WO2002018500A1

WO2002018500A1 - Anti-fouling agent extracted from marine algae

Info

Publication number: WO2002018500A1
Application number: PCT/KR2001/001460
Authority: WO
Inventors: Hyun-Woung Shin; Yong-Ki Hong
Original assignee: Nabichem Co., Ltd.
Priority date: 2000-08-31
Filing date: 2001-08-29
Publication date: 2002-03-07
Also published as: AU2001286280A1; KR20020017653A; KR100386417B1

Abstract

The present invention discloses an environment-friendly anti-fouling composition produced using extract from an alga with the scientific name of Phaeopyta phaeophyceae chordariales ishigeaceae ishige sinicola. The composition according to the present invention show strong anti-fouling activity against oceanic fouling organisms. It is expected that the present composition may replace tributyltin (TBT) which has a problem associated with disruption of ecosystems.

Description

ANTI-FOULING AGENT EXTRACTED FROM MARINE ALGAE

Technical Field

The present invention relates to an environment- friendly antifouling agent using extract of an alga. More particularly, antifouling activity of extract from an alga with the scientific name of Ishige sinicola, which has excellent antifouling ability.

Background Art

Antifoulant is an agent used in combination with paint to prevent adhesion and growth of marine adherent organisms (eg. sea animals, plants or microbes) on the surface of vessels and so on. The marine adherent organisms cause an increase in the rate of corrosion of the surface of the vessels and so on. Also, the irregular surface of a vessel has a direct correlation to the frictional resistance of a ship. Hence, when such oceanic fouling organisms adhere to the surface and grow, the frictional resistance of a ship is raised. As a result, the consumption of fuel increases to bring enormous loss in expense.

In the past, Tributiltin (TBT) , a type of organic tartar, mixed with paint was commonly used as an antifouling agent. TBT has been used since the 1960s and has an outstanding antifouling ability. However, it has been found that TBT is a main cause of coastal pollution and causes severe damage to the growth and reproduction of coastal fish and shellfish. Also, a recent report indicated that TBT is one of the environmental hormones (endocrine disruptor) . Eventually, in 1980, a movement towards usage regulation of TBT started. Most of the advanced countries strictly banned the usage of TBT starting from 1982 and application of TBT to vessels under 25 meters is banned by most of the countries. In the place of TBT, antifouling agents based on metals such as copper and others were marketed. However, with long-term usage, the toxicity of the metal ion pollutes the marine environment and even causes severe damage to nontarget organisms. Compared to TBT, heavy metal antifouling agents cause more environmental pollution and economic loss. U.S. Patent No. 5,607,741 (March 4, 1997) discloses an antifouling agent comprising the extract from a kind of angiosperm, Zostera marina, called Zosteric acid. Also, it has been reported that Ceratinamine, extracted from a kind of sponge called Psammaplysilla purpurea, has antifouling activity. However, it was found that the antifouling actions of these substances were inadequate for practical uses.

As the world's population increases, land resources are stretched to their limits, therefore, the importance of development and application of oceanic resources is escalating. In particular, antifouling agents are essential substances that protect and assist in the oceanic resource development, but continuing usage of currently available antifouling agents containing tin or copper will become a threat to the marine environment and bring about fatal effects to the oceanic ecosystem in the long run.

Disclosure of Invention

In order to overcome the foregoing problems occurring with the usage of conventional antifouling agents, the object of the present invention is to provide a novel antifouling agent using the extract from a marine alga, being environment-friendly since it is extracted from biomaterial, which is capable of substituting TBT and yet is environmentally safe.

Brief Description of Drawings The above and other objects, features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which: Fig. 1 is a schematic diagram illustrating the individual step to obtain active fractions from Ishige sinicola .

Best Modes for Carrying out the Invention

Collecting Ishege sinicola

Ishige sinicola belongs to Ishigeaceae family of Ichordariales order of Phaeophyceae class of Phaeopyta phylum on the classification system and has a scientific name of Ishige sinicola (Setchell et Gardner) Chihara. This plant has a heteromorphic alternation of generation in life cycle and thallus is composed of parenchyma with different cell structures interconnected. Aster is also like the thallus with a ramified formation. The color of the thallus is a dark yellow and its surface has a slippery secretion. Ishige sinicola is found in the tropical to temperate regions, especially in the tidal zone.

Ishige sinicola used in this invention was collected from October 1998 to July 2000 in different coastal areas of Korea, including Sokcho, Daechon, Koje Island, Wando, Busan, and Seonsanpo. The extraneous matters attached to the thallus of the collected ishige sinicola was carefully removed with a soft brush and the alga was washed in fresh water then dried completely at room temperature. Thereafter, the alga was finely ground, which was then .used for the test.

Separation and analysis of antifouling fractions After drying and pulverization of the collected

Ishigea sinicola, active ingredients were separated by the process illustrated in Fig. 1.

In the first step,l g of dry powdered Ishige sinicola was added to 10 ml of a solvent mixture (4:1) of methanol and ground for 5 minutes, followed by filtration. The residual solid substances were added to 5 ml of ethylacetate . By filtration, the insoluble fractions were separated. The ethylacetate extract was designated as Fraction II and the residual substance was designated as Fraction I. The extract of the mixture of methanol and water was concentrated to 1/10 of .the original volume at a temperature below 40 °C and acidified to a pH of 2 with 2M sulfuric acid. The resulting substance was extracted 3 times with chloroform (CHC1₃ 5 ml) . The combined organic layer was designated as Fraction III. The combined aqueous layer was treated with aqueous ammonia to pH 10 and extracted twice with a solvent mixture (3:1) of chloroform and methanol (5 ml) . The organic extract was designated as Fraction IV. The aqueous layer was designated as Fraction V. The respective fractions were then screened for their antifouling activities through examining the adhesion of Enteromorpha prolifera' s spores and the repulsion against attachment of Mytilus edulis (mussels) . Test of antifouling effect

In order to confirm the antifouling effects of the Ishige sinicola extract, Entero orpha prolifera and Mytilus edulis were used. These oceanic organisms are a representative marine alga and shellfish, respectively, which are widely distributed in the main oceans and are known to have a high adhesion frequency.

Enteromorpha prolifera was collected from a seaweed farm located on the coastal areas of Kimhae, Korea and Mytilus edulis was collected on the coastal areas of Busan. Enteromorpha prolifera was examined for the growth rate of its thallus and the adhesion of its spores. Mytilus edulis was examined for the repulsion against attachment of its pes and the adhesion in larvae stage.

In this test, Ishige sinicola was used in a powder form prepared by completely drying at room temperature for 3 to 7 days and finely grinding. 1 g of dry ground Ishige sinicola was added to 50 ml of methanol and left for 1 day at room temperature. The methanol layer was separated and concentrated. About 40 mg of the concentrate was dissolved in 1 ml of methanol to form a methanol fraction, which was used for the test. The substances insoluble in methanol were added to distilled water in the same amount as methanol and left for 1 day.

The resulting aqueous layer was separated and concentrated. A 40 mg of the concentrate was dissolved in 1 ml of distilled water to form an aqueous fraction, which was used for the test.

Experimental example 1

Test for growth rate of Enteromorpha prolifera :

A thallus of Enteromorpha prolifera was cut into pieces with a size of 5 mm and cultured for 15 days in PES (Provasoli's enrichment, Jap. Soc . Plant Physiol, 1968, 63-75) to determine its optimal growth time. The pieces of Enteromorpha prolifera were measured for their sizes every 2 days. On the 7^th day, the results showed the highest growth rate per day of 12.1 ± 2.7%, at which the size was 9.2 ± 0.7 mm.

The methanol fraction and aqueous fraction of ishige sinicola, prepared as above described, were added to the two separate PES culture media of Enteromorpha prolifera at a concentration of 200 μg/ml . While culturing for 7 days, the respective PES culture media containing the methanol fraction and aqueous fraction of Ishige sinicola were examined for the growth rate of Enteromorpha prolifera . After completion of the cultivation, the size of Enteromorpha prolifera pieces was measured. The methanol fraction of Ishige sinicola showed a significant antifouling effect. The results are shown in Table 1.

Experimental example 2

Test for adhesion of Enteromorpha prolifera spores :

The methanol fraction and aqueous fraction of Ishige sinicola, prepared as above described, were also examined for the adhesion rate of spores of Enteromorpha prolifera according to the method of Fletcher (Int. Biodeterior, 25, 407-422) . The methanol fraction of Ishige sinicola showed a significant preventive effect of spore adhesion. The results are shown in Table 1. Table 1

Experimental example 3

Test for repulsion against attachment of Mytilus edulis' pes:

The test for the repulsion against attachment of Mytilus edulis' pes was performed according to the method of Hyashi & Miki (J. Mar. Biotechnol . 4, 127- 130) . The used Mytilus edulis was cultivated and fully matured one having a size of 4.5 ± 0.2 cm. One μl of the Ishige sinicola extract (40 mg/ml) was diluted with a 10-fold volume of seawater and examined for repulsion against attachment of Mytilus edulis' pes. The methanol fraction of Ishige sinicola showed a significant repulsion effect against attachment of Mytilus edulis . The results are shown in Table 2.

Table 2

Experimental example 4

Test for adhesion of larvae of Mytilus edulis : The test for the adhesion of larvae of Mytilus edulis was performed according to the method of Kitamura (Noppon Suisan Gakkaishi, 58, 75-78). Whatman paper was used as an attachment substrate. The Whatman filter papers were absorbed with various concentrations of methanol extract from Ishige sinicola and soaked in seawater for 4 days . In the experiment , it was observed that the adhesion of larvae of Mytilus edulis was perfectly blocked by the methanol fraction of Ishige sinicola at a concentration of more than 0.8 mg/ml. The results are shown in Table 3. The data shows the proportion of the adhered larvae to the total larvae used in the initial seawater.

Table 3

Experimental example 5

Fractionation of antifouling substances: Following the procedures illustrated in Fig. 1, the dried and powdered Ishige sinicola was fractionated into Fractions I to V. Each Fraction was examined for its ability to prevent the adhesion of spores of Enteromorpha prolifera and to repulse the attachment of Mytilus edulis' pes. The results are shown in Table 4. Table 4

Industrial applicability

As described in the detailed description of the invention and the foregoing experimental examples, the antifouling agent containing extract from a marine alga according to the present invention shows an excellent antifouling effect against oceanic fouling organisms, such as Enteromorpha prolifera and Mytilus edulis . This environment-friendly antifouling agent is expected to substitute for TBT which has a problem associated with disruption of ecosystems.

Claims

1. An antifouling agent containing extracts of Ishige sinicola .

2. The antifouling agent according to claim 1, wherein the extracts is extracted with methanol .

3. The antifouling agent according to claim 2, the extract contains an active ingredient at a concentration of 0.1 to 4.0 mg/ml in methanol .

4. An antifouling agent containing the extracts of Ishige sinicola , in which the extracts is obtained by adding dry powder of Ishige sinicola to a solvent mixture of methanol and water, followed by filtration, concentrating the filtrate at a temperature below 40 °C, acidifying the concentrate to pH 1 to 2 by addition of acid, and extracting the acidified concentrate with chloroform to give an organic fraction (Fraction III) .

5. An antifouling agent containing the extracts from Ishige sinicola, in which the extract is obtained by adding dry powder of Ishige sinicola to a solvent mixture of methanol and water, followed by filtration, concentrating the filtrate with at a temperature below 40 °C, acidifying the concentrate to pH 1 to 2 by addition of acid, extracting the acidified concentrate with chloroform, alkalifying the aqueous extract to pH 10 by addition of aqueous ammonia, and extracting the alkalified extract with a solvent mixture of chloroform and methanol to give an organic fraction (Fraction IV) .