WO2005074965A1

WO2005074965A1 - Application of natural and synthetic eugenol and essential oils from cloves, pimentos and cinnamon leaves for the prevention and treatment of animal diseases caused by bacteria, fungi and parasites

Info

Publication number: WO2005074965A1
Application number: PCT/YU2004/000029
Authority: WO
Inventors: Vojin Gligovic
Original assignee: Vojin Gligovic
Priority date: 2004-02-06
Filing date: 2004-12-31
Publication date: 2005-08-18
Also published as: YU12504A; RS51374B

Abstract

Patent registration refers to applications of natural and synthetic eugenol, essential oils from plants containing eugenol: cloves (Eugenia caryophyllata), pimentos (Pimenta officinalis)and cinnamon leaves (Cinnamomum zeylanicum) in the prevention and treatment of animal diseases caused by bacteria, fungi and parasites. The technical problem of improvement of properties of veterinary preparations has been resolved so that natural and synthetic eugenol and eugenol from plant essential oils (cloves, pimentos and cinnamon leaves) have been added to them. This considerably prevents growth of bacteria, fungi and parasites or destroys them resulting in preventive effects or treatment of animals affected by some of the above disease causes. By applying eugenol in veterinary preparations in one of the above mentioned forms, negative effects and counter indications have been avoided and use of preparations has been made possible during the entire cycle of animal breeding.

Description

APPLICATION OF NATURAL AND SYNTHETIC EUGENOL AND ESSENTIAL OILS FROM CLONES, PIMENTOS AND CINNAMON LEAVES FOR THE PREVENTION AND TREATMENT OF ANIMAL DISEASES CAUSED BY BACTERIA, FUNGI AND PARASITES

TECHNICAL FIELD

The technical field to which this invention refers generally observed falls in the field of application of substances that are added in veterinary preparations and specifically refers to the application of natural and synthetic eugenol and essential oils from eugenol-containing plants (cloves, pimentos and cinnamon leaves) in the prevention and treatment of animal diseases caused by bacteria, fungi and parasites. According to the International patent classification (ICP, Intel. 7) the subject of this invention is classified and marked with the classification symbol A61K 31/775 that refers to medical preparations containing active organic ingredients and with the secondary classification symbols A6 IK 31/74 defining medical preparations containing synthetic polymer matters and C08K 5/105 referring to the use of organic matters as components for phenol mixtures.

TECHNICAL PROBLEM

The technical problem resolved with this invention consists of the following: how to make veterinary preparations with active properties of their ingredients that fully or considerably prevent the growth of bacteria, fungi and parasites and lead to their destruction. This results a more effective prevention of animal diseases or their treatment and allows a substitution of antibiotic, sulphonamide and coccidiostatic preparations.

Application of those ingredients allows that the preparations containing them have a high reliability level, can be used without interruption during the whole breeding cycle,

Animals tolerate them more easily and they have no any unwanted effects and counter indications. BACKGROUND

Modern intensive production of meat, milk and eggs, due to economic justification, requires that a large number of cattle be kept in as a small space as possible. Large concentrations of animals are a good basis for the outbreak of diseases, especially of gastrointestinal infections both with young cattle and older groups This is why antibiotic, sulphonamides and coccidiostatic preparations have been used over tens of years in order to prevent animal diseases. Adding some of the mentioned medications in animal food or water has given effective results to prevent diseases. Until 15-20 years ago, unfortunately, negative consequences that could have been caused by such prevention were not taken into account and they appeared in the form of bioresidues in milk, meat and eggs as a result of deposits of antibiotic and other preparations. Microorganisms against which those preparations were used have over time become resistant to those preparations. The man, by using animal produce with deposited bioresidues for his nutrition, has made a number of bacterial types in his body resistant to various kinds of antibiotics and sulphonamides. In order to prevent such and other unwelcome effects, economically developed countries of Europe, USA and Canada have brought a number of measures preventing or reducing the use of antibiotics and sulphonamides for nutritive and preventive purposes. At the same time they have introduced regulations preventing sales of produce (milk, meat, eggs) containing bioresidues. For the above reasons the author of this invention has opted for the development of preparations containing ingredients whose application considerably or fully can overcome the problems or limitations relating to use of antibiotic and sulphonamide preparations.

FUNDAMENTAL NATURE OF INVENTION

The fundamental nature of this invention is reflected in the finding and application of substances added to the preparations aimed at preventing and treating animal diseases caused by bacteria, fungi and parasites, make their effects different from the effects of traditional preparations. This is reflected primarily through their increased effectiveness and many other positive characteristics. It has been achieved by applying natural eugenol, eugenol in the form of sodium and potassium salts and essential oils from eugenol-containing plants. Its antiseptic and bactericide properties have been used and it actually acts as an antioxidant. The fundamentals nature of this invention is reflected in the fact that preparations containing natural eugenol, eugenol in the form of sodium and potassium salts and essential oils from eugenol-containing plants (cloves, pimentos and cinnamon leaves) affect bacteria, fungi and parasites in the following manner: 1. They damage the cytoplasmic membrane by separating membrane lipids; 2. In cytoplasm they prevent a synthesis of albumin that is responsible for transmitting oxygen to mitochondria; 3. They lead to the rapid dying of bacteria, fungi and parasites due to lack of oxygen, The fundamental nature of this invention is reflected in the fact that the peparations, in which natural eugenol, eugenol in the form of sodium and potassium salts and essential oils from eugenol-containing plants, represent potent enteroseptics and are a successful replacement for antibiotic, sulphonamide and coccidiostatic preparations. Basic advantages of application of natural eugenol, eugenol in the form of sodium and potassium salts and essential oils from eugenol-containing plants, according to this invention and with regard to other means used to prevent and treat diseases caused by bacteria, fungi and parasites, are reflected in the following: - active substances in preparations based on this invention are of natural and plant origin and they are not antibiotics. This is why they are not deposited in meat, milk and eggs and have no charenza that is a basic problem of antibiotic and sulphonamide preparations; - more effective and reliable effects with no risks of sudden and unexpected resistance; - animals take them easily with no harmful effects; - animals take them more easily in stronger doses that standard preparations widely used today; - preparations can be constantly used without breaks as they are not deposited in meat, milk and eggs so that animal protection is maintained during the whole cycle of their breeding. DESCRIPTION

Natural eugenol . Eugenol is widely present in aromatic plants and spices belonging to the family of

Myrtaceae and Lauracea. Eugenol is the main ingredient of the clove oil (Eugenia caryophyllata Thumb), pimento (Pimenta officinalis Lindley) and cinnamon leaves (Cinnamomum yeylanicum Nees). Eugenol is also an ingredient of the following essential oils: camphor tree (Cinnamomum camphora Sieb), (Acorus calamus L.), (Cymbopogon winterianus Jowitt),

(Canangium odoratum Baill.), (Cananga odorata Hook.), (Myristica fragrans Houtt),

(Sassafras albidum Nutt), (Commiphora myrrha Nees.), (Laurus nobilis L.), (Umbellularia californica Nutt.), (Alpinia officinarum Hanee), (Acacia farnesiana Willd.), (Ocimum minimum or Ocimum gratissimum L). and (Viola odorata L.) Natural eugenol is obtained from essential oils with a high percentage of eugenol. There is one characteristic of eugenol based on increased acidity of the phenol hydroxyl group that is used to separate eugenol from other accompanying components of essential oils. Together with diluted water solutions of alkalis it develops water-stable soluble salts that are not evaporable under the effect of water vapour.

Synthetic eugenol Apart from natural eugenol there is also synthetic eugenol like 2-methoxi-4 (2- propylene) phenol, CAS number 97-53-0.. This eugenol is also known as: 4-allyl- 2-methoxiphenol, allylguaiacol, eugenic acid and caryophyllic acid, molecular formula C_]0Hι₂O₂ of molecular mass 164.20. The eugenol structural formula is shown in drawing 1. OH 0^" Na+ (K+)

CH₂-CH=CH₂ CH₂-CH=CH₂

Eugenol Na-K eugenol salt Drawing 1.

Eugenol is a colorless liquid (it becomes darker in contact with light and air) and its boiling point is 252,60°C. It can be distilled at atmospheric pressure without any extensive decomposition. It has a clove spicy smell and the 'burning' taste. Its melting point is from -9.2°C to - 9.1°C. Density d 20/4 1.0664. Refraction index n 20/D 1.5410. Eugenol solubility in aquatic-alcoholic and alkali aquatic mediums is considerable. One eugenol volume dissolves in 5-6 volumes of 50% alcohol, 2-3 volumes of 60% alcohol or 1-2 volumes of 70% alcohol. One mL of eugenol dissolves in 4,27 mL alcohol at 25°C whereas to dissolve the same quantity at 15° 5,22 mL of alcohol of the same concentration is needed. 12,7 mL of 2% sodium hydroxide solution in water dissolves 1 mL of eugenol. The phenol hydroxyl group gives characteristics reactions. It produces brown cloudiness with ferrichloride in aquatic solution whereas in 2%alcoholic solution there is blue color. Hydroxyl group can be converted to ester or ether groups. In the immediate vicinity of the phenol hydroxide group, 2-metthoxi group, with its strong negative inductive effects, attracts the electronic pair of O-H bond and allows an easy departure of H⁺ ion and contributes to a higher acidity of molecules. Compared to phenol and its similar structures such as carvacrole and thymol , eugenol is much more acid, It reacts easily with carbonates and bicarbonates and colors blue litmus in red. Homolytic dissociation of the O-H bond is known and it results in creation of free oxygen radicals. This may be an advantage when used as a catalyst in some polymerizing reactions under radical mechanisms (for example in dentistry). Ionization of the O-H bnd, or creation of the O^" Me⁺ ionic pair prevents formation of free radicals. Metals that could form an ionic pair are sodium and potassium in the cases when the intention is to use eugenol per os or zinc if eugenol application is in dermatology. Such eugenol has bactericide and fungicide properties and is used as an antiseptic, preserving agent, antibiotic and antimycotic in pharmaceutical and veterinary preparations, perfumes and cosmetic preparations, toothpastes and soaps. It is used externally as pastes and by inhalation in the body as per oral preparations. Unlike some structurally very similar synthetic phenols (i.e. guaiacol) it is characterized with higher effectiveness, easy beaxableness and non-toxicity. Although in some cases pure eugenol can cause irritation in contact with skin and mucous membrane, it can be considered practically non-toxic during oral applications (LD₅₀ per os on rats 2680 mg/kg, on mice 3000 mg/kg). A method removing accompanying components of eugenol by extraction has been given to show invention feasibility. This method is based on eugenol property to develop soluble salt in water with a 3% aquatic solution of sodium hydroxide (NaOH) - sodium eugenolate that poorly dissolves in organic neprotic solvents such as diethyleter and petroleter. Accompanying components of essential oils are extracted with petroleter in a continuing liquid-liquid extraction process. Water solution is made acid (with ortho phosphorous acid) to pH value 4 and is distilled with water vapour. After separation of (the lower) oily phase the same gets dried and is distilled under a lower pressure. Table 1. shows dependence of eugenol boiling temperature from the pressure under which it is distilled.

Table 1. Eugenol boiling temperature under different pressures

Purity of the product obtained is > 99% (surface GC)

In order to prove invention feasibility a method is given how to obtain eugenol by removing accompanying components by distillation: This method is based on the eugenol property to develop in water a soluble salt with a 3% aquatic solution of sodium hydroxide (Na OH) - sodium eugenolate that is sufficiently stable at 100° C so that distillation with water vapour distills only accompanying essential oil components. Upon completion of distillation water solution gets acid (with ortho phosphorous acid) up to pH value 4 and is distilled with water vapour when pure eugenol is distilled. After separation of (the lower) oily phase the same gets dried and is distilled under a lower pressure. During distillation of accompanying components consisting of terpenes: α-pinene, camphene, β-pinene, limonene, cineol, p-cymene, linalool and β-caryophyllene products are lighter than water and it is necessary to use suitable separators for their separation. During eugenol distillation, after it becomes acid, the product is heavier than water and it is necessary to use separators for such products.

Eugenol in the form of sodium and potassium salts

In addition to application of natural and synthetic eugenol this invention also describes application of eugenol in the form of sodium and potassium salts. In order to prove invention feasibility, the process of obtaining eugenol sodium salts has been shown. This preparation is obtained by using the moist granulation technology. 40 g of aerosol is homogenized with 6,5 g of powdered unhydrated sodium carbonate (previously sifted through a sieve of 125 microns). A solution of clove oil in 96% ethyl alcohol (15 g oil with around 70% eugenol dissolved in 300 mL alcohol) is added to this homogenous mixture. Mass like dough should be completely mixed in the homogenizer and then sifted through a sieve of 2 mm. Moist granules should be dried at a temperature of up to 30°C. Drying (alcohol evaporation) produces granulated material with good mechanical properties. After drying the preparation is sifted through a sieve of 2 mm. Eugenol potassium salts are obtained in the following manner and this further proves invention feasibility: the preparation is obtained by using moist granulation technology. 40 g of aerosol is homogenized with 8,5 g of powdered unhydrated potassium carbonate (previously sifted through a sieve of 125 microns). A solution of clove oil in 96% ethyl alcohol (15 g oil with around 70% eugenol dissolved in 300 mL alcohol) is added to this homogenous mixture. Mass like dough should be completely mixed in the homogenizer and then sifted through a sieve of 2 mm. Moist granules should be dried at a temperature up to 30°C. Drying (alcohol evaporation) produces granulated material with good mechanical properties. After drying the preparation is sifted through a sieve of 2 mm. In accordance with the intention of this invention also shown is the process of obtaining eugenol sodium and potassium salts. The preparation is obtained by using the moist granulation technology. 40 g of aerosol is homogenized with 3,5 of powdered unhydrated sodium carbonate (previously sifted through a sieve of 125 microns) and 4,25 unhydrated potassium carbonate (previously sifted through a sieve of 125 microns).

A solution of clove oil in 96% ethyl alcohol (15 g oil with around 70% eugenol dissolved in 300 mL alcohol) is added to the homogenized mixture. Mass like dough should be completely mixed in the homogenizer and then sifted through a sieve of 2 mm. Moist granules should be dried at a temperature of up to 30°C. Drying (alcohol evaporation) produces granulated material with good mechanical properties. After drying the preparation is sifted through a sieve of 2 mm.

Application of essential oils from eugenol-containing plants: Cloves, pimentos and cinnamon leaves

Very good results are achieved by application of essential oils from eugenol- containing plants for improving preparations to prevent and treat animal diseases caused by bacteria, fungi and parasites:

1. Clove oil Clove tree (Eugenia caryophyllata Thumb, or Caryophyllus aromaticus L.) grows in humid hot tropic regions. Geographic territories are Zanzibar and the islands of Pemba, Madagascar, Ceylon, Malaysia and Indonesia. The fruit (dried flowers), plants in foliation and leaves are used for oil production. The fruit yields 17%-18% oil, plants in foliation 6% whereas the leaves yield 2-3%. Clove oil quality has been determined by eugenol contents. The highest eugenol content is found in the oil obtained from the fruit and ranges from 85-95%.

A similar oil quality is quality is also obtained from young plants in foliation. Oil obtained from leaves is of a slightly poorer quality, or with a smaller content of eugenol of 82-87%. Clove oil is heavier than water. Its specific weight (25°) ranges from 1.040 to 1.069 (oils with high phenol content have greater specific weight). Optical activity (rotation) ranges from -0° 36' to -1° 40' (oils with higher phenol content have a lower value of optical rotation). Refraction index (20°) ranges from 1.521 to 1.5391.

2. Pimento oil Pimento tree (Pimenta officinalis Lindley, Myrtus pimenta L. or Eugenia pimento D.C.) grows on the islands of West India and on the islands of coastal areas of Central and South America. It grows expansively on Jamaica. The largest oil quantity on the market is declared to originate from Jamaica - whether it is oil from fruits or leaves. Oil produced on other islands of Central and South America originates from various pimento sorts and differs by looks and aroma from Jamaican products. Pimento fruits yield is from 3.3% to 4.3%. Leaves produce less oil and to obtain oil with high phenol content a long distillation is necessary (over 10 hours). Such oil is usually finished off by redistillation in order to improve color and aroma. It is very difficult to make a difference between fruit and leave oils. Pimento oil is heavier than water. Oil specific weight (25°) ranges from 1.024 to 1.055, optical activity (rotation) ranges from -0° 32' to -5° 0', refraction index (20°) ranges from 1.525 to 1.536. Phenol content (soluble part in a 3% aquatic solution of sodium hydroxide) ranges from 65% to 89%.

3. Cinnamon leave oil There are several kinds of cinnamon. Most important, from the spice and essential oil production aspect, is Ceylon cinnamon (Cinnamomum zeylanicum Nees). Cinnamon oil considerably varies depending on whether it has been obtained from cinnamon bark or leave. Cinnamon bark of good quality (fineness 0000, gradation 00000 for the best bark and 0 for the worst) yields oil containing 62,2% to 75% of cinnamon aldehyde and only

6% to 13,2% of phenol (eugenol).. Best quality cinnamon leave oil coming from South Ceylon (south of Colombo) can contain 65% to 95% eugenol and only 4% cinnamon aldehyde. This oil has a specific weight (25°) ranging from 1.044 to 1.065, optical activity (rotation) from -0° 15' to +2° 20' and refraction index (20°C) from 1.531 to 1.540.

Qualitative and quantitative determination of eugenol For qualitative and quantitative determination of eugenol in commercial products (that are sold as eugenol), essential oils of cloves, pimentos and cinnamon, solid powdered premixes with around 20% eugenol as well as in fodder with 0,2- 0,04% eugenol (premix proportion: fodder 1:100 to 1:500), gas chromatography is used with flaming ionizing detector GC-FID.

Gas chromatograph HEWLETT PACKARD series II 5890 Column HP 1, 50 m x 0.2 mm x 0.33 μ Operating temperature after 5 minutes at 80° gradient 5°C/min, 5 min at 220°C Injection temperature 270°C Injecting volume 0,5 μL Injecting method automatic Carrying gas helium Detector ionizing flame Detector temperature 270°C

Calibration line has been used to present results — direct method The following have been taken as standard samples in this elaborate: Eugenol, Merck Company, Catalogue number 818455 CAS number 97-53-0 of 99% purity. The sample has been kept under nitrogen in the refrigerator at 4°C Iso- octane, Kemika Company Zagreb, purity for CHROMATOGRAPHY Naphtalene, Merck Company, catalogue number 820846 CAS Number 91-20-3, purity 99%. The standard solution is a standard naphthalene solution in is ø-octane, 14 mg in 25 mL. Eugenol calibration solutions in wo- octane have been prepared in a range from 20 to 160 mg of eugenol per liter.

Details for eugenol calibration line

Determination of eugenol in essential oils, deterpened essential oils and commercial products can be also carried out in the following manner: Two samples of essential oil solutions are prepared by dissolving approximately 100 mg of oil in 25 mL of t_o- octane. 0.5 mL of each solution is diluted in a volumetric vessel of 25 mL together with 1 mL of internal standard. Such a solution will contain around 80 mg of essential oil in one liter. By comparing Carrying gas helium Detector ionizing flame Detector temperature 270°C

Calibration line has been used to present results — direct method

The following have been taken as standard samples in this elaborate: Eugenol, Merck Company, Catalogue number 818455 CAS number 97-53-0 of 99% purity. The sample has been kept under nitrogen in the refrigerator at 4°C Iso- octane, Kemika Company Zagreb, purity for CHROMATOGRAPHY Naphtalene, Merck Company, catalogue number 82^846 CAS Number 91-20-3, purity 99%. The standard solution is a standard naphthalene solution in is o-octane, 14 mg in 25 mL. Eugenol calibration solutions in wo- octane have been prepared in a range from 20 to 160 mg of eugenol per liter.

Details for eugenol calibration line

Determination of eugenol in essential oils, deterpened essential oils and commercial products can be also carried out in the following manner: Two samples of essential oil solutions are prepared by dissolving approximately 100 mg of oil in 25 mL of iso- octane. 0.5 mL of each solution is diluted in a volumetric vessel of 25 mL together with 1 mL of internal standard. Such a solution will contain around 80 mg of essential oil in one liter. By comparing segments of the screened sample with the calibration segment it is possible to calculate eugenol content in essential oils.

The hydro distillation method to attain the calibration line is carried out in the following manner:

A series of eugenol dilutions in iso- octane from 200 to 1600 mg/L is prepared. Of those prepared solutions 2mL from each of them is put in the hydro distillation balloon (to determine essential oil content by Clavenger) and this is distilled for 2 hours. The obtained solution of eugenol in iso- octane is dried above anhydrated sodium sulphate. 1 mL of solution is placed a volumetric vessel of 10 mL, 0.4 mL of naphthalene solution is added and filled to the mark and analyzed with GC-FID.

Details for eugenol calibration line

Determination of eugenol in premixes with around 20% eugenol in the form of salts is carried out in the following manner: Nearly 0.1000 g of the sample is put in the hydro distillation balloon (to determine essential oil content by Clavenger), 200 mL demineralized water, 1 mL 25% H₂SO₄ (ρH<3) and 2 mL t_ø-octane are added and distilled for 2 hours. The obtained solution of eugenol in wo-octane is dried above unhydrated sodium sulphate. 0.5 mL of solution is placed in the volumetric vessel of 100 mL, 4 mL of naphthalene solution is added and analyzed with GC-FID.

Determination of eugenol in fodder with 0.2 to 0.04% eugenol in the form of salts is carried out in the following manner: Nearly 25 g of fodder sample is put in the hydro distillation balloon (to determine essential oil content by Clavenger), 200 mL demineralized water, 1 mL 25% H₂SO₄ (ρH<3) and 2 mL t_ø-octane are added and distilled for 2 hours. The obtained solution of eugenol in ώø~octane is dried above unhydrated sodium sulphate. 0.5 mL of solution is placed in the volumetric vessel of 100 mL, 4 mL of naphthalene solution is added and analyzed with GC-FID.

INDUSTRIAL OR OTHER APPLICATIONS OF THIS INVENTION

Industrial or other applications of eugenol, eugenol in the form of sodium and potassium salts in the matrix of hydrated aerosol and essential oils from eugenol- containing plants in the preparations for veterinary use is absolutely possible. Specialists in this field can without any problems produce preparations according to the processes described under DESCRIPTION. Preparations can also be produced in medicament producing factories and even in well-equipped pharmaceutical laboratories. Applications of this invention are especially recommended when it is necessary to achieve outstanding quality of meat, milk and eggs and for weak animals that do not take antibiotic and sulphonamide preparations easily. It should be stressed that its application is compulsory to prevent diseases of animals bred in bad climate and field conditions. Also, it must be emphasized that this invention has been laboratory tested and experimentally applied on many breeding poultry and pig farms.

Claims

PATENT CLAIMS

1. Application of natural eugenol in the prevention and treatment of animal diseases caused by bacteria, fungi and parasites is marked so that by integration of natural eugenol in veterinary preparations a synergetic effect is achieved, actually, in bacteria, fungi and parasites cytoplasmic membranes become damaged because of disintegration of lipids and prevention of synthesis of albumin responsible for transmission of oxygen to mitochondria and due to lack of oxygen the bacterial cell, fungi or parasites quickly die.

2. Application of synthetic eugenol in the prevention and treatment of animal diseases caused by bacteria, fungi and parasites is marked so that by integration of synthetic eugenol and eugenol in the form of sodium and potassium salts in veterinary preparations a synergetic effect is achieved, actually, in bacteria, fungi and parasites cytoplasmic membranes become damaged because of disintegration of lipids and prevention of synthesis of albumin responsible for transmission of oxygen to mitochondria and due to lack of oxygen the bacterial cell, fungi and parasites quickly die.

3. Application of essential oil from plants containing eugenol: cloves (Eugenia caryophyllata), pimentos (Pimenta officinalis) and cinnamon leaves Cinnamomum zeylanicum) in the prevention and treatment of animal diseases caused by bacteria, fungi and parasites is marked so that by integration of essential oils from plants containing eugenol - cloves, pimentos and cinnamon leaves - a synergetic effect is achieved, actually, in bacteria, fungi and parasites cytoplasmic membranes become damaged because of disintegration of lipids and prevention of synthesis of albumin responsible for transmission of oxygen to mitochondria and due to lack of oxygen the bacterial cell, fungi and parasites quickly die.