GB2159056A - Biocidal compositions comprising polyhydroxynaphthoquinones - Google Patents

Abstract

A biocidal composition comprises a naturally occurring or synthetic polyhydroxynaphthoquinone of formula <IMAGE> where at least two of A to F represent hydroxyl groups and the remainder represent H or other substituents (e.g. hydroxyl, etherified or esterified hydroxyl, alkyl, hydroxyalkyl, acyl, nitro, amino or halogen), or a mixture thereof with a carrier. The polyhydroxynaphthoquinones may be used:- topologically, incorporated with dermatologically acceptable carriers, as fungicides or bactericides; as antibiotics in pharmaceutical compositions with pharmacologically acceptable carriers; as biocides within wall treating compositions in fungicidally effective concentrations; herbicidally within suitable herbicidal compositions; and in antifouling compositions.

Description

SPECIFICATION Biocidal Agents and Biocidal Compositions Comprising the Same This invention relates to biocidal agents and to biocidal compositions comprising the same and more particularly, but not exclusively to anti-fouling compositions, i.e. paint applied to the underwater areas of ships in order to prevent the growth of marine organisms.

The marine organisms which lead to biological fouling of ships hulls may be of either plant or animal type. Fouling by plants is usually the result of algal attack which may be provided by a wide variety of seaweeds and like marine plants. However, fouling by bacteria and fungi may also be a problem.

Representatives of the animal kingdom which may lead to fouling are hydroids, polyzoa, tunicates, worms, mussels and barnacles. Fouling, which begins with the deposition of a slime film containing bacteria and diatoms can lead to a considerable loading of a ship and to roughening of its whole underwater surface, leading for both reasons to a reduction in speed. The object of anti-fouling compositions is to prevent development of the contents of the slime film into fully grown marine organisms attached to the hull.

A variety of anti-fouling compositions have been developed over the years to assist in minimising fouling. Such compositions are generally made up from solutions of polymers or mixtures of polymers, plasticisers, pigments and fillers which dry by solvent evaporation and contain biocides or mixtures of biocides. It is required that the biocidal materials should be leached from a film when structures coated therewith are immersed in seawater, at a rate sufficient to kill and/or repel the larvae and spores of fouling organisms. A wide variety of biocidal materials have been employed to varying effectiveness ranging, in this century, from copper salts as the basis of traditional anti-fouling to the new and highly efficacious organotin compounds which provide long-life anti-fouling.These latter compounds have the particular advantage of being biodegradable in ordinary environmental conditions.

Even the introduction of relatively harmless tin can, however, damage the ecological balance in the region of fixed structures such as harbour installations and may be found to be ecologically undesirable. It is an object of this invention to provide anti-fouling means whose biocidal component is ecologically safe under all conditions.

According to the present invention, there is provided a biocidal composition, which comprises a naturally occurring or synthetic polyhydroxynaphthoquinone dyestuff as biocide in association with a carrier appropriate to the intended use of the composition.

More specifically, this invention provides an anti-fouling composition which comprises a matrix which is slightly soluble in seawater and a biocide dissolved therein, which biocide is a naturally occurring or synthetic polyhydroxynaphthoquinone dyestuff.

The biocidal compounds to be used in the practice of this present invention may be made up into anti-fouling compositions in conventional manner. Such compositions will include fillers, suspending agents or thixotropes as are required to give the necessary application properties to the finished material.

Typical additives will be silicones, titanium dioxide, red iron oxide, bentonite, tributyl methacrylate. As resins there may be used acrylic resins including acrylic copolymers, phenolic resins, vinyl resins etc. Corn oil may be added to hold the pigments in suspension. Solvents which can be used as carriers for the biocides include xylene and toluene. The biocidal compounds may be present in concentrations appropriate to the rate of leaching from the resin bases of the anti-fouling compositions.

This invention also provides a fungicidal or batericidal composition for topological application which comprises a dermatologically acceptable carrier and an effective concentration therein of a naturally occurring or synthetic polyhydroxynaphthoquinone dyestuff. Such compositions may be for example powders with for example a talc for use in the treatment of fungal conditions such as Athlete's Foot, although they can also be made up in the form of aerosol spray compositions for such uses. For antibacterial topological uses the polyhydroxynaphthoquinones will usually be made up in the form of bactericidal creams or ointments with suitable antiseptic cream basis or unguents.

Insofar as their pharmaceutical uses are concerned, the polyhydroxynaphthoquinones also have antibiotic uses and may be made up into pharmaceutical compositions with pharmacologically acceptable carriers. For this purpose, they may be employed in the form of pills, capsules, suppositories, powders and sterile injectable solutions or suspensions. An appropriate suspending medium is propyleneglycol.

This invention further provides a wallpaper or wall-treating composition which comprises a fungicidally effective concentration of a naturally occurring or synthetic polyhydroxynaphthoquinone dyestuff in a base composition for application to a wallpaper or wall-treating composition. Such a composition may be, for example, a wallpaper paste or a surfactant solution which the dyestuff additive makes into an antifungal composition for application to walls as a preliminary step in a wall-coating treatment.

Another type of composition provided by this invention is a herbicidal composition which comprises a naturally occurring or synthetic polyhydroxynaphthoquinone dyestuff in association with an adjuvant therefor.

Polyhydroxynaphthoquinone dyestuffs are a class of substances of general structure:

wherein at least two of A to F represent hydroxyl groups and the remaining groups from A to F are, for example, hydroxyl, etherified or esterified hydroxyl, hydrogen, alkyl, hydroxyalkyl, acyl, nitro, amino or halogen.

Insofar as the naturally occurring polyhydroxynaphthoquinones are concerned, A two F are generally selected, subject to two of them being hydroxyl, from hydroxyl, acetyl, ethyl and hydroxyethyl.

In their simplest form, such compounds are to be derived from naphthazarin and juglone structures respectively of formulae

However of greater interest are those polyhydroxynaphthoquinones known as echinochromes and spinochromes which are known to occur in a wide range of echinoderms (Thomson R. H. "Naturally occurring quinones" page 302 et seq., Butterworths 195).The name echinochrome was coined by MacMunn (1883) "Studies in Animal Chromatology", Proceedings of the Birmingham Philosophic Society 3 351-407 who observed a red pigment in the perivisceral coelomic fluids of Echinus esculentus and Paracentrotuslividus. These are about twenty known polyhydroxynaphthoquinones obtainable from sea urchins and there has been devised a nomenclature system which is a semi-trivial system of substituted juglones or naphthazarins, in preference to the original alphabetical system.Thus the most important naturally occurring polyhydroxyhydroquinones are named thus in the two systems: Alphabetical Semi-trivial Spinochrome A 3-acetyl-2,7-dihydroxynaphthazarin Spinochrome B 2,3,7-trihydroxyjuglone Spi noch rome C 3-acetyl-2,6,7-trihyd roxynaphthaza rin Spinochrome D 2,3,6-trihydroxynaphthazarin Spi noch rome E 2,3,6,7-tetrahyd roxynaphthazarin Echinoch rome A 6-ethyl (or hydroxyethyl)-2,3,7-tri hydroxynaphthazarin (Sources at variance as to the character of the 6-substitution) The term spinochrome has been given to those pigments which occur particularly in the spines of sea urchins.The term echinochrome was previously employed to denote pigments occurring mainly elsewhere in the coelomic fluid of the sea urchin. However as the experimental results which follow show, echinochromes may also be found in the tests of sea urchins and indeed the term echinochrome is now redundant.

This invention is based on observations as to the sterility of the coelomic fluid and the lack of fouling on the tests of sea urchins, both of which are known to be sources of polyhydroxynaphthoquinones.

Polyhydroxynaphthoquinones are present in such skeletal material as tests, as salts of calcium and magnesium.

Although naturally occurring polyhydroxynaphthoquinones, especially Spinochrome A are of particular interest, this invention also extends to the use of synthetic polyhydroxynaphthoquinones, whether occurring in nature and produced synthetically, or entirely synthetic and not naturally occurring.

The following Examples illustrate the invention: EXAMPLE 1 Isolation of Spinoch rome A (3-acetyl-2,7-dihydroxynaphthazarin) Sea urchin (Echinus esculentus) tests were first immersed in 20% by weight aqueous sodium hypochlorite solution until all extraneous tissue was removed. The spines, Aristotles lantern, peristome and periproct, inter alia, were discarded. The denuded tests were washed repeatedly in distilled water and then dried in an oven at temperatures between 60"C and 80"C. The pigmented growth rings were drilled out of the genital and inter-ambulacral plates using an electrically powered foot operated drill with an abrasive burr fitted. A pink powder was obtained comprised of calcium and magnesium carbonate to which organic pigment was bound as a salt and was weighed.Any carbohydrates, lipids and proteins were removed during Soxhlet treatment for 3--4 hours using a chloroform solvent. The chloroform was evaporated off, leaving a pink coloured powder residue which was added to ether. 2N hydrochloric acid was titred until all CaCO3 and MgCO3 was dissolved, releasing CO2 gas, and leaving the organic pigment dissolved in the ether fraction and calcium chloride and magnesium chloride and any excess hydrochloric acid in the aqueous fraction.

The mixture thus obtained was separated in a separating funnel and the inorganic fraction was discarded. The ether fraction was washed once only in distilled water to remove inorganic ions. Repeated washing would have resulted in the pigment extract dissolving in water. Addition of HCI or CH3COOH pushes any quinone present back into the ether fraction. The ether fraction was placed in a flask and attached to a rotary evaporator. The ether was distilled leaving a red coloured residue. The residue was redissolved in methanol. Distillation was repeated and the residue was dissolved a second time in methanol. The extract in methanol (hereinafter termed "Echinus extract") was filtered using Whatman I filter paper prior to further analysis. The Spinochrome A shown to be present therein in the tests which follow was recoverable by distilling off the solvent.

Chemical analysis of the extract was carried out as follows: Firstly the colour of the extract dissolved in water was compared with the colour of solutions of standard spinochromes dissolved in water For this purpose, there were used standard solutions made up by dissolving spinochrome A, spinochrome B and echinochrome A in water. Spinochromes A and B are both isolatable from the test and spines of Echinus esculentus (L.) and Paracentrotus lividus (Lam.) and echinochrome Awas isolatable from the test and spines of Paracentotus lividus and Arbacia lixula (L.).

Then high performance liquid chromatography was carried out using a Pye Unicorn L.C.-U.V. detector and a dodecyl-silane column A Philips single pen recorder recorded the peaks and the peak times were determined using a supergrator I computing integrator. The solvent system used was 72 vol.% methanol 27 vol.% water and 1 vol.% acetic acid. The optimum wavelength selected was 270 nm. (The extract had to be filtered using a micro-pore filter prior to injection onto the column).

Finally, spectral data was compiled by running the extract dissolved in methanol in a S.P. 8100 ultraviolet spectrophotometer. The standard spinochromes were treated in the same manner. The pH of the spinochrome standards dissolved in water were measured and compared with the pH of the aqueous and ether fractions of the Echinus extract. pH was measured prior to dissolving the extract and spinochrome standards in methanol.

The following test results were obtained: A. Table I shows the pH and colour of solutions of the Echinus extract and of the standard spirochromes in water.

TABLE I Pigment Solution Colour pH Standard echinochrome A orange/yellow 4.6 Standard spinochrome A pink 5.4 Standard spinochrome B green/yellow 5.3 Echinus extract red/pink aqueous fraction 5.1 ether fraction 5.7 B. High Performance Liquid Chromatography Mixtures of naphthoquinones are difficult to separate by conventional thin layer chromatography. It was for this reason that high performance liquid chromatography (H.P.L.C.) was selected as a more suitable technique. A series of injections of standards and extract was run. The peak times and size of peaks were determined and the values obtained for the Echinus extract were compared with those from the standard spinochromes. Analyses of the peak magnitudes and times revealed that the major component of the extract was spinochrome A. Echinochrome A was also present in a small amount.

C. Spectral Data Table II shows the spectral maxima of spinochromes A and B and Echinochrome A, taken from recent publications, in addition to the spectral maxima of the standards used and the Echinus extract.

TABLE II Source Pigment Max (me OH/EtOH) 1) Goodwin Srisukh (1950) spinochrome A 271,310,518 2) Thomson (1971) spinochromeA 251,270(sh), 317, 490(sh), 520 3) Thomson standard spinochrome A 225, 262, 306, 502 Experimental Echinus extract 224,271,315,518 1) Goodwin Srisukh (1950) unnamed X 267, 323,468, 520 2) Thomson (1971) echinochrome A 260,343,470, 490(sh), 520 3) Thomson standard echinochrome A 254, 333, 463, 520 1) Goodwin Srisukh (1950) spinochrome B 272,320,388,480 2) Thomson (1971) spinochrome B 272,323,385,480 3) Thomson standard spinochrome B 268,315,384 N.B. The values at around 225 nm probably correspond to the absorption maxima of methanol which peaks at 225 nm, and in which the standard spinochromes and the Echinus extract were dissolved.

1) Goodwin T. W. and Srisukh S. (1950). A study of the pigments of sea urchins: Echinus esculentus and Paracentrotus lividus. Biochem. J. 47.

2) Thomson R. H. (1957) Naturally occurring quinones, Butterworths,302 etseq.

3) Samples specially made up by Professor R. H. Thomson for the experiments reported herein.

When extracted, spinochromes have a variety of colours as evidenced by the results in Table I. Most species yield up to 6 pigments and the proportions of the pigments determine the colour of the species and indeed individuals within a species. Thus experimental results set out hereinabove indicate that with an extract obtainable from the tests of Echinus esculentus, spinochrome A predominates, echinochrome A is probably present in small amount and in addition there are probably trace amounts of 3 or 4 other related compounds. More particularly spectral data and high performance liquid chromatography suggest that spinochrome A is the key naphthoquinone in the growth rings of Echinus esculentus.

EXAMPLE 2 Biocidal Activity of Polyhydroxynaphthoquinones Echinochrome A (Ech. A) was extracted from Echinus esculentus coelomic fluid. The total coelomic fluid from one Echinus individual was drained and collected by removing the peristome and Aristotle's lantern and inverting the animal over a 500 ml beaker. The coelomic fluid was centrifuged for 10 minutes at 2000 g and the supernatant discarded. Ech. A. was extracted in a 95% by vol. acetone:5% by vol. 0.1 N HCI solvent solution. After 30 minutes the solvent solution concentrate mixture was centrifuged for 10 minutes at 2000 G. The red/orange coloured supernatantwas collected and excess solvent evaporated off using a rotary evaporator.

Spinochrome A (Spin A) extracted from Echinus test plates by the procedure of Example 1 and redissolved in acetone and Echinochrome A from coelomic fluid as set out in the preceding paragraph were tested for cidal activity against baceteria, yeast and fungi. In the following results section the cidal activity on two bacterium species and three species of yeast were recorded. Also tested were standard Echinochrome A and Spinochrome A solutions. The starting materials for testing are summarised in Table 111.

TABLE Ill Test Material Echinus Esculentus Source Solvent 1. Spinochrome A Test plates Acetone 2. EchinochromeA Coelomicfluid Acetone 3. Echinochrome A Coelomic fluid Acetone/acid 4. EchinochromeA Standard Acetone 5. Spinochrome A Standard Acetone 6. Acetone control Acetone Control 7. Coelomicfluid as such Echinusesculentus none The above seven test materials were used in a series of bactericidal and fungicidal tests whose results are set out hereinafter, the test materials being referred to by number only. The distances set out indicate the size of zone maintainable free from the test microorganisms.

Cidal Effect A. Bacteria Horizontal Test Material Distance mm Candida R 1. 42 2. 48 3. 34 4. 24 5. 12 6.

7.

Candida M 1. 45 2. 46 3. 18 4. 20 5. 11 6.

7.

Horizontal Test Material Distance mm Candida Y 1. 44 2. 27 3. 16 4. 22 5. 10 6. 9 7.

Staphylococcus aureus 6571 1. 22 2. 18 3. 23 4. 15 5. 21 6.

7.

E. coli 1. 19 2. 15 3. 22 4. 10/12 5. 13 6. slight reduc tion in inten sity of colour 7.

B. Fungal Growth Inhibition A series of growth inhibition tests were run on two species of fungi: Aspergi//us niger and Trichophyton interdigitalae. The inhibition data are tabulated below.

Aspergillus niger Distance mm

1) +ve > very large zones 2) +ve - 3) 20 4) -ve 5) +ve-very large zone 6) No result obtained 7) -ve Trichophyton interdigitalae Distance mm

1) +ve very large zones 2) +ve J 3) +ve 4) +ve 5) -ve 6) -ve 7) slightly positive In the foregoing, 20 mm was the size of the smallest inhibition zone found, all other positive zones being huge. 20 mm is itself a highly significant growth inhibition zone size.

Hence the tested polyhydroxynaphthoquinones are very effective against a broad range of bacteria and against fungi.

EXAMPLE 3 Algal Toxicity An ethereal extract of spinochrome A from Echinus test plates was obtained by the procedure of Example 1 by separation of the ether fraction there described from the aqueous acid/salt fraction. The ether was then evaporated off and gradually replaced with 95% by volume filtered autoclaved sea water.

Spinochrome A is sparingly soluble in water to an extent of about 0.1 % by weight. When added to 20 ml of 95% by vol. filtered autoclaved sea water, the concentration of spinochrome A amounted to approximately 50,ug/ml. Approximately 501lg/ml spinochrome Asolution in sea water was added to a range of volumes of algal cultures. The range of concentrations of spinochrome A in the algal cultures then ranged from 0.5 to 4.51lg/ml. For the purpose of studying the toxicity of spinochrome A on algae, the following eukaryotic algae were investigated. The algae tested were Dunaliella primolecta (Chlorophycae) and Phaeodactylum tricornutum (Bacillophycae).

The concentration ranges employed were as follows: TABLE IV Range in Concentration Dunaliella Phaeodactylum Spinochrome culture Spinochrome culture (ml) (ml) (ml) (ml) 1 100 1 100 1 50 1 50 1 25 1 25 1 10 1 10 1 5 1 5 1 4 1 4 1 3 1 3 1 2 1 2 1 1 1 1 The absorption and percentage transmittance readings of the algal cultures used as starting materials were determined and are set out in Table V.

TABLE V % Transmittance Absorption Phaeodactylum 41 0.38 Dunaliella (1) 71 0.15 Dunaliella (2) 68 0.17 Likewise, absorption and percentage transmittance readings were made of solutions of spinochrome A in water in the volume to volume ratios set out in the following Table VI. All readings recorded in Tables V and VI were made using Bausch and Lomb Spectronic 20 spectrophotometer.

TABLE VI VN Ratio Absorption % Transmittance 1/1 1.3 7 1/2 0.85 15 1/3 0.75 18 1/4 0.52 32 1/5 0.44 37 1/10 0.22 60 1/25 0.095 80.5 1/50 0.05 90 1/100 0.03 94 As a final control experiment, absorption and percentage transmittance values of the algal culture control systems were determined after 6 days. The results obtained are shown in Table VII.

TABLE VII Culture Control Readings Phaeodactylum Dunaliella % Transmission Absorption % Transmission Absorption 100 ml 17 0.775 44 0.36 50 ml 46 0.38 75 0.13 25 mi 40 0.4 75 0.13 The algal toxicity experiments themselves, utilising the mixture of spinochrome/water and algal culture gave the following results: TABLE VIII Algal Toxicity Experiment 1) Dunaliella primolecta 2) Phaeodactylum triconutum spinochrome/culture spinochrome culture (volume/volume (volume/volume ratio) growth of algae ratio) growth of algae 1/100 +ve 1/100 +ve 1/50 +ve 1/50 +ve 1/25 +ve (reduction in 1/25 +ve numbers noted) 1/10 -ve 1/10 -ve 1/5 -ve 1/5 -ve 1/4 -ve 1/4 -ve 1/3 -ve 1/3 -ve 1/2 -ve 1/2 -ve 1/1 -ve 1/1 -ve Absorption and percentage transmittance readings of the spinochrome/algal growth inhibition cultures were made. The results obtained are set out in Tables IX and X.

TABLE IX Dunaliella % Transmittance Absorption 1/100 VN 20 0.7 1/50 VN 43 0.37 1/25 V/V 57 0.25 1/10 VN 73 0.14 TABLE X Phaeodactyulum % Transmittance Absorption 1/100 VN 32 0.5 1/50 VN 27 0.57 1/25 VN 25 0.6 1/10 VN 73 0.14 The series of experiments indicates that spinochrome A extracted from the sea urchin Echinus esculentus, has an algistaticfunction in addition to the bactericidal function and fungicidal activity recorded in the preceding Exampie.

Claims (6)

1. This invention relates to biocidal agents and to biocidal compositions comprising the same and more particularly to antifouling compositions. This invention provides for an antifouling means whose biocidal component is ecologically safe under all conditions. The present invention provides a biocidal composition which comprises a naturally occurring or synthetic polyhydroxynaphthoquinone dyestuff or mixture thereof as biocide in association with a carrier appropriate to the intended use of the composition.

2. This invention in addition to Claim 1 also provides a fungicidal composition for topological application which comprises a dermatologically acceptable carrier and an effective concentration therein of a naturally occurring or synthetic polyhydroxynaphthoquinone dyestuff.

3. This invention in addition to Claims 1 & 2 further provides an antibacterial composition for topological application where polyhydroxynaphthoquinones, whether naturally occurring or synthetic, will usually be made up in the form of bacetericidal creams or ointments.

4. In addition to Claims 1, 2 & 3 this invention provides a pharmaceutical usage of naturally occurring or synthetic polyhydroxynaphthoquinones as antibiotics in the form of pharmaceutical compositions with pharmacologically acceptable carriers.

5. In addition to Claims 1, 2, 3 & 4 this invention provides a wallpaper or wall treating composition which comprises a fungicidally effective concentration of a naturally occurring or synthetic polyhydroxynaphthoquinone dyestuff in a suitable suspension for the intended application.

6. This invention in addition to Claims 1,2,3,4 & 5 also provides a herbicidai composition incorporating a naturally occurring or synthetic polyhydroxynaphthoquinone dyestuff in association with an adjuvant therefor.