VETERINARY COMPOSITIONS FOR THE TREATMENT AND/OR PREVENTION OF PROTOZOAN DISEASES AND METHODS OF PREPARATION THEREOF

Abstract

Veterinary compositions comprising an essential oil such as parsley seed oil, lovage seed oil, and/or celery seed oil are disclosed. The essential oil is present in the form of a complex with a mixture of organic acids such as four acids of valeric acid, isovaleric acid, lactic acid, butyric acid, acetic acid, propionic acid, formic acid, benzoic acid, pelargonic acid, salicylic acid, malonic acid, citric acid, phthalic acid, tartaric acid, oxalic acid, malic acid, shikimic acid, fumaric acid, mandelic acid, cinnamic acid or derivatives thereof and a metal such as iron, bismuth, zinc, copper, manganese, cobalt, molybdenum, chromium, silver, gold, platinum, tungsten, germanium, tin, strontium, antimony, or salts or oxides thereof. The composition may be for the treatment and/or prevention of diseases caused by protozoa in animals. Methods of manufacturing the veterinary compositions are also disclosed.

Claims

1.-8. (canceled)

9. A veterinary composition to treat and/or prevent of diseases caused by protozoa in animals, the composition comprising: an essential oil including parsley seed oil, lovage seed oil, or celery seed oil, the essential oil present in a form of a complex with a mixture of organic acids and a metal, the of organic acids mixture including a plurality of acids selected from the group consisting of valeric acid, isovaleric acid, lactic acid, butyric acid, acetic acid, propionic acid, formic acid, benzoic acid, pelargonic acid, salicylic acid, malonic acid, citric acid, phthalic acid, tartaric acid, oxalic acid, malic acid, shikimic acid, fumaric acid, mandelic acid, cinnamic acid, or derivatives thereof and the metal including iron, bismuth, zinc, copper, manganese, cobalt, molybdenum, chromium, silver, gold, platinum, tungsten, germanium, tin, strontium, antimony, or salts or oxides thereof.

10. The composition of claim 9, wherein the plurality of acids includes at least four acids.

11. The composition of claim 10, wherein the plurality of acids includes acetic acid, propionic acid, lactic acid, and formic acid.

12. The composition of claim 11, wherein the acids in the mixture of organic acids are mixed in a ratio of 1:1:1:1.

13. The composition of claim 11, wherein the essential oil includes lovage seed oil or celery seed oil.

14. The composition of claim 11, wherein the metal is zinc or copper.

15. The composition of claim 9, wherein the essential oil includes lovage seed oil.

16. The composition of claim 9, wherein the essential oil includes celery seed oil.

17. The composition of claim 9, wherein the metal includes copper.

18. The composition of claim 9, wherein the metal includes zinc.

19. A method of manufacturing a veterinary composition to treat and/or prevent protozoan diseases in animals, the method comprising: a) mixing an essential oil and a plurality of organic acids at a ratio of 80:1 to 1:80 by weight, the essential oil including parsley seed oil, lovage seed oil, or celery seed oil and the plurality of organic acids including at least four acids selected from the group consisting of valeric acid, isovaleric acid, lactic acid, butyric acid, acetic acid, propionic acid, formic acid, benzoic acid, pelargonic acid, salicylic acid, malonic acid, citric acid, phthalic acid, tartaric acid, oxalic acid, malic acid, shikimic acid, fumaric acid, mandelic acid, cinnamic acid or derivatives thereof; b) adding a catalyst and a metal including iron, bismuth, zinc, copper, manganese, cobalt, molybdenum, chromium, silver, gold, platinum, tungsten, germanium, tin, strontium, antimony, or salts or oxides thereof to form a mixture; c) heating the mixture to form a reaction product; d) allowing the reaction product to cool; and e) filtering the reaction product.

20. The method of claim 19, wherein the essential oil is mixed with the plurality of organic acids at a ratio of 1:1 by weight.

21. The method of claim 19, wherein the plurality of organic acids includes acetic acid, propionic acid, lactic acid and formic acid.

22. The method of claim 21, wherein acids in the plurality of organic acids are mixed at a ratio of 1:1:1:1.

23. The method of claim 19, wherein the catalyst includes cobalt sulphate, ammonium molybdate, and manganese chloride or sulphate.

24. The method of claim 19, wherein the mixture is heated under reflux.

25. The method of claim 19, wherein the mixture is heated for 20 to 120 minutes.

26. The method of claim 19, wherein the reaction product is cooled for 10 to 24 hours.

27. The method of claim 19, wherein the mixture is heated to a boiling point of the mixture.

28. The method of claim 19, wherein the essential oil is celery seed oil.

Description

DETAILED DESCRIPTION

[0074] The compositions and methods are set out in detail in the following examples, wherein all tests and experimental procedures described below were carried out using commercially available test kits, reagents and devices, following the recommendations of the manufacturers of the kits, reagents and devices used, unless otherwise expressly indicated. All test parameters were measured using standard, well-known methods used in the field.

[0075] All raw materials used in the study are approved for both animal and human nutrition by the relevant directives and authorities. The selection of raw materials was made on the basis of Codex Alimentarius, i.e. the Codex Alimentarius established by FAO and WHO, Der Deutsche Arzneimittel-Codex (DAC), guidelines of the European Food Safety Authority (EFSA) and Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 Aug. 2003 on additives for use in animal nutrition. In addition, the essential oils used in the study met the requirements of the European Pharmacopoeia, the Swiss Pharmacopoeia and Der Deutsche Arzneimittel-Codex (DAC).

[0076] For in vitro tests of antiprotozoal activity of the composition five reference organisms representing taxonomic groups to which pathogenic protozoa belong were selected, i.e: [0077] Amoeba proteus—Chaos diffluens—a protozoan of the order Euamoebida, belonging to fifth supergroup of the Amoebozoa, living in waters. [0078] Paramecium caudatum—a slipper animalcule representing the Ciliata orachs, living in waters. [0079] Gregarina blattarum—gregarine isolated from cockroaches, representing the phylum Apicomplexa, living in the digestive tracts or body cavities of invertebrates. [0080] Euglena gracilis—a protozoan living in water, representing the flagellates—Mastigophora, family Euglenaceae. [0081] Trichomonas hominis—a protozoan living in the human colon, representing the Trichomonadidae.

[0082] Amoeba, Paramecium, Trichomonas and Euglena were observed under a microscope on watch glasses with viscose wool fibers (to facilitate observation) in a drop of water from the culture they came from. Different concentrations of the test compositions were introduced to the test samples, establishing an LD.sub.50 dose (50% mortality) and an LD.sub.100 dose (100% mortality). In all cases, 4-fold replicates of the test were used together with a blank test.

[0083] The gregarines were isolated from the cockroaches and, after being placed on a watch slide, were treated with the products at different concentrations in Ringer's solution. Each sample contained ten individuals. The lethal concentration of the substance for 50% and 100% of the individuals (LD.sub.50, LD.sub.100) within 3 minutes was determined. Isolation of gregarines from cockroaches was performed on the basis of the method of isolation of gregarines from beetles proposed by J. Moraczewski (Moraczewski J.: Exercises in the zoology of invertebrates. 1st Edition, PWN, Warsaw 1974, p. 29-31, p. 285-292).

[0084] Identification of individual protozoa was made on the basis of their descriptions and drawings after W. A. Dogiel and J. Hempel-Zawitkowska (Dogiel W. A.: Invertebrate zoology. 3rd edition, National Agricultural and Forest Publishing House 1972; Hempel-Zawitkowska J., Galka B., Kalińska B., Kamionek M, Komosińska H., Pezowicz E. Podsiadlo E., Sulgostowska T.: Zoology for agricultural universities. Scientific Publishing House PWN 2008).

[0085] The compositions, negative controls, and positive controls were dissolved in an aqueous solution of polysorbate 80 (0.05%) before application to a watch slide. No lethal effect of polysorbate 80 at the above concentration was observed.

EXAMPLE 1

A combination of a lovage seed Oil (Levisticum officinale W. D. J. Koch) with a Mixture of Acids in a Ratio of 1:1 by Weight and with Copper or Zinc

[0086] In this non-limiting example, the following two compositions were prepared: [0087] a) composition I—i.e., a composition of lovage seed oil with a mixture of acids and copper carbonate [0088] b) composition II—i.e., a composition of lovage seed oil with a mixture of acids and zinc carbonate

[0089] Although, in the non-limiting example the metal salts are in the form of carbonates, other salts (e.g. chlorides, sulfates) or other forms, e.g. oxides may also be used in the composition.

[0090] Furthermore, in this non-limiting example the mixture of organic acids is a mixture of acetic acid, propionic acid, lactic acid and formic acid. However, any combination of four acids selected from the group comprising valeric acid, isovaleric acid, lactic acid, butyric acid, acetic acid, propionic acid, formic acid, benzoic acid, pelargonic acid, salicylic acid, malonic acid, citric acid, phthalic acid, tartaric acid, oxalic acid, malic acid, shikimic acid, fumaric acid, mandelic acid, cinnamic acid or derivatives thereof, may be used for the preparation of the composition.

[0091] In order to prepare composition I, 100 ml of an acid mixture (containing acetic, propionic, lactic and formic acids mixed in a ratio of 1:1:1:1), 0.6 g of catalyst (which is cobalt sulfate, ammonium molybdate and manganese chloride mixed in a ratio of 1:1:1) and 5 g of copper carbonate were added to 100 ml of lovage seed oil. The mixture was heated at the boiling point, until the colour changed, under reflux for 20 minutes. The mixture was then left to cool (for 10 hours) to obtain a clear solution (one, two or three phase solution). After this time, the reaction product was filtered through filter paper. Composition II was prepared analogously to composition I, except that zinc carbonate was used as the metal. Compositions I and II were then analysed for their antiprotozoal properties. For this purpose, both compositions were diluted: 0.001% to 1%, after which the following protozoa were placed in each dilution: [0092] Amoeba proteus—Chaos diffluens—a protozoan of the order Euamoebida, belonging to fifth supergroup of the Amoebozoa, living in waters. [0093] Paramecium caudatum—a slipper animalcule representing the Ciliata orachs, living in waters. [0094] Gregarina blattarum—gregarine isolated from cockroaches, representing the phylum Apicomplexa, living in the digestive tracts or body cavities of invertebrates. [0095] Euglena gracilis—a protozoan living in water, representing the flagellates—Mastigophora, family Euglenaceae. [0096] Trichomonas hominis—a protozoan living in the human colon, representing the Trichomonadidae.

[0097] Individual acids, catalyst solution, essential oil and metal salt solutions were additionally analysed for protozoal properties. The tested preparations were dissolved in an aqueous solution of polysorbate 80 (0.05%) before being applied to a watch glass. No killing effect of polysorbate 80 in the above mentioned concentration was observed. Observation under a fluorescence microscope with phase contrast was carried out. Protozoicidal activity was considered effective when the death of 50% and 100% of individuals occurred within 3 minutes. The control antiprotozoal substances were CH—chloramphenicol and M—metronidazole.

[0098] The results obtained from the protozoal activity test are presented in Table 1. The results of the analysis showed that the killing and static activity in the compositions and after the reaction was higher than that of the individual substances in the reaction mixtures and complexes. Compositions I and II show many times stronger (potentiation) protozoal activity than each of these components separately. All the ingredients used in the compositions are approved for both animal and human nutrition by the relevant directives and authorities, which, combined with their high efficacy, allows their use in the treatment and/or prevention of parasitoses in animals, caused by protozoa, in particular histomonadiasis (caused by Histomonas meleagridis), coccidiosis (caused by Eimeria), cryptosporidiosis (caused by Cryptosporidium), trichomonadiasis (caused by Trichomonas), babesiosis (caused by Babesia), or amoebiasis (caused by Amoeba).

TABLE-US-00001 TABLE 1 LD.sub.50, LD.sub.100 values for compositions I and II, determined for selected protozoa. Sample Mixture Copper Zinc Lovage of Catalyst carbonate carbonate seed Composition Composition Protozoa CH* M** acids solution solution solution oil I II Euglena LD.sub.50: 0.05% LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: gracilis LD.sub.100: 0.1% LD.sub.100: 0.5% 0.05% 0.15% 0.1% 0.01% 0.006% 0.003% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 1.1% 0.1% 0.25% 0.3% 0.015% 0.008% 0.005% Gregarina LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: blattarum LD.sub.100: 0.13% 0.9% 0.07% 0.12% 0.2% 0.05% 0.007% 0.004% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.3% 1.1% 0.3% 0.37% 0.4% 0.06% 0.009% 0.006% Amoeba LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: proteus 0.07% 0.3% 0.6% 0.05% 0.09% 0.15% 0.05% 0.004% 0.006% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.15% 0.5% 1% 1% 0.17% 0.25% 0.06% 0.006% 0.009% Paramecium LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: caudatum 0.001% LD.sub.100: 0.8% 0.8% 0.35% 0.3 0.005% 0.004% 0.006% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.006% 1.25% 1.25% 0.5% 0.5% 0.01% 0.007% 0.008% Trichomonas LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: hominis LD.sub.100: 0.05% 0.8% 0.9% 0.1% 0.25% 0.06% 0.065% 0.03% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.1% 1% 1.1% 0.3% 0.4% 0.09% 0.085% 0.05% *CH—chloramphenicol; **M—metronidazole

EXAMPLE 2

A Combination of a Lovage Seed Oil (Levisticum officinale W. D. J. Koch) with a Mixture of Acids in a Ratio of 80:1 by Weight and with Copper or Zinc

[0099] In this non-limiting example, the metals used for preparing the composition according to the invention were copper and zinc. Nevertheless, other metals, e.g. iron, bismuth, zinc, copper, manganese, cobalt, molybdenum, chromium, silver, gold, platinum, tungsten, germanium, tin, strontium, antimony, salts thereof, or oxides thereof may also be used in the method according to the invention.

[0100] The following two compositions were prepared: [0101] a) composition III—i.e., a composition of lovage seed oil with a mixture of acids and copper carbonate [0102] b) composition IV—i.e., a composition of lovage seed oil with a mixture of acids and zinc carbonate

[0103] Although, in the non-limiting example of the implementation, metal salts in the form of carbonates were used, other salts (e.g. chlorides, sulfates) or other forms, e.g. oxides, may be used in the composition according to the invention.

[0104] In order to prepare composition III, 1 ml of a mixture of acids (containing acetic acid, propionic acid, lactic acid and formic acid mixed in a ratio of 1:1:1:1), 0.1 g of catalyst (which is cobalt sulfate, ammonium molybdate and manganese sulfate mixed in a ratio of 1:1:1) and 1 g of copper carbonate were added to 80 ml of lovage seed oil. The mixture was heated at the boiling point, until the color changed, under reflux for 120 minutes. The mixture was then left to cool (for 24 hours) to obtain a clear solution (one, two or three phase solution). After this time, the reaction product was filtered through filter paper. Composition IV was prepared analogously to composition III, except that zinc carbonate was added instead of copper carbonate.

[0105] Compositions III and IV were then analysed for their antiprotozoal properties analogously to Example 1, and the results confirming the antiprotozoal properties of the compositions tested are shown in Table 2.

TABLE-US-00002 TABLE 2 LD.sub.50, LD.sub.100 values for compositions IV and V, determined for selected protozoa. Sample Mixture Copper Zinc Lovage of Catalyst carbonate carbonate seed Composition Composition Protozoa CH* M** acids solution solution solution oil III IV Euglena LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: gracilis 0.05% LD.sub.100: 0.5% 0.05% 0.15% 0.1% 0.01% 0.008% 0.009% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.1% 1.1% 0.1% 0.25% 0.3% 0.015% 0.009% 0.01% Gregarina LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: blattarum LD.sub.100: 0.13% 0.9% 0.07% 0.12% 0.2% 0.05% 0.035% 0.025% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.3% 1.1% 0.3% 0.37% 0.4% 0.06% 0.045% 0.05% Amoeba LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: proteus 0.07% 0.3% 0.6% 0.05% 0.09% 0.15% 0.05% 0.035% 0.04% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.15% 0.5% 1% 1% 0.17% 0.25% 0.06% 0.05% 0.05% Paramecium LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: caudatum 0.001% LD.sub.100: 0.8% 0.8% 0.35% 0.3 0.005% 0.004% 0.003% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.006% 1.25% 1.25% 0.5% 0.5% 0.01% 0.008% 0.008% Trichomonas LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: hominis LD.sub.100: 0.05% 0.8% 0.9% 0.1% 0.25% 0.06% 0.045% 0.04% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.1% 1% 1.1% 0.3% 0.4% 0.09% 0.065% 0.06% *CH—chloramphenicol; **M—metronidazole

EXAMPLE 3

A Combination of a Celery Seed Oil (Apium graveolens L.) with a Mixture of Acids in a Ratio of 1:1 by Weight and with Copper or Zinc

[0106] In this non-limiting example, the following two compositions were prepared: [0107] a) composition V—i.e. a composition of celery seed oil with a mixture of acids and copper carbonate [0108] b) composition VI—i.e. a composition of celery seed oil with a mixture of acids and zinc carbonate

[0109] Although, in this non-limiting example, celery seed oil was used to produce the composition, parsley seed oil can also be used interchangeably in the composition.

[0110] Whereby, in order to produce composition V, 100 ml of an acid mixture (comprising acetic acid, propionic acid, lactic acid and formic acid mixed in a ratio of 1:1:1:1), 0.6 g of catalyst (which is cobalt sulphate, ammonium molybdate and manganese sulphate mixed in a ratio of 1:1:1) and 5 g of copper carbonate were added to 100 ml of celery seed oil. The mixture was heated at boiling point, until the colour changed, under reflux for 120 minutes. The mixture was then left to cool (for 24 hours) to obtain a clear solution (one, two or three phase solution). After this time, the reaction product was filtered through filter paper. Composition VI was prepared analogously to composition V, except that zinc carbonate was added instead of copper carbonate. Compositions V and VI were then analysed for their antiprotozoal properties as in Example 1, and the results are shown in Table 3. The results of the analysis showed that the killing and static activity in complex systems and after the reaction was higher than that of the substances separately, included in the reaction mixtures and complexes.

[0111] Compositions V and VI show many times stronger (potentiation) protozoal activity than each of these components separately. All the ingredients used in the compositions according to the invention are approved for both animal and human nutrition by the relevant directives and authorities, which, combined with their high efficacy, allows their use in the treatment and/or prevention of parasitoses in animals, caused by protozoa, in particular histomonadiasis (caused by Histomonas meleagridis), coccidiosis (caused by Eimeria), cryptosporidiosis (caused by Cryptosporidium), trichomonadiasis (caused by Trichomonas), babesiosis (caused by Babesia), or amoebiasis (caused by Amoeba).

TABLE-US-00003 TABLE 3 LD.sub.50, LD.sub.100 values for compositions V and VI, determined for selected protozoa. Sample Mixture Copper Zinc Celery of Catalyst carbonate carbonate seed Composition Composition Protozoa CH* M** acids solution solution solution oil V VI Euglena LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: gracilis 0.05% LD.sub.100: 0.5% 0.05% 0.15% 0.1% 0.02% 0.002% 0.003% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.1% 1.1% 0.1% 0.25% 0.3% 0.03% 0.003% 0.005% Gregarina LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: blattarum LD.sub.100: 0.13% 0.9% 0.07% 0.12% 0.2% 0.01% 0.003% 0.004% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.3% 1.1% 0.3% 0.37% 0.4% 0.02% 0.005% 0.006% Amoeba LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: proteus 0.07% 0.3% 0.6% 0.05% 0.09% 0.15% 0.01% 0.002% 0.002% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.15% 0.5% 1% 1% 0.17% 0.25% 0.02% 0.004% 0.005% Paramecium LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: caudatum 0.001% LD.sub.100: 0.8% 0.8% 0.35% 0.3 0.02% 0.003% 0.003% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.006% 1.25% 1.25% 0.5% 0.5% 0.03% 0.004% 0.005% Trichomonas LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: hominis LD.sub.100: 0.05% 0.8% 0.9% 0.1% 0.25% 0.01% 0.015% 0.01% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.1% 1% 1.1% 0.3% 0.4% 0.02% 0.03% 0.03% *CH—chloramphenicol; **M—metronidazole

EXAMPLE 4

A Combination of a Celery Seed Oil (Apium graveolens L.) with a Mixture of Acids in a Ratio of 1:80 by Weight and with Copper or Zinc

[0112] In this non-limiting example, the following two compositions were prepared: [0113] a) composition VII—i.e., a composition of celery seed oil with a mixture of acids and copper carbonate [0114] b) composition VIII—i.e., a composition of celery seed oil with a mixture of acids and zinc carbonate

[0115] In order to produce composition VII, 80 ml of acid mixture (containing acetic, propionic, lactic and formic acids mixed in the ratio 1:1:1:1), 1 g of catalyst (which is cobalt sulphate, ammonium molybdate and manganese chloride mixed in the ratio 1:1:1) and 5 g of copper carbonate were added to 1 ml of celery seed oil. The mixture was heated at the boiling point, until the colour changed, under reflux for 60 minutes. The mixture was then left to cool (for 12 hours) to obtain a clear solution (one, two or three phase solution). After this time, the reaction product was filtered through filter paper.

[0116] Composition VIII was prepared analogously to composition VII, except that zinc carbonate was added instead of copper carbonate. Compositions VII and VIII were then analysed for their antiprotozoal properties analogously to Example 1, and the results confirming the effectiveness of the compositions are shown in Table 4.

TABLE-US-00004 TABLE 4 LD.sub.50, LD.sub.100 values for compositions VII and VIII, determined for selected protozoa. Sample Mixture Copper Zinc Celery of Catalyst carbonate carbonate seed Composition Composition Protozoa CH* M ** acids solution solution solution oil VII VIII Euglena LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: gracilis 0.05% LD.sub.100: 0.5% 0.05% 0.15% 0.1% 0.02% 0.009% 0.01% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.1% 1.1% 0.1% 0.25% 0.3% 0.03% 0.015% 0.013% Gregarina LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: blattarum LD.sub.100: 0.13% 0.9% 0.07% 0.12% 0.2% 0.01% 0.009% 0.008% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.3% 1.1% 0.3% 0.37% 0.4% 0.02% 0.01% 0.009% Amoeba LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: proteus 0.07% 0.3% 0.6% 0.05% 0.09% 0.15% 0.01% 0.009% 0.008% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.15% 0.5% 1% 1% 0.17% 0.25% 0.02% 0.012% 0.01% Paramecium LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: caudatum 0.001% LD.sub.100: 0.8% 0.8% 0.35% 0.3% 0.02% 0.01% 0.015% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.006% 1.25% 1.25% 0.5% 0.5% 0.03% 0.02% 0.02% Trichomonas LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: LD.sub.50: hominis LD.sub.100: 0.05% 0.8% 0.9% 0.1% 0.25% 0.01% 0.009% 0.008% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.1% 1% 1.1% 0.3% 0.4% 0.02% 0.015% 0.01% *CH—chloramphenicol; **M—metronidazole