KR100373147B1 - Treatment agent of Scuticocilictida - Google Patents

Abstract

The present invention relates to a new use of δ-aminolevulinic acid (ALA), in particular, the use of environmentally friendly ALA for the prevention and treatment of infectious diseases of the fish parasite Scuticociliatida. It is related to the use, which can be usefully used to mass produce fresh fish without environmental pollution.

Description

Treatment agent of Scuticocilictida

The present invention relates to new uses of δ-aminolevulinic acid (hereinafter abbreviated as "ALA").

More specifically, the present invention relates to the use of the environmentally friendly ALA for the prevention and treatment of infectious diseases of the fish parasite Scuticociliatida, the ALA in the breeding tank to bathe or mix in the feed Oral administration to fish prevents parasitic infections or treats infected fish. As a result, it can be useful to produce aquaculture fish, etc. in large quantities without using environmental pollution in place of existing antibiotics.

In recent years, the fish farming industry is increasing rapidly as the consumption of live fish increases and the variety of the target fish is diversified. In response, technology for artificial seedling production is established, and research and development of filtration systems that enable high-density farming have been actively carried out, and the culture technology has been gradually advanced.

However, due to the diversification of farmed fish species and overcrowded farming to meet the supply of demand, as well as lower feeds and lower aquatic environments to lower production costs, diseases of farmed organisms continue to diversify. Damage ranges also tend to spread. In detail, the amount of damage caused by fish diseases is increasing day by day, and the countermeasures against fish diseases are emerging as an important task. In addition, the import and export of seafood is concerned about the influx of foreign pathogens, which will increase the incidence of diseases in domestic farms, increasing the need to protect the domestic aquaculture industry.

Marine fish diseases are caused by various pathogenic organisms such as parasites, bacteria, and viruses. The representative fish diseases that cause serious damage in our country are white spot disease and succinococciatiti, an infection of ciliary insects (Ichthiophthirius). Scuticosilia infections caused by Scuticosiliatida).

Scuticosiliatida is a parasite mainly infected with flounder, which is a parasitic species belonging to the siliophora and attaching to the oligophymenophora. Squatch caterpillars invade the surface and body organs of the body throughout all stages of growth, from seedlings of the flounder to sea urchins, causing parasitic deaths and causing the death of flounder. It is a parasite.

The study of Scotchka insects was first studied in Japan for the infection of cultured flounder. Specifically, the biological characteristics, morphological characteristics and pathogenicity of the Scoochi caterpillar have been reported, and in the case of Korea, studies on pathological studies and distribution of artificially infected flounder have been made. Specifically, in pathological studies, the carcasses were found to cause fatal damage by infection, passing through the epidermis and gills, and invading the nervous system in the body through the bloodstream. These pathological phenomena appear similar to those of flounder fry and freshwater fish.

In addition, studies by Mizuno and Yoshimizu et al. Have shown that treatment with formalin or veterinary medicines such as ecthesin and prezil have the effect of inhibiting the activity of Scotchka in vitro . However, it is known that in vivo infection does not have a therapeutic effect in the body, and in fact, there is no therapeutic effect even in fish that occur in fish farms. In Japan, research on formalin bathing method, which is commonly used as a method for controlling parasitic diseases, was conducted, but aquaculture protection team was based on reports of decreased activity at bathing concentration of about 100 ppm in case of exposed body. The results at this site showed no effect at all, demonstrating that the laboratory results do not actually apply.

In addition, studies have been conducted to investigate the pathogenicity of the S. chica on the cultured cells by the successful growth of the S. chica on the cell. However, although it has been successfully applied to flounder to confirm its pathogenicity and to remove its activity by using prezil and ecthesin as a method for inactivation of the in vitro in vitro, The study was not conducted. In addition, as a result of testing the treatment effect by using farmed flounder and medium rearing fish, it was found to be ineffective. The reason why there is no therapeutic effect in the body is that in the study of Mizuno and Yoshimi, it is possible to remove Protomont-based Scotch catworms exposed to the water system as in the case of formalin treatment, but the effect is not exerted because a series of experimental drugs cannot penetrate the body. It was judged not. In addition, the price of the drug is much higher than that of the general fishery medicine, which is more economical than the cheap formalin in the case of removing only the charges released in the water system exceeding 100,000 won per kg.

In the domestic aquaculture site, even though the parasite disease is not treated even if the drug is used, there is a tendency to overuse the drug. This imparts resistance to other aquatic pathogens, weakens the health of the fish, and poses risks such as environmental problems due to the accumulation of drugs in the water and in the poor. In addition, by causing problems such as drug residues in the fish, not only is it degrading the commerciality of fish farming, but there is also a concern about food hazards. Therefore, in order to develop a method capable of controlling the Scoochi caterpillar that is inhabited and propagated not only in the fish body but also in the aquatic water system and minimizing the impact on the environmental system, the search and development of a new environmentally friendly insecticide is necessary.

Recently, as the problem of environmental pollution arises due to the toxicity due to excessive use of chemical synthetic insecticides and herbicides, it is required to discover new materials of biological origin, especially microbial origin. In this respect, ALA is a biosynthetic precursor of tetrapyrrole compounds such as heme, bacteriochlorophyll and corrinoid in all living organisms, and is the only biological material known to date. Its effects have been widely reported as insecticides, plant growth promoters and cancer treatments. In addition, ALA has been regarded as an environmentally friendly herbicide and insecticide because it is easily broken down in nature. However, since the amount of ALA produced in microorganisms is low and its production by chemical synthesis is difficult to industrialize due to complex synthesis steps, it is currently toxic such as diphenyl ether (DPEs) and oxadiazole. Synthetic photoactive herbicides are used. Such synthetic photoactive herbicides cause problems of environmental pollution due to their harmfulness to animals and humans and their slow decomposition rate. In fact, the abuse of herbicides on golf courses has caused water pollution and the damage of nearby farmers to become social problems.

Δ-aminolevulinic acid (ALA), which is produced especially from Rhodobacter , selectively acts only on dicotyledonous plants when sprayed on plants, and is a strong oxidizing pchlide by the sun's rays. Forms. Pfylide induces a series of oxidation reactions in plants, thereby destroying the phospholipids of the leaves, resulting in herbicidal activity in which the leaves die. As such, ALA has been reported as an environmentally friendly herbicide because it selectively kills weeds without damaging people, animals and crops.

The inventors of the present invention, while searching for a compound to develop an environmentally friendly parasitic infectious agent, found that δ-aminolevulinic acid (ALA) has an excellent effect in the treatment and prevention of Scuticociliatida infection, a fish parasite. The present invention was completed by confirming.

It is an object of the present invention to provide a prophylactic and therapeutic agent for fish parasitic infections useful for mass production of cultured fish.

Figure 1 shows the toxicity to fish chemotactic cells of the ALA (δ-aminolevulinic acid) of the present invention.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment of Squatchka insect infection which is a fish parasite with δ-aminolevulinic acid as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for the prevention of infection with the Schizocartis, a fish parasite containing δ-aminolevulinic acid as an active ingredient.

Specifically, the present invention provides a pharmaceutical composition having a prophylactic and therapeutic effect against protozoan parasites, and preferably provides a pharmaceutical composition having a prophylactic and therapeutic effect against a succinacoptera, which is a fish parasite.

This invention is to determine the concentration of δ-aminolevulinic acid that does not have cytotoxicity after investigating the toxic effects of δ-aminolevulinic acid (ALA) on fish cells and the δ-amino The treatment of levulinic acid with Scoochi caterpillars in vitro confirms that δ-Aminolevulinic acid inhibits the growth of the Scotch caterpillar, and then the δ-aminolevulinic acid is pre-tanned or fish-administered to fish in vivo . Afterwards, the schizocaries were infected and mortality was examined over time to confirm the δ-aminolevulinic acid prevention of schizocaries infection. Aquatic or oral administration and mortality over time were examined to determine the effect of δ-aminolevulinic acid on the prevention and treatment of Squatica parasite infections on fish.

Hereinafter, the present invention will be described in detail.

The present invention provides the use of an environmentally friendly substance δ-aminolevulinic acid (ALA) as a therapeutic agent for parasitic infections.

Specifically, the ALA of the present invention can be used for the treatment and prevention of protozoan parasitic infections, and preferably for the treatment and prevention of parasitic parasitic infections, which are ciliary insects, and more preferably the infectious diseases of the fish parasite Scuticosiliatida. It can be used as a therapeutic and prophylactic for.

In order to confirm the therapeutic effect of ALA against parasitic infections, the present invention has the effects of (1) using fish cultured cells, (2) direct administration, (3) administration to parasites, (4) prevention, and (5) treatment. Investigate separately.

First, the present invention confirms the stable concentration and the toxic concentration in the treatment of ALA using fish coin cells, etc., which can be cultured Scoochi caterpillars.

The fish coin cells include RTG-2 (Rainbow trout gonad), FHM (Fathead minnow epithelioma) and CHSE-214 (Chinook salmon embryo), and the toxic effect is confirmed using MTT method.

As a result, it was confirmed that ALA is not toxic at the concentration of 10 mM or less when directly exposed to the cells, and ALA is not directly toxic to the fish at the concentration of 50 mM or less considering the absorption rate of the substance in the fish.

In addition, the present invention examines the toxicity of ALA to fish using (1) injection, (2) dipping and the like. As a result, the treatment of ALA by injection and dipping was not related to the survival rate and ALA concentration of fish species.

In addition, the present invention investigates the effect of ALA inactivating a Scotch.

As a result, the SLA was killed within 10 minutes when the ALA concentration was in the range of 500 mM to 125 mM, and within 20 minutes when it was in the range of 60 to 30 mM, and completely killed under the treatment conditions of 60 minutes or less in the range below 15 mM. In the range of 8 mM, more than 50% of the spuchica was killed by treatment within 60 minutes.

In addition, the present invention is to investigate the effect of the ALA to prevent the infection Sukuchika by artificially infecting the Sukuuchika worms by observing the death of the infection.

As a result, when the ALA concentration in the breeding water was maintained at 1.0 to 50 ppm, the survival rate was high for the infection, and when the ALA was added orally to the feed, the survival rate was higher.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are only examples of the present invention, and the scope of the present invention is not limited by the examples.

<Example 1> Toxicity of fish ALA (δ-aminolevulinic acid) to fish coin cells

First, the present invention confirmed the stable concentration and toxic concentrations of ALA treatment in fish coin cells used in the culture of Squatch caterpillars causing enormous damage to the marine cultured fish. Specifically, by directly exposing to ALA, toxicity experiments were performed on fish to establish ALA concentrations that did not affect fish cells.

ALA used in the present invention was prepared at 1.0 M concentration (0.167 g / 1 ml HBSS) and diluted by a 10-fold dilution method. Into a 96-well microplate, put 100 μl of the fish coin cell solution suspended at a concentration of 2.5 × 10 ⁵ cells / ml, and then add 100 μl of the ALA dilution to each of 4 wells per dilution unit. The experiment was performed.

In addition, fish chemotactic cells used in the cell experiments related to ALA toxicity include RTG-2 (Rainbow trout gonad), FHM (Fathead minnow epithelioma) and CHSE-214 (Chinooksalmon embryo). Culture was performed using MEM (Gibco) medium with ml of penicillin (Sigma), 100 μg / ml of streptomycin (Sigma) and 5% FBS.

The present invention investigated the toxic effects of ALA on fish coin cells using MTT method using the cells obtained in the above process.

Specifically, cells cultured in 96-well microplates were incubated for 3 hours in an incubator at 15 ° C., and then inoculated with 100 μl of ALA solution at 0 mM, 1 mM, 10 mM, 50 mM, and 100 mM concentrations at 18 ° C. Further incubated for 24 hours. On the other hand, the control group was charged with the same amount of HBSS buffer instead of ALA solution. After culturing in the above manner, MTT solution (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (DOJINDO Lot ZY903, Japan) was added to the PBS buffer at a concentration of 5 mg / ml. 100 μl was added to each well, and 100 μl of 0.04 N hydrochloric acid-isopropanol was added to each well, and the absorbance was measured at 570 nm with an ELISA reader. .

As a result, ALA toxicity to fish coin cells is as shown in FIG. When ALA was treated at the concentrations of 1 mM and 10 mM in all three coin cells used in the experiment, the cell proliferation was similar to that of the control, whereas the treatment of ALA at the concentrations of 50 mM and 100 mM was similar to that of the control. In comparison, it showed about half the low cell proliferation.

Therefore, when ALA was directly exposed to cells, the toxicity stability range was determined to be 10 mM or less in consideration of reaction conditions in the wells of cell culture conditions. This can be an experimental standard concentration standard under the culture conditions of the Scotch caterpillar, and can be used as a basic data for the study of the setting of the toxic concentration according to the application in the organism. In fact, it is assumed that the route of delivery to the target organ or cell unit by the body's absorption and metabolism of the substance (generally, the absorption rate of the substance in the fish body is 10-20%), and the fish concentration of the ALA of the present invention is 50 mM or less. Direct toxicity could not be expressed in fish.

Example 2 Toxicity Study of Fishes of ALA

As described in Example 1, the toxicity of the fish by the incorporation was confirmed with reference to the point of safety in ALA treatment at a concentration of 50 mM or less. Specifically, toxicity was investigated in two ways: (1) direct administration using injection method and (2) immersion method which is most easily applied as a method for the treatment of diseases in a large number of fish in fish farms.

Flounder (3 g), red sea bream (1.5 g) and zobololak (5 g) fry were used for experiments of the toxic effects, and ALA solutions were prepared at concentrations of 500 mM, 100 mM, 50 mM and 10 mM, respectively. First, the ALA solution was injected intraperitoneally into each fish by 100 μl by direct injection, followed by observation in a breeding tank for 1 week. In addition, ALA was prepared at a concentration of 167.6 ppm by dissolving 5.028 g in 30 L breeding seawater. In the same way, experimental seawater containing 10 and 100-fold dilution concentrations of 16.76 ppm and 1.676 ppm of ALA was prepared and placed in each tank to expose 10, 30, 60, 120 and 180 minutes. After each exposure, 20 fish were collected for each elapsed time and transferred to an ALA-free breeding tank, and survival rate was measured for 1 week.

As a result, in the ALA-treated experimental group by the intraperitoneal injection method (3 g flounder, 1.5 g red sea bream and 5 g sea bass), all the fish and species showed no change in survival rate regardless of ALA concentration.

In addition, in the ALA-treated experimental group by the immersion method, the survival rate of all the treated experimental groups and the control was confirmed to be 100%, and there was no difference between fish species, between treatment concentrations and immersion time.

In addition, it was judged that there was no direct toxicity to fish in the range of applicable concentrations considering the type of use and economics of applying ALA directly to fish. In other words, intraperitoneal injection, toxicity was not confirmed in the case of the flounder at the concentration of 50 mM or less at 3.3% or less, and in the case of red snapper and jeopbolak at the concentration of 50 mM or less at 2.0% or less of the body weight. No toxicity was confirmed when injected. When halibut, red snapper and sea bream were immersed in a concentration of 167 ppm for up to 180 minutes, they were not toxic to experimental fish species. Judging.

Example 3 Investigation of the Effect of ALA on Fish Scotch Caterpillars

In order to investigate the effects of the ALA of the present invention on the fish Squatchka worms, the Squatchica worms were isolated and cultured according to the following procedure, and the inactivation effect was assayed for the insects in the infection stage.

First, take a certain amount of epidermal mucus of the flounder infected with parasites, suspend it in HBSS buffer solution, make a suspension and leave it at room temperature for 30 minutes, then centrifuge at 800 rpm for 10 minutes to discard the supernatant, and precipitate the MEM-10 (minimum essential medium, 10% FBS, streptomycine, peniciline) was suspended for 30 minutes. Next, centrifugation was carried out and the supernatant was removed to obtain only cells, and a constant number of filling suspensions were prepared by counting them with a hemocytometer. The prepared suspension suspension was passaged in a laboratory, inoculated into FHM cells prepared in a predetermined number (2.5 × 10 ⁵ cells / ml) and incubated at 17 ^° C.

In order to test the inactivation effect of the worms, the optimum ALA concentration and treatment time for the control of the Scoccidae insects were examined by measuring the killing effect of the S against the insects according to the ALA concentration and the reaction time. The supernatant was discarded by centrifugation at 3000 rpm for 5 minutes, and then washed three times with PBS buffer. The 1.0 M ALA solution made with sterile tertiary distilled water was diluted twice with stock solution and diluted to 1000 times (1.0 mM). 50 μl of each of the Schizoca worms were added to each well in a 96-well microplate, and the ALA solution diluted in two steps was added thereto, followed by incubation for 120 minutes, and the survival of the Scoccichia worms was observed. The incubation time of the Scoochi caterpillars at each concentration was measured at 10 minute intervals for 120 minutes. After inoculation, 5 minutes were stirred and 5 minutes were left. After the reaction, the biopsied dye, 1% trypan blue, was added to each well in an equal amount of 100 μl and mixed. Next, the number of parasites was measured using a hemocytometer for the Scoochi caterpillar cultured with the ALA dilution.

As a result, parasites were completely killed within 10 minutes after treatment when the actual concentration of ALA was in the range of 500 mM to 125 mM, and when the actual concentration was 60 to 30 mM, the schizocarites were completely killed within 10 minutes of treatment. In addition, in the actual concentration range of 15 mM, at least 60 minutes of treatment conditions were completely killed, and at least 50% of the Scoochi caterpillar was killed by treatment within 60 minutes. In the case of the actual treatment concentration of 8 mM concentration, about 50% of the Scoochi-Chopper was killed in one hour of treatment. At least two hours of killing Scoochi caterpillars by treatment at a concentration range of 500 mM to 4 mM, killing more than 90% of the Scooter caterpillars was determined to be possible. At higher dilutions, the number of killed individuals rapidly decreased and no effective value was obtained.

Example 4 Investigation of the Prophylactic Effect of ALA Squaticus on Human Body Infection

In order to investigate the prophylactic effect of the ALA of the present invention against the scotch caterpillar, 20 rice flounders of 25 g before and after were grown in a circulating filtration system using two experiments and one control. Experimental group 1 was inoculated once every 3 days from the start of the experiment, and maintained for 3 weeks while maintaining the ALA concentration in the breeding water at 10 ppm. Experimental group 2 was used as artificial feed (EP, fish). 3% of the weight: 15 g) was sprayed (1.67 mg / day, 0.11 g / kg feed) of 10 mM ALA solution at a concentration of 1 ml each was dried and raised for 3 weeks while administering. During the experimental period, experimental group 1 and untreated group were used as artificial blended feed commercially available in the same amount as that of experimental group 2 without ALA treatment. As shown in Table 4, the experimental group 1 maintained at 10 ppm of ALA concentration in the breeding water and the experimental group 2 fed with 0.11 g of ALA per kg of feed were mortalized in the same way as the control group containing no ALA. Did not happen.

In order to investigate the prophylactic effect in the artificial infection of S. chicaworms, an artificial infection experiment was performed using a Scouchi carnacle purely cultured on FHM cells. Artificial infection of the carcinoma was carried out 3 weeks after the experiment breeding, artificial attack method was used to immersed in culture solution (1000 individuals / ml) directly exposed to aeration for 30 minutes. After the artificial infection, the experimental fish for each experimental group were transferred to the observation tank to observe the infection and the death due to the infection for two weeks. The water temperature was maintained at 20 ^° C. (± 2) during the whole experiment. As shown in Table 5, when the ALA concentration in the rearing water was maintained at 10 ppm, the artificial infection of the Scotch caterpillar showed a much higher survival rate of 85% compared to the control. In addition, the experimental group administered orally by adding ALA to the feed showed higher survival rate than the experimental group treated with ALA in the rearing water.

Therefore, ALA added at a concentration of 10 ppm in the rearing waters was excellent in preventing the infection of humans against Scotch caterpillars, and also prevented the flounder and the gecko rockac from Scotch cats in a wider range of concentrations. Similar to the above results, there was no mortality of experimental fish during the adaptation period of l3 weeks, and two-week breeding of artificially infected flounder and sea rockfish in the breeding water treated with 1, 5, 10, 20 and 50 ppm showed higher prevention than control. Effect appeared.

In addition, ALA added 0.11 g in 1 kg of feed showed a high prophylactic effect against anthropogenic infection of the Scotch caterpillars.

Example 6 Investigation of Disease Treatment Examples in Disease Occurrence Conditions

The ALA of the present invention was treated to the flounder fry of the seedling production site where the seedlings were produced at the flounder seedling production site, and the sale was stopped at the sales stage (5-6 cm in length) and the sale was stopped. Severe fish housed in the tank where only infected fish were collected through screening at the seedling production site were treated. The formalin was treated at 30 ppm concentration on the first day, and the ALA was treated for 2 hours at 1 ppm concentration on the second and third days.

As a result of treatment, the vitality of the treated fish was remarkably improved from the day after the treatment, and the mortality following the conventional treatment tended to decrease significantly. Afterwards, the animals were housed in breeding tanks in a kennel that applied an ozone treatment system to manage pathogens in the breeding water, and as a result, normal breeding was possible. As a result of confirming that the infection was not confirmed.

As described above, the ALA of the present invention exhibits a variety of toxicity and environmentally friendly substances to the Scuticociliatida, a fish parasite that has an excellent therapeutic and prophylactic effect on protozoan parasites and, in particular, causes enormous damage to the production of farmed fish. Therefore, as a prophylactic and therapeutic agent for fish parasitic infections, it can be usefully used to produce farmed fish in large quantities without environmental pollution.

Claims (4)

A pharmaceutical composition for the treatment of Squaticus worm infection, which is a fish parasite containing δ-aminolevulinic acid as an active ingredient. delete delete A pharmaceutical composition for the prevention of infection with a succinocarpus which is a fish parasite containing δ-aminolevulinic acid as an active ingredient.