JPH04360839A - Preventive and therapeutic agent for bacterial fish disease - Google Patents

Abstract

PURPOSE:To obtain a preventive and therapeutic agent, substitutive for medicines in fear of residual properties or safety for fish diseases caused by bacteria and having high safety. CONSTITUTION:A preventive and therapeutic agent containing at least one or a mixture of plural substances obtained by extracting raw or dried leaves, branches or tree barks of a eucalyptus with water, an organic solvent or a mixture of water with the organic solvent at various temperatures or further fractionating the active ingredient with various organic solvents or carrying out purification according to chromatography, etc.

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to a preventive and therapeutic agent for fish diseases caused by bacteria.

0002

[Prior Art] Currently, marine fish include yellowtail, amberjack, red sea bream, black sea bream, flounder, etc., and freshwater fish include rainbow trout, amago, yamame, sweetfish, carp, eel, etc.
Cultivation of crucian carp, tilapia, etc. is widely practiced, and the production volume is increasing year by year. As a result, damage caused by fish diseases has also increased, posing a major problem for the industry. Many fish diseases are caused by bacteria and viruses, and bacterial fish diseases are a particularly big problem. Typical bacterial fish diseases include streptococcosis, noduloid disease, vibrio disease, Sessou disease, paracolo disease, and Edward Gera disease.

Conventionally, antibiotics such as penicillin, tetracycline, and macrolide, and synthetic antibacterial agents have been used to prevent or treat these bacterial fish diseases. Methods of administration have been to administer these drugs together with feed or to bathe them in medicine. However, in recent years, the emergence of drug-resistant bacteria and the persistence and safety of drugs in fish have become a particular problem, and there is a growing trend that it is not desirable to administer antibiotics to farmed fish. . Therefore, there is a strong desire to develop a safe and effective preventive and therapeutic agent for bacterial fish diseases that can replace antibiotics and the like.

0004

SUMMARY OF THE INVENTION An object of the present invention is to provide a safe and effective preventive and therapeutic agent for fish diseases, specifically bacterial fish diseases of cultured fish.

[0005]

[Means for Solving the Problems] The present inventors have completed the present invention as a result of intensive research to solve the above problems. It is already known that eucalyptus extract has antibacterial effects (Egawa et al., Journal of Antibacterial and Antifungal Science, Vol. 7, No. 6,
P255-261, 1979; Egawa et al., same magazine, Vol.
．． 8, No. 2, P7-11, 1989; Nakay
ama et al., Agric. Biol. Chem. , Vol.
．． 54(1), P231-232, 1990; Yamakoshi et al., Journal of the Japanese Society of Agricultural Chemistry, VOL. 64, No. 3,19
However, these are reports of antibacterial properties against general bacteria, etc., and the present inventors discovered for the first time that the eucalyptus extract of the present invention exhibits antibacterial properties against bacteria that cause fish diseases. It is something that

[0006] The bacterial fish disease in the present invention refers to nodular disease (causative bacteria: Pasteurella piscicida).
), Vibrio disease (causative bacteria; Vibrio genus bacteria), Aeromonassis (causative bacteria; Aeromonas hydr
ophila), and Sessou disease (causative bacteria; Aerom
onas salmonicida), Edwardsiella tar
da). The eucalyptus used in the present invention refers to fresh or dried leaves, branches, and bark of a plant of the genus Eucalyptus of the family Myrtaceae.

[0007]Currently, eucalyptus is used in fields such as insect repellents, as it is known that the essential oil components of eucalyptus exhibit mosquito repellent effects and inhibit germination, rooting, and photosynthesis on plants. It is considered to be used as a pesticide (Nishimura et al., "Future biological resource eucalyptus - its biotechnology and bioscience", Uchida Rokakuba, 19
1989). Furthermore, for use in foods, ``eucalyptus leaf extract'' as an antioxidant and ``eucalyptus'' as a flavoring agent have been certified as food additives, so their practical application in the food field is expected (`` List of food additives other than chemically synthesized products,” Ministry of Health and Welfare, Environmental Sanitation Bureau, Food Chemistry Division, 1989
Year).

[0008] Eucalyptus extract is extracted from fresh or dried leaves, branches, and bark of eucalyptus using water, an organic solvent, or a mixture of water and an organic solvent at various temperatures, and further contains active ingredients. Refers to those fractionated with various organic solvents or purified by chromatography, etc.

Organic solvents used for extraction and fractionation include methanol, ethanol, propanol, isopropanol,
Examples include butanol, acetone, ethyl acetate, chloroform, petroleum ether, hexane, and benzene.

[0010] The preventive and therapeutic agent for bacterial fish diseases of the present invention includes:
Eucalyptus extract as it is, dissolved or dispersed in an appropriate solvent, or mixed or adsorbed with an appropriate solid carrier, and can be used in any form such as oil, emulsion, powder, gel, capsule, etc. Can be used in dosage form. In addition, pharmacologically acceptable emulsifiers, dispersants, spreading agents,
Penetrants, gelling agents, etc. can be added.

[0011] Examples of the solvent include water, alcohols,
Oils and fats (e.g. fish oil, vegetable oil, etc.) are suitable;
At least one kind or a mixture of two or more kinds of these can be used.

Examples of solid carriers include fish meal, egg powder, shell powder, egg shell powder, soybean flour, wheat flour, starch, calcium oxide, silicon oxide, magnesium oxide, alumina oxide, seaweed powder, cellulose, carboxymethyl cellulose, fibers, plastics, Wood and the like can also be used, and at least one or a mixture of two or more of these can be used.

As the emulsifier, food emulsifiers such as glycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, and soybean phospholipid can be used.

As the gelling agent, agar, alginic acid, gelatin, polyvinyl alcohol, dibenzylidene sorbitol, N-acylamino acid amine salt, etc. can be used. In addition, the preventive and therapeutic agent of the present invention may contain other preventive and therapeutic agents, feeding attractants and promoters, fragrances, pigments, and vitamins, minerals, antioxidants, etc. that are commonly used in fish feed. It is also possible to mix them appropriately.

The preventive and therapeutic agent of the present invention can be directly mixed into feed, dissolved in rearing water for medicinal bathing, or directly added to cages. The amount of eucalyptus extract used as an active ingredient is usually 0.01 to 20% per feed weight when mixed into feed as a preventive agent, and 100 pp in breeding water when added directly to medicinal baths or cages.
It is desirable to have it present at a concentration of m or more. Further, when used as a therapeutic agent, it is desirable that the amount used is larger than when used as a preventive agent. The details of the present invention will be explained below using Examples and Test Examples, but the present invention is not limited thereto.

[0016]

[Action] The antibacterial activity in the eucalyptus extract used in the present invention is that antibacterial activity is observed in the hexane-soluble fraction and water-soluble fraction of the methanol extract of eucalyptus, so the antibacterial component is mainly composed of terpene compounds. Possible components include essential oil components, polyphenol compounds, or aromatic carboxylic acids. However, it is unclear how these compounds act on fish disease bacteria and exert their antibacterial properties.

[0017]

【Example】

Example 1 Approximately 4 liters of methanol was added to 1,020 g of fresh eucalyptus leaves, and the mixture was allowed to stand at room temperature with occasional stirring for extraction for 10 days. This was repeated three times. After filtering the extract through filter paper, it was concentrated to dryness to obtain 266.62 g of eucalyptus extract. 100g of eucalyptus extract and 2g of emulsifier (Sunsoft Q-18B 0.3% Sunsoft 621B
Emulsified with a mixed solution of 0.2% (manufactured by Taiyo Kagaku Co., Ltd.), 0.5 g of xanthan gum, and 900 g of water, and
1,000 g of W-based emulsion was obtained.

Example 2 The eucalyptus extract obtained in Example 1 was used to prepare a sustained-release gelling agent for use in fish cages. polyvinyl alcohol 18
Add 720 g of 30% calcium chloride solution to 0 g (Poval UV manufactured by Unitika Co., Ltd.) and dissolve by heating at 120°C for 30 minutes to obtain 900 g of polyvinyl alcohol hydrogel.
was prepared. Next, 50 g of the eucalyptus extract obtained in Example 1
A suspension of 100 g of water containing 1 g of emulsifier (Sunsoft Q-182S, Taiyo Kagaku Co., Ltd.) was dispersed and mixed into polyvinyl alcohol hydrogel, poured into a cylindrical mold, solidified overnight at 5°C, and placed in a cage. Sustained release gel preparation 9
90g was obtained.

Example 3 1,000 g of fresh eucalyptus leaves were crushed, 15 liters of water was added, and the mixture was heated with occasional stirring and boiled for 30 minutes. After cooling, filtration was performed using filter paper, and the filtrate was freeze-dried to obtain 230 g of eucalyptus extract. Eucalyptus extract 100
g dissolved in 100 g of water and an emulsifier (Sunsoft Q
-818H Squid oil 8 containing 5g (manufactured by Taiyo Kagaku Co., Ltd.)
00 g to obtain 1,000 g of a W/O oil agent.

Test Example 1 The sensitivity of bacteria causing various fish diseases to eucalyptus extract was investigated. 10 each of the fresh leaves, branches, and bark of the collected eucalyptus
After applying 0 g to a mixer and pulverizing it, extraction was performed with methanol. After extraction, filtration was performed using filter paper, and only the liquid portion was concentrated to dryness to obtain 26.14 g of fresh leaves, 0.61 g of branches, and 0.52 g of bark, respectively, as methanol extracts. A 0.1% solution of these extracts was prepared and used as a test solution. 50 μl of the test solution was soaked into a paper disk with a diameter of 8 mm, and after drying, various fish disease bacteria were inoculated into 1.5
% NaCl supplemented brain heart infusion agar medium and cultured at 25°C for 24 hours, the diameter of the inhibition circle was measured. The tested bacteria were Vibrio anguillum, which was isolated from sweetfish with Vibrio disease, and Vibrio sp., which was isolated from flounder with Vibrio disease. ,
Edward isolated from flounder with Edwardian gelatosis.
siella tarda, Aeromonas hydrophila isolated from Aeromonas crucian carp
, Aeromona isolated from a rainbow trout with Sessou disease.
S salmonisida, and Pasteurella piscici isolated from tuberculous yellowtail.
It is da. The test results are shown in Table 1.

[0021]

[Table 1]

Test Example 2 100g of fresh eucalyptus leaves were crushed, and
Add twice the amount of water and coarsely grind with a mixer. After filtering through filter paper, the filtrate was freeze-dried to obtain 10.1 g of water extract (water extract). Additionally, 100 freshly crushed eucalyptus leaves
Water was added to the mixture, and the mixture was boiled for 30 minutes while stirring occasionally. After cooling, filtration was performed using filter paper, and the filtrate was freeze-dried to obtain 23.1 g of a hot water extract (hot water extract). Furthermore, 4 liters of methanol was added to 1,020 g of freshly crushed eucalyptus leaves, and the mixture was extracted at room temperature for 10 days. After the extraction, filtration was performed using filter paper, and the filtrate was concentrated to dryness to obtain 266.62 g of methanol extract (methanol extract). Add 1 liter of water to 150.11 g of the obtained methanol extract,
After dispersion and dissolution, 1 liter of hexane was added and the mixture was stirred vigorously and left overnight. The hexane layer was separated, hexane was distilled off, and then dried to obtain 27.85 g of a hexane soluble fraction (hexane soluble fraction). Next, the aqueous layer after separating the above-mentioned hexane layer was concentrated to dryness to obtain 73.53 g of a water-soluble fraction. Using these eucalyptus extracts as samples, an antibacterial test was conducted under the same test bacteria and conditions as in Test Example 1. The results are shown in Table 2.

[0023]

[Table 2]

Test Example 3 The minimum growth inhibitory concentration (μg/ml) of the hot water extract of the eucalyptus leaves obtained in Example 3 against various fish disease bacteria was determined by the agar dilution method. The test bacteria and the cultivation method thereof were the same as in Test Example 1. The results are shown in Table 3.

[0025]

[Table 3]

Test Example 4 The oil solution obtained in Example 3 was used to examine the effect of preventing infection in young yellowtail (Mojako) against nodular disease. Frozen squid locust containing 0.5% vitamin supplement and 4.5% squid oil was used as a basic sample. The test sample was prepared by adding 5% of the oil agent obtained in Example 3 to the basic sample without adding 4.5% of squid oil. Mojako 100 weighing about 20-30g
The tails were divided into two groups of 50 tails each, one group was fed with the test sample containing the oil solution obtained in Example 3, and the other group was fed with the basic sample. Breeding was carried out in a 200 liter polyethylene aquarium filled with 120 liters of seawater at a water temperature of 24 to 27° C. with circulation filtration and aeration. After 30 days of rearing, each group was treated with Pasteur, a bacterium that causes nodularity.
The bacteria of ella pscicida were artificially infected, and the mortality rate of both groups due to infection was observed for 14 days. Infection method is 1
．． Pasteurel cultured on brain heart infusion agar medium supplemented with 5% NaCl at 25°C for 24 hours.
The P. la pscicida bacterial solution was diluted with seawater to a concentration of 1×10 5 cells/ml, and the mojaco from each group was immersed in 20 liters of the diluted bacterial solution at 24° C. for 5 minutes under aeration. After artificial infection, the mortality rate of both groups for 14 days was 83% in the basic sample group and 2% in the test sample group containing the oil of the present invention.
It was 5%.

Test Example 5 The emulsion obtained in Example 1 was used to examine its effect on preventing Edward gerardiasis in flounder. Frozen locust containing 0.5% vitamin supplement was used as the basic sample. The test sample was prepared by adding 5% of the product of the present invention to the basic sample. 2 50 flounder weighing approximately 120-150g
The fish were divided into two groups of 5 fish each, one group was bred with the test sample containing the emulsion obtained in Example 1, and the other group was bred with the basic sample. Breeding is 2
120 liters of seawater was placed in a 0.00 liter polyethylene water tank, and the water temperature was 24°C while circulating filtration and aeration. After 20 days of rearing, each group was infected with Edwardsiea, the bacterium that causes Edwardsiosis.
llatarda bacteria was artificially infected, and the mortality rate of both groups due to infection was observed for 20 days. Infection method: 1.5% NaC
25 ml on Brain Heart Infusion Agar
Edwardsiella t cultured at ℃ for 48 hours.
Collect bacterial cells from Arda bacterial solution by centrifugation, wash twice with physiological saline, and reduce to 1.0 x 103 cells/ml with physiological saline.
The bacterial solution was adjusted so that Next, 1 ml of the bacterial solution was intraperitoneally injected into each group of flounder to perform artificial infection. The mortality rate of both groups 20 days after artificial infection was 72% in the basic sample group;
In the test sample group containing the emulsion obtained in Example 1, it was 16%.

[0028]

[Effects of the Invention] The eucalyptus extract used in the present invention exhibits antibacterial activity against various fish disease bacteria. Furthermore, eucalyptus grows quickly and is an abundant resource, making it easy to supply. Furthermore, since the active ingredient is derived from natural products and is used as a food additive, it is considered to be highly safe. Therefore, in situations where there are concerns about the safety and persistence of antibacterial agents such as antibiotics, it is considered that it is extremely effective industrially to use the product of the present invention as a preventive and therapeutic agent for bacterial fish diseases.

Claims (1)

[Claims] Claim 1: A preventive and therapeutic agent for bacterial fish diseases containing eucalyptus extract as an active ingredient.