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

Abstract

Veterinary compositions including an essential oil such as anise oil, cinnamon oil, tarragon oil, clove oil, dill oil, Balsam of Peru, Ballota oil, star anise oil, and/or calamus oil are disclosed. The essential oil may be present in the form of a complex with a mixture of organic acids such as four 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 or derivatives thereof and a metal such as molybdenum, cobalt, nickel, chromium, zinc, bismuth, copper, manganese, selenium, iron, and/or their salts or oxides. The compositions may be used for the treatment and/or prevention of protozoan diseases in animals. Methods of manufacturing the veterinary compositions are also disclosed.

Claims

1.-8. (canceled)

9. A veterinary composition to treat and/or prevent protozoan diseases in animals, the composition comprising: an essential oil including aniseed oil, cinnamon oil, tarragon oil, clove oil, dill oil, Balsam of Peru, Ballota oil, star anise oil, or calamus oil, the essential oil present in a form of a complex with a mixture of organic acids and a metal, the 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, or derivatives thereof and the metal including molybdenum, cobalt, nickel, chromium, zinc, bismuth, copper, manganese, selenium, iron, 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 at a ratio of 1:1:1:1.

13. The composition of claim 11, wherein the essential oil includes aniseed oil, clove oil, or cinnamon oil.

14. The composition of claim 9, wherein the essential oil includes aniseed oil.

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

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

17. The composition of claim 9, wherein the essential oil includes clove oil.

18. The composition of claim 9, wherein the essential oil includes cinnamon oil.

19. A method of manufacturing a veterinary composition to treat and/or prevent of 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 aniseed oil, cinnamon oil, tarragon oil, clove oil, dill oil, Balsam of Peru, Ballota oil, star anise oil, or calamus oil and the plurality of organic acids including at least four acids selected from the group consisting of valerian, isovalerian, lactic, butyric, acetic, propionic, formic, benzoic, pelargonic, salicylic, malonic, citric, phthalic, tartaric, oxalic, malic, shikimic, fumaric, mandelic, cinnamic acids or derivatives thereof; b) adding a catalyst and a metal including molybdenum, cobalt, nickel, chromium, zinc, bismuth, copper, manganese, selenium, iron, 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 a mixture of 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 aniseed, clove, or cinnamon 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

[0086] A combination of aniseed oil (Pimpinella anisum L.) with a mixture of acids in a ratio of 1:1 by weight and with copper or zinc.

[0087] In this non-limiting example, the following two compositions were prepared: [0088] a) composition I—i.e., a composition of aniseed oil with a mixture of acids and copper carbonate; [0089] b) composition II—i.e., a composition of aniseed oil with a mixture of acids and zinc carbonate;

[0090] Although, in the non-limiting example the metal salts are in the form of carbonates, other salts (e.g. chlorides, sulphates) or other forms, e.g. oxides, may also be used.

[0091] Furthermore, in this non-limiting example of implementation 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, isovaleric, lactic, butyric, acetic, propionic, formic, benzoic, pelargonic, salicylic, malonic, citric, phthalic, tartaric, oxalic, malic, shikimic, fumaric, mandelic, cinnamic acids or derivatives thereof may be used for preparation of the composition.

[0092] In order to prepare composition I, a 100 ml of an acid mixture (comprising acetic, propionic, lactic and formic acids mixed in a ratio of 1:1:1:1), 0.6 g of catalyst (which is cobalt sulphate, 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 essential oil. The mixture was heated at boiling point, until the change of colour, 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 component.

[0093] 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: [0094] Amoeba proteus—Chaos diffluens—a protozoan of the order Euamoebida, belonging to fifth supergroup of the Amoebozoa, living in waters. [0095] Paramecium caudatum—a slipper animalcule representing the Ciliata orachs, living in waters. [0096] Gregarina blattarum—gregarine isolated from cockroaches, representing the phylum Apicomplexa, living in the digestive tracts or body cavities of invertebrates. [0097] Euglena gracilis—a protozoan living in water, representing the flagellates—Mastigophora, family Euglenaceae. [0098] Trichomonas hominis—a protozoan living in the human colon, representing the Trichomonadidae.

[0099] Individual acids, catalyst solution, essential oil and metal salt solutions were also 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.

[0100] 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 Copper Zinc Mixture Catalyst carbonate carbonate Aniseed Compo- Compo- Protozoa CH* M** of acids solution solution solution oil sition I sition II 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% 0.2% 0.004% 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: 0.1% 1.1% 0.1% 0.25% 0.3% 0.4% 0.008% 0.004% 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% 0.4% 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: 0.3% 1.1% 0.3% 0.37% 0.4% 0.6% 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% 0.1% 0.003% 0.005% 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% 0.2% 0.006% 0.006% 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% 0.2% 0.002% 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% 0.3% 0.006% 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% 0.08% 0.045% 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: 0.1% 1% 1.1% 0.3% 0.4% 0.09% 0.075% 0.035% *CH—chloramphenicol; **M—metronidazole

Example 2

[0101] A combination of aniseed oil (Pimpinella anisum L.) with a mixture of acids in a ratio of 80:1 by weight and with copper or zinc.

[0102] In this non-limiting example, the metals used for preparing the composition were copper and zinc. Nevertheless, other metals may also be used in the method, e.g. molybdenum, cobalt, nickel, chromium, zinc, bismuth, copper, manganese, selenium, iron, salts thereof, or oxides thereof.

[0103] The following two compositions were prepared: [0104] a) composition III—i.e., a composition of aniseed oil with a mixture of acids and copper carbonate; [0105] b) composition IV—i.e., a composition of aniseed oil with a mixture of acids and zinc carbonate;

[0106] Although, in this non-limiting example, metal salts in the form of carbonates were used, other salts (e.g. chlorides, sulphates) or other forms, e.g. oxides can be used in composition.

[0107] In order to prepare composition III, 1 ml of an acid mixture (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 aniseed oil. The mixture was heated at the boiling point, until the change of colour, 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.

[0108] 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 Copper Zinc Mixture Catalyst carbonate carbonate Aniseed Compo- Compo- Protozoa CH* M** of acids solution solution solution oil sition III sition 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% 0.2% 0.03% 0.02% 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% 0.4% 0.08% 0.06% 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% 0.4% 0.055% 0.045% 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% 0.6% 0.2% 0.1% 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% 0.1% 0.03% 0.035% 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% 0.2% 0.1% 0.15% 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 0.2% 0.1% 0.08% 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% 0.3% 0.17% 0.13% 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% 0.08% 0.06% 0.05% 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% 0.07% 0.07% *CH—chloramphenicol; **M—metronidazole

Example 3

[0109] A combination of clove oil (Syzygium aromaticum (L.) Merr. & Perry) with a mixture of acids in a ratio of 1:1 by weight and with copper or zinc.

[0110] In this non-limiting example, the following two compositions were prepared: [0111] a) composition V—i.e., a composition of clove oil with a mixture of acids and copper carbonate; [0112] b) composition VI—i.e., a composition of clove oil with a mixture of acids and zinc carbonate;

[0113] Although, in this non-limiting example of implementation, the clove oil was used in the composition, another oil selected from the group comprising anise oil, cinnamon oil, tarragon oil, dill oil, Balsam of Peru, Ballota oil, star anise oil, calamus oil can be used in the composition.

[0114] In order to make composition V, a 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 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 essential oil. The mixture was heated at the boiling point, until the change of colour, 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.

[0115] 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. Compositions V and VI 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-00003 TABLE 3 LD.sub.50, LD.sub.100 values for compositions V and VI, determined for selected protozoa. Sample Copper Zinc Mixture Catalyst carbonate carbonate Compo- Compo- Protozoa CH* M** of acids solution solution solution Clove oil sition V sition 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% 0.001% 0.001% 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% 0.002% 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% 0.002% 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: 0.3% 1.1% 0.3% 0.37% 0.4% 0.02% 0.004% 0.005% 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% 0.001% 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% 0.003% 0.004% 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% 0.001% 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: 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% 0.004% 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: 0.1% 1% 1.1% 0.3% 0.4% 0.02% 0.006% 0.007% *CH—chloramphenicol; **M—metronidazole

Example 4

[0116] A combination of clove oil (Syzygium aromaticum (L.) Merr. & Perry) with a mixture of acids in a ratio of 1:80 by weight and with copper or zinc.

[0117] In this non-limiting example, the following two compositions were prepared: [0118] a) composition VII—i.e., a composition of clove oil with a mixture of acids and copper carbonate; [0119] b) composition VIII—i.e., a composition of clove oil with a mixture of acids and zinc carbonate;

[0120] In order to prepare composition VII, 80 ml of a mixture of acids (containing acetic, propionic, lactic and formic acids mixed in a ratio 1:1:1), 1 g of catalyst (which is cobalt sulphate, ammonium molybdate and manganese sulphate mixed in a ratio 1:1:1) and 5 g of copper carbonate were added to 1 ml of clove oil. The mixture was heated at the boiling point, until the colour has 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). After this time, the reaction product was filtered through filter paper.

[0121] 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 Clove oil (Syzygium Copper Zinc aromaticum Mixture Catalyst carbonate carbonate (L.) Merr. & Compo- Compo- Protozoa CH* M** of acids solution solution solution Perry) sition VII sition 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% 0.01% 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% 0.03% 0.02% 0.015% 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% 0.012% 0.01% 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% 0.009% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.15% 0.5% 1% 1% 0.17% 0.25% 0.02% 0.014% 0.012% 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% 0.009% LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: LD.sub.100: 0.006% 1.25% 1.25% 0.5% 0.5% 0.03% 0.018% 0.015% 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% 0.009% 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% 1.1% 0.3% 0.4% 0.02% 0.015% 0.017% *CH—chloramphenicol; **M—metronidazole

Example 5

[0122] A combination of cinnamon oil (Cinnamomum sp.) with a mixture of acids in a ratio of 1:1 by weight and with copper or zinc.

[0123] In this non-limiting example, the two following compositions were prepared: [0124] a) composition IX—i.e., a composition of clove oil with a mixture of acids and copper carbonate; [0125] b) composition X—i.e., a composition of clove oil with a mixture of acids and zinc carbonate;

[0126] In order to produce composition IX, 100 ml of a mixture of acids (containing acetic, propionic, lactic and formic acids mixed in a 1:1:1:1 ratio), 0.6 g of a catalyst (which is cobalt sulphate, ammonium molybdate and manganese sulphate mixed in a 1:1:1 ratio) and 5 g of copper carbonate were added to 100 ml of cinnamon oil. The mixture was heated at the boiling point, until the colour changed, under reflux for 120 minutes. The mixture was then allowed 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 X was prepared analogously to composition IX, except that zinc carbonate was used instead of copper carbonate.

[0127] Compositions IX and X were then analysed for their antiprotozoal properties analogously to Example 1, and the results confirming the effectiveness of the compositions are shown in Table 5.

TABLE-US-00005 TABLE 5 LD.sub.50, LD.sub.100 values for compositions IX and X, determined for selected protozoa. Sample Copper Zinc Mixture Catalyst carbonate carbonate Cinnamon Compo- Compo- Protozoa CH* M** of acids solution solution solution oil sition IX sition X 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% 0.005% 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% 0.007% 0.006% 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% 0.005% 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: 0.3% 1.1% 0.3% 0.37% 0.4% 0.035% 0.006% 0.005% 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% 0.003% 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: LD.sub.100: 0.15% 0.5% 1% 1% 0.17% 0.25% 0.06% 0.005% 0.006% 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% 0.002% 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: 0.006% 1.25% 1.25% 0.5% 0.5% 0.045% 0.005% 0.007% 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% 0.02% 0.05% 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% 0.04% 0.07% 0.065% *CH—chloramphenicol; **M—metronidazole