract or fraction of Kujippong as an active ingredient and an antibacterial composition against bacterial diseases in fish containing a compound derived from Kujippong as an active ingredient, which causes high mortality in fish. It has excellent antibacterial activity against pathogenic bacteria, and especially has excellent antibacterial activity against antibiotic-resistant fish disease bacteria.

Description

Antimicrobial composition against fish bacterial disease comprising Cudrania tricuspidata Bureau extract or fraction as an active ingredient}

The present invention relates to an antibacterial composition against bacterial diseases in fish containing an extract or fraction of Kujippong as an active ingredient, and an antibacterial composition against bacterial diseases in fish containing a compound derived from Kujippong as an active ingredient.

Looking at the domestic incidence of aquaculture disease, which is a worldwide problem in the aquaculture industry, the incidence rate in the early 1990s was less than 5%, but has increased to around 15% since the late 1990s, and the incidence rate has increased during periods of high temperature. Diseases that used to occur mainly are trending to occur throughout the year. Among them, the damage to farmed fish is increasing due to the increase in the occurrence of bacterial infectious diseases, and the treatment effect is decreasing due to the increase in the emergence of resistant bacteria due to drug misuse. In particular, in recent years, the risk of introducing new exotic infectious diseases has increased due to the increase in the amount of imported aquaculture seedlings, and there are concerns about the spread of diseases due to the movement of infected seedlings.

In particular, in the case of infectious diseases such as flounder and rockfish, which are important for domestic industry, there is a difference in the infectious diseases that mainly occur in fry and adult fish, and in fry weighing less than 100 g, scuticociliatosis, gill flukes, or Infectious diseases such as flavobacteriosis are common, and parasitic diseases mainly cause severe damage.

However, adult fish weighing more than 300 g are severely damaged by bacterial diseases such as Edwardsiellosis, Vibriosis, and Streptococcus.

There are currently commercially available vaccines to control these bacterial diseases, but they are mainly administered during the juvenile period, so when bacterial diseases occur in adult fish, there is no special treatment other than antibiotics.

Therefore, to treat these bacterial diseases, large amounts of antibiotics are used in fish farms, and in some cases, several antibiotics are used in combination. Due to this overuse of antibiotics, there are continued reports of the development of antibiotic resistance in fish disease bacteria.

In particular, looking at the antibiotic resistance trend for streptococci in farmed flatfish, resistance to oxytetracycline and tetracycline shows a steady antibiotic resistance of about 60% from 1999 to 2016. For penicillin-based antibiotics, the resistance rate was 70% in 2004 to 2005, showing that the antibiotic resistance rate has increased significantly over the past 5 to 6 years.

In other words, the continuous use of antibiotics will eventually cause resistance, which inevitably leads to the expansion of the use of new broad-spectrum antibiotics, which poses the biggest threat to the sustainability of aquaculture and the food safety of farmed fish. Therefore, the development of safe and effective antibiotic alternatives in aquaculture is urgently needed.

In the case of antibiotic substitutes using natural materials, the increased production cost compared to existing antibiotics is a weakness, so a low-cost production process is necessary, and therefore, it is important to derive the optimal extract that can contain the maximum amount of effective ingredients. In order to improve this process, the present invention uses Response Surface Methodology (RSM), a type of Design Of Experiment (DOE), to derive extraction conditions that maximize the target activity of the material to produce a cost-effective extract. The manufacturing method of the extract was designed to enable the derivation of .

Cudrania tricuspidata Bureau belongs to the Moraceae family and is a deciduous small tree or shrub widely distributed throughout East Asia, including Korea, Japan, and China. Cudrania tree leaves and fruits are used as food ingredients, and other parts, such as leaves and stems including fruits, are also used for medicinal purposes. Cudrania mulberry extract is known to have anti-inflammatory, antihypertensive, antidiabetic, antioxidant, and antimicrobial activities, but to date, no antibacterial activity against fish pathogenic bacteria has been reported.

Domestic published patent 10-2013-0019352 (2013.02.26) Domestic published patent 10-2012-0105776 (2012.09.26) Domestic published patent 10-2014-0129492 (2014.11.07)

Accordingly, the present inventors confirmed that Cudrania extract or fractions have excellent antibacterial activity against pathogenic bacteria that cause high mortality in fish, and in particular, have excellent antibacterial activity against antibiotic-resistant fish disease bacteria. In addition, using response surface analysis, extraction conditions optimized for antibacterial activity were obtained from Cudrania fruit, and an optimized extract was obtained from these.

Accordingly, the purpose of the present invention is to provide an antibacterial composition against bacterial fish diseases containing Cudrania quince extract or fractions as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bacterial diseases in fish containing a Cudrania extract or fraction as an active ingredient.

Another object of the present invention is to provide a composition for fish feed having fish bacterial activity containing a Cudrania extract or fraction as an active ingredient.

Another object of the present invention is to provide a composition for adding fish feed having fish bacterial activity containing Cudrania extract or fractions as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating bacterial diseases in fish using Cudrania extract or fractions.

Another object of the present invention relates to the use of Cudrania extract or fractions for preventing or treating bacterial diseases in fish.

Another object of the present invention relates to a method for producing an antibacterial composition against bacterial diseases in fish, comprising an extract or fraction of Kujippong as an active ingredient.

The present invention relates to an antibacterial composition against bacterial diseases in fish containing an extract or fraction of Kujippong as an active ingredient, and an antibacterial composition against bacterial diseases in fish containing a compound derived from Kujippong as an active ingredient.

Hereinafter, the present invention will be described in more detail.

An example of the present invention relates to an antibacterial composition against bacterial diseases in fish containing an extract or fraction of Kujippong as an active ingredient.

The bark of Kujippong is brown or grey-brown and shallowly cracked vertically, and bark buds develop on the branches. There are thick, sharp thorns that have turned into twigs in the leaf axils. The leaves, which grow alternately, are ovate, have smooth edges, and may be split into 2 or 3 branches. There are hairs on both sides, and the petioles also have hairs. It is a dioecious plant with light yellow flowers clustered in the leaf axils from May to June. The male flower spike is round and covered with light hair, and the male flower has 3 to 5 perianth pieces and 4 stamens. The female flower spike is oval-shaped, and the female flower has two styles that are split like threads. The fruit is hard and green, but becomes slightly soft and ripens to red in September or October. When you cut the leaves, a white liquid comes out and the fruit tastes sweet. It is a deciduous broad-leaved small tree or shrub that grows in sunny places in the mountains south of central Korea.

In the present invention, the term "kujimulberry" refers to a cudra tree, and "kujippong" and "kujimulberry tree" are interpreted as having the same meaning.

In the present invention, the root, stem, leaf, and fruit of Kujippong can be used. For example, it may be fruit or leaf, but is not limited thereto.

In the present invention, the kkujippong fruit may be an immature fruit or a mature fruit, for example, it may be a mature fruit.

In the present invention, the term 'immature fruit' means that the fruit is generally collected around September and has a redness (a*) of around -5 when measured with a colorimeter.

In the present invention, the term 'mature fruit' means that the fruit is generally collected around October and has a redness (a*) of around 25 when measured with a colorimeter.

When obtaining the Cudrania extract of the present invention by treating Cudrania with an extraction solvent, various extraction solvents can be used.

In the present invention, the extraction solvent may be a polar solvent or a non-polar solvent.

In the present invention, the polar solvent may be one or more selected from the group consisting of water, alcohol, acetic acid, dimethyl formamide (DMFO), and dimethyl sulfoxide (DMSO), but is not limited thereto.

In the present invention, the alcohol may be a straight-chain or branched alcohol having 1 to 5 carbon atoms, for example, methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, It may be one or more selected from the group consisting of 2-butoxyethanol and ethylene glycol, but is not limited thereto.

In the present invention, when a mixture of water and alcohol is used as the extraction solvent, the concentration is 10% or more to less than 100% (v/v), 20% or more to less than 100% (v/v), or 30% or more to 100% ( less than v/v), more than 40% to less than 100% (v/v), more than 50% to less than 100% (v/v), 10 to 90% (v/v), 20 to 90% (v/v) ), 30 to 90% (v/v), 40 to 90% (v/v), 50 to 90% (v/v), 10 to 80% (v/v), 20 to 80% (v/v) ), 30 to 80% (v/v), 40 to 80% (v/v), 50 to 80% (v/v) of an alcohol aqueous solution, for example, 50% (v/v) It may be an aqueous solution of a straight-chain or branched alcohol having 1 to 5 carbon atoms, but is not limited thereto.

In the present invention, nonpolar solvents include methylene chloride, acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, and diisobutyl. lene, 1-pentene, 1-chlorobutane, 1-chloropentane, o -xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide , chloroform, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF, but is not limited thereto.

According to one embodiment of the present invention, the extraction solvent and/or fractionation solvent used in the present invention is anhydrous or hydrous lower alcohol having 1 to 5 carbon atoms (e.g., methanol, ethanol, propanol, butanol, etc.), water, It may be one or more selected from the group consisting of a mixed solvent of lower alcohol and water, methylene chloride, ethyl acetate, chloroform, butyl acetate, 1,3-butylene glycol, hexane, and diethyl ether, but is not limited thereto. .

According to one embodiment of the present invention, the Cudrania extract of the present invention was obtained using an aqueous ethanol solution as an extraction solvent.

The term 'extract' used in this specification has the meaning commonly used in the art as a crude extract, as described above, but in a broad sense also includes fractions obtained by additional fractionation of the extract.

Therefore, the Cudrania extract of the present invention includes not only that obtained using the above-described extraction solvent, but also that obtained by additionally applying a purification process thereto. For example, fractions obtained by passing the Cudrania extract through an ultrafiltration membrane with a certain molecular weight cut-off value, separation by various chromatographs (designed for separation according to size, charge, hydrophobicity, or affinity), etc. Fractions obtained through various purification methods are also included in the Cudrania extract of the present invention.

The Cudrania extract of the present invention may be prepared in powder form by additional processes such as reduced pressure distillation, freeze drying, or spray drying, but is not limited thereto.

It is obvious to those skilled in the art that the Cudrania extract of the present invention can be obtained using not only the above-mentioned extraction solvent, but also other extraction solvents, showing substantially the same effect.

The Cudrania extract of the present invention may be obtained by ultrasonic treatment with an extraction solvent, but is not limited thereto.

As used herein, the term 'containing as an active ingredient' means containing a sufficient amount to achieve the efficacy or activity of the Cudrania root extract below.

The upper quantitative limit of the Cudrania extract included in the composition of the present invention can be selected and implemented by a person skilled in the art within an appropriate range.

The kkujippong extract according to the present invention may be a solvent fraction obtained by fractionating the solvent crude extract with an additional solvent. For example, the kkujippong solvent crude extract is mixed with one or more species selected from the group consisting of ethyl ether, ethyl acetate, butanol, and dichloromethane. It may be a solvent fraction using a solvent, for example, it may be a solvent fraction using dichloromethane as a solvent.

In the present invention, the fish may be farmed fish or ornamental fish, but is not limited thereto.

In the present invention, farmed fish include flounder, rockfish, bream, grouper, mullet, perch, shad, flounder, puffer, mackerel, yellowtail, tuna, croaker, yellowtail, horse mackerel, carp, mullet, persimmon, eel, catfish, loach and crucian carp. It may be one or more species selected from the group consisting of, for example, flounder ( Paralichthys olivaceus ), but is not limited thereto.

In the present invention, the ornamental fish may be one or more species selected from the group consisting of carp, catfish, goldfish, characins, minnows, cichlids, anavanthis, and nandus brackish water fish, but is limited thereto. That is not the case.

In the present invention, the bacteria may be gram-positive bacteria or gram-negative bacteria.

In the present invention, the Gram-positive bacteria may be one or more species selected from the group consisting of Streprococcus iniae, S. parauberis, Lactococcus garvieae, Staphylococcus epidermidis, Renibacterium salmoninarum, Nocardia seriolae and N. asteriodes , for example, Streprococcus iniae, S. parauberis and/or Lactococcus garvieae , but is not limited thereto.

In the present invention, the gram-negative bacteria include Aeromonas hydrophila, A. salmonicida, Edwardsiella tarda, E. ictaluri, Flavofacterium columnare, Photobacterium damselae ( or subsp. piscicida), Piscirickettsia salmonis, Pseudomonas anguilliseptica, P. fluorescens, Flexibacter maritimus, Vibrio alginolyticus, V It may be one or more species selected from the group consisting of anguillarum, V. cholerae, V. harveyi, V. ichthyoenteri, V. ordalii, V. salmonicida, V. vulnificus and Yersinia ruckeri , but is not limited thereto.

In the present invention, fish bacterial diseases include streptococcus disease, staphylococcus aureus, bacterial nephropathy, Nocardia disease, erythroderma disease, Eromonas disease, Edward's disease, sliding bacterial disease, nodular tuberculosis, red spot disease, pseudomonas disease, and rickettsia disease. , may be one or more types selected from the group consisting of vibriosis, for example, streptococci, but is not limited thereto.

In the present invention, compounds derived from kkujippong may be flavonoids or xanthones compounds, or derivatives thereof.

In the present invention, the compound derived from kkujippong may be one or more compounds selected from the group consisting of the following formulas 1 to 17, or a derivative thereof.

In one embodiment of the present invention, the composition of the present invention may include one or more compounds or derivatives thereof selected from the group consisting of Formulas 1 to 17 below:

[Formula 1]

In Formula 1,

R ₁ is -H or -OH,

R ₂ is -OH or -OCH ₃ ,

R ₃ and R ₄ are each independently -H, or am.

[Formula 2]

In Formula 2,

R ₁ is -H or -OH,

R ₂ is -OH or -OCH ₃ ,

R ₃ is -H or am.

[Formula 3]

In Formula 3 above,

R ₁ is -H or -OH,

R ₂ is -OH,

R ₃ is -H or am.

[Formula 4]

In Formula 4 above,

R ₁ is -H or -OH,

R ₂ is -OH or -OCH ₃ ,

R ₃ is -H or -OCH ₃ ,

R ₄ is -H or am.

[Formula 5]

[Formula 6]

In Formula 6, R is -H or -OH.

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

[Formula 15]

[Formula 16]

[Formula 17]

In one embodiment of the present invention, the compound in Formula ₁ is -OH, R ₂ is -OH, R ₃ and R ₄ may be -H, orobol.

In one embodiment of the present invention, _{the compound in Formula 1 is -H, R 2 is} -OCH ₃ , and R ₃ is and R ₄ may be -H, Gancaonin A.

In one embodiment of the present invention, the compound in Formula ₁ is -H, R ₂ is -OH, R ₃ and R ₄ is It may be phosphorus, 6,8-Diprenylgenistein.

In one embodiment of the present invention, the compound in Formula ₁ is -OH, R ₂ is -OH, R ₃ and R ₄ is It may be phosphorus, 6,8-Diprenylorobol.

In one embodiment of the present invention, _{the compound in Formula 1 is -H, R 2 is} -OCH ₃ , and R ₃ is and R ₄ is -H, 5,7-Dihydroxy-6-(2'p-hydroxy-3p-methylbut-3p-enyl)-4 -It may be methoxylisoflavone.

In one embodiment of the present invention, the compound in Formula ₁ is -H, R ₂ is -OH, and R ₃ is , and R ₄ is It may be Isoerysenegalensein E.

In one embodiment of the present invention, the compound in Formula ₁ is -H, R ₂ is -OH, and R ₃ is , and R ₄ may be -H, Wighteone.

In one embodiment of the present invention, the compound in Formula ₁ is -H, R ₂ is -OH, and R ₃ is , and R ₄ is It may be Erysenegalensein E.

In one embodiment of the present invention, the compound in Formula ₁ is -OH, R ₂ is -OH, and R ₃ is , and R ₄ is It may be phosphorus, Millewanin H.

In one embodiment of the present invention, the compound may be 4'-O-Methylalpinumisoflavone, where in Formula 2, R ₁ is -H, R ₂ is -OCH ₃ , and R ₃ is -H.

In one embodiment of the present invention, the compound in Formula 2 is 5,3',4'-Trihydroxy-6'', where R ₁ is -OH, R ₂ is -OH, and R ₃ is -H, It may be 6''-dimethylpyrano-[2'',3'';7,6]isoflavone.

In one embodiment of the present invention, the compound is 3'-Hydroxy-4'-O-methylalpinumisoflavone, where in Formula 2, R ₁ is -OH, R ₂ is -OCH ₃ , and R ₃ is -H. You can.

In one embodiment of the present invention, _{the compound in Formula 2 is -H, R 2 is} -OH, and R ₃ is It may be Euchrenone b8.

In one embodiment of the present invention, the compound may be Derrone, where in Formula 3, R ₁ is -H, R ₂ is -OH, and R ₃ is -H.

In one embodiment of the present invention, _{the compound in Formula 3 is -OH, R 2 is} -OH, and R ₃ is Phosphorus, 5, 3',4', 2'''-Tetrahydroxy-2'', 2''-dimethylpyrano-(5''6'':7,8)-6-(3'''-methyl- It may be 3'''-butenyl)isoflavone.

In one embodiment of the present invention, the compound is 4'-O-Methylerythrinin, where in Formula 4, R ₁ is -H, R ₂ is -OCH ₃ , R ₃ is -H, and R ₄ is -H. It may be C.

In one embodiment of the present invention, the compound is (±)-1" in Formula 4, where R ₁ is -H, R ₂ is -OH, R ₃ is -OCH ₃ , and R ₄ is -H. -O-Methylerythrinin F may be.

In one embodiment of the present invention, _{the compound in Formula 4 is -H, R 2 is} -OH, R ₃ is -H, and R ₄ is It may be Euchrenone b10.

In one embodiment of the present invention, _{the compound in Formula 4 is -OH, R 2 is} -OH, R ₃ is -H, and R ₄ is It may be Furowanin A.

In one embodiment of the present invention, the compound of Formula 5 may be 4'-O-Methyl-2''-hydroxydihydroalpinumisoflavone.

In one embodiment of the present invention, the compound may be Senegalensin, wherein in Formula 6, R is -H.

In one embodiment of the present invention, the compound may be Furowanin B, where in Formula 6, R is -OH.

In one embodiment of the present invention, compound 7 may be Macluraxanthone B.

In one embodiment of the present invention, Compound 8 may be Cudratricusxanthone A.

In one embodiment of the present invention, compound 9 may be Gerontoxanthone I.

In one embodiment of the present invention, compound 10 may be Macluraxanthone C.

In one embodiment of the present invention, compound 11 may be Cudracuspixanthone E.

In one embodiment of the present invention, compound 12 may be Alvaxanthone.

In one embodiment of the present invention, compound 13 may be Cudraxanthone L.

In one embodiment of the present invention, compound 14 may be Cudraxanthone M.

In one embodiment of the present invention, compound 15 may be Aromadendrin.

In one embodiment of the present invention, compound 16 may be 8-prenylnaringenin.

In one embodiment of the present invention, compound 17 may be Cudraflavanone A.

Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating bacterial diseases in fish, comprising an extract or fraction of Kujippong as an active ingredient.

In the present invention, the pharmaceutical composition may additionally include a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers in the present invention are those commonly used in preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline. It may include, but is not limited to, cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. .

In the present invention, the pharmaceutical composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. in addition to the above components, but is not limited thereto.

Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

In the present invention, the pharmaceutical composition may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, but is not limited thereto.

In the present invention, the pharmaceutical composition may additionally include a dispersing agent or stabilizing agent, but is not limited thereto.

In the present invention, the pharmaceutical composition can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., for example, orally. It may be administered by administration.

The appropriate dosage of the pharmaceutical composition in the present invention varies depending on factors such as formulation method, administration method, animal age, body weight, sex, pathological condition, administration time, administration route, excretion rate, and reaction sensitivity, and is usually A skilled doctor can easily determine and prescribe an effective dosage for desired treatment or prevention. For example, the composition of the present invention may be administered intraperitoneally or orally 1 to 5 times every 1 to 10 days at an active ingredient content of 1 to 300 mg per 1 kg of living body, but is not limited thereto.

Another aspect of the present invention relates to a composition for fish feed or a composition for adding fish feed having fish bacterial activity containing an extract or fraction of Kujippong as an active ingredient.

In the present invention, fish feed or a composition for adding fish feed can be prepared with various commonly used ingredients. For example, marine proteins (e.g. fish meal or krill meal), vegetable proteins (e.g. soy meal, wheat gluten, corn gluten, lupine meal, pea meal or sunflower seed meal), blood meal and bone meal. It may include one or more protein sources selected from the group, but is not limited thereto.

In the present invention, fish feed or a composition for adding fish feed is one or more energy sources selected from the group comprising fish oil (e.g., squid liver oil) or vegetable oil (e.g., rapeseed oil, soybean oil, soybean oil). , may include vitamin mixtures and mineral mixtures.

Since the pharmaceutical composition, the composition for feed, and the composition for adding fish feed of the present invention include the antibacterial composition, the description of common content between the two is omitted in order to avoid excessive complexity of the present specification.

Another aspect of the present invention relates to a method for producing a composition containing an extract or fraction of Kujippong as an active ingredient.

The manufacturing method according to the present invention is described in more detail as follows:

Kkujippong preparation stage to prepare kkujippong; and

Solvent extraction step of extracting kkujippong by adding an extraction solvent.

In the present invention, the roots, stems, leaves, and fruits of Kujippong can be used. For example, they may be fruits or leaves, but are not limited thereto.

In the present invention, the kkujippong fruit may be an immature fruit or a mature fruit, for example, it may be a mature fruit.

In the present invention, the preparation step may further include one or more steps selected from the group consisting of cutting the kkujippong, washing, and drying.

In the present invention, the extraction solvent is 10 to 50 times the volume, 15 to 50 times the volume, 20 to 50 times the volume, 25 to 50 times the volume, 10 to 45 times the volume, 15 to 45 times the volume, 20 to 45 times the weight of kkujippong. times the volume, 25 to 45 times the volume, 10 to 40 times the volume, 15 to 40 times the volume, 20 to 40 times the volume, 25 to 40 times the volume, 10 to 35 times the volume, 15 to 35 times the volume, 20 to 35 times the volume , it may be extracted with 25 to 35 times the volume, for example, 30 times the volume of the extraction solvent.

When the kkujippong extract of the present invention is obtained by treating kkujippong with an extraction solvent, various extraction solvents can be used.

In the present invention, the extraction solvent may be a polar solvent or a non-polar solvent.

In the present invention, the polar solvent may be water, alcohol, acetic acid, DMFO (dimethyl-formamide), and DMSO (dimethyl sulfoxide), but is not limited thereto.

In the present invention, nonpolar solvents include methylene chloride, acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, and diisobutyl. lene, 1-pentene, 1-chlorobutane, 1-chloropentane, o -xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide , chloroform, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF, but is not limited thereto.

According to one embodiment of the present invention, the extraction solvent and/or fractionation solvent used in the present invention is anhydrous or hydrous lower alcohol having 1 to 5 carbon atoms (e.g., methanol, ethanol, propanol, butanol, etc.), water, It may be one or more selected from the group consisting of a mixed solvent of lower alcohol and water, methylene chloride, ethyl acetate, chloroform, butyl acetate, 1,3-butylene glycol, hexane, and diethyl ether, but is not limited thereto. .

In the present invention, the alcohol may be a straight-chain or branched alcohol having 1 to 5 carbon atoms, for example, methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, It may be one or more selected from the group consisting of 2-butoxyethanol and ethylene glycol, for example, ethanol, but is not limited thereto.

In the present invention, when a mixture of water and alcohol is used as the extraction solvent, the concentration is 10% or more to less than 100% (v/v), 20% or more to less than 100% (v/v), or 30% or more to 100% ( less than v/v), more than 40% to less than 100% (v/v), more than 50% to less than 100% (v/v), 10 to 90% (v/v), 20 to 90% (v/v) ), 30 to 90% (v/v), 40 to 90% (v/v), 50 to 90% (v/v), 10 to 80% (v/v), 20 to 80% (v/v) ), 30 to 80% (v/v), 40 to 80% (v/v), 50 to 80% (v/v) of an alcohol aqueous solution, for example, 50% (v/v) It may be an alcohol aqueous solution (for example, an ethanol aqueous solution), but is not limited thereto.

The solvent extraction used in the present invention may be any commonly used method, for example, cold immersion, hot water extraction, ultrasonic extraction, or reflux cooling extraction, but is not limited thereto.

In the present invention, the temperature at which the solvent extraction step is performed is 20 to 100°C, 20 to 95°C, 20 to 90°C, 20 to 85°C, 20 to 80°C, 25 to 100°C, 25 to 95°C, 25 to 90°C. , 25 to 85°C, 25 to 80°C, 30 to 100°C, 30 to 95°C, 30 to 90°C, 30 to 85°C, 30 to 80°C, 35 to 100°C, 35 to 95°C, 35 to 90°C , 35 to 85°C, 35 to 80°C, 40 to 100°C, 40 to 95°C, 40 to 90°C, 40 to 85°C, 40 to 80°C, 45 to 100°C, 45 to 95°C, 45 to 90°C , 45 to 85°C, 45 to 80°C, 50 to 100°C, 50 to 95°C, 50 to 90°C, 50 to 85°C, 50 to 80°C, 55 to 100°C, 55 to 95°C, 55 to 90°C , 55 to 85°C, 55 to 80°C, 60 to 100°C, 60 to 95°C, 60 to 90°C, 60 to 85°C, 60 to 80°C, 65 to 100°C, 65 to 95°C, 65 to 90°C , 65 to 85°C, 65 to 80°C, 70 to 100°C, 70 to 95°C, 70 to 90°C, 70 to 85°C, 70 to 80°C, 75 to 100°C, 75 to 95°C, 75 to 90°C , 75 to 85°C, 75 to 80°C, for example, 80°C.

In the present invention, the performance time of the solvent extraction step is 5 to 10 hours, 6 to 10 hours, 7 to 10 hours, 5 to 9 hours, 6 to 8 hours, 7 to 10 hours, 5 to 9 hours, and 6 to 8 hours. , may be 7 to 8 hours, for example, 7.5 hours.

The extraction process can be repeated once or several times, and in a preferred example of the present invention, a method of re-extraction after the first extraction can be adopted, which means that even if the herbal medicine extract is effectively filtered when mass-producing the herbal medicine extract, the herbal medicine itself is damaged. This is to prevent loss as the moisture content is high, which reduces extraction efficiency through primary extraction alone. In addition, as a result of verifying the extraction efficiency at each stage, it was found that about 80 to 90% of the total extraction amount was extracted through secondary extraction.

In one example of the present invention, when the extraction step is repeated twice, 10 to 50 times the extraction solvent by volume, 15 to 50 times the volume, 20 to 50 times the volume, 25 to 50 times the volume, and 10 to 45 times the extraction solvent are added to the obtained residue. times the volume, 15 to 45 times the volume, 20 to 45 times the volume, 25 to 45 times the volume, 10 to 40 times the volume, 15 to 40 times the volume, 20 to 40 times the volume, 25 to 40 times the volume, 10 to 35 times the volume , reflux extraction may be performed at 15 to 35 times the volume, 20 to 35 times the volume, 25 to 35 times the volume, for example, 30 times the volume.

In one example of the present invention, a Cudrania extract is prepared by filtering after extraction, combining with the previously obtained filtrate, and concentrating under reduced pressure. In this way, extraction efficiency can be increased by performing two extractions and mixing the filtrate obtained after each extraction, but the extract of the present invention is not limited to extraction recovery.

The filtered extract obtained in this way is about 10 to 30 times, preferably 15 to 25 times, and more preferably about 20 times the total amount of the concentrate in order to adjust the content of remaining lower alcohol to be suitable for use as a raw material. It can be prepared as a powdery extract of Kujippong by azeotropically concentrating it with water 1 to 5 times, preferably 2 to 3 times, suspending it homogeneously by adding the same amount of water again, and then freeze-drying it.

The present invention relates to an antibacterial composition against bacterial diseases in fish containing an extract or fraction of Kujippong as an active ingredient and an antibacterial composition against bacterial diseases in fish containing a compound derived from Kujippong as an active ingredient, which causes high mortality in fish. It has excellent antibacterial activity against pathogenic bacteria, and especially has excellent antibacterial activity against antibiotic-resistant fish disease bacteria.

Figure 1 is a diagram showing extraction and solvent fractionation of Cudrania mulberry leaves according to an embodiment of the present invention.
Figure 2 is a diagram showing the extraction and solvent fractionation of immature fruits of Cudrania mulberry according to an embodiment of the present invention.
Figure 3 is a diagram showing the maturation of Cudrania mulberry and the extraction and solvent fraction of fruit according to an embodiment of the present invention.
Figure 4 is a diagram showing the correlation of antibacterial activity by structure according to an embodiment of the present invention.
Figure 5 is a diagram showing the correlation of antibacterial activity by structure according to an embodiment of the present invention.
Figure 6 is a diagram showing the correlation of antibacterial activity by structure according to an embodiment of the present invention.
Figure 7 is a diagram showing the structure of a compound isolated from the roots of Cudrania chinensis according to an embodiment of the present invention.
Figure 8 is a schematic diagram of the antibacterial activity according to the structure of a compound isolated from Cudrania chinensis according to an embodiment of the present invention.
9A to 9D are diagrams showing the main effect diagram and curvature effect according to an embodiment of the present invention.
Figure 10 is a residual plot (antibacterial activity) of a reduced model according to an embodiment of the present invention.
Figure 11 is a residual plot (cytotoxicity) of a reduced model according to an embodiment of the present invention.
Figure 12 is a diagram showing a response surface diagram of antibacterial activity against independent variables and optimal extraction conditions derived from multiple response surface analysis of antibacterial activity and cytotoxicity according to an embodiment of the present invention.
Figure 13 is a graph showing the results of observing the cumulative mortality rate after artificially infecting flounder administered with an optimized extract according to an embodiment of the present invention with fish disease bacteria.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Preparation Example 1. Preparation of extracts from each part of Cudrania mulberry tree

Grind the roots (CTR), leaves (CTL), stems (CTS), mature fruits (CTF), and immature fruits (CTU) of the dried Cudrania tricuspidata Bureau, and take 1 g of each at room temperature. Extracted with 10 mL of methanol. Next, it was concentrated using a rotary vacuum concentrator to obtain extracts for each part.

Preparation Example 2. Preparation of fractions for each part of Cudrania mulberry tree

2-1. Cudrania leaves

2-1-1. Extraction and solvent fractionation

0.8 kg of dried Cudrania mulberry leaves were extracted twice with 100% methanol at room temperature. Next, the extract was filtered and the filtrate was concentrated using a rotary vacuum concentrator to obtain 102.4 g of 100% methanol extract. This was suspended in distilled water and then fractionated in the order of n-hexane, CH ₂ Cl ₂ , ethyl acetate, and n-butanol, 12.2 g of n-hexane fraction, 15.2 g of CH ₂ Cl ₂ fraction, 4.7 g of ethyl acetate fraction, and n-butanol. 17.7 g of fraction was obtained. (Figure 1)

2-1-2. ingredient separation

The CH ₂ Cl ₂ fraction layer was divided into a total of 10 fractions (CTL-M-1 to CTL-M-10) by applying silica gel CC (CH ₂ Cl ₂ :MeOH=100:1~1:1, step gradient). did. The CTL-M-4 fraction was treated with Sephadex LH-20 (CH ₂ Cl ₂ :MeOH = 1:1) to produce a total of 5 subfractions (CTL-M-4-1 to CTL-M-4-5). got it Among these, the CTL-M-4-2 fraction was treated with Sephadex LH-20 ( n -hexane:CH ₂ Cl ₂ :MeOH = 10:10:1) to produce a total of 8 subfractions (CTL-M-4-2). -1 to CTL-M-4-2-8) were obtained. Among these, the CTL-M-4-2-3 fraction was used to obtain compound CTL-37 using semi-preparative HPLC (MeCN:H ₂ O=45:55), and CTL-M-4-2 -6 fractions were subjected to semi-preparative HPLC (MeCN:H ₂ O=60:40) to obtain compound CTL-45.

The CTL-M-4-4 fraction was treated with Sephadex LH-20 (n-hexane:CH ₂ Cl ₂ :MeOH = 10:10:1) to produce a total of 12 subfractions (CTL-M-4-4-1). to CTL-M-4-4-12) was obtained. Among these, the CTL-M-4-4-2 fraction was used as a semi-preparative HPLC (MeCN:H 2 O = 60:40) to obtain compound CTL-19, and the CTL-M-4-4-4 fraction was obtained as a semi-preparative HPLC (MeCN:H ₂ O = 60:40). -Compounds CTL-53 and 54 were obtained using preparative HPLC (MeCN: H2O=60:40).

The CTL-M-4-4-9 fraction was semi-prepared to obtain compound CTL-40 using HPLC (MeCN:H ₂ O = 60:40), and the CTL-M-4-4-10 fraction was semi-prepared. Compounds CTL-28, 55, and 56 were obtained using HPLC (MeCN:H ₂ O = 55:45), and the CTL-M-4-4-11 fraction was subjected to semi-preparative HPLC (MeCN:H ₂ O = 55:45). Compound CTL-32 was obtained using 45).

2-2. Cudrania unripe fruit

2-2-1. Extraction and solvent fractionation

2.8 kg of fresh unripe Cudrania tree fruits were extracted twice with 75% ethanol at room temperature. Next, after filtering the extract, the filtrate was concentrated using a rotary vacuum concentrator to obtain 508.2 g of 75% ethanol extract, which was suspended in distilled water and fractionated in the following order: n -hexane, CH ₂ Cl ₂ , ethyl acetate, and n- butanol. Thus, n -hexane (30.1 g), CH ₂ Cl ₂ (44.6 g), ethyl acetate (7.5 g), and n- butanol (35.8 g) were obtained. (Figure 2)

2-2-2. ingredient separation

The CH ₂ Cl ₂ fraction layer was divided into a total of 11 fractions (CTU-M-1 to CTU-M-11) by applying silica gel CC (n-hexane:EtOAc = 50:1 to 0:100, step gradient). . The CTU-M-1 fraction was defated (CH ₂ Cl ₂ :MeOH=1:1 add 0.1% H2O) to obtain a total of 2 small fractions (CTU-M-1MC, CTU-M-1M). Among these, the CTU-M-1M fraction was subjected to semi-preparative HPLC (MeCN:H ₂ O=90:10) to obtain compound CTU-33. The CTU-M-6 fraction was subjected to MPLC (CH ₂ Cl ₂ :MeOH=50:1 to 0:100, step gradient) to obtain a total of 11 subfractions (CTU-M-6-1 to CTU-M-6- 11) was obtained. Among these, the CTU-M-6-4 fraction was subjected to semi-preparative HPLC (MeCN:H2O=57:43) to obtain compound CTU-37.

The CTU-M-7 fraction was subjected to recrystallization and Sephadex LH-20 (MeOH=100) to obtain four small fractions (CTU-M-7-1 to CTU-M-7-4). Among these, the CTU-M-7-3 fraction was subjected to semi-preparative HPLC (MeCN:H2O=55:45) to obtain the CTU-M-7-3-1 subfraction and compounds CTU-18 and 43. The CTU-M-8 fraction was treated with Sephadex LH-20 (MeOH=100) to obtain a total of 4 small fractions (CTU-M-8-1 to CTU-M-8-4). Among these, the CTU-M-8-3 fraction was subjected to semi-preparative HPLC (MeCN:H2O=55:45) to obtain compound CTU-34.

The CTU-M-9 fraction was treated with Sephadex LH-20 (MeOH=100) to obtain a total of 4 small fractions (CTU-M-9-1 to CTU-M-9-4). Among these, the CTU-M-9-2 fraction was treated with Sephadex LH-20 (CH ₂ Cl ₂ :MeOH=1:1) to produce a total of 2 small fractions (CTU-M-9-2-1 to CTU-M). -9-2-2) was obtained. Among these, the CTU-M-9-2-2 fraction was subjected to semi-preparative HPLC (MeCN:H2O=57:43) to obtain compound CTU-21. The CTU-M-9-4 fraction was subjected to semi-preparative HPLC (MeCN:H2O=45:55) to obtain compounds CTU-36 and 46.

2-3. Cudrania tree maturity and fruit

2-3-1. Extraction and solvent fractionation

1.2 kg of fresh mature and fruit of Cudrania mulberry were extracted twice with 100% MeOH at room temperature. After filtering the extract, the filtrate was concentrated using a rotary vacuum concentrator to obtain 486.5 g of 100% MeOH extract, which was suspended in distilled water and fractionated in the order of n-hexane, CH ₂ Cl ₂ , ethyl acetate, and n-butanol to obtain n- Hexane (8.8 g), CH ₂ Cl ₂ (14.4 g), ethyl acetate (4.3 g), and n-butanol (19.5 g) were obtained. (Figure 3)

2-3-2. ingredient separation

The CH ₂ Cl ₂ fraction layer was subjected to silica gel CC (n-hexane:EtOAc=20:1 to 0:100 to EtOAc:MeOH=100:0 to 0:100, step gradient) to obtain a total of 11 fractions (CTF-M). -1 to CTF-M-11). CTF-M-7 fraction and CTF-M-8 fraction were recrystallized to obtain compounds CTF-11 and 10, respectively. The CTF-M-10 fraction was treated with Sephadex LH-20 (CH ₂ Cl ₂ :MeOH=1:1) to produce a total of 5 subfractions (CTF-M-10-1 to CTF-M-10-5). got it Among these, the CTF-M-10-4 fraction was subjected to semi-preparative HPLC (MeCN:H2O=80:20) to obtain compound CTF-12.

The ethyl acetate fraction layer was divided into a total of 5 fractions (CTF-E-1 to CTF-E-5) by performing RP-MPLC (MeOH:H2O=50:50 to 100:0, step gradient). The CTF-E-1 fraction was treated with Sephadex LH-20 (MeOH=100) to obtain a total of 5 subfractions (CTF-E-1-1 to CTF-E-1-5). Among these, CTF-E-1-5 fraction was recrystallized to obtain compound CTF-2.

2-4. Cudrania root

2-4-1. Extraction and solvent fractionation

7.5 kg of dried Cudrania root was extracted twice with 80% methanol at room temperature. After filtering the extract, the filtrate was concentrated using a rotary vacuum concentrator to obtain 461.4 g of 80% methanol extract, which was suspended in distilled water and fractionated in the following order: n-hexane, CH ₂ Cl ₂ , ethyl acetate, and n-butanol. Hexane (1.2 g), CH ₂ Cl ₂ (66.9 g), ethyl acetate (69.4 g), and n-butanol (130.1 g) were obtained.

2-4-2. ingredient separation

The CH ₂ Cl ₂ fraction layer was divided into a total of 17 fractions (C-1 to C-17) by applying silica gel CC (n-hexane > CH ₂ Cl ₂ > MeOH gradient). Among these, the C-15 fraction was subjected to MPLC (n-hexane:CH ₂ Cl ₂ , gradient) to obtain a total of 5 subfractions (C15A to C15E), and the C15B fraction was subjected to RP-MPLC (MeOH:H ₂ O = A total of 14 small fractions (C15B1 to C15B14) were obtained by performing a 45:55, MeOH gradient.

Among the small fractions, C15B9 was treated with Sephadex LH-20 (MeOH=100) to obtain a total of 5 small fractions (C15B9A to C15B9E). Among these, C15B9D was subjected to semi-preparative HPLC (MeCN: 0.1% aqueous acetic acid solution = 65:35) to obtain compounds CTR-X13 and F36.

Among the small fractions, the C15B10 fraction was subjected to Sephadex LH-20 (MeOH=100) to obtain a total of 5 small fractions (C15B10A to C15B10E), and the C15B10E fraction was subjected to semi-preparative HPLC (MeCN:H ₂ O with 0.1% acetic). acid=60:40) to obtain compounds CTR-X14, 15, 17, and 19.

Among the small fractions, the C15B12 fraction was subjected to Sephadex LH-20 (MeOH=100) to obtain a total of 4 small fractions (C15B12A to C15B12D), and the C15B12B fraction was subjected to MPLC (CH ₂ Cl ₂ :MeOH) to obtain a total of 3 small fractions. Small fractions (C15B12B1 to C15B12B3) were obtained. Among these, the C15B12B2 fraction was subjected to semi-preparative HPLC (MeCN:H2O with 0.1% acetic acid=60:40) to obtain compound CTR-X16.

The C-16 fraction was subjected to MPLC (CH ₂ Cl ₂ :MeOH=90:1, gradient) to obtain a total of 5 small fractions (C16A to C16E), of which the C16B fraction was subjected to RP-MPLC (MeOH:H2O= 1:1) to obtain a total of 18 small fractions (C16B1 to C16B18).

Among the small fractions, C16B12 was subjected to Sephadex LH-20 (MeOH=100) to obtain a total of 9 small fractions (C16B12A to C16B12I), and the C16B12F fraction was subjected to semi-preparative HPLC (MeCN:H ₂ O=45:55). Compound CTR-F38 was obtained using .

Among the small fractions, C16B17 was treated with Sephadex LH-20 (MeOH=100) to obtain a total of 5 small fractions (C16B12A to C16B12E), and the C16B12E fraction was treated with Sephadex LH-20 (CH ₂ Cl ₂ :MeOH = 1: 1) was performed and divided into a total of two small fractions (C16B12E1 to C16B12E2). The C16B12E2 fraction was labeled with compound CTR-X11 using semi-preparetive HPLC (MeCN:H ₂ O = 80:20). Got 23.

The ethyl acetate fraction layer was subjected to silica gel CC (CH ₂ Cl ₂ :MeOH=90:1, gradient) to obtain a total of 7 fractions (E1 to E7), of which the E4 fraction was subjected to RP-MPLC (MeOH:H ₂ O = 20:80) was performed to obtain 7 subfractions (E4A to E4G). Compound CTR-F40 was obtained through recrystallization of E4C in the small fraction.

Experimental Example 1. Measurement of antibacterial activity

The antibacterial activity of extracts from each part of Cudrania tree and the isolated compounds was tested using microdilution assay. Streptococcus iniae KCTC3657, S. iniae DSJ19, S. iniae BS9, S. parauberis KCTC3651, Lactococcus garvieae KCTC3772, Edwardsiella tarda KCTC12267, Vibrio anguillarum KCTC2711, Aeromonas salmonicida KCCM40239 in BHIB (brain heart infusion broth) 10 After inoculation in ml, 25℃ After culturing according to 0.5 Mcfarland standard for 24 hours, the final bacterial count was adjusted to ¹ After the extract and compound were well dissolved in DMSO, BHIB was added to adjust the final DMSO concentration to 10%, and the mixture was diluted two-fold. Afterwards, equal amounts of sample dilution solution and bacterial solution were added to a 96-well plate to bring the final volume to 200 ul, and cultured in an incubator at 25°C for 24 hours. To check the minimum bactericidal concentration (MBC), the plate cultured for 24 hours was visually observed, 20 ul was taken from the section corresponding to the upper concentration from the part where bacteria did not grow, and 100 ul BHIB was dispensed. It was inoculated into a 96-well plate and cultured in an incubator at 25°C for 24 hours. The final minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by injecting 30 ul of p-INT at a concentration of 0.2 mg/ml per well and reacting in dark conditions for 2 hours, then removing the unstained section. Determined by MIC and MBC. Oxytetracycline and amoxicillin were used as positive controls.

The MIC of extracts from each part of Cudrania tree for 3 Gram-positive and 3 Gram-negative strains was determined, and the results are shown in Table 1. The MIC was determined for 5 Gram-positive strains (standard strains and clinical isolates) and 1 Gram-negative strain. The MIC and MBC for the compounds isolated from each extract were determined, and the results are shown in Table 2.

Sample S. iniae KCTC3657 S. parauberis
KCTC3651 L. garvieae KCTC3772 E. tarda
KCTC12267 V. anguillarum KCTC2711 A. salmonicida KCCM40239 ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml CTR 31.25 125 62.5 1000 >250 500 CTL 250 1000 1000 2000 N.T. 1000 CTS 1000 >2000 >500 >2000 N.T. 1000 CTF 62.5 250 125 >2000 >2000 2000 CTU >2000 >2000 >1000 >2000 N.T. 2000 OTC 0.25 0.5 One 0.5 <0.125 0.125 DMSO vehicle 10% >10% 5% 10% 10% 10% Cell No. (CFU/ml) 11.6*10 ⁵ 11.9*10 ⁵ 5.2*10 ⁵ 6.5*10 ⁵ 6.3*10 ⁵ 7.7*10 ⁵

OTC, oxytetracycline; DMSO, dimethyl sulfoxide; NT, not tested

As can be seen in Table 1, the extracts of Cudrania root, leaves, stems, mature fruits, and immature fruits showed antibacterial activity against major Gram-positive and Gram-negative fish disease bacteria, especially against Gram-positive bacteria compared to Gram-negative bacteria. It showed relatively excellent antibacterial activity. In particular, it was confirmed that the antibacterial activity of roots and mature fruits was remarkable.

Sample S. iniae KCTC3657 S. iniae DSJ19 S. iniae BS9 L. garvieae KCTC3772 S. parauberis KCTC3651 V. anguillarum KCTC2711 ㎍/ml ㎍/ml ㎍/ml ㎍/ml ㎍/ml ㎍/ml Genistein >500 >500 N.T. N.T. N.T. N.T. CTF-2 500 N.T. N.T. >250 N.T. N.T. CTF-10 ^62.5a ( ^500b ) N.T. N.T. >250 N.T. N.T. CTF-11 3.91(7.81) 1.95 1.95 3.91 3.91(15.63) >250 CTF-12 7.81(31.25) 7.81 3.91 31.25 31.25(250) >250 CTU-18 31.25(125) N.T. N.T. 250 N.T. N.T. CTU-21 1.95(3.91) 1.95 1.95 N.T. 1.95(15.63) >250 CTL-19 7.81(15.63) 3.91 3.91 7.81 15.63(62.5) >250 CTL-28 7.81(15.63) 3.91 3.91 7.81 15.63(125) >250 CTL-32 15.63(62.5) N.T. N.T. 15.63 N.T. N.T. CTU-33 250(>500) N.T. N.T. >250 N.T. N.T. CTU-36 31.25(250) N.T. N.T. >250 N.T. N.T. CTU-37 250(500) N.T. N.T. 250 N.T. N.T. CTU-43 62.5(250) N.T. N.T. 250 N.T. N.T. CTU-34 >500 N.T. N.T. >250 N.T. N.T. CTU-46 62.5(250) N.T. N.T. >250 N.T. N.T. CTL-37 125(500) N.T. N.T. 250 N.T. N.T. CTL-40 250(>500) N.T. N.T. 250 N.T. N.T. CTL-53 7.81(15.63) 7.81 7.81 15.6 125(>250) >250 CTL-55 62.5(250) N.T. N.T. 125 N.T. N.T. CTL-45 125(500) N.T. N.T. >250 N.T. N.T. CTL-54 7.81(31.25) 3.91 3.91 31.25 62.5(250) >250 CTL-56 31.25(125) N.T. N.T. 62.5 N.T. N.T. CTR-X11 125(>500) N.T. N.T. >125 250(>250) >250 CTR-X13 1.95(7.81) N.T. N.T. 15.6 7.81(62.5) >250 CTR-X14 7.81(31.25) N.T. N.T. >125 31.25(250) >250 CTR-X15 7.81(15.63) N.T. N.T. >125 31.25(250) >250 CTR-X16 7.81(15.63) N.T. N.T. >125 15.63(250) >250 CTR-X17 7.81(31.25) N.T. N.T. >125 31.25(250) >250 CTR-X19 7.81(31.25) N.T. N.T. >125 62.5(250) >250 CTR-X23 3.91(15.625) N.T. N.T. >125 31.25(250) >250 CTR-F36 500(>500) N.T. N.T. >125 >250 250 CTR-F38 15.63(31.25) N.T. N.T. 62.5 31.25(250) >250 CTR-F40 1.95(7.81) N.T. N.T. 31.25 3.91(62.5) >250 OTC 0.25(1) 0.5 0.25 One 0.5(1) 0.25 AMX 0.0078 0.0156 0.0156 One N.T. N.T. DMSO vehicle 10% 10% 10% 5% >10% 10% Cell No. (CFU/ml) 7.2*10 ⁵ 18.4*10 ⁵ 18.8*10 ⁵ 5.5*10 ⁵ 1.16*10 ⁵ 2.08*10 ⁵

a, MIC; b, MBC; NT, not tested; OTC, oxytetracycline; AMX, amoxicillin sodium, DMSO, dimethyl sulfoxide

As can be seen in Table 2, the antibacterial activity of a total of 34 compounds was confirmed, and the compounds isolated from each region showed excellent antibacterial activity against Gram-positive bacteria. Similar to the results of the MIC of the extract, it can be seen that the antibacterial activity of the compounds isolated from the roots and mature fruits is excellent, and the MIC range of the compounds with excellent activity among the compounds isolated from the roots and mature fruits is 1.95-7.81㎍/ml. It was confirmed that it showed activity similar to the MIC of OTC, 0.25-0.5㎍/ml.

Looking at this by structure, as can be seen in Figure 4, the MIC of genistein, which is the basic structure, was >500 ㎍/ml, while when it is prenylated at the R3 and 4 positions in the basic structure, such as F11 F12 U21 L19, 28, and 32, it is 1.95 to 7.81 ㎍. It was confirmed that the MIC value was lowered with /ml. In comparison with F11, 12 and L28, 32, the MIC value doubled when OH was substituted for H at the R1 position, and it was confirmed that the activity increases as the polarity of the structure decreases.

A structure-activity correlation existed even in the cyclized structure, and as can be seen in Figure 5, when OH at the 7th position and phenyl at the 6th position are cyclized, the antibacterial activity is 4 to 8 times lower (CTU-33 (250㎍/ml) and CTF-10 (62.5㎍/ml)/ CTU-43 (62.5㎍/ml) and CTL-28 (7.81㎍/ml)) Similarly, the phenylated structure (U43, U46) Activity increased.

Likewise, as can be seen in Figure 6, when cyclized, the antibacterial activity is lowered by 2-4 times (CTL-53 (7.81㎍/ml) and CTU-21 (1.95㎍/ml)/CTL-45 (125㎍/ml) (Comparison of CTU-18 (31.25㎍/ml)/CTL-56 (31.25㎍/ml) and CTL-32 (15.63㎍/ml)) Likewise, the activity of prenylated structures appears to increase (L53, 55, 54, 56) ), the activity decreased as H was replaced with OH, and the activity increased as the polarity of the structure decreased.

Experimental Example 2. Measurement of antibacterial activity against antibiotic-resistant fish disease bacteria

The antibacterial activity of extracts from each part of Cudrania tree and isolated compounds that showed activity in the previous antibacterial activity test was examined for their antibacterial activity against Streptococcus parauberis KSP44, an amoxicillin-resistant fish pathogenic bacterium.

Streptococcus parauberis KSP44 (J14) MIC (ug/ml) MBC (ug/ml) CTF 62.5 125 CTR 62.5 250 CTL 500 1000 Genistein 250 >250 CTF-11 1.95 1.95 CTF-12 31.25 125 CTU-21 1.95 1.95 CTL-19 15.625 31.25 CTL-28 7.81 125 CTL-53 7.81 125 CTL-54 15.625 62.5 Amoxicillin 0.5 4 Cell No. (CFU/ml) ^5.6*105

MIC, minimum inhibitory concentration MBC, minimum bactericidal concentration

As can be seen in Table 6, CTF and CTR showed MICs of 62.5 ug/ml, and the MIC of the compound (CTF-11) isolated from CTF was confirmed to be 1.95 ug/ml. In particular, looking at the MBC results, it can be seen that CTF-11 and CTU-21 act as disinfectants, and the MBC value is about two times lower than that of amoxicillin (4ug/ml), showing excellent antibacterial activity even in antibiotic-resistant bacteria. It was confirmed that it appears.

Experimental Example 3. Optimization of extraction of antibacterial activity for mature fruits of Cudrania chinensis

3-1. Main effects plot and curvature effect analysis

A 2 ³ full factorial design (center point: 3) was performed to identify the main effects of each factor and their interactions. At each experimental point, 2g of sample was extracted using a sample-to-volume ratio of 30. The extract was concentrated under reduced pressure, transferred to a 50ml vial, freeze-dried, and examined for antibacterial activity. Based on the antibacterial activity results and extraction yield, the levels of independent variables for experimental design were determined. The results are shown in Table 5 below.

No. Solvent composition (ethanol %) Extraction temperature (℃) Extraction time (h) One 0 90 10 2 50 65 7.5 3 0 90 5 4 0 40 5 5 0 40 10 6 50 65 7.5 7 100 90 5 8 100 40 5 9 100 90 10 10 50 65 7.5 11 100 40 10

Ethanol content (%) Extraction temperature ( ) Extraction time (h) antibacterial activity
MIC (ug/ml) Extraction yield (%) 0 90 10 >1000 54.1 50 65 7.5 62.5 57.2 0 90 5 >1000 54.8 0 40 5 >1000 50.8 0 40 10 >1000 51.4 50 65 7.5 62.5 57.3 100 90 5 125 56 100 40 5 62.5 43 100 90 10 125 57 50 65 7.5 62.5 56.4 100 40 10 62.5 47.4 OTC - - 0.5 - DMSO - - 10% - Cell No.
(CFU/ml) - - ^1.5*106 -

MIC, minimum inhibitory concentration; OTC, oxytetracycline; DMSO, dimethyl sulfoxide

As can be seen in Figure 9, in the main effect analysis on antibacterial activity, the ethanol content appears to have an effect on antibacterial activity, but it was confirmed that there was no statistical significance. However, in all factors, the antibacterial activity of the central point was found to be the highest, so it was determined that a curvature effect exists. In the main effects analysis on extraction yield, extraction temperature was confirmed to be a significant factor, and ethanol content was not a significant factor, but was confirmed to have an interaction with the temperature factor. Likewise, in the extraction yield, the yield was highest at the center point, so it was determined that a curvature effect exists.

Therefore, through the above experiment, three factors that determine the characteristics of the extract were identified, and the presence of a curved surface in the reaction was confirmed, and response surface analysis was performed. The dependent variables of the response surface analysis were determined to be antibacterial activity and cytotoxicity, and cytotoxicity was selected as the final dependent variable as it was judged to be a major item related to the toxicity of the extract due to long-term use in the in vivo antibacterial activity test. However, in the case of extraction yield, it was excluded from the dependent variable because there was no significant difference between each condition in the above experiment.

3-2. Optimization of antibacterial activity and cytotoxicity extraction from mature Cudrania chinensis using Box-Behnken Design (BBD)

BBD of response surface methodology (RSM) was used to analyze the optimal conditions for antibacterial activity and cytotoxicity of Cudrania mulberry ripening samples and their antibacterial activity and cytotoxicity against S. iniae . The independent variables of the extraction conditions are ethanol content (X ₁ ), extraction temperature (X ₂ ), and extraction time (X ₃ ) through main effects and curvature effect analysis. The experiment was designed at three levels. Antibacterial activity and cytotoxicity tests were performed by extracting according to 15 designed experimental conditions. To predict the optimal extraction conditions that maximized antibacterial activity and lowered cytotoxicity, the analysis was performed using the MINITAB® release 14 (Minitab INC., Pennsylvania, USA) program, and the results are shown in Table 6.

order of experiment independent variable dependent variable Ethanol content % (X ₁ ) Extraction temperature (X ₂ ) Extraction time
(X ₃ ) Antibacterial activity (MIC)
(ug/ml, Y ₁ ) Cytotoxicity (CC ₅₀ )
(ug/ml, Y ₂ ) actual value predicted value ^a actual value predicted value ^a One 80 (+1) 60 (0) 5 (-1) 62.5 86.0 114.5 120.5 2 80 (+1) 80 (+1) 7.5 (0) 62.5 31.3 140.2 123.8 3 20 (-1) 80 (+1) 7.5 (0) 2000 1984.4 200.0 203.7 4 50 (0) 80 (+1) 5 (-1) 62.5 70.3 129.6 140.0 5 20 (-1) 60 (0) 10 (+1) 2000 1976.6 200.0 194.0 6 50 (0) 40 (-1) 10 (+1) 125 117.2 151.6 141.2 7 80 (+1) 40 (-1) 7.5 (0) 125 140.7 135.0 131.3 8 50 (0) 60 (0) 7.5 (0) 62.5 62.5 133.9 141.2 9 20 (-1) 60 (0) 5 (-1) 2000 2007.8 200.0 185.9 10 50 (0) 80 (+1) 10 (+1) 62.5 101.6 130.2 132.5 11 50 (0) 60 (0) 7.5 (0) 62.5 62.5 146.0 141.2 12 50 (0) 40 (-1) 5 (-1) 250 211.0 154.0 151.7 13 80 (+1) 60 (0) 10 (+1) 62.5 54.7 80.4 94.5 14 20 (-1) 40 (-1) 7.5 (0) 2000 2031.3 200.0 216.4 15 50 (0) 60 (0) 7.5 (0) 62.5 62.5 143.7 141.2

MIC, minimum inhibitory concentration; CC ₅₀ , 50% cytotoxic concentration ^a , predicted value is derived from the quadratic regression equation for the dependent variable of the independent variable

3-2-1. Establishment of antibacterial activity model

The statistical significance of the model equation was evaluated by T -test, and as can be seen in Table 10, the terms with P < 0.05 are significant, so the terms X ₁ , X ₂ and (X ₁ ) ² were confirmed as significant factors in this model.

port Coefficient standard error of coefficient T P a constant 62.500 21.35 2.928 0.033 ^* linear term X ₁ -960.938 13.07 -73.507 0.000 ^* X ₂ -39.063 13.07 -2.988 0.031 ^* X ₃ -15.625 13.07 -1.195 0.286 square term (X ₁ ) ² 945.313 19.24 49.126 0.000 ^* (X ₂ ) ² 39.062 19.24 2.030 0.098 (X ₃ ) ² 23.437 19.24 1.218 0.278 interaction term X ₁ X ₂ -15.625 18.49 -0.845 0.437 X ₁ X ₃ -0.000 18.49 -0.000 1.000 X ₂ X ₃ 31.250 18.49 1.690 0.152

R ² =99.9% R ² (adj)=99.8%*: P < 0.05

To confirm the final fit model, the model was reduced by sequentially removing non-significant terms starting from the higher order terms. However, the main effects included in the significant higher-order terms were included in the final model to maintain the hierarchical model even if the P value was greater than 0.05.

Through the residual plot in Figure 10, it was confirmed that the normal distribution of the reduced model, the equality of variance in all fitted values, and the randomness over time were appropriate, and the analysis of variance showed that all terms of the reduced model were significant.

item DF M.S. F P regression equation 3 3568048 1881.83 0.000 linear term 2 3699707 1951.27 0.000 square term One 3304729 1742.95 0.000 residual error 11 1896 lack of suitability 5 2609 2.00 0.211 pure error 6 1302 total 14

R ² =99.8% R ² (adj)=99.8%DF: degrees of freedom; MS: sum of squares/DF

As can be seen in Table 8, the lack-of-fit value indicating the suitability of the model is P > 0.05, indicating that the reduced model is suitable, and R ² explains the variation in the response variable of the regression model. The (adj) value was confirmed to be 99.8%. The final reduced model (1) is as follows.

[Reduced final model (1)]

Y = 4430.43 - 136.57X ₁ - 1.95X ₂ + 1.05(X ₁ ) ²

3-2-2. Establishment of cytotoxicity model

port Coefficient standard error of coefficient T P a constant 141.197 9.456 14.932 0.000 ^* linear term X ₁ -41.236 5.791 -7.121 0.001 ^* X ₂ -5.075 5.791 -0.876 0.421 X ₃ -4.499 5.791 -0.777 0.472 square term (X ₁ ) ² 17.484 8.524 2.051 0.095 (X ₂ ) ² 10.117 8.524 1.187 0.289 (X ₃ ) ² -9.951 8.524 -1.167 0.296 interaction term X ₁ X ₂ 1.293 8.189 0.158 0.881 X ₁ X ₃ -8.525 8.189 -1.041 0.346 X ₂ X ₃ 0.752 8.189 0.092 0.930

R ² =92.4% R ² (adj)=78.6%*: P < 0.05

As can be seen in Table 9, the statistical significance of the model equation was evaluated by T -test, and since the term with P < 0.05 was significant, only X ₁ was confirmed as a significant factor in this model. To confirm the final fit model, the model was reduced by sequentially removing non-significant terms starting from the higher order terms. However, the main effects included in the significant higher-order terms were included in the final model to maintain the hierarchical model even if the P value was greater than 0.05.

item DF M.S. F P regression equation 2 7371.6 31.43 0.000 linear term One 13603.4 58.00 0.000 square term One 1139.7 4.86 0.048 residual error 12 234.5 pure error 12 total 14

R ² =84.0% R ² (adj)=81.3%DF: degrees of freedom; MS: sum of squares/DF

Through the residual plot in Figure 11, it was confirmed that the normal distribution of the reduced model, the identity of variance in all fitted values, and the randomness over time were appropriate. As can be seen in Table 13, the variation in the response variable of the regression model was explained. The R ² (adj) value was confirmed to be 81.3%. The final reduced model is as follows (2)

[Reduced final model (2)]

Y = 258.553 - 3.31591X ₁ + 0.0194137(X ₁ ) ²

3-2-3. Multiple response optimization

Through the final model of antibacterial activity and cytotoxicity of Cudrania mulberry ripening and extract, multiple response surface analysis was performed on two response variables: antibacterial activity below 40 ug/ml and cytotoxicity above 140 ug/ml, and optimized from this. The reaction conditions were derived as 50.9884% ethanol content and 79.9598°C extraction temperature.

As can be seen in Figure 12, the predicted response values of the dependent variables were predicted to be 28.5919 ug/ml for antibacterial activity and 139.9518 ug/ml for cytotoxicity, and the overall satisfaction (D) at this time was 0.63207. Because the extraction time was not a significant factor for both antibacterial activity and cytotoxicity, it was excluded from optimization, but the final extraction conditions were determined to be the median value of 7.5 hours, which showed the best activity. The ethanol content and extraction temperature were adjusted to 50% and 80°C, respectively, for the convenience of mass production of extracts in the future. Therefore, the final extraction conditions selected for extract preparation were ethanol content of 50%, extraction temperature of 80°C, and extraction time of 7.5 hours, and the sample to solvent ratio was fixed at 30 times for extraction (50.80.7.5 extract).

reaction Individual optimal extraction conditions Overall satisfaction (D) actual value
(ug/ml) predicted value ^a
(ug/ml) predicted capacity
(%) ethanol
Content % (X ₁ ) extraction
Time (X ₂ ) extraction
Temperature (X ₃ ) cytotoxicity
(CC ₅₀ , ug/ml) 50 P >0.05 P >0.05 1.0000 NT ^b 141.29 - Antibacterial activity (MIC, ug/ml) 51 63 P >0.05 1.0000 N.T. 61.37 - Multi-response optimization 0.7365 cytotoxicity
(CC ₅₀ , ug/ml) 51 80 7.5 0.3995 153.18±2.93 139.95 109.45% antibacterial activity
(MIC, ug/ml) 1.0000 40.00±0.00 28.59 71.48%

^a , the predicted value is derived from a quadratic regression equation for the dependent variable of the independent variable; ^b , not tested

As can be seen in Table 11, as a result of a verification experiment on the optimized extract, the antibacterial activity was confirmed to be 40 ug/ml and the cytotoxicity was confirmed to be 153.18±2.93 ug/ml. The predicted capacity was 71.5% for antibacterial activity and 109.5% for cytotoxicity, confirming that the final regression model explains the variation in response variables, and similarly, the reliability of the model was confirmed to be high.

Experimental Example 4. In flounder administered Cudrania mulberry ripening and optimized extract in vivo effect

In order to administer the optimized extract, 5 fish (10 fish per group) were placed in static water tanks (30L) and fed with extract-adsorbed feed at 10 to 50 mg/kg bw once a day at 0.5% of fish body weight for 5 weeks. It was administered orally. Amoxicillin, a treatment for flounder streptococci, was used as a control drug and was administered at 40 mg/kg bw once a day at 0.5% of fish body weight for 5 days. At this time, the control group was administered regular feed (Flounder Maru No. 2, Cheonha Jeil Feed). During the adsorption feed administration period, the water temperature was maintained at 20 ± 0.1°C, and the daily water exchange amount was 100% of the tank capacity. ^Through preliminary experiments, the infection concentration of S. iniae DSJ19 was determined to be ² The results are shown in Figure 13 and Table 12.

infection concentration
(CFU/fish) group Administration concentration
(mg/kg bw/day) Cumulative mortality rate
(%) relative survival rate
(%) P value Asterisk - NC - 0 - - - ² B.C. 0 80 - - - R.C. 40 10 87.5 0.0008 *** O.E. 10 90 -12.5 0.9391 - 25 50 37.5 0.0362 * 50 30 62.5 0.0133 * ¹ B.C. 0 70 - - - R.C. 40 0 100.0 0.0016 ** O.E. 10 80 -14.3 0.6464 - 25 50 28.6 0.2225 - 50 10 85.7 0.0054 **

As can be seen in Figure 13 and Table 12, the relative survival rate of the optimized extract administered at 10, 25, and ⁵⁰ mg/kg was -12.5, 37.5, and 62.5, respectively, in the 2 %, and the 25 and 50 mg/kg administration groups showed significant survival rates compared to the infection control group ( P < 0.05). In addition, the relative survival rates of the ¹ In particular, the 50 mg/kg dose group showed similar results to the relative survival rate of amoxicillin (87.5 and 100%) used as a control drug, and was confirmed to be as effective as antibiotics.

Preparation Example 4. Mass production of Kkujippong ripening and optimized extract

After drying, 20 g of the coarsely mature Cudrania chinensis fruit sample was extracted according to the above optimized extraction conditions, and was stirred every 2.5 hours during extraction. The extract was filtered through Whatman No. 4 filter paper, concentrated under reduced pressure, and the remaining residue was freeze-dried for 48 hours. The extraction yield after freeze-drying was confirmed to be 62.4%, and it was stored in a -20°C freezer until use.

Claims (8)

An antibacterial composition against fish bacterial diseases caused by one or more species selected from the group consisting of Streptococcus iniae , Streptococcus parauberis , Lactococcus garvieae and Vibrio anguillarum , comprising one or more compounds selected from the group consisting of the following formulas 15 to 17:
[Formula 15]
;
[Formula 16]
; and
[Formula 17]
. The antibacterial composition for fish bacterial diseases according to claim 1, wherein the fish is farmed fish or ornamental fish. The method of claim 2, wherein the farmed fish include flounder, rockfish, sea bream, grouper, mullet, sea bass, gizzard shad, flounder, puffer, mackerel, yellowtail, tuna, croaker, yellowtail, horse mackerel, carp, persimmon, eel, catfish, loach, and An antibacterial composition against bacterial diseases in fish, which is one or more species selected from the group consisting of crucian carp. The antibacterial composition for fish bacterial diseases according to claim 1, wherein the fish bacterial diseases are at least one selected from the group consisting of streptococcus disease and vibriosis. A pharmaceutical for the prevention or treatment of fish bacterial diseases caused by one or more species selected from the group consisting of Streptococcus iniae , Streptococcus parauberis , Lactococcus garvieae and Vibrio anguillarum , including one or more compounds selected from the group consisting of the following formulas 15 to 17. Composition:
[Formula 15]
;
[Formula 16]
; and
[Formula 17]
. A composition for fish feed having antibacterial activity against one or more types of fish pathogenic bacteria selected from the group consisting of Streptococcus iniae , Streptococcus parauberis , Lactococcus garvieae and Vibrio anguillarum , comprising at least one compound selected from the group consisting of the following formulas 15 to 17 :
[Formula 15]
;
[Formula 16]
; and
[Formula 17]
. Addition of fish feed having antibacterial activity against one or more types of fish pathogenic bacteria selected from the group consisting of Streptococcus iniae , Streptococcus parauberis , Lactococcus garvieae and Vibrio anguillarum , including one or more compounds selected from the group consisting of the following formulas 15 to 17 Composition for:
[Formula 15]
;
[Formula 16]
; and
[Formula 17]
. Method for preventing or treating bacterial diseases in fish caused by one or more species selected from the group consisting of Streptococcus iniae , Streptococcus parauberis , Lactococcus garvieae and Vibrio anguillarum , using one or more compounds selected from the group consisting of the following formulas 15 to 17:
[Formula 15]
;
[Formula 16]
; and
[Formula 17]
.