KR101388047B1 - Composition for preventing and treating scuticociliatosis in fishes - Google Patents

Abstract

The present invention relates to a composition for preventing and treating squaticosis of fish.
The composition for preventing and treating squaticosis of fish of the present invention is characterized by containing limonene as an active ingredient.
According to the present invention, there is provided a composition for preventing and treating squaticosis of fish, which has an excellent insecticidal effect against the squatica insect and which can prevent or treat squaticosis of fish.

Description

COMPOSITION FOR PREVENTING AND TREATING SCUTICOCILIATOSIS IN FISHES}

The present invention relates to a composition for preventing and treating squaticosis of fish, and more particularly, because it has an excellent insecticidal effect against Scutika insects, and thus prevents and cures the disease of the fish. A therapeutic composition.

Jeju Island has good water quality and natural sea water temperature in the range of 12 ~ 26 ℃ throughout the year, which is suitable for turbulent fish farming.

In addition, clean underground seawater, which maintains a water temperature of around 17 ° C throughout the year, is being developed in most areas, making it ideal for farming flounder.

However, with the long history of the flounder farming and the growing number of farms, various kinds of fish disease have occurred. This is because the farms are densely populated and the continuous farming has created an environment suitable for the growth and propagation of pathogens.

Representative fish diseases include streptococcosis of the genitals and scuticasis of the larvae, in particular the skutikasis is one of the diseases that fishers suffer the most difficulties in the flounder farm due to difficult treatment during infection.

As one of the ciliate called Scutica insects to cause the Scutica. Dozens of species are also known here. In other words, the seahorse parasitic miamienses avidus, the parasitic and tuna parasitic parasites (Uronema marinum, Uronema nigricus), and the terrestrial cultivation of the pilaterisides (Philasterides dicentrarchi) The Scutika insects infected with cultured flounder in Korea and Japan have been reported as Eurone insects.

In Europe, there have been cases of Scutika insects infected with ornamental fish, tanks, and turtles. In Japan, the first case of aquatic flounder seedlings was confirmed in 1986. Since then, it has been increasing rapidly.

When Scutika worms are infected by flounder fry, the body surface, fins and kidneys, blood vessels, and brain tissues are variously induced. Most of the flounders are killed through the destruction of plastic connective tissue.

The current treatment method is hydrogen peroxide bath or other compounds or ocher baths, but infiltrated in the gill epidermis, eye, brain, and muscle is difficult to remedy.

In addition, Scutika insects are also detected in rotifers, which are the food organisms of the flounder, and are parasitic from fry to sperm, which are difficult to treat if not detected early and cause massive mortality.

Therefore, in recent years, as a way to solve this problem, a study on the insecticide Skutika insects using weak toxic components have been made.

Korean Patent Publication No. 10-0279281 (Fish parasite Scutica insecticide comprising alginate oligosaccharide derivative) as an active ingredient, low molecular weight of alginate as a polymer and the production of alginate oligosaccharide, development of fish parasite Scutica insecticide and electric insecticide Disclosed is a manufacturing method.

Korean Patent Laid-Open No. 10-0379026 (Method for Controlling Scutika Insects of Cultured Flounder) discloses a method for controlling Scutica insects infected inside and outside of cultured flounder using ketoconazole.

However, the above inventions are difficult to manufacture and the oral administration method used as a method for controlling Scutika insects is a method that is practically difficult to use, and there is a problem in that fish pass and fish toxicity occur due to the toxicity of the components used as insecticides.

Korean Patent Publication No. 10-0279281 "Fish parasite Scutika insecticide using alginate oligosaccharide derivative as active ingredient" Korean Registered Patent Publication No. 10-0379026 "Method for controlling Scutika insects of cultured flounder"

It is an object of the present invention to provide a composition for preventing and treating squaticosis of fish, which has an excellent insecticidal effect against scuticartis, which can prevent or treat squaticosis of fish.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.

To achieve the above object, the composition for preventing and treating squaticosis of fish of the present invention is characterized by containing limonene as an active ingredient.

The limonene is characterized in that it is prepared by dissolving with a solvent and a surfactant, followed by dilution with water, and using dimethyl sulfoxide as the solvent.

The fish is characterized in that the flounder.

The limonene is characterized in that it contains 10 to 90% by weight based on the total weight of the composition.

The composition is characterized in that it further comprises at least one selected from cinnamic aldehyde (eugenol) and eugenol (eugenol).

The composition is characterized by having an insecticidal effect against Scutica.

A method for preventing and treating squaticosis of fish of the present invention is to bathe fish in seawater containing the composition.

As described above, the composition for preventing and treating squaticosis of fish according to the present invention has no toxicity by containing limonene (limonene) as an active ingredient, and exhibited excellent insecticidal effect against Scutika insects.

1 is a view showing the chemical structural formula of limonene.
FIG. 2 is a diagram showing the process of obtaining and treating Scutica insects by separating and culturing Scutica insects from infected flounder and observed microscopic results of Scutica insects.
A: infected flounder
B: Scutika insects
C: fish coin cell line
D: Scutica insect culture
Figure 3 is a view showing the morphological changes of the Scutika insects during treatment of the composition for preventing and treating Skutikasis of the present invention.
A: Normal Scutika insect
B: Vaccuolation of Scutika insects
C: Lymphocyte lysis

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

In the present invention, in order to develop a composition for preventing and treating squaticosis, which can replace formalin, which is currently prohibited for use due to toxic ingredients, the insecticidal effect against Scutika insects was confirmed in various organic compounds.

That is, formalin, benzyl aldehyde, cinnamic aldehyde, eugenol, salicylaldehyde, acetaldehyde, dimethyl sulfoxoide, Organic compounds such as vanillin, camphor, limonene, cinnayl acetate, cinnaol, rotenone, fotenoen, ivermec, and hinokitiol As a result of observing the insecticidal effect on Scutica insects, Limonene was selected as an organic compound having the best insecticidal effect against Scutica insects and having no fish toxicity, and thus preventing and preventing Scuticasis of the present invention. It has been used as an active ingredient in the preparation of therapeutic compositions.

Limonene (Limonene) is an organic compound consisting of a structure of C ₁₀ H ₁₆ as shown in Figure 1, D-type and L-type optical isomers are present and D-limonene is contained as about 90% as the main component of orange oil In addition, it is contained in plant essential oils such as lemon oil and lime oil bergamot oil, and is widely distributed in the vegetable kingdom.

The composition for preventing and treating squaticosis of the present invention is characterized by including the limonene as an active ingredient, it is preferable to contain 10 to 90% by weight based on the total weight of the composition. If the content of limonene is less than 10% by weight, it is difficult to exhibit the expected insecticidal effect of Scutica insects. If the content of limonene is more than 90% by weight, the amount of surfactant and solvent that is added to dissolve when using limonene is used. This becomes so small that it becomes difficult to dissolve. For surfactants at least 10% by weight or more should be used.

More preferably, it is preferable to use 20 to 300 ppm as the content used when spraying limonene in the water tank, which is less than 20 ppm, it is difficult to exhibit the expected insecticidal effect of Scutica insects, and when the content exceeds 300 ppm, the effect of the change is minimal. This is because there is a risk of poisoning.

In addition, it has been confirmed that the cinnamic aldehyde (eugenol) and eugenol (eugenol) of the organic compound also exhibits the insecticidal effect against Scutica, for the prevention and treatment of the skuticiosis of the present invention to obtain a higher insecticidal effect The composition may further include one or more selected from cinnamic aldehyde and eugenol.

The cinnamic aldehyde is an organic compound composed of a structure of C ₉ H ₈ O, and is mainly used in the perfume industry as a main component of cinnamon, and is used as various product additives such as food, beverages, and pharmaceuticals.

Eugenol is an organic compound composed of C ₁₀ H ₁₂ O _{2. It} is an oil widely distributed in essential oils such as cloves and pimento. It is used in fillers, ulcers and the like.

These organic compounds are preferably used after being dissolved in an acid solution, in consideration of the stability to fish, dissolved in dimethyl sulfoxide as a solvent, 0.5 to 2 times based on the weight of the organic compounds Preference is given to using the content.

In addition, in order to help the organic compound is dissolved in a solvent, a surfactant is added and dissolved when dissolving the organic compound in a solvent, and preferably, a nonionic surfactant is used.

As the nonionic surfactant, at least one selected from ethylene oxide, ethanolamine, ethanolamine, sorbitol, and glycerin is used, and in particular, sorbitol It is preferred to use at least one selected from SPAN-20 (Sorbitan Monolaurate) and TWEEN-80 (PEO (20) -Sorbitan Monooleate).

When the dissolved organic compounds are used substantially as described above, it is preferable to dilute them in seawater, and more preferably, use sterile seawater. Sterilized seawater is collected by filtering the seawater coming through the fish pond collection pipe, sterilized for 15 minutes at 121 ℃, when using the insecticidal composition diluted with sterilized seawater in the bath method, by controlling the osmotic pressure to kill fish It also serves to prevent.

The composition for the prevention and treatment of scuticasis of fish of the present invention comprises an organic compound of the above form as an active ingredient, the composition may be used in the form of bathing the fish is added to water like other drugs In addition, it may be administered in addition to the feed for oral administration into the body of the fish for the purpose of convenience of work, and preferably the flounder as the fish.

In the case of oral administration, the composition of the present invention is used within 200 mg per kg of body weight, and an appropriate amount of 40 to 100 mg / day of the composition of the present invention is preferably administered for 4 to 5 days. In this case, the insecticidal effect is insufficient in this range, and the improvement of the insecticidal effect is insignificant.

In the case of bathing by adding the composition of the present invention to seawater, an appropriate insecticide effective concentration is 25 to 100 ppm. This is because the insecticidal effect and reproduction inhibition effect is insufficient in the case below this range, and further improvement of the insecticidal effect is insignificant even if it exceeds this range. It can be usefully used as an environmentally friendly hygiene pest with action.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited thereto.

<Example 1> Preparation of the composition for preventing and treating skutikaosis of fish of the present invention 1

Prepare 300 ml of limonene, dimethyl sulfoxide 300 ml, tween 80 2.5 g, span 80 6.25 g, and dilute 400 ml of sterile seawater to prevent squaticosis of the fish of the present invention. And a therapeutic composition.

<Example 2> Preparation of the composition for preventing and treating skutikaosis of fish of the present invention 2

Prepare 300 ml of limonene, 100 ml of cinnamic aldehyde, 300 ml of dimethyl sulfoxide, 2.5 g of tween 80, 6.25 g of span 80, add 300 ml of sterile seawater and dilute To prepare a composition for the prevention and treatment of skutikaosis of the fish of the present invention.

<Example 3> Preparation of the composition for preventing and treating skuticiosis of fish of the present invention 3

Prepare 300 ml of limonene, 100 ml of eugenol, 300 ml of dimethyl sulfoxide, 2.5 g of tween 80, 6.25 g of span 80, add 300 ml of sterile seawater and dilute To prepare a composition for preventing and treating squaticosis of fish of the invention.

<Example 4> Preparation of the composition for preventing and treating skutikaosis of fish of the present invention 4

Prepare 300 ml of limonene, 100 ml of cinnamic aldehyde, 100 ml of eugenol, 300 ml of dimethyl sulfoxide, 2.5 ml of tween 80, 6.25 g of span 80 300 ml of sterilized seawater was added thereto to dilute to prepare a composition for preventing and treating squaticosis of fish of the present invention.

<Comparative Example 1> Preparation of the composition for preventing and treating squaticosis of fish using formalin

The composition was prepared as in Example 1, but using formalin instead of limonene.

<Comparative Example 2> Preparation of the composition for preventing and treating squaticosis of fish using benzyl aldehyde

The composition was prepared as in Example 1, but using benzyl aldehyde instead of limonene.

<Comparative Example 3> Preparation of a composition for preventing and treating scuticasis of fish using cinnamic aldehyde

The composition was prepared as in Example 1, but using cinnamic aldehyde instead of limonene.

<Comparative Example 4> Preparation of the composition for the prevention and treatment of squatosis of fish using eugenol

The composition was prepared as in Example 1, but using eugenol instead of limonene.

<Comparative Example 5> Preparation of the composition for the prevention and treatment of squatosis of fish using salicylic aldehyde

The composition was prepared as in Example 1, but using salicylic aldehyde (salicylaldehyde) instead of limonene.

<Comparative Example 6> Preparation of the composition for preventing and treating scuticasis of fish using acetaldehyde

Prepared as in Example 1, but instead of limonene (limonene) to prepare a composition using acetaldehyde (aceataldehyde).

<Comparative Example 7> Preparation of the composition for preventing and treating squaticosis of fish using dimethyl sulfoxide

Prepared as in Example 1, but instead of limonene (limonene) to prepare a composition using dimethyl sulfoxoide (dimethyl sulfoxoide).

<Comparative Example 8> Preparation of the composition for the prevention and treatment of squatosis of fish using vanillin

The composition was prepared as in Example 1, but vanillin was used instead of limonene.

<Comparative Example 9> Preparation of the composition for preventing and treating squaticosis of fish using camphor

The composition was prepared as in Example 1, but using camphor instead of limonene.

<Comparative Example 10> Preparation of the composition for the prevention and treatment of squatosis of fish using cinnamil acetate

Prepared in the same manner as in Example 1, but instead of limonene (limonene) to prepare a composition using cinnayl acetate (cinnamyl acetate).

<Comparative Example 11> Preparation of the composition for preventing and treating squaticosis of fish using cineol

Prepared as in Example 1, but instead of limonene (limonene) to prepare a composition using a cineole (cineole).

<Comparative Example 12> Preparation of the composition for preventing and treating squaticosis of fish using rotenone

The composition was prepared as in Example 1, but using rotenone instead of limonene.

<Comparative Example 13> Preparation of the composition for the prevention and treatment of squatosis of fish using worker bee mek

The composition was prepared as in Example 1, but using ivermec instead of limonene.

<Comparative Example 14> Preparation of the composition for the prevention and treatment of squatosis of fish using the hinokithiol

The composition was prepared as in Example 1, but using hinokitiol instead of limonene.

Experimental Example 1 Confirming Insecticidal Effect of Scutika

1. Experimental Method

1) Isolation and cultivation of Scutika insects

Scutica insects were directly isolated from halibut disease infected with Scutica ciliated larvae and cultured.

Referring to Figure 2, first removed a portion of the tissue of the infected site from the flounder diseased and placed on a slide glass and microscopic observation, it was confirmed that the flounder disease is infected with Scutika insects.

The Scutica fungus thus confirmed was inoculated in P1Y1 (1% proteose peptone, 1% yeast extract) medium to perform primary culture at 15 ° C.

Since many bacteria exist in the primary culture of Scutica insects, pure cultures using fish coin cell lines were performed due to the contamination of bacteria and the limitation of the number of growth.

For pure cultivation of Scutika, fish seed cells CHSE-214 (Chinook salmon embryo) were added to 7.5% sodium bicarbonate in a modified eagle (Modified Eagle's Medium) medium adjusted to pH 7.3 at 10% FBS and penicillin-strepto Mycin (penicillin-streptomycin) was added and incubated at 20 ° C.

The fish cultured cell line grown in monolayer was inoculated with the first cultured Scutica locus and the culture was continued at 20 ° C. until the Scutica insects completely consumed the cell line.

After observing that the Scutica insects completely ingested the cell line, the number of grown cells was counted by counting the number of Scutica cells in 1 mm × 1 mm using the hematocytometer. It was.

2. Experimental results

As a result, when incubation after the incubation of the Scutika insects, after 5-7 days, all fish coin cells were used as food and disappeared and the density of Scutika insects reached 5.1 × 10 ₆ ciliate / ml. This was collected by centrifugation and used for the following experiment after dilution.

Experimental Example 2 Observation of Morphological Changes and Motility Changes of Scutica Insects

0.1 ml of each of the compositions for Example 1 and Comparative Examples 1 to 14 were placed in a 24 well plate, mixed with 0.9 ml of the cultured Scutica insect culture, and left at 20 ° C. for 1 hour and then under a microscope. Changes in morphology and motility were observed by association. .

In the case of Scutika insects, the phenomenon occurs when contact with the pesticidal substance is a stage of motility, bulging morphological changes, morphological changes burst through.

Experimental results were determined by motility and morphology in 0, 1, 2 stages as shown in Table 1 below, and the insecticidal rate was based on the sputum of the Scutika.

Division score
(Score) Interpretation motility
(Motility) 0 No effect on motility: Cilia are highly mobile and highly motile.
(No effect on motility: ciliates are highly motile with many moving across the microscopic field) One Low Mobility: No ciliary movement or very few cilia.
(Low to moderate motility: cilia movement present with no or very few ciliates moving across the microscopic field) 2 No mobility
(No evidence of motility) shape
(Morphologic) 0 No morphological changes: The ciliary worms retain their ellipical shape.
(No evidence of morphological changes: ciliates retain ellipical shape) One The largest number of ciliary insects have little or no cell division.
Majority of ciliates in the microscopic field are round with little or no lysis 2 Large-scale cell division: few or no intact cell traces.
(Extensive lysis: few or no intact cells evidence in all microscopic fields)

2. Experimental results

The experimental results are shown in Table 2 and FIG.

turn Target Active substance concentration (ppm) 100 morphology
(shape) motility
(motility) contrast formalin
(formalin)-Comparative Example 1 0 2 One Benzyl aldehyde
(benzyl aldehyde)-Comparative Example 2 One 0 2 Cinnamic aldehyde
(cinnamic aldehyde)-Comparative Example 3 2 One 3 Eugenol-Comparative Example 4 2 One 4 Salicylic Aldehyde
(salicylaldehyde)-Comparative Example 5 0 0 5 Acetaldehyde
(acetaldehyde)-Comparative Example 6 One One 6 Dimethyl sulfoxide
(dimethyl sulfoxoide)-Comparative Example 7 0 0 7 vanillin
(vanillin)-Comparative Example 8 0 One 8 Camper
(camphor)-Comparative Example 9 0 0 9 Limonene
(limonene)-Example 1 2 2 10 Cinnamil acetate
(cinnamyl acetate)-Comparative Example 10 0 One 11 Cineol
(cineole)-Comparative Example 11 0 0 12 Roteon
(fotenoen)-Comparative Example 12 0 0 13 Worker bee
(ivermec)-Comparative Example 13 0 0 14 Hinoki Thiol
(hinokitiol)-Comparative Example 14 0 One

As shown in Table 2 and FIG. 3, there were limonene, cinnamic aldehyde, and eugenol as organic compounds having better insecticidal effects than conventional used formalin (Comparative Example 1). Limonene was found to exhibit the most excellent pesticidal effect.

Thus, the organic compounds exhibiting the insecticidal effect was confirmed the pesticidal effect according to each concentration.

As a result, it appeared as Table 3 below.

turn
Active substance Active substance concentration (ppm) 100 75 50 25 morphology
(shape) motility
(motility) morphology
(shape) tility
(motility) morphology
(shape) tility
(motility) morphology
(shape) tility
(motility) contrast formalin
(formalin) 0 2 0 One 0 One 0 One One Cinnamic aldehyde
(cinnamic aldehyde) 2 One 2 One One One 0 0 2 Eugenol
(eugenol) 2 One 2 One 2 0 0 0 3 Limonene
(limonene) 2 2 2 2 2 2 2 One

As shown in Table 3, it was confirmed for each concentration of the substance showing an excellent insecticidal effect against Scutica among the various organic compounds, Cynamic aldehyde (cinnamic aldehyde) and eugenol (eugenol) from 50 ppm Skutika insect Insecticidal capacity is reduced, but limonene (limonene) was found to be hardly reduced insecticidal capacity even at 25 ppm.

As a result, the composition for preventing and treating squaticosis of fish is not toxic by containing limonene (limonene) as an active ingredient, and it can be seen that it exhibits excellent insecticidal effect against Scutika insects. It can be predicted to show higher pesticidal effect by further including eugenol.

The present invention has been described with reference to preferred embodiments and experimental examples, and those skilled in the art to which the present invention pertains within the essential technical scope of the present invention and other embodiments of the detailed description of the present invention Examples and experimental examples may be implemented. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.

Claims (8)

Contains limonene as an active ingredient,
The limonene is characterized by exhibiting an insecticidal effect against Scutica insects at a concentration of 25 to 100 ppm,
Composition for the prevention and treatment of skutikaosis of fish. The method of claim 1,
The limonene is prepared by dissolving with a solvent and a surfactant and then diluting with water,
Composition for the prevention and treatment of skutikaosis of fish. 3. The method of claim 2,
Using dimethyl sulfoxide as the solvent,
Composition for the prevention and treatment of skutikaosis of fish. The method of claim 1,
The fish is characterized in that the culture flounder,
Composition for the prevention and treatment of skutikaosis of fish. delete The method of claim 1,
Characterized in that it further comprises one or more selected from cinnamic aldehyde (eugenol) and eugenol (eugenol),
Composition for the prevention and treatment of skutikaosis of fish. delete delete

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