ANTIBACTERIALLY ACTIVE MARINE ALGAE EXTRACTS FROM THE CLASS DIATOMATAE AND METHODS OF USE

Abstract: The present invention relates to the use of extracts obtained from marine algae of the class Diatomatae as antibacterially active agents and to compositions containing such agents for use against pathogenic bacteria. This invention also relates to a method of depurating an aquiculture using as a feed source marine algae from the class Diatomatae.

Description: ANTIBACTERIALLY ACTIVE MARINE ALGAE EXTRACTS FROM THE CLASS DIATOMATAE AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of co-pending application Serial No. 08/618,861, filed on March 20, 1996. The parent application cited above is herewith incorporated by reference. FIELD OF THE INVENTION: The present invention relates to the use of marine algae extracts obtained from members of the class Diatomatae as antibacterially active agents and to compositions containing such agents for use against pathogenic bacteria. This invention also relates to a method of depurating crustaceans and mollusks by using certain marine algae from the class Diatomatae as part of the feed. This invention also relates to a method of maintaining a pathogenic vibrio free environment for the cultivation of marine aquatic animals, particularly oysters and shrimp, by maintaining sufficient density of certain marine algae in the cultivating water system. BACKGROUND OF THE INVENTION: The contamination of seabeds has become more problematic as the environment has gradually become more polluted. Pathogenic microorganisms are well known to infect seabeds, which are exposed to toxic waste and other undesirable pollutants. Controlled cultivation offish, mollusks, crustaceans and other aquianimals has become more prevalent in view of the ever growing scarcity of uncontaminated sources. Such contamination concerns are further complicated by the fact that a number of bacteria have become multi- drug resistant as a result of improper and/or overuse of antibiotics. The emergence of antibiotic-resistant bacteria has become more widespread and has resulted in the necessity to track the spread of such resistant strains and to the initiation of research efforts which seek alternative antibacterial agents which can be used to combat such resistant strains. Pathogenic Vibrio vulnificus is commonly found in raw oysters and has been associated with the occurrence of shrimp diseases, either as a direct cause of disease, as a carrier thereof or both. Additionally, bacteria such as Staphylococcus aureus has become resistant to methicillin, leaving only vancomycin as the remaining effective antibiotic against this strain. Virulent strains of enterococcus have emerged which are vancomycin resistant causing an alarming realization of the need to find new antibiotics against this organism. Such pathogenic bacteria are known to be extremely dangerous to humans. The present invention provides a solution to depuration of arthropods and mollusks by using marine algae from the class Diatomatae as a food source for the aquianimals. Marine algae from this class of organisms provide natural antibiotic properties which cleanse the aquianimal from pathogenic vibrio, thereby making the harvested seafood safe for consumption. Additionally, there is a need for new antibiotics and pharmaceutical compositions based thereon which are effective against a broad spectrum of bacteria and which are particularly useful against known multi-drug resistant strains of bacteria, including methicillin resistant Staphylococcus aureus. vancomycin resistant enterococcus. Vibrio cholerae and Vibrio vulnificus. Additionally, there is a need for the development of new antibacterially effective agents which can be made inexpensively and which are safe for use in mammals, aquianimals and the like. The present invention provides antibacterial agents which are based on the extracts of marine algae that are members of the class Diatomatae. Fractions and isolates of these extracts have been shown to provide antibacterial activity and, in particular are active against certain pathogenic bacteria, including for example, methicillin resistant Staphylococcus aureus. vancomycin resistant enterococcus. Vibrio vulnificus. Corvnebacterium xerosis. Streptococcus dvsenteriae. Streptococcus mitis. Streptococcus faecalis. Bacillus subtilis and Bacillus cereus. The antib , Serially effective extracts of these algae are believed to either occur naturally in the unicellular algae or are produced by the algae in response to the presence of bacterium and other algae that compete against them for living space. Pharmaceutical compositions and methods of treating mammals by administering antibacterially effective amounts of one or more extracts from the isolates of Diatomatae class marine algae are claimed herein. SUMMARY OF THE INVENTION: The present invention relates to the use of marine algae extracts obtained from the class Diatomatae as antibacterial agents and novel pharmaceutical compositions based thereon using one or more of the antibacterially effective isolates from these extracts. Furthermore, a method of treating mammals having bacterial infection by administering an antibacterially effective amount of one or more isolates or compounds extracted from Diatomatae class marine algae cells is also contemplated. Moreover, a method of depurating aquianimal cultures such as oysters, clams, mussels, scallops, shrimp and the like is also contemplated. Thus, in one embodiment a composition including an antibacterially active extract obtained from Diatomatae class marine algae is contemplated. Specific marine algae from this class include, for example, Chaetoceros. Thalassiosira. Phaedactylum. Navicula and mixtures thereof. The extracts obtained from these algae have been found to be active against a number of virulent bacteria including methicillin resistant Staphylococcus aureus. vancomycin resistant enterococcus. Vibrio vulnificus. Corvnebacterium xerosis. Streptococcus dvsenteriae. Streptococcus mitis. Streptococcus faecalis. Bacillus subtilis and Bacillus cereus. The extract itself, which includes a number of compounds, has been shown to have activity against bacteria, including those mentioned above. Additionally, a number of isolates have been extracted or fractionated from the crude extract. These isolates have also demonstrated antibacterial activity against bacteria including the particularly virulent strains mentioned above. Three compounds have been specifically identified which have antibacterial activity. The compounds, labeled II, III and IV are fatty acids which have heretofore not been known to have antibacterial activity against the above bacteria. The present invention also contemplates pharmaceutical compositions which are made to include the extract of one or more of the isolates obtained from marine algae of the class Diatomatae together with a pharmaceutically accepted carrier. The isolates may be used singly or one or more of the pure compounds may be combined as a mixture. Pharmaceutically acceptable derivatives and salt ' s of these compounds and isolates are contemplated. A method of treating bacterial infections by administering a pharmaceutical composition containing one or more antibacterially active isolates is also contemplated. In addition to the novel compounds and pharmaceutical compositions described herein, the present invention also contemplates a method of depurating crustaceans and mollusks which are contaminated with bacteria. The method includes introducing marine algae from the class Diatomatae as a feed for an aquianimal in a sufficient amount and for sufficient time to effectuate depuration. Due to the natural antibacterial activity of these marine algae, the exposure and/or ingestion of the algae as feed by the aquianimal produces a depurating effect and cleanses the aquianimal of bacteria such that the aquianimal can be further cultured and grown in a healthy environment, and finally be consumed as a food source. Thus, co-culturing of these marine algae along with the aquianimal is included in the present invention in order to bring about the desired effect of both feeding the aquianimal and depurating the aquiculture of bacteria. The present methods are useful in feeding and depurating aquianimal cultures of marine members of the phyla Mollusca, Arthropoda and mixtures thereof. Members of these phyla which can be used with the present invention include oysters, clams, scallops, shrimp, crabs, lobsters, abalone, mussels and krill. The eluant used to obtain the fractions and measure their activity against the specified bacteria is tabulated in Tables II, III and IV herein. Various fractions obtained from gradient elution have been shown to have activity. DETAILED DESCRIPTION OF THE INVENTION: Depurating aquianimals, especially those from the Phyla Mollusca and Arthropoda which include oysters, mussels, clams, snails, scallops, shrimp and the like have generally not been effective with regard to the elimination of pathogenic vibrio. The use of antibiotics in the depuration of an aquianimal has not received approval from the FDA. The present invention provides both a food source and simultaneous cleansing of an aquianimal due to the natural antibiotic properties exhibited by marine algae of the class Diatomatae in the presence of bacteria. During experimentation in a controlled- environment with oyster cultures which were contaminated with bacteria, it was discovered that the introduction of Diatomatae class marine algae as a food source, when used in a sufficient amount and for a sufficient time, caused depuration of the oysters and decontamination of pathogenic bacteria, including Vibrio vulnificus and Vibrio cholerae present therein. In the course of analyzing this effect, members of the class Diatomatae including Chaetoceros. Thalassiosira. Phaedactylum. and Navicula marine algae were studied closely for their contribution to the natural antibacterial cleansing of the oysters in the culture medium. Typical of the marine algae classified in the Diatomatae class, the Chaetoceros algae is a single celled organism, the main body of which is shaped liked a petri dish. When viewed from the side, individual cells appear square, having dimensions of about 12 to about 14 microns long and about 15 to about 17 microns wide, with spines protruding from the corners. The cells may form chains of about 10 to 20 cells which may reach up to 200 microns in length. The chains are formed by interconnecting the spines of adjacent cells. When cultured with strong aeration, Chaetoceros does not form colonies. Large cultures are brown in appearance and individual cells are golden brown in color, surrounded by a translucent cell wall. The algae is known to be a food source for members of the Mollusca and Arthropoda phyla including, for example, rotifers, clams, oysters, and larval shrimps. The taxonomy of Chaetoceros is described as follows: Division: Bacillariophyta Class: Diatomatae Order: Centrales Suborder: Biddulphiincae Family: Chaetoceraccae Genus: Chaetoceros The genus Chaetoceros is described usually as an oval or circular section which, in girdle view, appears quadrangular with straight sides and concave, flat or weakly convex ends. The cell has a valve consisting of more or less a flat end surface (the valve surface) and a cylindrical part (the valve mantle) which are bound together without a seam. The cell is valve-bearing at each end of the long axis (apical axis) on the corners, and has a long thick or thin bristle, sometimes called an awn. The opposite bristles of neighboring cells touch one another, usually directly but sometimes by a bridge, and become firmly fused together. By fusion of the bristles, the cells are formed into chains, usually with large or- small apertures or foramina between the cells. In some species the length of the chain is limited by the formation of special end-cells, their outer bristles not being connected with neighboring cells and differing from the others, often being shorter and thicker and nearly more parallel with the chain axis. These end-cells are believed to serve certain purposes when the chain floats. The cell wall of the Chaetoceros algae is formed of two halves and one or two girdle bands. Intercalary bands are present in some species, but are usually difficult to see without special methods. The valve or chain axis runs through the valves from center to center. The cell cytoplasm forms either only as a thin sack along the cell wall or it can fill the greater part of the inside of the cell. The nucleus may be against the cell wall or be central to the cell. Chromatophores are present, numbering from one to several and may be small or large. Resting spores are formed in most of the neritic species, usually in the cylindrical part near the girdle band of the mother cell, one to each cell. The valves of the spores are often armed with spines or spicules, sometimes only on one valve, sometimes on both, but rarely at the sides. After observing the antibacterial depuration of oysters when marine algae of the Diatomatae class, including for example Chaetoceros. Thalassiosira. Phaedactylum. and Navicula. were used as a feed for an oyster culture, an algal culture was developed for the purpose of determining an activity profile against bacterial microorganisms and to determine the efficacy of different fractions and extracts from the algae on bacteria. The algal cells, for example Chaetoceros cells, were harvested and ground to a wet algae-paste, which was then taken into water and centrifuged to obtain a supernatant liquid. The supernatant liquid was then loaded onto a chromatography column and eluted with an aqueous gradient eluant. Various fractions were identified and tested for antibacterial activity, as was the mixture as a whole. The details of such testing will be further described herein. For purposes of this invention, the term "extract" will include compounds, isolates, fractions, as well as, mixtures thereof obtained from marine algae cells of the class Diatomatae including for example, Chaetoceros. Thalassiosira. Phaedactylum. and Navicula. The extract can be obtained using any known method of extraction or fractionation, including the use of chromatography columns and eluants, solvent extraction and the like. For purposes of this disclosure, column chromatography has been used for its ease of use and accuracy, but other commercial separation techniques which may vary from laboratory techniques are contemplated herein. Among the isolates that have been identified as having antibacterial activity are those which correspond to the following general formula: R'-(CH 2 -CH=CH-) n R 2 -COOH (I) wherein R 1 and R 2 are independently alkyl groups of 1-100 carbon atoms; n is a positive integer from 1 to 100; and their pharmaceutically acceptable salts and derivatives thereof. Among the specific compounds corresponding to this formula which have shown antibacterial properties include the following: (Z,Z,Z)-6,9,12-Hexadecatrienoic acid (16:3Δ6,9,12) of the formula: CH 3 -CH 2 -CH 2 -CH=CH-CH 2 -CH=CH-CH 2 -CH=CH-CH 2 -CH 2 -CH 2 -CH 2 -COOH (II); (Z,Z,Z)-9,12-Hexadecadienoic acid (16:2Δ9,12) of the formula: CH 3 -(CH 2 ) 2 -CH=CH-CH 2 -CH=CH-(CH 2 ) 8 -COOH (III); and (Z,Z,Z,Z,Z)-5,8,l l,14,17-Eicosapentaenoic acid (20:5Δ5,8, 11 ,14,17) of the formula: CH 3 -(CH 2 -CH=CH) 5 -(CH 2 ) 3 -COOH (IV). The Minimum Inhibition Concentration (MIC) for Compound II with respect to methicillin resistant Staphylococcus aureus fMRSA) is 10-15 g/disk and 20μg/disk for vancomycin resistant enterococcus (VRE). It has been found that lOOμg/disk of Compound II of the present invention is active against Proteus vulgaris. Corvnebacterium xerosis. Corvnebacterium diptheriae. Shigella dvsenteriae. Streptococcus mitis. Streptococcus pyogenes. Streptococcus facaelis. Bacillus subtilis. Bacillus cereus. methicillin resistant Staphylococcus aureus (MRS A), and vancomycin resistant enterococcus (VRE), Micrococcus smegmatis. Micrococcus luteus and Vibrio vulnificus. It was also found that 1 OOμg/disk of Compound II is not effective against Salmonella tvphimurium. Klebsiella pneumoniae and Pseudomonas aeruginosa. Moreover, with respect to fungi, Compound II showed activity at 100 μg/dish for the following: Aspargillus niger. Candida tropicalis. Candida glabrata. Rhodotorula rubra. Trichosporon beigelii and Candida albicans. Furthermore, lOOμg/disk of Compound III of the present invention has been found to be active against Bacillus subtilis. MRSA, VRE, Micrococcus smegmatis. Micrococcus luteus. Streptococcus pyogenes. Streptococcus mitis. Streptococcus faecalis. Listeria monocvtogenes. Proteus vulgaris. Corvnebacterium xerosis. Corvnebacterium diptheriae. Bacillus cereus. Staphylococcus epidermidis and Vibrio vulnificus. Compound III at lOOμg/disk, however, is not active against Shigella dvsenteriae. Salmonella tvphimurium. Pseudomonas aeruginosa and Klebsiella pneumoniae. With respect to fungi, Compound III is active at 100 μg/disk for the following: Aspargillus niger. Candida tropicalis. Candida glabrata. Rhodotorula rubra and Trichosporon beigelii. Compound III, however, is not active against Candida albicans at a concentration of 1 OOμg/disk. Moreover, lOOμg/disk of Compound IV of the present invention has been found to be active against Bacillus subtilis. MRSA, VRE, Streptococcus mitis. Streptococcus faecalis. Streptococcus dvsenteriae. Corvnebacterium xerosis. Bacillus cereus and Vibrio vulnificus. Compound IV at 1 OOμg/disk, however, is not active against Streptococcus tvphimurium. Pseudomonas aeruginosa. Proteus vulgaris. and Streptococcus pneumoniae. With respect to fungi, Compound IV is active against Candida glabrata at a concentration of lOOμg/disk. Compound IV, however, is not active at 100 μg/disk for the following: Aspargillus niger. Candida tropicalis. Rhodotorula rubra. Trichosporon beigelii and Candida albicans. TABLE I-A MINIMUM INHIBITORY CONCENTRATION OF COMPOUND II ON METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS πVIRSA AFTER 48 HOURS TABLE I-B MINIMUM INHIBITORY CONCENTRATION OF COMPOUND II ON VANCOMYCIN RESISTANT ENTEROCOCCUS (VRE) AFTER 48 HOU TABLE I-C MINIMUM INHIBITORY CONCENTRATION OF COMPOUND III ON METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS (MRSA AND VANCOMYCIN RESISTANT ENTEROCOCCUS (VRE) AFTER 24 HOURS * The 20 μg/disk data point was used in 2 experiments yielding an average of 1.13 mm. In one experiment, the Inhibition Zone was clear; in the second experiment, it was fuzzy. iBLE I-D MINIMUM INHIBITORY CONCENTRATION OF COMPOUND IV ON METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS (MRSA^ AND VANCOMYCIN RESISTANT ENTEROCOCCUS rVRE) AFTER 24 HOURS These MIC experiments indicate that all three compounds (II, III and IV) effectively inhibit the ability of MRS A and VRE to grow. Moreover, Compounds II and III have been isolated in pharmaceutically useful quantities in Chaetoceros. Thalassiosira and Navicula. but not Phaedactylum. Compound IV, however, has been isolated in pharmaceutically useful quantities from Thalassiosira. Phaedactylum and Navicula. but not Chaetoceros. The compounds of the present invention are capable of forming both pharmaceutically acceptable acid addition and/or base salts. Base salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Pharmaceutically acceptable acid addition salts are formed with organic and inorganic acids. Examples of suitable acids for salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, gluconic, fumaric, succinic, ascorbic, maleic, methane sulfonic and the like. The salts are prepared by contacting the free base form with a sufficient amount of the desired acid to produce either a mono-, di-, etc. salt in the conventional manner. The free base forms may be regenerated by treating the salt form with a base. For example, dilute solutions of aqueous base may be utilized. Dilute aqueous sodium hydroxide, potassium carbonate, ammonia, and sodium bicarbonate solutions are suitable for this purpose. The free base forms differ from their respective salt forms somewhat in certain physical properties such a solubility in polar solvents, but the salts are otherwise equivalent to their respective free base forms for purposes of the invention. Certain compounds of the invention may exist in optically active forms. All such isomers, as well as, mixtures thereof are intended to be included in the invention. The compounds of the invention can be prepared and administered in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms comprise as the active component either a compound of formulae I through IV or a corresponding pharmaceutically acceptable salt of a compound of formulae I through IV. For preparing pharmaceutical compositions from the compounds described by this invention, inert, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, cachets, and suppositories. A solid carrier can be one or more substance which may also act as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, or tablet disintegrating agent; it can also be an encapsulating material. In powders, the carrier is a finely divided solid which is an admixture with the finely divided active compound. In the tablet, the active compound is mixed with a carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain from about 5 or 10 to about 70 percent of the active ingredient. Suitable solid carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the formulation of the active compound with an encapsulating material acting also as a carrier and providing a capsule in which the active component (with or without other carriers) is thereby surrounded. Similarly, cachets are included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration. Liquid form preparations include solutions, suspensions and emulsions. As an example, water or water-propylene glycol solutions for parenteral injection may be used. Such solutions are prepared so as to be acceptable to biological systems (isotonicity, pH, etc.). Liquid preparations can also be formulated in solution in an aqueous polyethylene glycol solution. Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, i.e., natural or synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose, and other well-known suspending agents. Preferably, the pharmaceutical preparation is in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, for example, packeted tablets, capsules, and powders in vials or ampules. The unit dosage form can also be a capsule, cachet, or tablet itself or it can be the appropriate number of any of these packaged forms. The quantity of active compound in a unit dose of preparation may be varied or adjusted from 1 mg to 100 mg according to the particular application and the potency of the active ingredient. In therapeutic use as agents for treating bacterial infections, the compounds utilized in the pharmaceutical method of this invention are anticipated to be administered at the initial dosage of about 3 mg to about 40 mg per kilogram body weight daily. A daily dose range of about 6 mg to about 14 mg per kilogram body weight is preferred. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. The following in vitro examples are given to illustrate certain embodiments of the invention, but are not intended in any way to be limiting of the effective scope or spirit of the invention as described and claimed. Observations as to activity were taken after 12 hours unless otherwise specified. EXAMPLE I From 201.4 liters of Chaetoceros algae culture having a cell density ranging from lxl 0 6 to 7x10 6 cells per milliliter of water, 22.5 grams of ground wet algae-paste was obtained. The wet paste was taken into 600 milliliters of water and centrifuged at 10,000 RPMs for approximately 20 minutes. The supernatant liquid was then loaded onto an Amberlite XAD-2 column (sigma, 30x7 cm) and eluted with 2.0 liters of the following gradient: (1) water, (2) 10% aqueous MeOH, and (3) 30% aqueous MeOH, (4) 50% aqueous MeOH, (5) 70% aqueous MeOH, (6) 90% aqueous MeOH, (7) MeOH and (8) CH 2 C1 2 . These fractions were tabulated in Table II below and show that identified fractions were placed in petri dish cultures of methicillin resistant Staphylococcus aureus. As can be seen from Table II, activity against Staphylococcus aureus was observed in fractions 5-7 with fraction 6 yielding the highest activity. Activity is indicated by the dark portion around the white circle of antibiotic which was introduced into the petri dish. The observations as to activity were taken after 12 hours. CO c O O c H m co I m m c r- m r σ> ractions 5 through 8, with the highest activity being present various fractions against Vibrio vulnificus can be seen ngest in fractions 5 throu h 8, with the highest activity le II, the most active fractions against each of the o 8, with fraction 6 obtained from a 90% by volume ing the highest activity. 1 concentrated under reduced pressure to provide the crude s observed in fractions 5 through 8, these fractions were C-ODS-120A (reverse-phase silica gel, C18; 20 X 2.5 cm) nt, the fraction number in the table being indicated with ueous MeOH, (2.1) 40% aqueous MeOH, (3.1) 60% 1 ous MeOH, and (5.1 ) MeOH. Five fractions of 250 to 300 ted for activities using previously prepared cultures of methicillin resistant Staphylococcus aureus. vancomycin resistant enterococcus and Vibrio TABI E HI ACTIVE AGAINST Fraction No |3) Eluent Staphylococcus aureus ( " Fnterococcus (2) Vibrio vulnificus I 1 20% aq MeOH 2 1 40% aq MeOH ++ 3 1 60% aq MeOH ++ 4 1 80% aq MeOH ++ 5 1 MeOH CO CONTROL None c σ 1 1 20% aq MeOH 3 H 2 1 40% aq MeOH ++ C 3 1 60% aq MeOH + m CO x 4 1 80% aq MeOH m m 5 I MeOH H NJ 2 1 40% aq MeOH 3 I 60% aq MeOH 4 1 80% aq MeOH 5 1 MeOH CON! ROI None 1 It'l 40% aq MeOH ( 1 ) Methiullm Resistant Staph> lococciis aureus (3) Combination Of Fractions (2) Vcincoi απ Resistant 1 nterococciis (4).! ratlion No 2 I alter 24 H As indicated in Table III, the active fractions were 2.1, 3.1 and 4.1. Fractions 2.1 and 3.1 were combined because they exhibited very similar UV spectra. Fraction 4.1 was not combined with other fractions because its UV spectra indicated that it was different from the other fractions. Further separation of the combined fractions of 2.1 and 3.1 by HPLC reverse-phase silica gel (C8,250 x 10 mm, 10 μ; 40% MeOH: 50% MeOH: 10% MeCN: H 2 O: MeOH, at a flow rate of 2.0 milliliter per minute and UV detection at 220 nm) provided five additional major fractions 1.2-5.2. Fractions 2.2, 3.2, 4.2 and 5.2 appear to be effective against methicillin resistant Staphylococcus aureus. while fractions 4.2 and 5.2 are effective against vancomycin resistant enterococcus and fractions 2.2 and 3.2 are effective against Vibrio vulnificus 32. The results of this are tabulated in Table IV below. TAB! E IV AC1 IVE AGAINST Fraction No (3> Eluent Staphylococcus aureus (l1 Enterococcus ' Vibrio vulnificus 1 2 30% aq MeOH 2 2 40% aq MeOH 4 + 3 2 60% aq MeOH ++ 4 2 80% aq MeOH ++ 5 2 None ++ O CONTROL c CD 1 2 20% aq MeOH O H H 22 40% aq MeOH C H cn 32 60% aq MeOH m co 42 80% aq MeOH x m m 52 MeOH H 3 CONTROL None c r rπ 12 20% aq MeOH +- σ> 22 40% aq MeOH ++ 32 60% aq MeOH 42 80% aq MeOH 82 MeOH CONTROL None ( 1 ) Mh. I I IICILLIN RLSIS1 AN P S I API IYLOCOCCUS AURLUS + Activity (2) VANCOMYCIN RESIST AN I FNTLROCOCCUS ++Strong Act * Negligible EXAMPLE II The following experiment demonstrates the ability of the Chaetoceros algae to be used both as a nutrient source and as a means to depurate various aquianimals such as mollusks and crustaceans including, for example oysters, clams, shrimp and the like. A closed recirculating system was provided as a culture medium for oysters. Chaetoceros algae was co-cultured along with the oyster to achieve a cell density of the algae in the range of 10 4 to 10 6 cells per liter of culture medium (sea water). Each of the oysters was known to be infected with Vibrio vulnificus. The water in the system was circulated for a sufficient time such that the bivalves would be exposed to and ingest the Chaetoceros algae as food. After three days the oysters were checked for the Vibrio pathogen and were found to be completely free of the bacteria. The natural antibacterial activity of the algae served to cleanse the oyster as it was feeding on the algae. No additional source of antibiotics was incorporated into the circulating water medium. Example II was repeated using shrimp as the aquianimal to be cultured. It is known that pathogenic Vibrio bacteria have been associated with the occurrence of shrimp diseases and that this bacteria is often a carrier of virus as well. The shrimp to be cultured were carriers of the bacteria and were placed in the culturing water having a desirable level of Chaetoceros algae present. The cell density of the algae was approximately 10 4 to 10 5 algae cells per liter of sea water. The culture water was circulated in a closed system for sufficient time to allow ingestion of the algae by the shrimp as a nutrient and subsequent depuration of the Vibrio bacteria. After seven days in the culture medium, the shrimp were tested for the presence of Vibrio bacteria. The results indicated that the shrimp had been cleansed of the bacteria. EXAMPLE IV Example I was repeated, this time substituting Vibrio cholerae as the pathogenic bacteria. The results were identical to Example I. EXAMPLE V Examples II - V were repeated using water that contained Chaetoceros at densities varying from 10 4 to 10 6 cells per milliliter, but with algae removed. No reduction in the presence of Vibrio bacteria was detected. EXAMPLE VI 1.2 Kg (wet weight) of algae paste derived from the sample method of Example 1 was homogenized, in MeOH, filtered and concentrated into a crude extract. This crude . extract was then loaded onto an Amberlite XAD-2 column (Sigma, 30x7cm) and step- eluted with the following reagents: Fraction (1) Hexanes, Fraction (2) Toluene, Fraction (3) CH 2 C1 2 , Fraction (4) EtOAc (ethylacetate). Fractions 1 and 2 contained most of the antibacterial activity and were pooled. The pooled material was then loaded onto a silica gel flash column and eluted with 5:1 to 2: 1 Hexanes: ethyl acetate. The eluent was collected into four fractions. Fractions two and three contained most of the reactive material and were pooled. The pooled fractions were then loaded onto a column and eluted in five fractions with ultracarb 5 ODS; 90: 10 MeCN:H 2 0. Fraction 3 was then loaded onto a Silica HPLC column and eluted with 99:1 Hexanes:2-propanol. The major peak was collected and yielded purified Compound II referred to earlier in this application. Fraction 4 was loaded onto a reversed-phase HPLC column and eluted with either Econosil C8. 65% aqueous MeCN or Ultracarb 5 ODS 30, 80% aqueous MeCN. The major peak was collected and yielded purified compound III referred to earlier in this application. EXAMPLE VII Examples I-V are repeated with Thalassiosira substituted for Chaetoceros. The results obtained are identical. EXAMPLE VIII Examples I-V are repeated with Phaedactylum substituted for Chaetoceros. The results obtained are identical. EXAMPLE IX Examples I-V are repeated with Navicula substituted for Chaetoceros. The results obtained are identical. EXAMPLE X The in vitro cytotoxicity of compounds II, III and IV was determined in NIH 3T3 (mouse fibroblast) cells. Approximately 8.0 X 10 3 NIH 3T3 cells were introduced into a multi-welled tissue culture dish containing approximately 0.5 ml of growth supporting medium. The dish was placed in a humidity controlled tissue culture incubator set at 37°C and the cells were allowed to attach to the bottom of the wells for several hours. Thereafter, the medium was changed and new medium containing 80μg/ml compound II was added. The dish was returned to the tissue culture incubator and remained there for 24 hours. Thereafter, the dish was removed from the incubator and washed several times with phosphate buffered saline at 37°C. The cells were then stained with 0.1% trypan blue. Under a microscope, the number of living cells, i.e., those which did not stain blue, were counted. The above-described procedure was carried out with 20 μg/ml of compounds III and IV, as well as, 12 μl of methanol and cells only as a control. As shown in Table V below, no cytotoxic effects were observed in the NIH 3T3 cells due to the presence of compounds II, III and IV at concentrations which are active against pathogenic bacteria and fungi. y The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention and, all such modifications are intended to be included within the scope of the following claims.

Claims: WHAT IS CLAIMED IS: 1. A composition comprising an antibacterially active marine algae extract obtained from the class Diatomatae. 2. The composition of claim 1, wherein said marine algae are selected from the group consisting of Chaetoceros. Thalassiosira. Phaedactylum. Navicula and mixtures thereof. 3. The composition of claim 1 which is active against methicillin resistant Staphylococcus aureus. vancomycin resistant enterococcus. Vibrio vulnificus. Corvnebacterium xerosis. Streptococcus dvsenteriae. Streptococcus mitis. Streptococcus faecalis. Bacillus subtilis and Bacillus cereus. 4. The composition of claim 1 including one or more isolates of said extract having the formula R 1 -(CH 2 -CH=CH-) n R 2 -COOH (I) wherein R 1 and R 2 are independently alkyl groups of 1-100 carbon atoms; n is a positive integer from 1 to 100; and their pharmaceutically acceptable salts and derivatives thereof. 5. An isolate from the composition of claim 4 having the formula CH 3 CH 2 CH 2 CH=CHCH 2 CH=CHCH 2 CH= HCH 2 CH 2 CH 2 CH 2 COOH (II). 6. An isolate from the composition of claim 4 having the formula CH 3 (CH 2 ) 2 CH=CHCH 2 CH=CH(CH 2 ) 8 COOH (III). 7. An isolate from the composition of claim 4 having the formula CH 3 -CH 2 -CH-CH-CH 2 -CH=CH-CH 2 -CH=CH-CH 2 -CH=CH-CH 2 -CH=CH-CH 2 .CH 2 -CH 2 -COOH (IV). 8. A pharmaceutical composition comprising an antibacterially effective amount of the extract of claim 1 together with a pharmaceutically acceptable carrier. 9. A pharmaceutical composition comprising an antibacterially effective amount of one or more isolates of claim 4 together with a pharmaceutically acceptable carrier. 10. The composition of claim 1 wherein column chromatography and aqueous eluant is used to obtain said extracts from the supernatant liquid of marine algae of said Diatomatae class. 11. The composition of claim 10 wherein said column is a cationic ion exchange column and said aqueous eluant is methanol. 12. The composition of claim 1 wherein solvent extraction is used to obtain said extract. 13. A pharmaceutical composition comprising an antibacterially effective amount of a compound selected from the group consisting of CH 3 CH 2 CH 2 CH=CHCH 2 CH=CHCH 2 CH=CHCH 2 CH 2 CH 2 CH 2 COOH (II), CH 3 (CH 2 ) 2 CH=CHCH 2 CH=CH(CH 2 ) 8 COOH (III) CH r (CH 2 CH=CH) 5 -(CH 2 ) 3 COOH (IV), mixtures thereof and their pharmaceutically acceptable derivatives and salts, together with a pharmaceutically acceptable carrier. 14. A method of treating bacterial infections in mammals comprising administering a pharmaceutical composition comprising one or more antibacterially active isolates obtained from extracts of marine algae in the class Diatomatae. 15. A composition comprising at least one isolate from Diatomatae class marine algae having antibacterial activity obtained by eluting a crude extract of cells from said algae off a chromatography column using an aqueous eluant. 16. The composition of claim 15 wherein said eluant is an aqueous solution of methanol. 17. The composition of claim 15 wherein said eluant is a 10%-90% by volume aqueous solution of methanol. 18. A pharmaceutical composition comprising an antibacterially effective amount of one or more isolates obtained by eluting a supernatant of cells obtained from Diatomatae class marine algae off a chromatography column with an aqueous eluant, together with a pharmaceutically acceptable carrier. 19. The pharmaceutical composition of claim 18 wherein said eluant is an aqueous solution of methanol. 20. The pharmaceutical composition of claim 18 further including the pharmaceutically acceptable derivatives and salts of the compound of formula I. 21. A method of treating bacterial infections in mammals comprising administering to said mammals a pharmaceutical composition comprising one or more isolates obtained by eluting a crude cell extract obtained from Diatomatae class algae off a chromatography column with an aqueous eluant. 22. The method of claim 21 wherein the eluant is aqueous methanol. 23. A compound having antibacterial properties derived from marine algae of the class Diatomatae, said compound having the formula R'-(CH 2 -CH=CH-) n R 2 -COOH (I) wherein R 1 and R 2 are independently alkyl groups of 1-100 carbon atoms; n is a positive integer from 1 to 100; and their pharmaceutically acceptable salts and derivatives thereof. 24. A pharmaceutical composition comprising an antibacterially effective amount of a compound of the formula R'-(CH 2 -CH=CH-) n R 2 -COOH (I) wherein R' and R 2 are independently alkyl groups of 1-100 carbon atoms; n is a positive integer from 1 to 100; and their pharmaceutically acceptable salts and derivatives thereof. 25. The pharmaceutical composition of claim 24 wherein said compound is selected from the group consisting of CH 3 CH 2 CH 2 CH=CHCH 2 CH=CHCH 2 CH=CHCH 2 CH 2 CH 2 CH 2 COOH (II), CHj(CH 2 ) 2 CH=CHCH 2 CH=CH(CH 2 ) 8 COOH (III), CH 3 -(CH 2 CH=CH) 5 -(CH 2 ) 3 COOH (IV) mixtures thereof and their pharmaceutically acceptable derivatives and salts, together with a pharmaceutically acceptable carrier. 26. A method of treating bacterial infections in mammals comprising administering to said mammals the pharmaceutical composition of claim 25. 27. A method of depurating an aquianimal culture contaminated with bacteria comprising introducing a marine algae from the class Diatomatae as a feed for said aquianimal in a sufficient amount and for a sufficient time to effectuate said depuration. 28. The method of claim 27, wherein said marine algae is present at a cell density of about 10 4 to about 10 6 cells per litter of culture medium. 29. The method of claim 27, wherein said aquianimal culture is selected from the group consisting of marine members of the phyla Mollusca, Arthropoda and mixtures thereof. 30. The method of claim 27, wherein said aquianimal culture is further selected from the group consisting of oysters, clams, snails, scallops, shrimp, crabs, prawns, lobsters, abalone, mussel and krill. 31. A method of controlling the growth of bacteria in an aquiculture comprising the steps of co-culturing marine algae of the Diatomatae class and an aquianimal in said aquiculture. whereby said algae are ingested as food by said aquianimal and provide antibacterial activity in the presence of said bacteria. 32. The method of claim 31 , wherein said algae are present in said aquiculture at a cell density of about 10 4 cells to about 10 6 cells per liter of water.

Inventors: WANG JAW-KAI, WANG, JAW-KAI
Applicants: AQUACULTURE TECHNOLOGY INC, WANG JAW KAI, AQUACULTURE TECHNOLOGY, INC., WANG, JAW-KAI
Application year: 1997
Main classification: A01N63
IPC codes: A01N37, A01N63, A01N65, A61K31, A61K36, A61P31