COMPOSITION FOR EXTERMINATING FISH PARASITES

Abstract

The present invention relates to a composition for exterminating fish parasites comprising an extract of Juglans mandshurica bark as an active ingredient, making it possible to safely and effectively exterminate scuticociliates using an extract derived from a natural material, instead of formalin that is highly harmful, which has been conventionally used to treat scuticociliatosis in fish.

Claims

1. A composition for exterminating a fish parasite comprising an extract of Juglans mandshurica bark as an active ingredient.

2. The composition according to claim 1, wherein the parasite is a scuticociliate.

3. The composition according to claim 1, wherein the extract of Juglans mandshurica bark is an extract using a solvent selected from the group consisting of ethanol, methanol, chloroform, and water.

4. The composition according to claim 1, wherein the extract of Juglans mandshurica bark is an ethanol extract.

5. A method of exterminating a fish parasite comprising orally administrating or bath administrating the composition according to claim 1.

6. The method according to claim 5, wherein the fish parasite is a scuticociliate.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0013] FIG. 1 shows herbal materials used for screening aquatic anthelmintic agent.

[0014] FIG. 2 shows results confirming the cytotoxicity of Juglans mandshurica bark extracts depending on the concentration of ethanol that is used for extraction.

[0015] FIG. 3 shows the mortality rates (A: LC.sub.50, B: LD.sub.50) after administration of the Juglans mandshurica bark extract to olive flounder.

[0016] FIG. 4 shows results of blood biochemical analysis of an acute toxicity test (LC.sub.50) after bath administration of olive flounder with the Juglans mandshurica bark extract (ALP, alanine aminotransferase; GOT, glutamic oxalacetic transaminase; GPT, glutamic pyruvate transaminase; GLU, glucose; BUN, blood urea nitrogen).

[0017] FIG. 5 shows scuticociliate extermination effects depending on the type of administration of the Juglans mandshurica bark extract to olive flounder.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS OF THE INVENTION

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those typically understood by those skilled in the art to which the present invention belongs. In general, the nomenclature used herein is well known in the art and is typical.

[0019] In the present invention, in order to exterminate scuticociliates, which are fish parasites causing mass mortality in aquaculture olive flounder, using a natural extract that is free from residual problems and is safe and highly efficacious, herbal extracts are prepared, and extracts having scuticociliate killing activity are screened, confirming that the ethanol extract of Juglans mandshurica bark exhibits high scuticociliate killing activity.

[0020] Accordingly, in an aspect, the present invention relates to a composition for exterminating fish parasites comprising an extract of Juglans mandshurica bark as an active ingredient.

[0021] In the present invention, the fish parasite may be a scuticociliate.

[0022] In the present invention, the extract of Juglans mandshurica bark may be an extract using a solvent selected from the group consisting of ethanol, methanol, chloroform, and water, and is preferably an ethanol extract.

[0023] The ethanol extract of Juglans mandshurica bark according to the present invention may be a 100-10% ethanol extract, preferably a 90-20% ethanol extract, more preferably an 80-30% ethanol extract, much more preferably a 70-40% ethanol extract, even much more preferably a 60-45% ethanol extract.

[0024] In the present invention, the Juglans mandshurica bark is the bark of Juglans mandshurica, and there are 15 species of ‘Juglandaceae’ and ‘Juglans’ worldwide, and 4 species including those cultivated in Korea.

[0025] The four species cultivated in Korea are Juglans mandshurica (Juglans cathayensis Dode, Juglans draconis Dode, Juglans formosana Hayata), Juglans regia (walnut tree, Persian walnut), Juglans cordiformis, and Juglans sieboldiana (Juglans mandshurica var. sieboldiana Makino). Juglans mandshurica contains large amounts of hydrojuglone and tannins, along with quinone, phenol, flavonoids, coumarine, lignan, and steroids. It is known that the bark, root, leaf, unripe fruit peel, etc. of Juglans mandshurica have anthelmintic agent and sterilization effects, and particularly hydrojuglone secreted from the root has cytotoxic and anti-inflammatory effects, and tannin has disinfecting and anti-inflammatory effects through astringent action.

[0026] In another aspect, the present invention relates to a method of exterminating fish parasites comprising orally administrating or bath administrating the composition described above.

[0027] In the present invention, the fish parasite may be a scuticociliate.

[0028] A better understanding of the present invention may be obtained through the following examples. These examples are merely set forth to illustrate the present invention, and are not to be construed as limiting the scope of the present invention, as will be apparent to those skilled in the art.

Example 1: Production of Aquatic Anthelmintic Agent Candidates Derived from Natural Materials

[0029] Herbal materials and seaweeds were chosen and extracted as candidates for aquatic anthelmintic agent. Herbal extracts were prepared using different solvents (hot water and ethanol). A solvent was added to 50 g of the herbal material, followed by ultrasonic extraction (three times), concentration under reduced pressure, and freeze-drying to eliminate the effect of the extraction solvent. Then, the resulting product was dissolved in dimethyl sulfoxide (DMSO), administered at different concentrations, and used to measure antiparasitic efficacy.

[0030] Extracts of 15 herbal materials (FIG. 1) known to have antiparasitic efficacy were prepared using solvents (ethanol and hot water), concentrated under reduced pressure, and freeze-dried, after which the efficacy against scuticociliates depending on the concentration (0 to 10,000 mg/L) thereof was measured. Therefore, antiparasitic efficacy was confirmed in Ostericum koreanum, Juglans mandshurica bark, Torilis japonica, and Lappula echinata, and the criterion for efficacy was determined to be 100 mg/L, similar to the aquatic formalin dosage concentration (Table 1).

Example 2: Confirmation of Antiparasitic Efficacy of Herbal Materials and Investigation of Drug Effect Concentration

[0031] The ability of the herbal extracts prepared in Example 1 to kill scuticociliates was confirmed.

[0032] Scuticociliates were isolated from the abdominal cavity of subjects infected therewith, inoculated into Hirame natural embryo (HINAE) cells, and cultured in MEM (minimum essential media, Welgene) supplemented with 1% antibiotics (Gibco) and 10% FBS (Gibco) in an incubator at 20° C. After culture for 3 to 4 days to full confluency, scuticociliates were centrifuged at 3000 g and 4° C. for 10 minutes, and the supernatant was removed to separate scuticociliates, which were then dispensed at 1×10.sup.4/well into a 96-well plate and used in experiments. The extract was added at different concentrations to the cultured scuticociliates, and the killing efficacy (extent of killing of scuticociliates, movement, and extent of cytoplasmic destruction) was observed at intervals of 10 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours in an incubator at 20° C.

[0033] The efficacy of killing scuticociliates was evaluated to be the highest in Ostericum koreanum, but strong fish toxicity was confirmed upon oral administration of Ostericum koreanum and thus histopathologic examination was carried out. Consequently, exfoliation of the gill epithelium, vacuolar degeneration of the kidneys, and liver atrophy were observed, and drug toxicity response appeared generally, and the efficacy was ultimately determined in the order of Juglans mandshurica bark>Lappula echinata>Ostericum koreanum.

TABLE-US-00001 TABLE 1 Confirmation of ability of herbal materials to kill scuticociliates 100% EtOH extract dosage Treatment concentration (mg/L) Candidate time Control 1 10 100 1,000 10,000 Ostericum 10 min 100% 100% 100%  80% Death Death koreanum 30 min 100% 100% 100%  50% Death Death 1 h 100% 100% 100% Death Death Death 2 h 100% 100% 100% Death Death Death 4 h 100% 100% 100% Death Death Death Juglans 10 min 100% 100% 100% 100% Death Death mand- 30 min 100% 100% 100%  50% Death Death shurica 1 h 100% 100% 100%  10% Death Death bark 2 h 100% 100% 100% Death Death Death 4 h 100% 100% 100% Death Death Death Torilis 10 min 100% 100% 100% 100% 100% Death japonica 30 min 100% 100% 100% 100%  50% Death 1 h 100% 100% 100%  50%  50% Death 2 h 100% 100% 100%  30% Death Death 4 h 100% 100% 100% Death Death Death Lappula 10 min 100% 100% 100% 100% Death Death echinata 30 min 100% 100% 100% 100% Death Death 1 h 100% 100% 100% 100% Death Death 2 h 100% 100% 100%  50% Death Death 4 h 100% 100% 100%  50% Death Death

[0034] Thus, the Juglans mandshurica bark extract was chosen as the final candidate, and a 50% ethanol extract was ultimately determined for commercialization of safe aquatic anthelmintic agent according to alcohol methods.

TABLE-US-00002 TABLE 2 100% EtOH extract dosage Treatment concentration (mg/L) Candidate time Control 1 10 100 1,000 10,000 Juglans 10 min 100% 100% 100% 100% Death Death mand- 30 min 100% 100% 100%  70% Death Death shurica 1 h 100% 100% 100%  20% Death Death bark 2 h 100% 100% 100% Death Death Death 100% EtOH 4 h 100% 100% 100% Death Death Death Juglans 10 min 100% 100% 100%  20% Death Death mand- 30 min 100% 100% 100%  20% Death Death shurica 1 h 100% 100% 100% Death Death Death bark 2 h 100% 100% 100% Death Death Death 70% EtOH 4 h 100% 100% 100% Death Death Death Juglans 10 min 100% 100% 100%  20% Death Death mand- 30 min 100% 100% 100%  20% Death Death shurica 1 h 100% 100% 100% Death Death Death bark 2 h 100% 100% 100% Death Death Death 50% EtOH 4 h 100% 100% 100% Death Death Death

[0035] In addition, experimentation was conducted to determine a specific active concentration of the 50% ethanol extract of Juglans mandshurica bark, confirming that scuticociliates were killed at a final concentration of 40 mg/L. Accordingly, experimentation was performed at 40 mg/L.

TABLE-US-00003 TABLE 3 Confirmation of scuticociliate killing activity of 50% ethanol extract of Juglans mandshurica bark depending on dosage concentration and treatment time 50% EtOH extract dosage Treatment concentration (mg/ L) Candidate time Control 10 20 40 60 80 100 200 Juglans 10 min 100% 100% 100% 100% 90% 90% 60% 60% mandshurica 30 min 100% 100%  90%  60% 50% 40% 50% 50% bark 1 h  100% 100%  80%  50% 30% Death Death Death 50% EtOH 2 h  100% 100%  80% Death Death Death Death Death 4 h  100% 100%  80% Death Death Death Death Death

Example 3: Confirmation of Cytotoxicity of Herbal Extract

[0036] Measurement was performed on cytotoxicity of the prepared extracts using HINAE cells. Cells at 1×10.sup.5/well were cultured to 80% density at 20° C. for 24 hours, and then treated with the extracts at different concentrations (0 to 1,000 mg/L). After incubation for 48 hours, absorbance was measured at 450 nm using a cytotoxicity measurement drug (CCK-8, Dojindo). Based on results, as shown in FIG. 2, cytotoxicity was exhibited at concentrations of 40 mg/L or more when using the 100% ethanol extract of Juglans mandshurica bark, but did not appear even at a concentration of 200 mg/L when using the 50% ethanol extract of Juglans mandshurica bark.

Example 4: Fish Acute Toxicity Test of Natural Aquatic Anthelmintic Agent

[0037] In order to evaluate the safety of Juglans mandshurica bark, an acute toxicity test (lethal concentration 50%: LC.sub.50, lethal dose 50%: LD.sub.50) after bath administration or oral administration was conducted in 10 olive flounder (13.9±0.9 cm, 27.1±4.6 g) for each experimental group. A 50% ethanol extract of Juglans mandshurica bark was prepared at 0 to 1,000 mg/L and used up to 1,000 mg/L, including the control, for bath administration, and was prepared at concentrations of up to 400 mg/kg B.W. and used for oral administration. After addition of the extract at each concentration, mortality rates were measured every day for 96 hours, and blood and histopathological examinations of surviving subjects were performed. For histopathological analysis, the gills, heart, intestines, kidneys, liver, and spleen of experimental fish were fixed in 10% neutral formalin, dehydrated, and paraffin-embedded, and the sliced tissues were stained with hematoxylin-eosin (H&E) and observed under a microscope.

[0038] Based on the results, as shown in FIG. 3, LC.sub.50 (96 h) was measured to be 509.06 mg/L, and LD.sub.50 (96 h) was measured to be 805.8 mg/kg B.W.

[0039] Based on results of blood biochemical analysis of the acute toxicity test (LC.sub.50) after bath administration, as shown in FIG. 4, the items that are functional indicators of the liver and kidneys, which are the main target organs for drug toxicity, were not different compared to the control. Thereamong, GOT and GPT levels showed a tendency to greatly decrease at 40 mg/L, which was judged to be closely related to the hepatoprotective effect among the effects of Juglans mandshurica bark.

[0040] Based on results of acute toxicity evaluation, in the acute toxicity test (LC.sub.50, LD.sub.50) after bath or oral administration of olive flounder using the Juglans mandshurica bark extract, this extract did not show any harmful effects on blood biochemistry and histopathology at 160 mg/L and 160 mg/kg B.W., indicating safety.

Example 5: Confirmation of Efficacy of Juglans mandshurica Bark Extract as Anthelmintic Agent

[0041] Healthy olive flounder without disease were purchased from olive flounder farms in Chungcheongnam-do, Korea, and used as experimental fish after acclimatization for 2 weeks at a water temperature of 20±1° C. in a seawater flow-through land tank. On the 2.sup.nd day after artificially infecting 10 experimental fish for each group with 1×10.sup.5 cell/fish of scuticociliates (I, II) stored in the Pathology Research Department of the National Institute of Fisheries Science (Type I and II scuticociliates), the extract was added to feed pellets at a concentration of 40 mg/L, and the fish were anesthetized and subjected to oral administration or bath administration therewith once or twice. To this end, a powdery feed mixture was formed into feed pellets (about 0.25 g), after which a Juglans mandshurica bark powder was added to a concentration of 40 mg/kg B.W., followed by coating with a feed paste once more. In the control for oral administration, feed pellets, prepared in the same manner with the exception that the extract was not added, were orally administered to fish that were anesthetized. Then, fish death by scuticociliates was confirmed.

[0042] The results of scuticociliate extermination effect are shown in FIG. 5. There was no significant pathogenicity due to a difference in the type of scuticociliates, and based on results of fish death caused by scuticociliates for 17 days, the oral administration group showed a relative survival rate of 60-80%, and the bath administration group showed a relative survival rate of 50-80%.

INDUSTRIAL APPLICABILITY

[0043] According to the present invention, it is possible to safely and effectively exterminate scuticociliates using an extract derived from a natural material, instead of formalin that is highly harmful, which has been conventionally used to treat scuticociliatosis in fish.

[0044] Although specific embodiments of the present invention have been disclosed in detail above, it will be obvious to those skilled in the art that the description is merely of preferable exemplary embodiments and is not to be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents. Simple modifications or alterations of the present invention may be easily used by those of ordinary skill in the art, and all such modifications or alterations may be considered to be included in the scope of the present invention.