KR20110078053A - Composition for Parasitology of Cultured Fishes Using Conifer Leaf Extract - Google Patents

Abstract

PURPOSE: An anthelmintic composition containing conifer leaf extract is provided to eliminate parasites from cultured fishes by an environmentally-friendly method. CONSTITUTION: An anthelimintic composition for eliminating parasites from cultured fishes contains one or more water extract selected from Pinus densiflora, Cryptomeria japonica, and Chamaecyparis obtuse. The composition is used together with hydrochloric acid, acetic acid, or pyroligneous liquor to adjust pH concentration.

Description

{Anthelmintic composition for farming fish parasite using needles leaf plant extracts}

The present invention relates to a parasitic insect repellent composition using coniferous leaf extract, and more particularly, to a composition comprising a water extract of coniferous leaf composed of pine, cypress and cedar as an active ingredient. The present invention relates to a composition for killing parasites of farmed fishes, which has a strong fighting effect against parasitic philasterides dicentrarchi , Ichthyophthirius multifiliis , and the like.

China's aquaculture industry has been rapidly developed along with general industrialization. The share of aquaculture fish in total fishery output has risen from 26.0% in 2000 to 34.6% in 2005, among which the production of seawater fish continues to increase. Sea fish species include flounder, sea bass, rockfish, perch, defense, dome, and abalone.

When fish are farmed, unlike the natural state, they are kept in a closed environment, and various diseases are caused by the breeding environment. That is, various diseases are caused by water pollution, aging of fish farms, cultivation of varieties, overcrowding, etc., and the death rate of farmed fish is 10 to 20% of the total farmed fish.

Diseases affecting farmed fish can be divided into bacterial and parasitic diseases.

Bacterial diseases are typical of vibrio bacteria, slide bacteria, streptococci, and Edward bacteria, which are caused by single infection or mixed infection. Antibiotic and antimicrobial agents are the most common for bacterial diseases of farmed fish. However, repeated and excessive use of antibiotics and antimicrobials, which have been used until now, leads to the acquisition of fish resistance, making it more difficult to remedy diseases, and chemicals accumulate in fish, which adversely affects the human body and causes environmental pollution. There is a problem.

A parasitic disease may be a disease caused by such charge Surgical urticae (philasterides dicentrarchi), baekjeomchung (Ichthyophthirius multifiliis). For parasitic diseases caused by farmed fish, chemicals such as formalin and hydrogen peroxide are mainly used. However, repeated use of such chemicals leads to fish stress, which slows growth and is known to have very low effects on parasite control. As an antiparasitic method for the prevention of parasitic diseases, Korean Patent No. 379026 discloses a method for controlling Scutica insects parasitic on cultured flounder by ketoconazole drug, and Korean Patent No. 820247 extracts pine nodes with methanol. Also disclosed is a composition for controlling Scutica insects by extracting Songjeol extract obtained by extracting with hexane.

Meanwhile, conifer extracts are already well known to have antimicrobial, antibiotic and antioxidant activity. For example, the extract of cedar ( Cryptomeria japonica ) has an antimicrobial activity against bacteria and the like [Korean Journal of Oriental Ophthalmology & Otolaryngology & Dermatology 19 (3), 68-74; J. Agric. Food Chem. 53, 614-619 (2005); J. Wood Sci ., 52, 552-556 (2006); Phytotherapy Research , 21, 295-299 (2007); J. of Agricultural and Food Chemistry , 53, 614-619 (2005), silverfish ( Lepisma saccharina ), insecticidal action against mosquitoes [ J. Wood Sci ., 522-526 (2006); Bioresource Technology 100, 465-470 (2009). Antioxidant Activity of Chamaecyparis obtusa Extracts [ Forest , No.194, pp.6-7 (2007)], Antibacterial Activity [ Fitoterapia , 78, 149-152 (2007). Antioxidant Activity of Pinus densiflora Extracts [ Journal of Life Science , vol 14 (5), 863-867 (2004); Korean J. Food Sci. Technol. , vol 31 (2), 527-534 (1999).

According to the literature published so far, there is no case in which coniferous water is extracted and used as it is using water sole solvent as the extraction solvent. Most of the extracts are extracted using a polar solvent such as alcohol or water firstly, and then reextracted with a nonpolar organic solvent as a second active ingredient. Therefore, the extract solid material is dissolved in a nonpolar organic solvent prior to use.

On the contrary, the conifer extract, which is characterized by the present invention, is a water extract, which is obtained by using no organic solvent at all, and shows a considerable difference in its composition from the conventional conifer extract using a nonpolar organic solvent. In addition, extract solids can be dissolved in water and applied to aquaculture farms in aqueous solution.

It is an object of the present invention to provide a composition for parasite control parasites which is excellent in controlling parasites of cultured fish.

An object of the present invention is to provide a composition for parasite repellent natural farmed fish that environmentally combats parasites that cause fatal damage to the aquaculture industry, such as flounder, sea bass, rockfish, perch, defense, dome, abalone.

An object of the present invention is to provide a water extract of the antioxidant coniferous leaves as an active ingredient of the composition for aquaculture parasites.

In order to solve the above problems, the present invention includes a water (Water) extract extracted with one or two or more coniferous leaves selected from pine ( Pinus densiflora ), cedar ( Cryptomeria japonica ) and cypress ( Chamaecyparis obtusa ) as water as an active ingredient. Characterized by the composition for parasite repellent fish farming.

In addition, the present invention is an aqueous solution composition comprising a water extract of the conifer leaf, characterized in that the composition for controlling parasites parasites in which the pH of the aqueous solution is adjusted to the range of 1.5 to 7.0.

The composition of the present invention has the effect that can be fully controlled of the Scotica ( philasterides dicentrarchi ), white bug (Ichthyophthirius multifiliis).

The composition of the present invention is applied to the site of fish farming has the effect of increasing the survival rate of the flounder infected with Scutika insects more than 70%.

The composition of the present invention has the effect of increasing the production of fish due to combating fish parasites.

The composition of the present invention is safe for fish and workers as a natural medicine, and does not cause environmental pollution, so it has excellent environmental friendliness.

The composition of the present invention is used in combination with the cultured water in the liquid form has an effect that is convenient to use.

The present invention is a water extract extracted from coniferous leaves, such as pine ( Pinus densiflora ), Cypress ( Chamaecyparis obtusa ), Cedar ( Cryptomeria japonica ) collected in June-October 2009, as an active ingredient The present invention relates to a cultured parasite repellent.

Pine, cypress, and cedar are known to have antioxidant and antimicrobial activities as conifers and to have antioxidant activities in the order of cypress> cedar> pine. The active ingredients contained in pine, cypress, and cedar, respectively, are as follows. Pine contains oinene, pinene, camphene, quercetin, kaempferol, and antioxidant furanone derivatives as active ingredients. Cypress has active ingredients such as pinene, tujone, limonne, mycene, sabinene, terpineol, and terpinene-4ol. -ol), iso-bomyl acetate, terpinyl acetate and the like. Cedar has active ingredients such as guiacol derivatives, eugenol derivatives, vanillin, lignin, elemol, terpineol, pinene and savinen (sabinene), terpinene-4-ol, 10-cadinem-4-ol, cardinene, isoledene, mulorene Numerous compounds such as muurolene are included.

However, it is the first fact revealed through the present invention that the water extract of pine, cypress, or cedar has an excellent effect on the control of cultured fish parasites such as Scutika, white spotworm. In particular, the conventional natural extract is obtained by using an organic solvent such as alcohol, hexane to obtain an industrially usable level of extraction yield, the extract of the present invention can extract the active ingredient in high concentration with water alone.

In addition, the present invention is characterized in that the final acidity (pH) of the solution to prepare a composition for aquaculture parasites parasitic insect repellent in the range of 1.5 to 7.0, preferably 1.5 to 4.5. In general, Scutica is strongly dependent on pH, so it is difficult to grow under strong acid conditions of pH 5 or lower, or strong base conditions of pH 10 or higher, and survival in neutral regions of pH 6 to 9 is easy. The composition of the present invention has a remarkable effect, even when prepared and treated in a neutral region of pH 7, and when the final pH is maintained in an acid range of 1.5 to 4.5 using an inorganic acid, such as hydrochloric acid, The effect can be much improved. The acid solution to be used for the acidity control of the present invention is an acidic solution such as hydrochloric acid, acetic acid or wood vinegar, and may be diluted with water.

The present invention as described above will be described in more detail based on the following Preparation Examples, Examples and Test Examples, but the present invention is not limited thereto.

Preparation Example Preparation of Extract

Preparation Example 1 Preparation of Cypress Leaf Extract

The leaves of the cypress ( Chamaecyparis obtusa ) were collected and pulverized, 1 kg of nonwoven fabric was added, 10 L of purified water was added, and extracted at 1 pressure and 95 ° C. for 3 hours. The extract was cooled to room temperature for 1 hour and then filtered to give 9 L of a bluish brown solution. The pH of this solution was 3.90. The solution was evaporated under reduced pressure for 2 hours while gradually raising the temperature of the water bath from 10 ° C. to 60 ° C. to obtain 57 g (0.63%) of a brown solid.

[HPLC analysis]

In order to confirm the active ingredient of the cypress tree extract obtained by the above method, a mixture of 0.1% phosphoric acid and acetonitrile was analyzed by HPLC at 210 nm and 254 nm conditions in the concentration gradient of Table 1 below.

Residence time Solvent Gradient (%)  0.1% phosphoric acid Acetonitrile 0 min 90 10 10 min 30 70 20 min 30 70

The HPLC analysis of the cypress tree extract is attached to FIG. 1. According to the results of FIG. 1, the retention time was 1.3 to 11.5 minutes in the UV 210 nm condition, and the retention time was 1.3 to 9.2 minutes in the UV 254 nm condition.

Preparation Example 2 Preparation of Cedar Leaf Extract

25 Kg of leaves of cedar ( Cryptomeria japonica ) were collected and pulverized, and then divided into 1 kg of nonwoven fabric, 230 L of purified water was added, and extracted at 1 pressure and 95 ° C. for 3 hours. The extract was cooled to room temperature for 2 hours and then filtered to yield 220 L of a bluish brown solution. The pH of this solution was 4.08. The solution was evaporated under reduced pressure for 5 hours while slowly raising the temperature of the water bath from 10 ° C to 70 ° C. At this time, since the evaporation while raising the temperature to about 70 ℃ from the beginning to form a bubble (overflow), at first the evaporation under reduced pressure at low temperature and gradually raise the temperature to obtain a solid residue of all components. The solid residue was further dried for 2 days in a vacuum pump to give 1826 g (0.83%) of a brown solid.

In order to confirm the active ingredient of the cedar tree extract obtained by the above method, HPLC analysis was carried out under the conditions of Preparation Example 1, and the results were attached as FIG. 2. According to the results of FIG. 2, the retention time was 3.1 to 13 minutes in the UV 210 nm condition, and the retention time was 2 to 15 minutes in the UV 254 nm condition.

Preparation Example 3 Preparation of Water Extract of Pine ( Pinus densiflora ) Leaves

The leaves of pine ( Pinus densiflora ) were collected and pulverized, 1 kg of nonwoven fabric was added, 10 L of purified water was added, and extracted at 1 pressure and 95 ° C. for 3 hours. The extract was cooled to room temperature for 1 hour and then filtered to give 9 L of a bluish brown solution. The pH of this solution was 4.10. The solution was evaporated under reduced pressure for 2 hours while gradually raising the temperature of the water bath from 10 ° C. to 60 ° C. to obtain 57 g (0.63%) of a brown solid.

In order to confirm the active ingredient of the pine tree extract obtained by the above method, HPLC analysis was carried out under the conditions of Preparation Example 1, and the results were attached as FIG. 3. According to the results of FIG. 3, the retention time was 1.8 to 9 minutes in the UV 210 nm condition, and the retention time was 1.3 to 6.8 minutes in the UV 254 nm condition.

Preparation Example 4 Preparation of Leaf Extract of Cypress and Cedar

Cypress leaves and cedar leaves were mixed in a 1: 1 weight ratio, pulverized by taking a total weight of 1.5 kg, and then put into an extractor, 17 L of purified water was added, and extracted at 1 pressure and 95 ° C. for 3 hours. The extract was cooled to room temperature for 3 hours and then filtered to obtain 16 L of the extract mixture. The pH of this mixed solution was 3.79. 1 L of the mixed extract was evaporated under reduced pressure for 2 hours while gradually raising the temperature of the water bath from 10 ° C. to 70 ° C. to obtain 7.1 g (0.71%) of a brown solid.

Preparation Example 5 Preparation of Pine, Cypress and Cedar Leaf Extracts

Pine, cypress and cedar leaves were mixed in a 1: 1: 1 weight ratio, pulverized by taking a total weight of 3 kg, and then put into an extractor, 30 L of purified water was added, and extracted at 1 pressure and 95 ° C. for 5 hours. The extract was cooled to room temperature for 12 hours and then filtered to give 28 L of an off-brown extract mixture. The pH of this mixed solution was 4.03. Take 1 L of the extraction mixture and evaporate under reduced pressure for 2 hours while gradually raising the temperature of the water bath from 10 ° C. to 70 ° C. with a vacuum evaporator, and dry for 3 days with a vacuum pump for 1 day at room temperature and 6.9 g of a brown solid material ( 0.69%).

In order to confirm the active ingredients of pine, cypress and cedar leaf extracts obtained by the above method, HPLC analysis was carried out under the conditions of Preparation Example 1, and the results are attached as FIG. 4. According to the results of FIG. 4, the retention time was 1.4 to 11.5 minutes in the UV 210 nm condition, and the retention time was 1.3 to 9 minutes in the UV 254 nm condition.

EXAMPLES Preparation of a Parasitic Insect Repellent for Cultured Fishes

To prepare a composition for parasite repellents for farmed fish at a composition ratio as shown in Tables 2 and 3.

division Compositions for Parasitology of Cultured Fishes extract PH (pH) Example 1  Cypress Extract (Manufacturing Example 1) 3.90 Example 2  Cedar Extract (Manufacturing Example 2) 4.08 Example 3  Pine Extract (Manufacturing Example 3) 4.10 Example 4  Cypress + Cedar Extract (Manufacturing Example 4) 3.79 Example 5  Pine + Cypress + Cedar Extract (Manufacturing Example 5) 4.03

division Compositions for Parasitology of Cultured Fishes extract Acid solution PH (pH ^* ) Example 6  Cypress Extract (Manufacturing Example 1) Hydrochloric acid  1.88 Example 7  Cedar Extract (Manufacturing Example 2) Acetic acid 1.97 Example 8  Cypress + Cedar Extract (Manufacturing Example 4) Wood vinegar 2.07  Final pH of the pesticidal composition adjusted by addition of acid solution

[Test Example] Confirm the insecticidal effect of the composition

Test Example 1 In Vitro Insecticidal Insecticidal Effect Test ( In vitro )

The antiparasitic effect on the antiparasitic composition of the present invention was performed by the following procedure according to the treatment concentration of the antiparasitic composition and the time elapsed after the treatment.

Scutika insects were isolated from the gills of 16 cm Scutika infected halibut in the Jeju fish farm. Scutika worms were ovate-droplets with a pointed cilia, and their movement was very agile and active. Petri dishes (Petri dish, 8.7 cm in diameter) were filled with 20 mL of sterile seawater and the harvested Scutica worms were suspended.

Insect repellent effect test was injected into the sterile seawater and filling mixture liquid concentrations of 10 ppm, 20 ppm, 30 ppm. Scutica insects were taken at each time period, placed on a slide glass, covered with a cover glass, and the movement of cells and cell destruction were observed using a 400 magnification microscope, comparing the degree of movement and the morphological characteristics of the insects. The control group was not treated with the antiparasitic composition.

Elapsed time (min.) Example 1 Insecticidal Effect by Treatment Concentration of Composition  10 ppm  20 ppm 30 ppm Control 0 +++ +++ +++ +++ 10 ++ ++ ++ +++ 30 ++ + + +++ 40 + + + +++ 50 + + + +++ 60 + + + +++ +++: caries movement active (proliferation) ++: caries movement normal (survival)
+: Weak tooth movement (decrease)-: Loss of tooth movement (death)
-: Ovum Drip Formation Destruction (Destruction)

Elapsed time (min.) Example 2 Insecticidal Effect by Composition  10 ppm  20 ppm 30 ppm Control 0 +++ +++ +++ +++ 10 ++ ++ ++ +++ 30 ++ ++ + +++ 40 + + + +++ 50 + + + +++ 60 + + + +++ +++: caries movement active (proliferation) ++: caries movement normal (survival)
+: Weak tooth movement (decrease)-: Loss of tooth movement (death)
-: Ovum Drip Formation Destruction (Destruction)

Elapsed time (min.) Example 3 Insecticidal Effect by Treatment Concentration of Composition  10 ppm  20 ppm 30 ppm Control 0 +++ +++ +++ +++ 10 ++ ++ ++ +++ 30 ++ ++ + +++ 40 ++ + + +++ 50 + + + +++ 60 + + + +++ +++: caries movement active (proliferation) ++: caries movement normal (survival)
+: Weak tooth movement (decrease)-: Loss of tooth movement (death)
-: Ovum Drip Formation Destruction (Destruction)

Elapsed time (min.) Example 4 Insecticidal Effect by Composition  10 ppm  20 ppm 30 ppm Control 10 ++ ++ ++ +++ 30 ++ ++ + +++ 40 + + + +++ 50 + + - +++ 60 + + - +++ +++: caries movement active (proliferation) ++: caries movement normal (survival)
+: Weak tooth movement (decrease)-: Loss of tooth movement (death)
-: Ovum Drip Formation Destruction (Destruction)

Elapsed time (min.) Example 5 Insecticidal Effect by Composition  10 ppm  20 ppm 30 ppm Control 10 ++ ++ ++ +++ 30 ++ ++ + +++ 40 + + - +++ 50 + + - +++ 60 + + - +++ +++: caries movement active (proliferation) ++: caries movement normal (survival)
+: Weak tooth movement (decrease)-: Loss of tooth movement (death)
-: Ovum Drip Formation Destruction (Destruction)

Elapsed time (min.) Insecticidal effect at 30 ppm concentration of composition  Example 6 Composition Example 7 Composition Example 8 Composition Control 10 ++ ++ ++ +++ 30 + + + +++ 40 - - - +++ 50 - - - +++ 60 - - - +++ +++: caries movement active (proliferation) ++: caries movement normal (survival)
+: Weak tooth movement (decrease)-: Loss of tooth movement (death)
-: Ovum Drip Formation Destruction (Destruction)

As a result of the in vitro test of Tables 3 to 9, the Scutica insects before the treatment of the insecticidal composition were in an oval transparent liquid membrane tube, but the Scutica insects that were stopped as time passed after the treatment of the insecticidal composition were transformed into a circular state. It was observed that the protein surface was damaged and crumbled and disappeared due to its disappearance. 5 and 6

Test Example 2 Insect Repellent Insect Effects in Flounder in a Small Tank ( In vivo )

Flounder fry (approximately 6 cm) About 1 to 2 months (about 10 to 15 cm in length) after stocking, those classified as lung flounder due to Scutika insects, 60 cm wide, 1 m long, 15 high gathered in a small FRP tank of cm. Seawater was collected, filtered, sterilized at 80 ° C. for 1 hour, cooled to room temperature, and filled in two tanks each about 10 cm high. Here, Scutika infected flounder was divided into control and experimental groups, and 6 animals were put in each.

The pesticidal composition was added to 30 ppm and bathed for 2 hours. At this time, the temperature of the tank was 21 ℃, pH was 6.5. After 24 hours, the same bath solution was repeatedly bathed. During bathing, the Scutica worms were collected from the flounder gill or epidermis, and the movement and cell destruction of Scutica worms were observed under a 400 magnification microscope.

Halibut number Example 8 Insecticidal Effect of the Composition (30 ppm) 30 minutes 60 minutes 90 minutes 120 minutes One ++ + - - 2 + + + - 3 ++ - - - 4 ++ + - - 5 ++ + - - 6 ++ - - - Control
(6 flounder) +++
1 dead +++
1 horse +++
1 dead +++
1 dead +++: caries movement active (proliferation) ++: caries movement normal (survival)
+: Weak tooth movement (decrease)-: Loss of tooth movement (death)
-: Ovum Drip Formation Destruction (Destruction)

In Example 8 composition in vitro test results of Table 9, the Scutica insects at 30 ppm concentration was gradually reduced, weak and destroyed in 30 minutes, but in the in vitro test of Table 10 Skutika at 30 ppm concentration The caries died in 90 to 120 minutes. The reason for the specific effect after a certain period of time after bathing is that the skin of the flounder is made of mucus protein and the Scutika insects are parasitic in the skin of the mucus, so the insect repellent composition penetrates the skin of the flounder to control the Scutika insects. I need time.

Experimental Example 3. Scutica insect repellent effect test in flounder farm

10 m long, 10 m wide, and 1.2 m high, each tank was adjusted to 10 tons (10 cm tall) of seawater, and then flocks of about 10 to 15 cm in size were infected. In addition, the pesticidal composition was added to the bath solution at a concentration of 30 ppm, and bathed for 3 hours. At this time, the temperature of the bath was W 23 ℃ A, W 25 ℃ W, pH was 6.0 W 6.0, B 6.9. After the bath was filled with about 30 to 50 tons of seawater while returning to fresh seawater. After 24 hours, the bath was repeated twice more in the same manner as above. There was no stress on the flounder during the test, and the feeding condition before and after bathing was good, so there was no need for a separate test for fish safety. The control was also performed in the same way. After the bath was finished, the mucus epidermis in the ulcer area or the back was taken at a magnification of 400 times under a microscope and placed on a slide glass and covered with a cover glass.

Example 1 Composition Bathing Treatment exam
Halibut
(Mari) Our flounder (mari) Cumulative Death
(Mari) Olive flounder survival rate (%) 1 day 2 days 3 days 5 days Test Wando A 500 68 72 20 3 163 67.4 Wando B 500 49 33 28 22 132 73.6 Experimental Average Survival Rate 1,000 117 105 48 25 295 70.5 Control 500 47 39 141 217 444 11.2

As a result of the microscopic observation of Table 11, the control was observed to be very active in the mucosa of the scuticaticidal movement, but the test was observed that after the treatment of the drug is significantly slowed or weakened and the movement of the squatica insect finally stopped and destroyed. Before the bath treatment, the active Scutika insects were in the oval transparent liquid membrane tube, but after treatment, the Scutica insects that had stopped after the treatment were deformed into a circular state, and the surface of the protein was damaged and broken. As a result, the number of flounder mortality in the test plot was significantly reduced, whereas the flounder mortality in the control was increased.

As described above, the cultured fish parasite repellent composition of the present invention has an excellent effect on combating such as parasitoids ( philasterides dicentrarchi ), whiteworm ( Ichthyophthirius multifiliis ) parasitic halibut , rockfish, abalone. In addition, the active ingredient included in the composition of the present invention is a coniferous leaf water extract (water) has excellent environmental friendliness, significantly reducing water pollution and exhibiting a selective control effect on parasites while maximally inhibiting the death of farmed fish. The composition of the present invention is actually applied to aquaculture farms to obtain an effect of increasing the survival rate of the flounder infected with Scutika insects to 70% or more. The survival rate of the control was 11%.

Therefore, the composition of the present invention has the utility that can be applied to the aquaculture industry to create a high profit.

1 is an HPLC analysis result of the water extract of the leaves of Chamaecyparis obtusa measured at 210 nm and 254 nm.

Figure 2 is an HPLC analysis of the water extract of the leaves of cedar ( Cryptomeria japonica ) measured at 210 nm and 254 nm.

Figure 3 is an HPLC analysis of the water extract of pine ( Pinus densiflora ) leaves measured at 210 nm and 254 nm.

Figure 4 is an HPLC analysis of the water-soluble extract mixture of pine leaves, cypress leaves and cedar leaves measured at 210 nm and 254 nm.

5 is a micrograph (× 400) of a Scutika caterpillar active before bathing.

6 is a micrograph (× 400) of the Scutica insects killed after bathing.

Claims (3)

A composition for insect repellent parasites including water extracts of one or two or more coniferous leaves selected from pine ( Pinus densiflora ), cedar ( Cryptomeria japonica ) and cypress ( Chamaecyparis obtusa ). The method according to claim 1, The composition for parasite insect repellents prepared in an aqueous solution of pH 1.5 to 7.0 range. The method according to claim 2, A composition for parasite insect repellents using hydrochloric acid, acetic acid or wood vinegar to adjust the pH of the aqueous solution.

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