PL243785B1 - Veterinary composition for the treatment and/or prevention of diseases caused by protozoa in animals, containing eucalyptus oil, and a method for preparing the same - Google Patents

Abstract

The subject of the invention is a veterinary composition for the treatment and/or prevention of diseases caused by protozoa in animals, containing eucalyptus oil, characterized by the fact that eucalyptus oil is in the form of a complex with an organic acid and a metal. Another subject of the invention is a method for preparing a veterinary composition for the treatment and/or prevention of diseases caused by protozoa in animals, containing eucalyptus oil, which includes the following steps: a) mixing eucalyptus oil with an organic acid in a weight ratio of 80:1 to 1:80; b) adding a catalyst to the mixture from step a); c) adding a metal selected from the group consisting of copper, zinc, manganese, salts, or oxides thereof; d) heating the mixture with the catalyst obtained in step c) to the boiling point and continuing heating at the reflux temperature for 20 - 120 minutes; e) allowing the reaction product to cool for a period of 10 to 24 hours; f) filtering the cooled reaction product.

Description

The subject of the invention is a veterinary composition for the treatment and/or prevention of diseases caused by protozoa in animals, containing eucalyptus oil, and a method of preparing it.

The literature on the subject indicates that some secondary plant metabolites, such as phytoncides and phytoalexins, have potential killing and static properties against protozoa, bacteria, viruses and fungi.

Phytoncides (Greek phyton - plant; cid - a syllable meaning killing properties) were first detected by Soviet researchers in 1928-1930. The greatest achievements in phytoncide research are made by: G.I. Nilovv, B. P. Tokin (1900-1984), A. Filatov and I. Toroptsev. The term and definition of phytoncide were introduced by B.P. Tokina. Phytoncides are substances secreted and excreted by higher plants (Cormophyta) with antibacterial, protozoal and fungicidal properties. Phytoncides are the equivalent of antibiotics produced by bacteria, fungi and lichens (Tariq, S., S. Wani, W. Rasool, K. Shafi, M. A. Bhat, A. Prabhakar, A. H. Shalla and M. A. Rather (2019). A comprehensive review of the antibacterial, antifungal and antiviral potential of essential oils and their Chemical constituents against drug-resistant microbial pathogens. Microbial Pathogenesis 134: 103580).

Chemically, they are very diverse. Phytoncides come in gaseous, crystalline and liquid forms. Many of them sublimate and boil at low temperatures, around 30°C. According to B.M. Kozo-Poliański, volatile fractions of phytoncides are the first line of plant defense, the second line of defense are non-volatile tissue phytoncides. There is confusion in the literature regarding the terminology of chemical compounds involved in plant disease resistance processes. The concept of phytoncides is related to the concepts of phytoalexins, prohibitin, inhibitorin and postinhibitin. In 1960, Cruickshank and Perrin first isolated from Pisum sativum (pea) and identified the phytoalexin - pizatin (pisatin). In 1973, J.L. Ingham published an interesting division of the factors of resistance of higher plants to infections (J.L. Ingham J.L., Disease resistance in higher plants. The concept of pre-infectional and post-infectional resistance, "Phytopath. Z. " 1973, 78, p. 314-335):

Prohibitins are metabolites that limit or completely inhibit the growth of microorganisms. They exist constantly in plant tissues in constant concentration, e.g. berberine (alkaloid), isoflavones, catechins.

Inhibitins are metabolites whose content in cells increases after infection, e.g. chlorogenic acid, coumarins.

Postinhibitins are substances formed from existing but phytoncide-inactive compounds, e.g. as a result of hydrolysis or oxidation. These include cyanogenic glycosides (e.g. prunazin in bird cherry, sambunigrin in elderberry), tuliposides, glucosinolates of garlic and onion. Ingham's concept of the definition of phytoalexins is not entirely correct. For example, benzoic acid, coniferyl alcohol, scopoletin, resveratrol, safinol are among the typical phytoalexins. According to Ingham's hypothesis, these substances are formed de novo, after contact with a pathogen. Meanwhile, many plants have these compounds constantly in their chemical composition, regardless of whether they are infected or not, e.g. Myroxylon balsamum (L.) Harm. Prohibitins, inhibitins and post-inhibitins are present both in healthy plants and those attacked by pathogenic microorganisms, making it possible to isolate these ingredients from plant raw materials and introduce them into preparations for animals and humans. The concept of phytoalexins was developed in 1941 by K.O. Muller and H. Borger. According to her, phytoalexin is a compound that inhibits the development of a pathogen (Guest, D. I. (2017). Phytoalexins, Natural Plant Protection. Encyclopedia of Applied Plant Sciences (Second Edition). B. Thomas, B. G. Murray and D. J. Murphy. Oxford, Academic Press: 124 -128).

An inhibitory factor is an isolated chemical compound, a product of the host cell. Phytoalexin is a non-specific compound in its toxic effect on the pathogen, however, pathogenic organisms may show varying sensitivity to this compound. Phytoalexins include substances with various chemical structures, e.g. resveratrol (stylbe n), cyclobrassinine sulfoxide, momilactone A (diterpene), safinol (polyacetylene), scopoletin (coumarin), 7-hydroxycalamenene (sesquiterpene). Not every phytoncide is also a phytoalexin (it does not meet Muller's and Borger's rules), but every phytoalexin is a phytoncide. Currently, attention has been paid to phytoalexins due to their strong anticancer properties, e.g. brassinin, resveratrol. The simplest phytoalexin is benzoic acid, produced by many plants when pathogens invade tissues. Phytoalexins, prohibitins and inhibitins play a defensive role in plants against pathogens, just like antibodies and interferon in humans and animals.

When phytoncides and then phytoalexins were discovered, work immediately began to isolate, stabilize and use them in medicine. This was largely prevented by intensively developed research on antibiotics and sulfonamides. The cultivation of antibiotic-producing bacteria and fungi and the synthesis of sulfonamides were undoubtedly simpler, cheaper and more technologically accessible than phytoncides and phytoalexins. However, due to the increasing problem of growing resistance of bacteria and protozoa to commonly used chemotherapy drugs, now in some countries (Switzerland, Germany, USA, France) the concept of using phytoncides in medicine has been revived. At the same time, new therapeutic properties of these compounds were discovered, e.g. anti-atherosclerotic, hypotensive, hypoglycemic, oncostatic and estrogenic properties. Already in the 1950s and 1960s, many studies showed that sulfur phytoncides have a stronger and faster antibacterial effect on Gram-positive and Gram-negative bacteria than some known antibiotics (e.g. bacitracin, neomycin). Additionally, sulfur and isosulfurcyanide phytoncides have antispasmodic, cholagogic, cholagogenic and hypotensive effects (they reduce high blood pressure). They enhance the penetration of nutrients from the intestines into the blood. They inhibit the development of putrefactive bacteria and pathogenic fungi. They have a protozoal-killing effect. They stimulate the secretion of digestive juices, increase appetite, and lower cholesterol and glucose levels in the blood. Ajoenes (garlic oils) inhibit blood cell aggregation, preventing thrombosis. The volatile phytoncide of hooves, elecampane, marigold, celandine, garlic or asturtium kills tuberculosis bacilli within 3 minutes, which is faster than carbolic acid (phenol). The stilbene phytoncide resveratrol has an anticancer effect, reduces the risk of myocardial infarction, improves coronary circulation and inhibits blood cell aggregation and the formation of atherosclerotic plaques. Additionally, it inhibits the growth of bacteria and fungi and reduces the elevated level of glucose in the blood (H. Różański, J. Kilar, M. Ruda, The influence of phytoncide plants on maintaining the health of deer animals in organic farming. LXXV Scientific Meeting of the Polish Zootechnical Society. Conference materials, Poznań 2011, p. 209; E. Strzelec, R. Niżnikowski, H. Różański, M. Klockiewicz, K. Głowacz, G. Czub, A. Darkowska, K. Szymański, A. Pokrop, Effect of use of herbal feed additive on coccidian invasion level and performance traits in goats, "Annals of Warsaw University of Life Sciences - SGGWAnimal Science" 2011, no. 49, pp. 11-20).

With the development of analytical chemistry and phytochemistry in the 19th and 20th centuries, new natural compounds with antiparasitic, including protozoal, properties were discovered. At that time, quinine, berberine, pelletierine, allantolactone, ascaridol, santonine and others were introduced into medicine, most often in the form of salts with inorganic acids (Skowroński W., Farmakologja. Published by the Polish Society "Bratnia Pomoc" Students of the Academy of Veterinary Medicine, Lviv 1932, p. 183191 ).

In the second half of the 20th century, sulfonamides began to be used to treat protozoal diseases. Unfortunately, with the increasing use of chemical drugs, especially synthetic ones, an increase in parasite resistance to the administered drugs has been observed. Phytoncides and phytoalexins can be a valuable alternative to antibiotics and sulfonamides. Therefore, this will include prohibitins, inhibitorins, post-inhibitins and actual phytoalexins, as well as secondary plant metabolites that exert antimicrobial, disinfecting and antiseptic effects in vivo and in vitro, including compounds that are not classified (at the current stage of research) as phytoalexin-type immunity factors. Initially, phytoncides were defined as antibiotics produced by higher plants (B. Czerwiecki, Lexicon specificorm, FIWNIA Warszawa 1950, pp. 320-323).

Strong phytoncides are produced by, among others: bloodwort - Sanguisorba, wormwood - Artemisia Absinthium L., nettle - Urtica, beetroot - Beta, onion - Allium cepa L., corn - Zea, garlic - Allium sativum L. or Allium ursinum L., mustard - Sinapis, borscht - Heracleum, Turkish pepper - Capsicum annuum L., bird cherry - Prunus padus L., hornbeam - Carpinus, poplar - Populus, oak - Quereus, jasmine - Jasminus, dogwood - Cornus, yew - Taxus, radish - Raphanus, birch - Betula, horseradish - Cochlearia armoracia L., juniper - Juniperus communis L. The volatile phytoncide of garlic kills tuberculosis bacilli within 3-5 minutes, therefore faster than carbolic acid. Phytoncides are a powerful factor changing the composition of microflora in the atmosphere and soil. According to B.P. Tokina and G.I. Nilowa 1 hectare of juniper emits 3 kg of volatile phytoncides per day; this amount is enough to sterilize the area of a large city (A. Danysz, Pharmakologia i recipe, Ministry of National Defense, Warsaw 1955, pp. 41-46). The development of research by Soviet biologists on phytoncides dates back to 1928. The most research on phytoncides was carried out by Boris Tokin, professor of biology, author of the work published in 1942 entitled "Bactericides rastitielnovo proischożdenia (phytoncides)" and the work "Phytoncides" published in 1948, as well as "Medicinal plant agents (phytoncides)" in 1949.

In recent years, the search for new antimicrobial substances has led to a significant increase in interest in compounds of plant origin. Reviews of the use of phytoncides in recent years have been presented in the works of: Degtyarik et al., Duka and Ardelean, Ahuja et al. (Degtyarik S. M., Slobodnitskaya, G. V., Grebneva, E. I., Benetskaya, N. A., Macksimyuk, E. V., & Bespalyi, A. V. (2017). Effect of phytoncides of plants on the viability and virulence of etiologies of agents of bacterial infections in fish. Becuj HaąbinHanbHU aKad3Mii HaByK Benapyci. Cepblx spapHblx Ha6yK; Duka, R., & Ardelean, D. (2010). Phytoncides and phytoalexins-vegetal antibiotics. Jurnal Medical Aradean (AradMedical Journal), 13(3), 19-25; Ahuja, I., R . Kissen and A. M. Bones (2012). Phytoalexins in defense against pathogens. Trends in Plant Science 17(2): 73-90).

A significant increase in review and research work on the use of essential oils can also be observed. They concern their multi-directional use, from veterinary practice and use in improving animal health and combating bacterial and parasitic diseases, through food protection, to the use of their medicinal and bactericidal properties (Aleksic Sabo, V. and P. Knezevic (2019). Antimicrobial activity of Eucalyptus camaldulensis Dehn, plant extracts and essential oils: A review. Industrial Crops and Products 132: 413-429; Vergis, J., G. Palanisamy, R. Agarwal and A. Kumar (2013). Essential Oils as Natural Food Antimicrobial Agents: A Review. Critical reviews in food science and nutrition 55; Singh, A., A. K. Dwivedy, V. K. Singh, N. Upadhyay, A. K. Chaudhari, S. Das and N. K. Dubey (2019). Essential oils based formulations as safe preservatives for stored plant masticatories against fungal and mycotoxin contamination: A review. Biocatalysis and Agricultural Biotechnology 17: 313-317; Pateiro, M., F. J. Barba, R. Dominguez, A. S. SanfAna, A. Mousavi Khaneghah, M. Gavahian, B. Gómez and J. M. Lorenzo (2018). Essential oils as natural additives to prevent oxidation reactions in meat and meat products: A review. Food Research International 113:156-166; Raut, J. S. and S. M. Karuppayil (2014). A status review on the medicinal properties of essential oils. Industrial Crops and Products 62: 250-264; Nerio, L. S., J. OliveroVerbel and E. Stashenko (2010). Repellent activity of essential oils: A review. Bioresource Technology 101(1): 372-378; Deyno, S., A. G. Mtewa, A. Abebe, A. Hymete, E. Makonnen, J. Bazira and P. E. Alele (2019). Essential oils as topical anti-infective agents: A systematic review and meta-analysis. Complementary Therapies in Medicine 47: 102224).

Regulation (EC) No 1831/2003 retained cokeydiostats and introduced histomonostats as a new category of feed additives, while establishing the withdrawal of existing antibiotics from use (and placing on the market) as feed additives from 1 January 2006, taking into account that the use of antimicrobials as growth stimulants involves the risk of selecting bacterial strains resistant to drugs used to treat humans or animals. This issue was closely related to the National Antibiotic Protection Program in Poland for 2006-2010, supervised by the Ministry of Health. According to the Program guidelines, in order to protect the therapeutic effectiveness of antibiotics, special control should also be placed on veterinary antibiotic therapy, the use of which should be subject to regulations analogous to those introduced in medicine. Antibiotics used in animal breeding promote selection and the spread of resistance among microorganisms living there.

Drug-resistant strains can move through the food chain and colonize the human gastrointestinal tract, creating reservoirs of potential pathogens, including resistance genes, e.g. Salmonella sp., Campylobacter sp., Enterococcus sp., which can then be transmitted to the etiological factors of human infections ( H. Różański, W. Drymel, Herbal preparations in the prevention of malabsorption syndrome and liver cirrhosis in animals. Polskie Drobiarstwo, part I 6/2010, pp. 44-46; part II 7/2010, pp. 28-30; part III 8/2010, pp. 43-44).

Some official coccidiostats also have an antibiotic nature with antibacterial activity, e.g. lasalocid is an ionophore polyether with anticoccidial and antibacterial activity, isolated from Streptomyces lasaliensis in 1951. Monensin (ionophore antibiotic) isolated in 1967 from Streptomyces cinnamonensis also has coccidiostatic and antibacterial properties . Maduramycin produced by Actinomadura rubra additionally inhibits the growth of gram-positive bacteria. These antibiotics, although they were obtained for use in human medicine, were not used there due to toxicity and side effects that exceeded their therapeutic value. Despite their known toxicity and the possibility of cross-resistance with other antibiotics and accumulation in products of animal origin if misused, EFSA has not yet collected sufficient evidence to withdraw them from animal production. Nevertheless, discussions on this subject continue and are fueled from time to time by protests of various consumer and ecological organizations (Różański H., Drymel W.: Adicox as a source of phytoalexins and phytoncides. Polskie Drobiarstwo. 12/2010, pp. 17-20 ).

The basic problem limiting the effectiveness of antibiotics, sulfonamides and antibiotic growth stimulants is antibiotic and sulfonamide resistance, i.e. the development of resistance of microorganisms to the static or killing effects of chemotherapeutics. Acquisition of resistance by bacteria (including fungi and pathogenic protozoa) occurs as a result of selection or adaptation. The development of resistance of microorganisms may involve changes in their metabolism, as a result of which the metabolic pathway "blocked" by the chemotherapy drug is bypassed, or the production of enzymes that degrade antimicrobial drugs, e.g., a penicillin-resistant strain of Staphylococcus aureus produces an enzyme - penicillinase, which decomposes penicillin. This is chromosomal immunity. Resistance to chemotherapeutics (e.g. fluoroquinolones, antibiotics, sulfonamides) may be caused by inhibition of the drug's penetration into the pathogen's cell, e.g. in the case of tetracyclines. Resistance to generally used chemotherapeutics is also transferred between microorganisms via an extrachromosomal route (plasmids). Antibiotic-sulfonamide- or fluoroquinolone-resistance is a property of microorganisms that is passed on to subsequent generations, and is often the so-called cross-resistance, i.e. a pathogen resistant to one chemotherapeutic agent simultaneously becomes resistant to many others, most often with a similar mechanism of action. Cross-resistance is observed, for example, in relation to tetracyclines, partly to penicillins and cephalosporins, and to macrolide antibiotics (A. Danysz, W. Buczko, Compendium of pharmacology and pharmacotherapy, Urban and Partner, Wrocław-Warszawa 2008).

Due to the introduction and uncontrolled use of an increasing range of chemotherapy drugs, often incorrectly, the danger of fungal, viral and Actinobacter or Chlamydia infections is increasing. The second danger of chemotherapy is enzymes that inactivate antimicrobial and antiparasitic drugs. In addition to beta-lactamase and dehydropeptidase I, enzymes that inactivate aminoglycosides were detected. Most antibiotics used in medicine have an unfavorable (immunosuppressive) effect on the immune system. Therefore, the idea of using additional immunostimulating agents in chemotherapy of infections arose (A. Danysz, Compendium of pharmacology and pharmacotherapy. Volumed, Wrocław 1994, p. 110).

Many phytoncides have antimicrobial, antiparasitic and immunostimulating properties at the same time, e.g. Tanacetum sesquiterpene lactones, capsaicin, piperine, or latreoside from Lathraea (H. Różański, History of research and use in medicine of domestic parasitic plants from the Scrophulariaceae and Cuscutaceae families, K. Medical University Marcinkowskiego, Poznań 2004; W. Roeske, Zarys fitoterapia. Pharmacology and recipe of medicinal herbs, PZWL Warszawa 1955, pp. 76-78; D. Korniewicz, H. Różański, Effectiveness of active substances of plant origin in pigs feeding, "Mag "Wet.", Supl. Świnie, 2006, 22-24).

This is evidenced by recent disturbing reports about the particular virulence of some strains of E. coli and Enterococcus faecalis. E. faecalis are resistant to vancomycin (VRE), the "antibiotic of last resort" produced by Amycolatopsis orientalis. Enterococcal antibiotic resistance genes are transferred to other bacteria, e.g. staphylococci and coli bacteria. In the 20th century, linezolid was discovered - a synthetic antibiotic that inhibits protein synthesis in bacteria. However, linezolid-resistant strains have already appeared among vancomycin-resistant VRE strains. Clinical resistance to metronidazole has been documented in protozoa, e.g. trichomonas vaginalis, giardia, and many anaerobic bacteria. In vitro, an increase in resistance among dysentery trophozoites was also observed as a result of gradually increasing the doses of metronidazole (Brunton L.L., Lazo J., S., Parker K.L., Farmkologia Goodmana and Gilmana, volume II. Wydawnictwo Czelej, Lublin 2007, pp. 1127-1129).

Phytoncides can help solve the problem of resistance to chemotherapy drugs not only in bacteria, but also in protozoa.

Chemoprophylaxis has become dangerous in animal production. When properly indicated, it can be useful and valuable, but in many cases it is useless and even dangerous (infection with drug-resistant bacteria and protozoa, masking disease symptoms). Chemoprophylaxis should not be used in circumstances of zootechnical and nutritional neglect, as it undoubtedly leads to the defeat of veterinary and human chemotherapy.

The biggest problem is the isolation of phytoncides from plant raw materials and their identification and stabilization. To date, little research has been done on the antimicrobial properties of pure chemical forms of phytoncides. The antibacterial and fungistatic properties of phytoncides are identified with whole fractions of substances or extracts from medicinal plants, and not with specific compounds (R. Niżnikowski, E. Strzelec, H. Różański, M. Klockiewicz, K. Głowacz, G. Czub, A. Darkowska , K. Szymański, A. Pokrop: The effect of addition of phytoncides treatment to concentrate on growth performance and dairy traits in goats. IDF International Symposium on Sheep, Goat and other non-Cow Milk, Athens, May 2011; W. Drymel, H . Różański, The use of phytoalexins in improving the health of farm animals. The Polish Branch of Worlds Poultry Science Associaton. XXII International Poltry Symposum PB WPSA, "Science for poultry practice - poultry practice for science. Olsztyn 2010, p. 151).

The AdiFeed Research and Development Department has developed a number of preparations based on phytoncides. Despite their introduction to the market, in vitro and in vivo scientific research is still being carried out, as well as field tests on a larger population of farm animals (poultry, fur animals, pigs, ruminants). The production technology of phytoncide preparations is complicated because these compounds are labile (unstable) and reactive (they react and undergo spontaneous transformations). Some of them are lipophilic (they dissolve well in organic solvents, e.g. fats, alcohols), others are hydrophilic (well soluble in water). Therefore, many phytoncide preparations are two-phase and take the form of an emulsion.

Phytoncides belong to various chemical compounds and therefore their preparations may be alkaloid, polyphenolic, phenolic, terpene, anthraquinone, iridoid, coumarin, polyacetylene, saponin or phenylalkylamine. Phytoncides belonging to different chemical groups may enhance and complement each other's antimicrobial activity, or they may act antagonistically and cancel each other's activity.

The addition of various metals, e.g. iron, in low concentrations, enhances the antibacterial and antiparasitic effects of phytoncides. The mechanism of antiseptic and antiparasitic action uses the oligodynamic effect. He noted that metals can inhibit the growth of microorganisms and plants if they are in the right concentration in the environment. In the 19th century, the mechanism of the oligodynamic effect could not be explained. Such antimicrobial and antiseptic metals include, among others: copper, iron, silver, manganese, mercury, bismuth, tin, zinc (Różański H. Antiseptics and disinfectants used in ancient and modern medicine. Lek w Polsce, vol. 14 no. 3'04, pp. 66-77. Vol 13 (154) No. 10/2003, pp. 68-81, vol. 13 (155) No. 11/2003, pp. 94-110; Penzoldt F.: Textbook of clinical pharmacology for the use of doctors and students. Printed by Maryi Ziemkiewiczowa, Warsaw 1891, p. 942).

Many of them have found permanent use in medicine. Soon, attention was paid to the "cleanliness" of metal door handles (e.g. brass and steel) in hospitals, which, despite being touched by many sick patients, do not contain active pathogenic bacteria on their surface, which in turn occur in large numbers on wooden objects and floors. , plastics or bedding. This phenomenon is explained by the oligodynamic effect. Water, including spa waters, rich in various metals is also very low in bacteria. Before antibiotics and sulfonamides began to be used in medicine, commonly used chemotherapeutics and antiseptics were preparations of bismuth, silver, mercury, iron, copper, gold, platinum, tin and zinc (Butkiewicz T.: Chirurgiageneral. PZWL Warszawa 1954; pp. 31-45 ). In the 20th century, antibiotics (e.g. bacitracin with zinc) and sulfonamides (e.g. silver sulfadiazine) were even combined with metals to obtain a more effective bacteriostatic effect (Chruściel T., Gibiński K. (ed.): Lexicon of Drugs. PZWL Warszawa 1991, p. .484-485). The dual mechanism of antibacterial action of silver sulfadiazine salt makes it difficult to develop resistant strains (Ibidem, p. 485). A similar benefit is obtained when phytoncides are combined with metals (Drymel W., Różański H.: The use of phytoalexins in improving the health of farm animals. The Polish Branch of Worlds Poultry Science Association. XXII International Poultry Symposium PB WPSA, "Science for poultry practice - poultry practice for science. Olsztyn 2010, p. 151; Korniewicz D., Różański H., Effectiveness of active substances of plant origin in pigs feeding. Mag. Wet., Supl.-Świnie., 2006, 22-24).

If metallic silver is added to distilled water, it acquires bactericidal properties, even though the ion concentration in these conditions is only 1: 20,000,000. This action is called the oligodynamic effect, and its mechanism is not clear, despite many hypotheses put forward (Kostowski W., Herman Z. (ed.): Pharmacology. Basics of pharmacotherapy. PZWL Warszawa 2003; 3rd ed.; Volume II, p. 271; Kostowski W., Kubikowski P.: Pharmacology. Basics of pharmacotherapy and clinical pharmacology. 3rd ed. .PZWL Warszawa 1991, pp. 740-741). One of the hypotheses puts the oligodynamic effect in disturbing the distribution of ionic charges within cell membranes, disturbing the polarity of the cell. Many metals also destabilize (by attaching) the structure of key proteins (enzymes, channel proteins) and nucleic acids.

Animal and human diseases caused by protozoa cause significant morbidity and mortality worldwide. The use of chemotherapeutics in the treatment of protozoal infections has proven to be problematic due to increasing drug resistance, variable effectiveness between strains or species, and toxicity. There is a strong need to find new, effective solutions to treat these diseases.

During the analysis of literature reports and the description of the state of the art, it was observed that published research is mainly based on the analysis of properties inhibiting the development of protozoa (IC50 and IC100) over a period of 24 to 72 hours. A much smaller percentage of researchers analyzed the lethal dose for protozoa.

The use of plant raw materials in the treatment of parasitoses is common in natural and traditional medicine. Previous research has shown that medicinal plants contain active compounds that have strong anti-protozoal properties. An example of a commonly used natural antiparasitic agent of plant origin is quinine - an alkaloid from the bark of the cinchona tree, artymisinin - a sesquiterpene from Artemisia annua and Artemisia indica (Hygeia Public Health 2014, 49(3): 442-448).

Many scientific experiments have also been published, showing (in vitro and in vivo) inhibition of the development of protozoa by the use of plant extracts and selected secondary metabolites from them: essential oils, alkaloids, phenolic compounds (Natural products as sources of antiprotozoal drugs. Current Opinion in Anti -infective Investigational Drugs 2000; 2, 47-62).

Copper-based complexes are known to act by interacting with the DNA of protozoa, e.g. Trypanosoma cruzi. Becco et al. showed an inhibitory effect on the development of 50% of the population (IC50) for the compounds they synthesized at the level of 3.9 ± 1.5 to 11.3 ± 3.8 μΜ, compared to the drug Nifurtimox (6 μΜ). The IC100 value was achieved for the analyzed compounds at a concentration of >20 μM (Becco, L., Rodriguez, A., Bravo, M. E., Prieto, M. J., Ruiz-Azuara, L., Garat, B., Moreno V., Gambino, D. (2012). New achievements on biological aspects of copper complexes Casiopeinas®: Interaction with DNA and proteins and anti-Trypanosoma cruzi activity. Journal of inorganic biochemistry, 109, 49-56).

Other researchers have proposed complexes of vanadium with 2,2'-bipyridine or dipyridino[3,2-a: 2',3'-c]phenazine and salicylaldehyde semicarbazide or its derivative 5-bromosalicylaldehyde semicarbazide. As in the case of previous researchers, the substance to which the authors related their results was nifurtimox. They obtained IC50 results for four variants of the complexes, in the range of 13-84 μM (Benitez, J., L. Guggeri, I. Tomaz, G. Arrambide, M. Navarro, J. Costa Pessoa, B. Garat and D. Gambino (2009). Design of vanadium mixed-ligand complexes as potential anti-protozoa agents. Journal of Inorganic Biochemistry 103(4): 609-616).

Similar analyzes were conducted by two teams of researchers: Martins et al. and Paixao et al.. In their research, they focused on the use of copper ions to create complexes with properties against Trypanosoma cruzi. The first group of researchers successfully used commonly used antibiotics (levofloxacin and sparfloxacin) to create complexes (Martins, DA, Gouvea, LR, Batista, DDJ, Da Silva, PB, Louro, SR, Maria de Nazare, CS, & Teixeira, LR ( 2012).Copper (II)-fluoroquinolone complexes with anti-Trypanosoma cruzi activity and DNA binding ability. BioMetals, 25(5), 951-960). However, Paixao et al., like Benitez et al., created complexes with the general formula [Cu(N_O)(N_N)] ²⁺ , using 2-methoxybenzhydrazide, 4-methoxybenzhydrazide and three α-diimine ligands: 1,10- phenanthroline, 2,2'-bipyridine and 4-4'-dimethoxy-2-2'-bipyridine (Paixao, DA, Lopes, CD, Carneiro, ZA, Sousa, LM, de Oliveira, LP, Lopes, NP, Pivatto M ., Chaves JDS, de Almeida MV, Ellena J., Moreira MB, Netto AVG, de Oliveira RJ, Guilardi S., de Albuquerque S., Guerra W Moreira, MB (2019). In vitro anti-Trypanosoma cruzi activity of ternary copper (II) complexes and in vivo evaluation of the most promising complex. Biomedicine & Pharmacotherapy, 109, 157-166).

Sulfoaminoamide complexes with an 8-aminoquinoline moiety of copper and zinc showed effectiveness against pathogenic strains of Leishmania braziliensis, chagasi and Trypanosoma cruzi. Their lowest IC50 was determined to be 0.35 mM (approximately 0.034%) under laboratory conditions (Everson da Silva, L., Teixeira, D. S. J., Nunes Maciel, E., Korting Nunes, R., Eger, I., Steindel, M. , & Andrade Rebelo, R. (2010). In vitro antiprotozoal evaluation of zinc and copper complexes based on sulfonamides containing 8-aminoquinoline ligands. Letters in Drug Design & Discovery, 7(9), 679-685).

Other synthetic metal complexes, i.e. manganese, cobalt, nickel in the form of 4'-(2-ferrocenyl)-2,2':6'2-terpyridinium derivatives were very effective in vitro at a concentration of 1.1 mM against Plasmodium falciparum . The authors proved the effectiveness of mixtures of manganese, iron, cobalt, nickel and copper salts (Al-Khodir, F. A. I., & Refat, M. S. (2017). Investigation of coordination ability of Mn (II), Fe (III), Co (II), Ni (II), and Cu (II) with metronidazole, the antiprotozoal drug, in alkaline media: Synthesis and spectroscopic studies. Russian Journal of General Chemistry, 87(4), 873-879).

The possibility of effective formation of complexes with a commonly used antibiotic with strong antiprotozoal activity (Metronidazole) with metals, including: Mn(II), Fe(III), Co(II), Ni(II), and Cu(II) (Al-Khodir, F. A. I. and M. S. Refat (2017). Investigation of coordination ability of Mn(II), Fe( III), Co(II), Ni(II), and Cu(II) with metronidazole, the antiprotozoal drug, in alkaline media: Synthesis and spectroscopic studies. Russian Journal of General Chemistry 87(4): 873-879).

The enhancement of the antiprotozoal activity of copper and zinc metal ions in synthetic organic imidazopyridinium and diarylpiperidinium complexes also gained patent protection. Thus, in the patent US6291480B1 and US20060178358 the activity of diarylpyridyl derivatives against Toxoplasma gondii, Trypanosoma cruzi and Emeria species: tenella, acervulina, necatrix, brunetti maxima was proven. Another example of the use of antiprotozoal metal complexes, including copper and low molecular weight bioorganic compounds (US20110207701A1).

So far, research on the antimicrobial properties has shown the very strong effects of essential oils. Escobar, P et al. conducted tests on the antiprotozoal properties of 5 plants of the Lippi genus. They analyzed the obtained oils for inhibiting the development of protozoa on Trypanosoma cruzi and Leishmania chagasi, in relation to nifurtimox. They obtained IC50 values from 4.4 to >100 μg/ml, while in the case of nifurtimox this value was 0.30.4 μg/ml (Escobar, P., Milena Leal, S., Herrera, L. V., Martinez, J. R., & Stashenko, E. (2010). Chemical composition and antiprotozoal activities of Colombian Lippia spp essential oils and their major components.Memórias do Instituto Oswaldo Cruz, 105(2), 184-190).

Similarly, from application WO2008101131 A1 a composition for killing or repelling ectoparasites and/or pests is known, comprising at least 3% of Lippia javanica essential oil and at least one other essential oil.

Another group of researchers demonstrated the effect of essential oils from Annona coriacea on Trypanosoma cruzi and various species of leishmania (Leishmania (L.): amazonensis, braziliensis, chagasi, major). Two commonly used compounds in the case of leishmaniasis, pentamidine and benznidazole, were used for comparative analysis. The values obtained for essential oils (39.93-261.20 μg/mL) were much higher than for the tested drugs (0.06022 μg/mL and 45.02 μg/mL, respectively) (Siqueira, C. A. T., J. Oliani, A. Sartoratto, C. L. Queiroga, P. R. H. Moreno, J. Q. Reimao, A. G. Tempone and D. C. H. Fischer (2011). Chemical constituents of the volatile oil from leaves of Annona coriacea and in vitro antiprotozoal activity. Revista Brasileira de Farmacognosia 21: 0-0).

Perez et al. in their review, they collected information on antiprotozoal properties, IC50 (Giardia lamblia, Trichomonas vaginalis, Leishmania sp, Trypanosoma cruzi) for, among others, thyme, garlic, basil, lavender, tea or yarrow oil. They demonstrated the antiprotozoal properties of essential oils in a very wide concentration range, from 8.3 ng/ml to 8 mg/ml (Perez, S., M. Ramos-Lopez, E. Sanchez-Miranda, M. Fresan-Orozco and J. Perez- Ramos (2012). 'Antiprotozoa activity of some essential oils. Journal of medicinal plant research 6: 2901-2908).

Monzote et al. collected in their work literature reports on the antiparasitic properties of essential oils, which were created over the years 1988-2012. They represent a significant increase in interest and research on the use of essential oils in the control of protozoa (Monzote, L., O. Alarcón and W. Setzer (2012). 'Antiprotozoal Activity of Essential Oils. Agriculturae Conspectus Scientificus 77:167-175).

Moon et al., in their research work, presented the protozoal properties of two lavender oils against Giardia duodenalis, Trichomonas vaginalis and Hexamita inflata. They showed that the concentration of 0.1% lavender oil has a killing effect against the analyzed protozoa (Moon, T., J. Wilkinson and H. Cavanagh (2006). 'Antiparasitic activity of two Lavandula essential oils against Giardia duodenalis, Trichomonas vaginalis and Hexamita inflata. Parasitology research 99: 722-728).

However, from application EP2070427 A1 it is known to use at least one essential oil compound selected from the group consisting of cinnamaldehyde, 2-decenal and nerolidol as or in a histomonastat preparation. Preferably, the essential oil compound is further combined with at least one compound selected from the group consisting of p-cymene, thymol, salicylaldehyde, tea tree oil, peppermint oil, cumic aldehyde, cinnamic acid, cinnamic alcohol, farnesal and farnesyl acetone.

Application EP2119363 A2 discloses an antimicrobial composition based on essential oils of plants, with increased antimicrobial effectiveness, containing: at least two essential oils from plants as the main ingredient; and a small but antimicrobially effective amount of an enhancer selected from the group consisting of polyionic organic enhancers (e.g., polyethyleneimine) and polyionic inorganic enhancers (e.g., sodium tripolyphosphate, sodium hexametaphosphate).

From document US2014106012 AA a composition is known containing: an essential oil selected from the group consisting of anise oil, rosemary oil, calendula oil, tea tree oil, sassafras oil, quasi oil, cinnamon oil, clove oil, eucalyptus oil, lavender oil, peppermint oil, or combinations thereof; from about 10 percent to about 30 percent (v/v) isopropyl alcohol; from about 30 percent to about 50 percent (v/v) isopropyl myristate; from about 5 percent to about 20 percent (v/v) silicone oil; and from about 5 percent to about 25 percent (v/v) capric triglyceride.

Document EP1512409 B1 discloses an aqueous composition for combating head lice and their eggs, which contains at least one essential oil as active ingredients, characterized in that the composition also contains an infusion of: dried peppermint leaves, tea and garlic. However, the disclosed method of preparing the said composition includes the following steps: making an infusion of peppermint leaves, tea and garlic in boiling water and allowing it to cool, adding essential oils to the cooled infusion, and then mixing the cooled infusion with surfactants and thickening agents to form a gel.

EP 1089745 B1 discloses the use of an oregano extract or a metabolic product of an oregano extract in the preparation of a medicament for reducing or eliminating intestinal amoeba selected from the group consisting of Entamoeba hartmanni, Blastocystis hominis, Endolimax nana and Entamoeba histolytica in humans in need of antiprotozoal treatment. The disclosed medicament is adapted for administration in the form of an emulsified sustained-release tablet containing carvacrol as an active ingredient.

However, from document US2014037698 AA (EP2666364 (B1) an animal feed additive is known containing a combination of an organic acid salt with at least one active ingredient of plant origin, this combination partially covered with vegetable oils and/or fats. The active ingredients of plant origin include essential oils selected from the group consisting of ginger, piperine, oregano, thymol, carvacrol, cinnamaldehyde, garlic and combinations thereof, while the organic acid is preferably selected from the group consisting of butyric, propionic, formic, lactic, citric, lauric, capric, caprylic , capric and acetic. The additive has antiprotozoal properties.

The purpose of the invention is to provide a new composition for combating protozoa with killing properties.

The essence of the invention is a veterinary composition for the treatment and/or prevention of diseases caused by protozoa in animals, containing eucalyptus oil, characterized in that the eucalyptus oil is in the form of a complex with an organic acid selected from the group consisting of acetic acid, propionic acid, lactic acid and metal. selected from the group consisting of copper, zinc, manganese, oxides or salts thereof.

The preferred ratio of eucalyptus oil to organic acid is from 1:80 to 80:1.

The preferred ratio of eucalyptus oil to organic acid is 1:1.

Another essence of the invention is a method for preparing a veterinary composition for the treatment and/or prevention of diseases caused by protozoa in animals, containing eucalyptus oil, according to the invention, characterized by the following steps:

a) Mixing eucalyptus oil with an organic acid selected from the group consisting of acetic acid, propionic acid, lactic acid in a weight ratio of 80:1 to 1:80;

b) adding a catalyst to the mixture from step a);

c) adding a metal selected from the group consisting of copper, zinc, manganese, salts, or oxides thereof;

d) heating the mixture with the catalyst obtained in step c) to boiling point and continuing heating at reflux temperature for 20-120 minutes;

e) allowing the reaction product to cool for a period of 10 to 24 hours;

f) filtering the cooled reaction product.

Preferably, a mixture of cobalt sulfate, ammonium molybdate and manganese chloride or sulfate is used as a catalyst.

The invention provides the following advantages:

• the protozoanicidal properties of the composition ensure complete elimination of infection;

• the composition according to the invention works at low concentrations;

• the composition according to the invention has a broad spectrum of action - i.e. it has a good killing effect against many species of protozoa;

• the composition according to the invention may be an alternative to protozoal chemotherapeutics (Antiprotozoal), such as metronidazole, albendazole, tinidazole, amprolium, lasalocid, salinomycin, robenidin, nicarbazin, monensin, decoquinate, diclazuril, or it may also be an addition to antiprotozoal chemotherapeutics, reducing the risk development of resistance to a given drug.

The invention is described in detail in the following embodiments, and all tests and experimental procedures described below were performed using commercially available test kits, reagents and apparatus, following the recommendations of the manufacturers of the kits, reagents and apparatus used, unless expressly indicated otherwise. All test parameters were measured using standard, well-known methods used in the art to which this invention belongs.

All raw materials used for research are approved for both animal and human feeding by appropriate directives and authorities. The selection of raw materials was made on the basis of Codex Alimentarius, i.e. the Codex Alimentarius created 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 August 2003 on additives for use in animal nutrition. Moreover, the essential oils used for the research met the requirements of the European and Swiss Pharmacopoeias and Der Deutsche Arzneimittel-Codex (DAC).

However, 5 organisms representing taxonomic groups that include pathogenic protozoa were selected for in vitro testing of the antiprotozoal activity of the composition according to the invention, i.e.:

• Amoeba proteus - a misfit amoeba - a protozoan from the order Euamoebida, belonging to the supergroup 5 Amoebozoa, living in water.

• Paramecium caudatum - a tailed paramecium representing the ciliate Ciliata, living in water.

• Gregarina blattarum - gregarin isolated from cockroaches, representing the Apicomplexa phylum, living in the digestive tracts or body cavities of invertebrates.

• Euglena gracilis - a protozoan living in water, representing flagellates - Mastigophora, family Euglenaceae.

• Trichomonas hominis - a protozoan living in the human large intestine, representing the Trichomonadidae.

Amoeba, Paramecium, Trichomonas and Euglena were observed under a microscope on watch glasses with viscose cotton fibers (to facilitate observation) in a drop of water from the culture from which they came. Various concentrations of the tested compositions were introduced into the test samples, determining the LD50 dose (50% mortality) and LD100 dose (100% mortality). In all cases, the test was repeated four times with a blank test.

Gregarins were isolated from cockroaches and, after placing on a watch glass in Ringer's solution, they were exposed to the products at various concentrations. Each sample included ten individuals. The lethal concentration of the substance was determined for 50% and 100% of individuals (LD50, LD100) within 3 minutes. The isolation of gregarins from cockroaches was based on the method of isolating gregarins from beetles proposed by J. Moraczewski (Moraczewski J.: Exercises in invertebrate zoology. 1st edition, PWN, Warsaw 1974, pp. 29-31, 285-292).

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. Third Edition, National Agricultural and Forest Publishing House 1972; Hempel-Zawitkowska J., Gałka B., Kalińska B., Kamionek M., Komosińska H., Pezowicz E. Podsiadlo E., Sulgostowska T.: Zoology for agricultural universities. Wydawnictwo Naukowe PWN 2008).

All tested preparations (i.e. compositions according to the invention and control samples), before being applied to the watch glass, were dissolved in an aqueous solution of polysorbate 80 (0.05%). No killing effect of polysorbate 80 was found in the above-mentioned products. concentration.

Example 1

A combination of eucalyptus oil (Eucalyptus globulus Labill.) with acids (acetic, propionic, lactic) in a 1:1 ratio and metal (copper, manganese and zinc).

In this non-limiting embodiment, the following nine compositions were prepared:

a) composition I - i.e. a composition of eucalyptus oil with acetic acid and copper

b) composition II - i.e. a composition of eucalyptus oil with acetic acid and manganese

c) composition III - i.e. a composition of eucalyptus oil with acetic acid and zinc

d) composition IV - i.e. a composition of eucalyptus oil with propionic acid and copper

e) composition V - i.e. composition of eucalyptus oil with propionic acid and manganese f) composition VI - i.e. composition of eucalyptus oil with propionic acid and zinc g) composition VII - i.e. composition of eucalyptus oil with lactic acid and copper h) composition VIII - i.e. a composition of eucalyptus oil with lactic acid and manganese

i) composition IX - i.e. a composition of eucalyptus oil with lactic acid and zinc.

However, in this non-limiting embodiment, metal salts in the form of carbonates are used, while other salts (e.g. chlorides, sulfates) or other forms, e.g. oxides, can also be used in the composition according to the invention.

To prepare composition I, 100 ml of acetic acid, 0.6 g of catalyst (which consists of cobalt sulfate, ammonium molybdate and manganese chloride mixed in a 1:1:1 ratio) and 5 g of copper carbonate were added to 100 ml of eucalyptus oil. The whole was heated under reflux for 20 minutes until the color changed. The mixture was then left to cool (for 10 hours) and obtain a clear solution (single, two or three-phase). After this time, the reaction product was filtered through filter paper. Compositions II-IX were obtained analogously to composition I.

Compositions I-IX were then analyzed for their protozoanicidal properties. For this purpose, both compositions were diluted: 0.001% to 1%, and then the protozoa were placed in individual dilutions.

• Amoeba proteus - a changeling amoeba - a protozoan from the order Euamoebida, belonging to the Amoebozoa supergroup, living in water;

• Paramecium caudatum - a tailed paramecium representing the Ciliata ciliates, living in water;

• Gregarina blattarum - gregarin isolated from cockroaches, representing the Apicomplexa phylum, living in the digestive tracts or body cavities of invertebrates;

• Euglena gracilis - a protozoan living in water, representing flagellates - Mastigophora, family Euglenaceae.

• Trichomonas hominis - a protozoan living in the human large intestine, representing the Trichomonadidae.

The protozoanicidal properties of individual acids, catalyst solution, tea tree oil and metal salt solutions were analyzed. The tested preparations were dissolved in an aqueous solution of polysorbate 80 (0.05%) before being applied to the watch glass. No killing effect of polysorbate 80 was found in the above-mentioned products. concentration. Observation was performed under a fluorescence microscope with phase contrast. Protozoal activity was considered effective when 50% and 100% of individuals died within 3 minutes. Control antiprotozoal substances were CH - chloramphenicol and M - metronidazole.

The results of the analysis are presented in Table 1 and Table 2. Table 1 contains the results of the antiprotozoal activity of control samples, th. control antiprotozoal substances and individual components of compositions I-IX. The activity of compositions I-IX is presented in Table 2.

The analysis showed that the killing and static effects in complex systems and after the reaction were higher than the effects of the substances individually included in the reaction mixtures and complexes. Compositions I-X show many times stronger (potentialization) protozoanicidal activity than each of these ingredients separately. All ingredients used in the compositions according to the invention are approved for both animal and human feeding by appropriate directives and authorities, which, combined with their high effectiveness, allows their use in the treatment and/or prevention of parasitoses in animals caused by protozoa. In particular, histomonadosis (caused by Histomonas meleagridis), coccidiosis (caused by Eimeria), cryptosporidiosis (caused by Cryptosporidium), trichomonadosis (caused by Trichomonas), babesiosis (caused by Babesia), or amoebiasis (caused by Amoeba).

PL 243785 BI

Cucalyplus oil (Eucalyptus giobulus Labill.) LDso: 0.1% LDioo: 0.2% LDso: 0.08% LDioo: 0.1% LDso: 0.05% LDioo: 0.07% LDso: 0.1% LDioo: 0.3% LDso: 0.09% LDioo: 0.1% Catalyst solution LDso: 0.05% LDioo: 0.1% LDso: 0.07% LDioo: 0.3% LD ₅₀ : 0.05% LDioo: 1% LDso: 0.8% LDioo: 1.25% LDso: 0.9% LDioo: 1.1% Zinc carbonate solution LDso: 0.1% LDioo: 0.3% LDso: 0.2% LDioo: 0.4% _x © 'η oy ó o aa « uuo LDso: 0.3 LDioo: 0.5% LDso: 0.25% LDioo: 0.4% Copper carbonate solution LDso: 0.15% LDioo: 0.25% LDso: 0.12% LDioo: 0.37% LDso: 0.09% LDioo: 0.17% LDso: 0.35% LDioo: 0.5% LDso: 0.1% LDioo: 0.3% Manganese chloride solution LDso: 0.05% LDioo: 0.1% LDso: 0.07% LDioo: 0.3% LD50: 0.05% LDioo: 1% © 05 °· o S? 3 3 LDso: 0.9% LDioo: 1.1% Lactic acid LDso: 0.6% LDioo: 1.3% LDso: 1% LDioo: 1.1% LDso: 0.9% LDioo: 1.4¾ LDso: 1% LDioo: 1.5% LDso: 0.95% LDioo: 1.3% Propionic acid LDso: 0.5% LDioo: 1.1% Ο ^χ ® T • - ooo QQJ u LDso: 0.6% LDioo: 1% p © 05 - oh oh 8 8 8' X© X© 00 c - about . . o a a a Q Q J J Acetic acid Q « Q J o J a x.° o x° 3 S* S 8- X© O x© o 'Λ — 3 at 8 LDso: 1% LDioo: 1.3% xQ and—H o o X© Ά - ©% 2 5% * K p 8 Q Q U J s s a q J u R 5 Q Q J J = 8 Q Q 8 3 Q Q J J CH* o ·> ? 1 Q Q U J • - o o a Q Q £s? o o (5 Q LDso :0.001% LDioo :O.OO6% Q Q J J 5 S ££ χ/ ii .X £ Euglena gracilis Gregarina blattamm Amoeha proteus Paramecium caudatum Trichomonas hominis

*CH - chloramphenicol, M** - metronidazole

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Table 2. LD50, LD100 values of Ι-ΙΧ compositions, determined for selected protozoa Eucalyptus oil Lactic acid zinc Composition IX LDso: 0.002% LDioo: 0.004% LDso: 0.003% LDioo: 0.006% LDso: 0.005% LDioo: 0.006% LDso: 0.006% LDioo: 0.008% LDso: 0.025% LDioo: 0.035% manganese Composition VIII LDso: 0.001% LDioo: 0.003% LDso: 0.002% LDioo: 0.005% LDso: 0.007% LDioo: 0.009% LDso: 0.004% LDioo: 0.007% LDso: 0.02% LDioo: 0.03% copper Composition VII* LDso: 0.002% LDioo:0.003% LDso: 0.002% LDwo: 0.004% LDso: 0.005% LDioo: 0.007% LDso: 0.004% LDioo: 0.008% LDso: 0.03% LDioo: 0.04% Propionic acid zinc Composition VI LDso: 0.002% LDioo: 0.003% LDso: 0.004% LDioo: 0.007% LDso: 0.005% LDioo: 0.006% LDso: 0.006% LDioo: 0.009% LDso: 0.02% LDwo: 0.035% manganese Composition V LDso: 0.001% LD™: 0.002% LDso: 0.003% LDioo: 0.005% LDso: 0.007% LDioo: 0.009% LDso: 0.003% LDioo: 0.007% LDso: 0.015% LDioo: 0.025% copper Composition IV LDso: 0.001% LDioo: 0.003% LDso: 0.003% LDioo: 0.004% LDso: 0.007% LDioo: 0.008% LDso: 0.003% LDioo: 0.008% LDso: 0.035% LDioo: 0.04% Acetic acid zinc Composition III LDso: 0.003% 1 LDioo: 0.007% LDso: 0.003% LDioo: 0.006% LDso: 0.005% LDioo: 0.009% LDso: 0.004% LDioo: 0.007% LDso: 0.05% LDioo: 0.09% manganese Composition II * LDso: 0.003% LDioo: 0.006% LDso: 0.003% LDioo: 0.007% LDso: 0.004% LDioo: 0.008% LDso: 0.002% LDioo: 0.007% LDso: 0.03% LDioo: 0.055% copper Composition I LDso: 0.004% LDioo: 0.008% LDso: 0.004% LDioo: 0.005% LDso: 0.003% LDioo: 0.006% LDso: 0.002% LDioo: 0.006% LDso: 0.045% LDioo: 0.075% « 's 1 and E Euglena gracilis Gregarina blattarum Amoeba proteus Paramecium caudatum Trichomonas hominis

Example 2

A combination of eucalyptus oil (Eucalyptus globulus Labill.) with acids (acetic, propionic, lactic) in a ratio of 80:1 and metal (copper, manganese and zinc).

In this non-limiting embodiment, the following nine compositions were prepared:

a) composition X - i.e. a composition of eucalyptus oil with acetic acid and copper

b) composition XI - i.e. a composition of eucalyptus oil with acetic acid and manganese

c) composition XII - i.e. a composition of eucalyptus oil with acetic acid and zinc d) composition XIII - i.e. a composition of eucalyptus oil with propionic acid and copper

e) composition XIV - i.e. a composition of eucalyptus oil with propionic acid and manganese

f) composition XV - i.e. a composition of eucalyptus oil with propionic acid and zinc

g) composition XVI - i.e. a composition of eucalyptus oil with lactic acid and copper h) composition XVII - i.e. a composition of eucalyptus oil with lactic acid and manganese

i) composition XVIII - i.e. a composition of eucalyptus oil with lactic acid and zinc.

However, in this non-limiting embodiment, metal salts in the form of oxides are used, while other forms such as salts (e.g. chlorides, sulfates, carbonates) can also be used in the composition according to the invention.

To prepare composition The whole was heated at reflux for 120 minutes until the color changed. The mixture was then left to cool (for 24 hours) and obtain a clear solution (one-, two- or three-phase). After this time, the reaction product was filtered through filter paper.

Compositions XI-XVIII were prepared analogously to composition X. Compositions

PL 243785 Β1

LDso: 0.035% LDioo: 0.08% LDso: 0.048% LDioo: 0.08% LDso: 0.03% LDioo: 0.06% LDso: 0 ₍ 08% LDioo: 0.12% LDso: 0.065% LDioo: 0.08% LDso: 0.03% LDioo: 0.09% LDso: 0.07% LDwo: 0.079% LDso: 0.045% LDioo: 0.064% LDso: 0.08% LDioo: 0.15% LDso: 0.068% LDioo: 0.087% LDso: 0.04% LDioo: 0.07% LDso: 0.05% LDwo: 0.086% LDso: 0.028% LDioo: 0.05% LDso: 0.07% LDioo: 0.1% LDso: 0.05% LDioo: 0.08% LDso: 0.036% LDioo: 0.08% LDso: 0.07% LDwo: 0.088% LDso: 0.037% LDioo: 0.058% LDso: 0.084% LDioo: 0.2% • O O' Ά ABOUT about S Ό Q Q Q J J o 3 8 Ξζ Q Q o J J o LDso: 0.065% LDioo: 0.09% LDso: 0.039% LDioo: 0.057% LDso: 0.082% LDioo: 0.22% LDso: 0.074% LDioo: 0.085% LDso: 0.032% LDioo: 0.074% LDso: 0.063% LDioo: 0.092% LDso: 0.047% LDioo: 0.064% LDso: 0.075% LDioo: 0.15% LDso: 0.072% LDioo: 0.089% LDso: 0.029% LDwo: 0.059% LDso: 0.071% LDioo: 0.094% LDso: 0.037% LDioo: 0.062% LDso: 0.078% LDioo: 0.17% LDso: 0.077% LDioo: 0.083% χθ G'· 8 o .. ^ Q Q o LDso: 0.069% LDioo: 0.093% LDso: 0.035% LDioo: 0.055% LDso: 0.084% LDioo: 0.12% LDso: 0.067% LDioo: 0.084% LDso: 0.045% LDioo: 0.078% LDso: 0.065% LDioo: 0.087% LDso: 0.028% LDioo: 0.048% LDso: 0.08% LDioo: 0.19% LDso: 0.07% LDioo: 0.08% Euglena gracilis Gregarina blat larum Amoeba proteus Paramecium caudatum Trichomonas hominis

Example 3

A combination of eucalyptus oil (Eucalyptus globulus Labill.) with acids (acetic, propionic, lactic) in a ratio of 1:80 and metal (copper, manganese and zinc).

In this non-limiting embodiment, the following nine compositions were prepared:

a) composition XIX - i.e. a composition of eucalyptus oil with acetic acid and copper

b) composition XX - i.e. a composition of eucalyptus oil with acetic acid and manganese c) composition XXI - i.e. a composition of eucalyptus oil with acetic acid and zinc

d) composition XXII - i.e. a composition of eucalyptus oil with propionic acid and copper

e) composition XXIII - i.e. a composition of eucalyptus oil with propionic acid and manganese

f) composition XXIV - i.e. a composition of eucalyptus oil with propionic acid and zinc

g) composition XXV - i.e. a composition of eucalyptus oil with lactic acid and copper h) composition XXVI - i.e. a composition of eucalyptus oil with lactic acid and manganese

i) composition XXVII - i.e. a composition of eucalyptus oil with lactic acid and zinc.

However, in this non-limiting embodiment, metal salts in the form of carbonates are used, while other salts (e.g. chlorides, sulfates) or other forms, e.g. oxides, can also be used in the composition according to the invention.

To prepare composition XIX, 80 ml of acetic acid, 1 g of catalyst (i.e. cobalt sulfate, ammonium molybdate and manganese chloride mixed in a 1:1:1 ratio) and 5 g of copper carbonate were added to 1 ml of eucalyptus oil. The whole was heated under reflux for 60 minutes until the color changed. The mixture was then left to cool (for 12 hours) and obtain a clear solution (single, two or three-phase). After this time, the reaction product was filtered through filter paper.

Compositions XX-XXVII were prepared analogously to composition XIX. Compositions XIX-XXVII were then analyzed for their protozoanicidal properties analogously to Example 1, and the results are presented in Table 4.

PL 243785 Β1

LDso: 0.04% LDioo: 0.08% LDso: 0.067% LDioo: 0.09% LDso: 0.045% LDioo: 0.067% LDso: 0.09% LDioo: 0.24% LDso: 0.08% LDwo: 0.09% LDso: 0.047% LDioo: 0.09% LDso: 0.06% LDioo: 0.085% LDso: 0.047% LDioo:O.O68% LDso; 0.089% LDioo: 0.2% LDso: 0.078% LDioo: 0.085% LDso: 0.039% LDwo: 0.089% LDso: 0.065% LDioo: 0.089% about'· ? at S Ξ 3 n q q -Jo LDso: 0.088% LDioo: 0.19% LDso: 0.079% LDwo: 0.084% LDso: 0.045% LDioo: 0.079% LDso: 0.063% LDioo: 0.084% o ^x ł § z = 7 QQ o -J -io LDso: 0.09% LDioo: 0.27% LDso: 0.085% LDioo: 0.09% LDso: 0.043% LD wo: 0.084% ABOUT = . . 5? at 8 o'clock Q Q o L o and about ° s sat Q Q o -Jo LD ₅ o; 0.093% LDioo: 0.26% LDso: 0.075% LDwo: 0.086% LDso: 0.047% LDioo: 0.085% LDso: 0.062% LDioo: 0.083% LDso: 0.037% LDioo: 0.06% LDso; 0.088% LDioo: 0.23% LDso: 0.077% LDwo: 0.83% LDso: 0.043% LDioo: 0.083% LDso: 0.064% LDioo: 0.077% LDso: 0.047% LDioo: 0.065% LDso: 0.085% LDioo: 0.21% LDso; 0.088% LDioo: 0.094% LDso: 0.04% LDioo: 0.08% LDso: 0.062% LDioo: 0.08% O Q O L U o LDso: 0.089% LDioo: 0.18% LDso: 0.085% LDwo: 0.09% LDso: 0.044% LDioo: 0.089% LDso: 0.065% LDioo: 0.086% LDso: 0.043% LDioo: 0.059% LDso; 0.082% LDiode: 0.25% LDso: 0.082% LDioo: 0.089% Euglena gracilis Gregarina blattarum Amoeba protetis Paramecium caudatum Trichomonas hominis

Claims (5)

1. A veterinary composition containing eucalyptus oil, characterized in that the eucalyptus oil is in the form of a complex with an organic acid selected from the group consisting of acetic acid, propionic acid, lactic acid and a metal selected from the group consisting of copper, zinc, manganese, their oxides or salts for use in the treatment and/or prevention of diseases caused by protozoa in animals. 2. The composition according to claim 2. 1, characterized in that the ratio of eucalyptus oil to organic acid is from 1:80 to 80:1. 3. The composition according to claim 1. 2, characterized in that the ratio of eucalyptus oil to organic acid is 1:1. 4. A method for preparing a veterinary composition for the treatment and/or prevention of diseases caused by protozoa in animals, containing eucalyptus oil, according to any of the preceding claims. from 3, characterized in that it includes the following stages: a) mixing eucalyptus oil with an organic acid selected from the group consisting of acetic acid, propionic acid, lactic acid in a weight ratio of 80:1 to 1:80; b) adding a catalyst to the mixture from step a); c) adding a metal selected from the group consisting of copper, zinc, manganese, salts, or oxides thereof; d) heating the mixture with the catalyst obtained in step c) to boiling point and continuing heating at reflux temperature for 20-120 minutes; e) allowing the reaction product to cool for a period of 10 to 24 hours; f) filtering the cooled reaction product. 5. The method according to claim 1. 4, characterized in that a mixture of cobalt sulfate, ammonium molybdate and manganese chloride or sulfate is used as a catalyst.