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WO1997034747A1 - Agent de preservation du bois - Google Patents

Agent de preservation du bois Download PDF

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Publication number
WO1997034747A1
WO1997034747A1 PCT/CA1997/000177 CA9700177W WO9734747A1 WO 1997034747 A1 WO1997034747 A1 WO 1997034747A1 CA 9700177 W CA9700177 W CA 9700177W WO 9734747 A1 WO9734747 A1 WO 9734747A1
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WO
WIPO (PCT)
Prior art keywords
wood
organism
wood preserving
composition
growth
Prior art date
Application number
PCT/CA1997/000177
Other languages
English (en)
Inventor
Michael Risk
Paul Harrison
J. Cam Lewis
Ocky Karna Radjasa
Original Assignee
Michael Risk
Paul Harrison
Lewis J Cam
Ocky Karna Radjasa
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Michael Risk, Paul Harrison, Lewis J Cam, Ocky Karna Radjasa filed Critical Michael Risk
Priority to AU20893/97A priority Critical patent/AU2089397A/en
Publication of WO1997034747A1 publication Critical patent/WO1997034747A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B27—WORKING OR PRESERVING WOOD OR SIMILAR MATERIAL; NAILING OR STAPLING MACHINES IN GENERAL
    • B27K—PROCESSES, APPARATUS OR SELECTION OF SUBSTANCES FOR IMPREGNATING, STAINING, DYEING, BLEACHING OF WOOD OR SIMILAR MATERIALS, OR TREATING OF WOOD OR SIMILAR MATERIALS WITH PERMEANT LIQUIDS, NOT OTHERWISE PROVIDED FOR; CHEMICAL OR PHYSICAL TREATMENT OF CORK, CANE, REED, STRAW OR SIMILAR MATERIALS
    • B27K3/00—Impregnating wood, e.g. impregnation pretreatment, for example puncturing; Wood impregnation aids not directly involved in the impregnation process
    • B27K3/34—Organic impregnating agents
    • B27K3/36—Aliphatic compounds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/10—Animals; Substances produced thereby or obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D15/00—Woodstains
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
    • C09D5/16—Antifouling paints; Underwater paints
    • C09D5/1606—Antifouling paints; Underwater paints characterised by the anti-fouling agent
    • C09D5/1612—Non-macromolecular compounds
    • C09D5/1625—Non-macromolecular compounds organic

Definitions

  • Biofouling is characterized as the attachment to a solid surface and metabolic breakdown of a substrate by microorganisms (microbial fouling) and macroorganisms (macrofouling) .
  • microbial fouling microorganisms
  • macroorganisms macroorganisms
  • Marine biofilms typically consist of two major components, microorganisms that embed within the substratum of a substrate, and a matrix of extra-polymeric substances produced by bacteria that aid in adsorption and/or adhesion by other organisms.
  • Surface films of marine fouling bacteria play a role in the settlement, attachment and metamorphosis of marine invertebrate larvae such as shipworms and pholad bivalves.
  • the surface films resulting from the activity of microorganisms may also influence settlement by providing a roughness to the surface of the substrate to facilitate adhesion.
  • Biofilms range in form from a monolayer of cells covering a portion of the substratum to an algal mat that can be as thick as 300-400 mm.
  • zymogenous chemoorganotrophs with low substrate affinities are the first species to attach to a substrate, usually within hours after immersion of the substrate in an aqueous environment.
  • one marine bacterium, Vibrio as part of a mixed microbial flora, forms a primary film on substrates submersed in marine and estuarine environments that attracts fouling and boring organisms.
  • the primary colonizers are replaced by oligotrophic organisms such as Calobacter and Saprospira after about 48-72 hours. This formation of initial bacterial film is followed by the attachment of larger organisms such as cyanobacteria, diatoms, ciliated and stalked protozoa, and eventually invertebrates.
  • fouling organisms do not colonize the surfaces of many marine organisms including, for example, sponges and octocorals.
  • Biologically-active compounds that inhibit settlement by fouling organisms have been isolated from those organisms.
  • barnacle settlement inhibitors have been identified in extracts of sea pansies, Renilla reniformis (Rittschof et al., Bulletin of Marine Science 39 (2) .-376-382 (1986)) .
  • Other inhibitors have been isolated from sponges, gorgonians and alcyonaceans that have activity against mytilids and barnacles (Mizobuchi et al., 1992 ASOMPS VII, W017, Coll. 1991 Chem Revs. (1992); Sears et al. , J. Che . Ecol . 16:791-799 (19 )) .
  • none of the compounds identified to date have been shown to be effective antifouling and/or protective agents for wood materials.
  • Soft corals are a diverse group of marine invertebrates that dominate many Indo-Pacific reefs. Many of the soft corals belong to the order Alcyonaceae, a diverse group of benthic colonial invertebrates that vary in form from soft, fleshy members to prickly members to hard, leather-like forms. Alcyonacean soft corals produce . secondary metabolites that are released into surrounding water and play a role in defense against predators, in competition for space between species, and in reproduction. It has also been observed that fouling organisms usually do not colonize the surface of alcyonacean soft corals . Various compositions containing.
  • an object of the invention is to provide an antifouling composition containing an active agent that is a natural product derived from alcyonacean soft coral. Another object is to identify and isolate the active antimicrobial and/or antifouling compound(s) of alcyonacean soft coral, and to develop a protocol for purification of the active inhibitory compound(s) .
  • the wood preserving agent can be naturally derived from a soft coral (octocoral) of the Family Alcyonaceae, for example, Sinularia spp. which occur on shallow reefs throughout the tropical Indo- Pacific, including for example, Sinularia polydactyla, S. dura , S . flexibilis , S . variabilis, S . conferta , and S. leptoclados .
  • Other genera of soft corals useful according to the invention include Sarcophyton and Lobophytum.
  • the wood preserving agent possesses activity to inhibit the growth of a biofilm-forming organism and/or wood boring organism on and in wood, plastic, synthetic resins, and other substrates subjected to humid, damp or wet conditions.
  • the wood preserving agent is identified as a fatty acid ester of a long chain C 10 -C 30 alcohol, preferably a C 16 -C 18 alcohol, having the structural characteristics and properties of hexadecyl palmitate and/or octadecyl stearate, preferably with a molecular weight (m.w.) of about 480-536, a relative mobility on silica in hexane-1% ethyl acetate of about 0.8, with the region of mobility of 0.8 having an EC S0 growth inhibition value of about 0.08 mg/10 ml.
  • m.w. molecular weight
  • the invention also provides a wood preserving composition made essentially of a commercial or naturally- derived fatty acid ester which is hexadecyl palmitate (cetyl palmitate) and/or octadecyl stearate, and/or isomers thereof, in combination with an organic solvent as an inert carrier, for example, hexane or other like hydrocarbon.
  • a commercial or naturally- derived fatty acid ester which is hexadecyl palmitate (cetyl palmitate) and/or octadecyl stearate, and/or isomers thereof, in combination with an organic solvent as an inert carrier, for example, hexane or other like hydrocarbon.
  • Synthetic forms of hexadecyl palmitate and octadecyl stearate can be obtained from a commercial source such as Aldrich Chemical Company.
  • composition made solely of hexadecyl palmitate (cetyl palmitate) and/or octadecyl stearate, or of the described soft coral tissue extract, effectively inhibits the growth of film-forming microorganisms and wood boring organisms on and in wood and/or other substrate.
  • the wood preserving agent in the naturally- derived or synthetic form, can be used to inhibit the growth of a biofilm-forming organism and/or wood boring organism on a substrate such as a deck, patio, poles, wood planks, timbers and the like, exposed to damp or humid conditions; and/or a boat, raft, pier or other floating platform, dock, and the like, that is partially or completely submerged in water including marine, estuarine, or freshwater environment.
  • the wood preserving agent is effective in inhibiting the growth of marine biofilm-forming organisms such as Vibrio harveyi , and other Vibrio spp . , and the growth of freshwater biofilm-formers.
  • the wood preserving agent is also effective in preventing penetration into wood by wood boring organisms, such as, shipworms ( Teredo sp . ) , and members of the family Pholadidae, or pholads, and inhibiting the settlement of zebra mussels (Dreissena polymorpha) on submerged surfaces in fresh water.
  • a composition containing the wood preserving agent is applied to the substrate to be treated.
  • the substrate can be coated with or soaked in the composition.
  • the composition is formulated with the wood preserving agent and an inert carrier.
  • Suitable carriers include, for example, an organic solvent that will provide dispersion of an even layer of the active esters of the wood preserving agent onto the substrate surface or enhance distribution of the active esters into a matrix such as a paint.
  • Suitable organic solvents include hexane, heptane, cyclohexane, gasoline, or other hydrocarbons and petroleum compounds, and ethanol, chloroform, acetone, methanol, ether, and the like, and mixtures thereof.
  • a preferred composition containing the naturally- derived agent is formulated with about 50 wt-% Sinularia tissue containing the wood preserving agent, and about 50 wt-% organic solvent.
  • Another useful composition comprises the wood preserving agent in substantially pure form obtained by extracting and isolating the active agent from its natural association with other proteins, lipids, and other like substances and elements of the soft coral tissue, or by dissolving a commercially available source of hexadecyl palmitate (cetyl palmitate) and/or octadecyl stearate in the organic solvent.
  • Such separation techniques are known and used in the art, as described, for example, in W.C. Still, M. Kahn and A. Mitra, J. Org. Chem.
  • composition can also be formulated with the synthetic wood preserving agent, hexadecyl palmitate, octadecyl stearate, or isomers and mixtures thereof, and an organic solvent.
  • the wood preserving agent can also be combined with an alkyd paint, and a compatible pigment and solvent as desired.
  • a paint formulation can be prepared with a trace to about 10 wt-% of the wood preserving agent, about 10-40 wt-% organic solvent, with the balance as the alkyd paint, optionally including pigment.
  • Pigments that can be used include, for example, titanium dioxide, iron oxides, chromium oxides, cadmium lithopones, carbon blacks, azo pigments, anthraquinones, and the like.
  • Suitable organic solvents for use in the paint composition include, for example, toluene, naphtha, cyclohexane, mineral spirits, methyl ethyl ketone, ethyl acetate, CellosolveTM mono- and dialkyl ethers of ethylene glycol and their derivatives, and the like.
  • the paint composition can further include a binder such as mica, talc, silica, and the like; a drier such as tung oil, soya, castor, tall, safflower, cottonseed, coconut, fish, and other like oils, modifications thereof; and/or a release agent such as metallic soaps of lead, manganese, cobalt, iron, zinc or compounds of fatty, rosin or higher monobasic acids.
  • a binder such as mica, talc, silica, and the like
  • a drier such as tung oil, soya, castor, tall, safflower, cottonseed, coconut, fish, and other like oils, modifications thereof
  • a release agent such as metallic soaps of lead, manganese, cobalt, iron, zinc or compounds of fatty, rosin or higher monobasic acids.
  • the process includes treating the tissue of a soft coral with an organic solvent such as hexane, ethanol, chloroform and the like, and mixtures thereof, to extract the wood preserving agent as a crude extract.
  • an organic solvent such as hexane, ethanol, chloroform and the like, and mixtures thereof
  • the crude extract is separated by chromatography, for example, thin layer chromatography (TLC) , high performance liquid chromatography (HPLC) , flash column chromatography, or other technique involving separation on silica gel using a sequential treatment with different organic solvents, preferably with hexane, then chloroform, and then a methanol/chloroform mixture, in order to elute the partially purified ester from the column which retains other natural products, and to provide separate fractions.
  • the fraction containing the active wood preserving agent is then separated by chromatography with an organic solvent, preferably 5% ethyl acetate in hexane, to further purify the agent, and provide a second extraction fraction.
  • the second extraction fraction is separated by chromatography with an organic solvent, preferably 1% ethyl acetate in hexane, to further purify the agent, and provide a third extraction fraction.
  • the third fraction is separated by chromatography, preferably HPLC, with an organic solvent, preferably 1% ethyl acetate in hexane, to further purify the agent, and provide an extract containing the purified wood preserving agent.
  • the process can also include testing the crude extract and/or one or more of the extraction fractions for antibacterial activity against a film-forming bacterium.
  • the wood preserving agent or composition can be packaged as part of an article of manufacture, or kit.
  • the kit can include in association, for example, (a) the wood preserving agent, carrier, and optional additives for forming the composition, or the prepared wood ' preserving composition, placed in containing means such as a vial, jar, pouch, can, bottle, and the like; and (b) means for instructing as to the formulation and/or the use of the composition for inhibiting growth of an organism such as a biofilm former or wood borer, on wood or other substrate.
  • the components in part (a) may further include ingredients such as a carrier and pigment for preparing the composition as a paint, or the composition formulated as a paint.
  • the parts of the kit can be contained or separately packaged within a packaging material, such as a box or bag.
  • the present wood preserving agent in either the natural or synthetic form, provides a natural material that is environmentally safe and effective as a marine or freshwater antifoulant to inhibit growth of biofilm forming and/or wood boring organisms on wood as well as other substrates.
  • the agent is also useful in inhibiting growth of zebra mussels on submerged surfaces .
  • the invention also provides a wood preserving composition which is a simple formulation containing a commercially- available synthetic compound that surprisingly has been found to provide effective antifouling activity against biofilm formers and wood borers, without the presence of other active ingredients.
  • a further advantage of the present compositions is its simple formulation of a wood preserving agent and a carrier, with the wood preserving agent as the only active agent so that the composition can be prepared at a low cost.
  • the composition contains the wood preserving agent at a high level of activity which is not reduced due to dilution or interaction by the addition of multiple ingredients or other active agents.
  • FIGURE 1 is a schematic diagram of the bioassay- directed isolation and purification of the inhibitory compound of tissue extract of Sinularia polydactyla .
  • FIGURE 2 is a graphical depiction of the growth inhibition of Vibrio harveyi by soft coral hexane crude extract. The absorbance at each concentration was compared to that for zero concentration to obtain % inhibition for each data point.
  • FIGURE 3 is a graphical depiction of the percent inhibition of Vibrio harveyi by soft coral hexane crude extract. The data points are the average inhibition of days 1, 2, and 3 from FIGURE 1, calculated for each concentration and replotted.
  • FIGURE 4 is a graphical depiction of the percent inhibition of Vibrio harveyi by the chloroform fraction.
  • FIGURE 5 is a graphical depiction of the percent inhibition of Vibrio harveyi by the fraction of 5% ethyl acetate in hexane.
  • FIGURE 6 is a graphical depiction of the percent inhibition of Vibrio harveyi by the fraction of 1% ethyl acetate in hexane.
  • FIGURE 7 is a graphical depiction of the percent inhibition of Vibrio harveyi by the fraction collected from high performance liquid chromatography.
  • FIGURE 8 is a graphical depiction of the growth inhibition of Vibrio harveyi by synthetic esters.
  • Sinularia polydactyla was collected at a depth of about 5 meters in the vicinity of Panjang island, Jepara region, Central Java, Indonesia.
  • Sinularia polydactyla is characterized by J. Verseveldt, "A revision of the genus Sinularia May” (Octocorallia, Alcyonacea) , Zosammlung Verhandelingen No. 179, 128 pp., E. J. Brill, Leiden (1980) .
  • Sinularia polydactyla forms small colonies with distinct polyps (i.e., stalks) fused into a common mass of coenchyme, the stalks sometimes encrusting, with the lobes crowded, arborescent, and with fingerlike branches.
  • the surface layer of the lobes and the sterile stalk usually containing club-shaped sclerites, with many of the clubs having a central wart.
  • the length of the clubs is about 0.09- 0.19 mm, and the length of the coenenchymal spicules is about 3-5.5 mm.
  • the colonies of S. polydactyla were placed into a container of seawater immediately after collection and transported to the laboratory for preparation.' The tissues of S . polydactyla were washed with distilled water, cut into small pieces, weighed, and placed into a beaker for extraction.
  • the S. polydactyla tissue was homogenized in hexane (750 ml/500 gm fresh weight) in a Waring blender. The mixture was filtered using vacuum filtration. The filtrate was reduced to a dried crude extract on a rotary evaporator (B ⁇ chi RE-111) , and then weighed.
  • tissue extract from Example 1 above was chemically analyzed and purified according to a bioassay-directed purification by repetitive column chromatography using Vibrio harveyi as the test strain. The results showed that S. polydactyla possesses antibacterial properties against this bacterium. Purification of tissue extracts gave a mixture of esters that inhibited the growth of V. harveyi with an EC S0 value of 0.075 mg/10 ml culture medium.
  • the character of the active agent in the tissue extract was determined by comparing spectrographs of the active agent with those of synthetically prepared esters.
  • the esters hexadecyl palmitate, hexadecyl stearate, octadecyl palmitate and octadecyl stearate, were chemically synthesized from alcohols and fatty acid acyl halides.
  • Each purified ester was characterized by NMR and mass spectrometry (MS) .
  • MS mass spectrometry
  • harveyi It was found that hexadecyl palmitate was active, octadecyl stearate was less active, and hexadecyl stearate and octadecyl palmitate were inactive at the concentrations tested.
  • Standard Deviation of cuvettes A standard deviation was performed to calculate the deviation of the cuvettes used in the assays described below. The absorbance of thirty (30) cuvettes were measured using a Perkin-Elmer Lambda 9 UV spectrophotometer at 324.9 nm. The data were then used to generate the standard deviation. The results showed the standard deviation among the cuvettes at 0.00889 AU (Absorbance Unit) .
  • Vibrio harveyi (American Type Culture Collection, ATCC 33868) was cultured as the test organism for assaying the antibacterial activity of the Sinularia tissue extract. Vibrio harveyi is a halophile found in marine aquatic habitats with a wide range of salinities.
  • the luminous broth medium recommended by ATCC to grow the bacterium, was formulated as follows:
  • the ingredients were weighed into an Erlenmeyer flask, and distilled water was added up to 1 liter.
  • the medium was heated on a hot plate and homogenized with a magnet stirrer.
  • the medium was adjusted to pH 7.1 with NaOH (I N) , and sterilized in an autoclave.
  • a test tube containing 10 ml luminous broth medium was inoculated with Vibrio harveyi and incubated overnight at 30°C.
  • Standard deviation of the antibacterial assay A standard deviation of the various assays for anti-bacterial activity was performed as follows. Two rows of tubes containing culture medium were inoculated with Vibrio harveyi and potentially inhibiting esters at a range of concentrations from 0-10 mg/ml, and incubated for 3 days at 30°C. The absorbance of the medium was measured at 324.9 nm using a Perkin-Elmer Lambda nine UV spectrophotometer. The data were used to generate the standard deviation of the assay. The results showed that standard deviation of the assay was 0.0763 AU (Absorbance Unit) .
  • Assay for antibacterial properties (crude assay) . As shown in the protocol diagrammed in FIGURE 1, a preliminary assay was performed on the crude hexane extract of Sinularia tissue to determine whether the tissue extract possessed antibacterial properties against Vibrio harveyi .
  • the preliminary assay was an antibacterial bioassay that provided a profile of the antibacterial properties of the extract.
  • the assay on the crude extract was as follows. A range of concentrations of the crude
  • S. polydactyla tissue extract was prepared by diluting the extract into chloroform (0, 2, 4, 6, 8, 10 mg tissue extract/per tube) . The chloroform was then evaporated. Luminous broth medium (10 ml) was added to each test tube. Broth medium with no crude extract was the control. Each tube was inoculated with test strain V. harveyi (0.2% v/v) , and incubated for 3 days at 30oc.
  • FIGURE 2 Growth inhibition of the test strain Vibrio harveyi by the hexane crude extract of tissue from S. polydactyla is shown in FIGURE 2.
  • the EC 50 value of the growth inhibition was 5.5 mg/10 ml of the culture medium, as shown in FIGURE 3.
  • the hexane crude extract was first separated by flash column chromatography on silica gel using three different solvents: hexane, followed by chloroform, followed by
  • Antibacterial assay The following assay was used to test for antibacterial activity of the extract fractions against Vibrio harveyi to guide further purification.
  • a range of concentrations of the active extract (0, 0.05, 0.25, 0.50, 0.75 and 1.0 mg/tube) were prepared by diluting the extract in chloroform. The chloroform was then evaporated, and 10 ml luminous broth medium added to each test tube. The medium with no extract was the
  • Each tube was inoculated with the bacterial test strain Vibrio harveyi (0.2% v/v) .
  • the test tubes were then incubated for 3 days at 30 ⁇ c.
  • the growth curves were measured daily using a Perkin-Elmer lambda 9 UV spectrophotometer. These data were used to generate percent inhibition curves and to determine the EC S0 for growth inhibition to identify the active fraction that inhibited the growth of Vibrio harveyi .
  • the assay results showed that the active compound that inhibited V. harveyi was contained in the chloroform fraction with an EC 50 value of 0.715 mg/10 ml of the culture medium.
  • the dried fractions were then weighed and examined for antibacterial activity against V. harveyi to guide further purification of the active fractions, as follows.
  • a range of concentrations of the active dried fractions (0, 0.05, 0.25, 0.5 and 0.75 mg/tube) were prepared by dilution into chloroform. The fractions were evaporated to dryness, and 10 ml luminous broth medium was added to each tube. Medium with no dried fraction was used as a control.
  • Each tube was inoculated with the test strain V. harveyi (0.2% v/v), and incubated for 3 days at 30oC.
  • the growth curves were measured daily using a Perkin-Elmer lambda 9 UV spectrophotometer. These data were used to generate percent inhibition curves and to determine EC 50 for growth inhibition.
  • the active compound was contained in the 5% ethyl acetate fraction.
  • the fraction possessed an EC S0 value of 0.669 mg/10 ml of the culture medium.
  • the 5% active dried fraction was analyzed using TLC and rechromatographed on a silica gel column using a mixture of hexane and ethyl acetate as the eluent. The percentages of ethyl acetate in the mixture were increased as the elution proceeded (0, l, 2, 5, 7%) . Each fraction was collected from each different percentage of ethyl acetate, evaporated and weighed. The dried fractions were tested according to the above-described antibacterial assay against V.
  • the results showed that the active compound of the 5% fraction was contained in the fraction eluted with 1% ethyl acetate in hexane.
  • the 1% fraction showed growth inhibition in the antibacterial bioassay and was found to have an EC S0 value of 0.129 mg/10 ml of the culture medium, as shown in FIGURE 6.
  • the 1% ethyl acetate/hexane active fraction was further purified on preparative high performance liquid chromatography (HPLC) using hexane:ethyl acetate (99:1) as the eluent. HPLC was performed using a Beckman HOB solvent delivery module with a Beckman 4.6 mm x 15 cm column of silica gel.
  • the active fraction was injected into the HPLC column and the eluted fractions were collected using a Gilson microfraction collector (Model 203) . Each fraction was evaporated and tested for activity against the bacterium V. harveyi . The separation of the 1% active fraction by HPLC yielded several active fractions that were combined to provide a product that inhibited the growth of V. harveyi and possessed an EC 50 value of 0.075 mg/10 ml of the culture medium, as shown in FIGURE 7.
  • the EC S0 value was 5.5 mg/10 ml; the chloroform fraction showed an EC 50 value of 0.715 mg/10 ml; the 5% ethyl acetate in hexane fraction showed an EC S0 value of 0.669 mg/10 ml; the 1% ethyl acetate in hexane fraction showed an EC 50 value of 0.129 mg/10 ml; and the HPLC fraction showed an EC 50 value of 0.075 mg/10 ml.
  • the active fractions from the HPLC separation of Example 2 were combined, evaporated and weighed, and a structural analysis of the active compounds in those fractions was conducted as follows.
  • the fractions were structurally analyzed by nuclear magnetic spectroscopy using -NMR spectroscopy in CDCI 3 at 500 Mhz using a Br ⁇ cker AM-500 NMR spectrometer, and by mass spectrometry (MS) using a ZAB-E mass spectrometer (Varian VG) .
  • Palmitoyl chloride (1.492 ml; 0.00492 mol) was added to a mixture of 1-hexadecanol (1 g; 0.0041 mol) in ether (10 ml) in a round bottom flask with stir bar. After the reaction was completed (about 16 hours) , the mixture (12 ml) was added to water (25 ml) and adjusted to pH 7 with NaOH (1 N; 5 ml) . The aqueous phase (30 ml) was extracted with ether (2 x 20 ml) . The ether layers (50 ml) were collected, magnesium sulphate (5 g) was added to remove the water, and the organic phase was evaporated to dryness.
  • Palmitoyl chloride (1.328 ml; 0.00438 mol) was added to a mixture of 1-octadecanol (1 g; 0.00365 mol) in ether (10 ml) in a round bottom flask with stir bar. After the reaction was completed (about 16 hours) , the mixture (12 ml) was added to water (25 ml) and adjusted to pH 7 with NaOH (1 N; 4.5 ml), and extracted as described above for the hexadecyl palmitate synthesis procedure to give a substantially pure product of octadecyl palmitate. The purity of the product was tested as described above. The results were as follows: NMR (Hi CDC1 3 , 200 MHz), ⁇ as above except 1.25 (br m, 54H, CH 2 ) ; MS (El) m/e 508, 257.
  • EXAMPLE 4 Assay of anti-bacterial activity of synthetic esters The four synthetic esters, hexadecyl palmitate, hexadecyl stearate, octadecyl palmitate and octadecyl stearate, were tested for anti-bacterial activity against Vibrio harveyi .
  • esters were tested according to the antibacterial assay described above. The results of the tests are shown in TABLE 1, below.
  • Sinularia polydactyla possesses antibacterial properties as a result of a mixture of esters, with hexadecyl palmitate as the principal inhibitory compound.
  • the inhibitory compound prepared from Sinularia tissue extract from Example 1 above was tested for its effectiveness in inhibiting penetration by wood borers into wood.
  • the Sinularia tissue was extracted with gasoline or a methanol/chloroform mixture to extract nonpolar materials with potential biological activity, and coated onto teak blocks that were submerged for an extended time in seawater.
  • the teak sandwich blocks (3 x 6 x 12 cm) were prepared by band sawing standard air-dried untreated teak from a local lumberyard. Individual teak pieces were then dowelled together to make composite sandwiches. A monofilament suspensory line was threaded through a hole drilled into the block.
  • Sin/gasoline block was soaked in composition made of a filtered extract of Sinularia tissue with gasoline (1:1 ratio) ;
  • Meth/Chlor block was soaked in a mixture of absolute methanol and chloroform (1:1 ratio) ; and Sin/meth/chl : block was soaked in composition made of a filtered extract of Sinularia tissue prepared with the methanol/chloroform mixture (1:1) .
  • Wood preserving paint composition to inhibit wood borers and barnacle encrustation
  • Tissue extracts of Sinularia and Sarcophytum were added to an alkyd paint and coated onto teak blocks to test the wood preserving properties of the extracts as a paint coating.
  • Solid teak blocks 2 x 5 x 10 cm were prepared.
  • the blocks were coated (1 coat) with the paint compositions described below, and immersed in seawater (about 1 meter) for 6 months to test the effectiveness of the paint composition to inhibit penetration by wood borers into the wood and barnacle encrustation.
  • the filtered extracts of Sinularia tissue and Sarcophytum tissue were prepared as in Example 1.
  • Blocks were also coated with shark liver oil, a commonly used antifouling agent for wooden boats, alone and as an additive in the paint.
  • the shark liver oil was obtained by rendering a fresh piece of shark liver over a gas ring.
  • the teak blocks were coated as follows Control : untreated, no coating;
  • the results show the effectiveness of a paint containing the Sinularia and Sarcophytum extracts in inhibiting penetration by wood boring organisms and settlement of barnacles on submerged wood.
  • Paint compositions were prepared by combining a commercial, alkyd paint ("paint") with an extract of Sinularia tissue alone and separately combined with the following: a) gasoline, b) hexane, c) chloroform, and d) a mixture of chloroform and absolute methanol. Each of the paint compositions were coated onto teak blocks to test the effectiveness of the paint compositions to inhibit penetration by wood borers into the wood.
  • Teak blocks prepared as described in Example 6, were coated with the paint compositions that are described below, and immersed in seawater for 6 and 8 months.
  • the filtered extract of Sinularia tissue was prepared as in Example 1.
  • the teak blocks were coated as follows: Control : untreated, no coating; Paint block coated (1 coat) with white GLO-TEX semi-gloss enamel exterior paint (Nippon Paint Co. ) ;
  • Paint/Sin/Hex block coated (1 coat) with a mixture of
  • Paint/Sin/Chl block coated (1 coat) with a mixture of
  • Paint/Sin/Chi/Met block coated (1 coat) with a mixture of 75% paint + 25% of a mixture of filtered extract of Sinularia tissue in a 1:1 vol/vol mixture of chloroform and absolute methanol.
  • Table 4 shows the estimate from visual examination of external surfaces of the teak blocks of the damage by wood boring organisms in percent (%) intactness.
  • results show that an effective treatment for preserving wood from bioerosion and biofilm formation can be prepared using filtered extract of Sinularia tissue.
  • the block treatments were as follows.
  • the cetyl palmitate (hexadecyl palmitate) was an off-the-shelf brand (Aldrich Chemical Co., Wisconsin, U.S.A.) .
  • the white enamel was a commercial, thick alkyd enamel purchased locally in Indonesia.
  • the painted blocks were white in appearance.
  • the total number of bivalve apertures (equivalent to live, wood-boring bivalves, i.e., teredos and pholads) were counted in each block using a binocular microscope and a plastic overlay sheet. Algae cover was evaluated using a non-parametric 5-point scale in which 1 represents total coverage by algae and 5 represents 0 algae. The results are shown in Table 5 below (average of 3 blocks/treatment) .
  • barnacle numbers were frequently lower on treated panels, there appeared to be little or no effect of cetyl palmitate treatment on settlement by barnacles. This may be attributed to the settling behavior of larvae which is governed by the roughness of the surface to ensure that the adult form will remain adhered to the substrate.
  • the algae results were not statistically significant.

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Plant Pathology (AREA)
  • Environmental Sciences (AREA)
  • Agronomy & Crop Science (AREA)
  • Dentistry (AREA)
  • Pest Control & Pesticides (AREA)
  • Materials Engineering (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Forests & Forestry (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

L'invention porte sur un agent de préservation du bois dérivant de la main de mer alcyonacéane Sinularia sp. et sur sa méthode d'utilisation qu'il soit extrait du mollusque ou produit industriellement pour empêcher la croissance d'organismes générateurs de biofilms et/ou ou d'organismes xylophages sur des substrats de bois ou autres. L'invention porte en outre: sur un procédé de séparation de l'agent de préservation d'avec le mollusque, sur une composition préservatrice composée essentiellement d'ester d'acide gras, d'hexadécyle palmitate et/ou d'octadécyle stéarate, et sur une trousse comportant la composition sous forme d'ingrédients séparés ou de préparation finie dans des récipients, en association avec des instructions pour préparer et/ou utiliser la composition comme agent de préservation du bois et/ou comme préparation antisalissure.
PCT/CA1997/000177 1996-03-15 1997-03-14 Agent de preservation du bois WO1997034747A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU20893/97A AU2089397A (en) 1996-03-15 1997-03-14 Wood preserving composition

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US1470696P 1996-03-15 1996-03-15
US60/014,706 1996-03-15

Publications (1)

Publication Number Publication Date
WO1997034747A1 true WO1997034747A1 (fr) 1997-09-25

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WO (1) WO1997034747A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003035342A1 (fr) * 2001-10-25 2003-05-01 Teredo Marine Protection Aps Procede de prevention contre les attaques de bernacles
US7214407B2 (en) 2001-10-25 2007-05-08 Teredo Marine Protection Aps Method for the prevention of barnacle attacks
CN103728405A (zh) * 2013-12-24 2014-04-16 中国检验检疫科学研究院 一种木制儿童用品中木材防腐剂迁移规律的研究方法

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FR2156903A1 (fr) * 1971-10-21 1973-06-01 Fosroc Ag
JPH06345611A (ja) * 1993-06-04 1994-12-20 Yokohama Rubber Co Ltd:The 防汚エラストマー組成物
JPH0789814A (ja) * 1993-09-27 1995-04-04 Kaiyo Bio Technol Kenkyusho:Kk 水中有害付着生物防除剤

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FR2156903A1 (fr) * 1971-10-21 1973-06-01 Fosroc Ag
JPH06345611A (ja) * 1993-06-04 1994-12-20 Yokohama Rubber Co Ltd:The 防汚エラストマー組成物
JPH0789814A (ja) * 1993-09-27 1995-04-04 Kaiyo Bio Technol Kenkyusho:Kk 水中有害付着生物防除剤

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CHEMICAL ABSTRACTS, vol. 113, no. 15, 8 October 1990, Columbus, Ohio, US; abstract no. 129561, LIU, ZHUJIN ET AL: "A study on chemical constituents of South China Sea soft coral Sinularia microclavate" XP002034850 *
CHEMICAL ABSTRACTS, vol. 126, no. 5, 3 February 1997, Columbus, Ohio, US; abstract no. 57564, RAO, T. SUDHAKARA ET AL: "Investigations on a soft coral of Sinularia species of the coasts of the Andaman and Nicobar Islands" XP002034849 *
CHEMICAL ABSTRACTS, vol. 67, no. 1, 3 July 1967, Columbus, Ohio, US; abstract no. 2370, RUDMAN, PETER ET AL: "The causes of natural durability in timber. XXI. The anti-termitic activity of some fatty acids, esters, and alcohols" XP002034854 *
CHEMICAL ABSTRACTS, vol. 96, no. 9, 1 March 1982, Columbus, Ohio, US; abstract no. 65950, LAI, ZUOQI ET AL: "Studies on the chemical constituents of Chinese soft Coral. (II)" XP002034851 *
DATABASE WPI Section Ch Week 9510, Derwent World Patents Index; Class A60, AN 95-070184, XP002034853 *
HOLZFORSCHUNG (1967), 21(1), 24-6 CODEN: HOLZAZ, 1967 *
INDIAN J. CHEM., SECT. B: ORG. CHEM. INCL. MED. CHEM. (1996), 35B(12), 1356-1358 CODEN: IJSBDB;ISSN: 0376-4699, 1996 *
PATENT ABSTRACTS OF JAPAN vol. 095, no. 007 31 August 1995 (1995-08-31) *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003035342A1 (fr) * 2001-10-25 2003-05-01 Teredo Marine Protection Aps Procede de prevention contre les attaques de bernacles
US7214407B2 (en) 2001-10-25 2007-05-08 Teredo Marine Protection Aps Method for the prevention of barnacle attacks
CN103728405A (zh) * 2013-12-24 2014-04-16 中国检验检疫科学研究院 一种木制儿童用品中木材防腐剂迁移规律的研究方法
CN103728405B (zh) * 2013-12-24 2016-12-07 中国检验检疫科学研究院 一种木制儿童用品中木材防腐剂迁移规律的研究方法

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