WO2021255646A1 - A copper based composition for animal consumption - Google Patents

A copper based composition for animal consumption Download PDF

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Publication number
WO2021255646A1
WO2021255646A1 PCT/IB2021/055277 IB2021055277W WO2021255646A1 WO 2021255646 A1 WO2021255646 A1 WO 2021255646A1 IB 2021055277 W IB2021055277 W IB 2021055277W WO 2021255646 A1 WO2021255646 A1 WO 2021255646A1
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WIPO (PCT)
Prior art keywords
composition
acid
solution
sample
water
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PCT/IB2021/055277
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French (fr)
Inventor
Mark Litvak
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Margana Bio Technologies Ltd.
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Publication of WO2021255646A1 publication Critical patent/WO2021255646A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/105—Aliphatic or alicyclic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/20—Inorganic substances, e.g. oligoelements
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • A61K31/10—Sulfides; Sulfoxides; Sulfones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/20—Elemental chlorine; Inorganic compounds releasing chlorine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/34—Copper; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B11/00—Oxides or oxyacids of halogens; Salts thereof
    • C01B11/02—Oxides of chlorine
    • C01B11/022—Chlorine dioxide (ClO2)

Definitions

  • the present invention relates to the field of special feed additives, specifically to the field of supporting and enhancing the immune system in animals.
  • Zoonoses also known as zoonosis and as zoonotic diseases
  • zoonosis infectious diseases caused by bacteria, viruses and parasites that spread between animals (usually vertebrates).
  • Animal disease usually relates to an impairment of the normal state of an animal that interrupts or modifies its vital functions.
  • veterinary medicine deals with the study, prevention, and treatment of diseases not only in domesticated animals but also in wild animals and in animals used in scientific research.
  • the prevention, control, and eradication of diseases of economically important animals are agricultural concerns.
  • composition for boosting the immune system of an animal comprising Dimethyl sulfoxide (DMSO), a vegetable oil, a fatty acid, Chlorine dioxide and a surfactant.
  • DMSO Dimethyl sulfoxide
  • the vegetable oil may be sesame oil
  • the fatty acid may be heptanoic acid
  • the surfactant may be Tween 80.
  • the composition may further include an immune- boosting formulation including Copper containing nanoparticles and a hydrophobic substance.
  • the hydrophobic substance may be selected from a group containing vegetable oil, vegetable oil with unsaturated connections, vegetable oil with dienic unsaturated connections.
  • the vegetable oil may be sesame oil.
  • the immune-boosting formulation may further include polyalkoxylated butyl ether.
  • the vegetable oil of the immune-boosting formulation may be sesame oil
  • the fatty acid of the immune-boosting formulation may be heptanoic acid
  • the surfactant of thee immune-boosting formulation may be Tween 80.
  • the composition may preferably also include polyalkoxylated butyl ether.
  • composition of the present invention for boosting the immune system of farm animals and poultry.
  • the use of the present invention may include adding the above composition to the drinking water of said animals and poultry.
  • the use of the present invention may include adding the composition to the drinking water according to the following regimen:
  • Week 6 1.5 liters of said composition to a 1000 liters of drinking water for 3-5 days.
  • FIG. 1-4 illustrates a laboratory analysis of particle size, as measured by a Malvren Zetasizer device.
  • Figure 1 illustrates laboratory analysis results from a sample of 70mg/L of AR-DEO, as measured by a Malvren Zetasizer device in accordance with some demonstrative embodiments.
  • Figure 2 illustrates laboratory analysis results from a sample of 125 mg/L of AR-DEO, as measured by a Malvren Zetasizer device in accordance with some demonstrative embodiments.
  • Figure 3 illustrates laboratory analysis results from a sample of AR-DEO + con + vitkanol 1 , as measured by a Malvren Zetasizer device in accordance with some demonstrative embodiments.
  • Figure 4 illustrates laboratory analysis results from a sample of AR-DEO concentrate, as measured by a Malvren Zetasizer device in accordance with some demonstrative embodiments.
  • compositions for oral consumption by an animal including Dimethyl sulfoxide (DMSO), a vegetable oil, a fatty acid, Chlorine dioxide and a surfactant.
  • DMSO Dimethyl sulfoxide
  • Chlorine dioxide tends to quickly decompose upon exposure to air. According to some demonstrative embodiments, mixing the chlorine dioxide with DMSO preserves the Chlorine dioxide and diminishes the decomposition and/or absorbance of humidity, e.g., upon exposure to air.
  • the composition of the present invention allows for the formation of chlorinic acid, e.g., upon ingestion of the composition.
  • the chlorinic acid allows for the effective neutralization of harmful bacteria and/or viruses within a living organism.
  • the unique combination of components of the composition of the present invention allows for a stable composition, which does not readily decompose upon exposure to the environmental conditions (such as air, humidity, elevated temperature etc.), while exhibiting antibacterial and antiviral properties.
  • the vegetable oil may include sesame oil, cocoa oil, castor oil and the like. According to some embodiments, the vegetable oil may preferably be sesame oil.
  • the fatty acid may be selected from the group including heptanoic acid, lauric acid, myristic acid, palmitic acid, palmitate, palmitoleate, hydroxypalmitate, arachidic acid, oleic acid, stearic acid, behenic acid, sodium stearate, calcium stearate, magnesium stearate, hydroxyoctacosanyl hydroxystearate, oleate esters of short-chain, esters of fatty acids, fatty alcohols, esterified fatty diols, hydroxylated fatty acid, hydrogenated fatty acid, triesters of fatty acids, triglycerides received from fatty acids and fatty acid eutectics.
  • the fatty acid of the composition is preferably a short chain hydrophobic acid such as heptanoic acid.
  • the surfactant may preferably be Polysorbate 80 (Tween 80) Polysorbate 20 and/or Sorbitan monooleate (Span 80) and their analogs from Arlacel or BRIJ detergent lines or their mixtures that provide adjustment of predetermined specific HLB values.
  • the composition of the present invention may exhibit various useful characteristics including, for example, where it improves microcirculation in brain tissue and other areas of the body of the animal; improves microcirculation in the tissues of the joints; prevents the development of DIC syndrome, especially its local manifestations.
  • the composition of the present invention may also reduce coagulation time, improving microcirculation in the lesion. Reduces the tendency of red blood cells to aggregate. Normalizes the process of fibrin formation, which improves tissue trophism. Improves the nutritional conditions of the transplanted skin flap by expanding the capillary network, increasing the total number of functioning vessels. It creates the most favorable conditions for the healing of skin flaps and preventing the development of necrosis in them.
  • the composition may have a vasodilating effect, has a moderately pronounced hypotensive effect. Reduces peripheral vascular resistance and improves the rheological properties of blood, reduces the adhesion and aggregation of blood cells.
  • the composition may increase the permeability of the walls of venules. It easily penetrates the skin and cell membranes without damaging them. Penetrating through intact skin, it carries with it into the cells and intercellular tissue various medicinal substances.
  • the composition of the present invention may also increase the permeability of the histohematological barriers of the lungs and liver, however, without affecting the permeability of the histohematic barriers of the kidneys. Increases the permeability of the blood-brain and blood-brain barrier. Deposits medicinal substances in tissues for 1-2 weeks. It has a unique ability to conduct (transport) the substances dissolved in it through the skin and mucous membranes without damaging them. It breaks the hydrophobic bonds and lipoprotein complexes of cell membranes, which underlies the increase in their permeability under the influence of the composition. Promotes prolonged deposition of drugs in the tissues.
  • the composition of the present invention may also possess anti-inflammatory activity which may be comparable to non-steroidal anti inflammatory drugs. It has not only local, but also systemic anti-inflammatory effects.
  • the composition of the present invention may have an antiexudative effect, inhibits the activity of proteases, the synthesis of prostaglandins. At the same time, it increases the secretion of cortisol, strengthens the membrane of lysosomes. Reduces cellular infiltration in the focus of inflammation.
  • the composition of the present invention may have a direct antiproteolytic effect, prevents the development of edema and necrosis, inhibits platelet aggregation. It has an antipyretic effect, suppresses exudation processes in the focus of inflammation.
  • the composition of the present invention including at least 33% DMSO may quickly lead to a decrease in soft tissue edema. Stimulates reparative processes in tissues, proliferative processes in the focus of inflammation.
  • the composition of the present invention may also quickly eliminate the phenomena of inflammation of the soft tissues. It accelerates the development of granulation tissue, slowing down the processes of scar formation. Gentle scars form. Promotes resorption of inflammatory infiltrates, including in the stroma of the lungs and pleura. It resolves rough scars, playing the role of a “chemical orderly” in the area of inflammation, a biochemical cleaner.
  • the composition of the present invention may interact with protein breakdown products, forming intermolecular (hydrogen and hydrophobic) bonds with functional groups of polypeptides - OH, NH, SH, NH 2 . Promotes their removal from the focus of inflammation.
  • the composition of the present invention may inhibit the proliferation of fibroblasts, and in combination with corticosteroids completely suppresses it.
  • the composition of the present invention may inhibit collagen synthesis. Reliably and for a long time reduces the level of prostaglandins PgE b, PgF 2a by suppressing the intensity of the synthesis of the latter. According to some embodiments, the composition of the present invention may also reduce and slow down the development of radiation and drug pneumonitis.
  • the composition of the present invention may not have an allergenic effect.
  • the composition of the present invention may not cause contact sensitization with cutaneous application. On the contrary, it is characterized by desensitizing and antiallergic activity. Inhibits, according to various sources, anaphylactic reactions by 41 - 72%.
  • the antiallergic activity of the composition of the present invention may be proportional to the concentration of the drug used.
  • the basis of antiallergic activity is the ability to suppress B-lymphocytes and plasma cells, the blockade of histaminergic serotonergic receptors, antihistamine action, the ability to inactivate histamine released from mast cells, and deplete the pool of histamine-producing cells.
  • the composition of the present invention may be a long-acting anesthetic with a pronounced local analgesic effect. It has a long analgesic effect. Selectively blocks nerve fibers conducting a pain impulse. It can be considered as a symptomatic analgesic for pain syndromes of various origins.
  • a prolonged blockade of nerve endings and trunks may be achieved by the introduction of the composition of the present invention with an anesthetic, and the final concentration of the solvent should be 30-50%.
  • composition of the present invention may potentiate the effect of antibiotics.
  • the composition of the present invention may also inhibit bacterial lactamases.
  • the phenomenon of a selective increase in the concentration of antibiotics in the affected tissue with the combined administration of antibiotics with dimexide, their selective accumulation was discovered.
  • the concentration of the antibiotic in the lesion increases by 2.5-3 times; thus, the composition of the present invention may enhance the antibacterial effect of antibiotics.
  • the introduction of antibiotics together with the composition of the present invention may intravenously and/or orally open up new paths in antibiotic therapy and anti-inflammatory therapy.
  • the composition of the present invention may have an anticoagulant effect.
  • the plasma volume decreases, its viscosity, as well as the viscosity of whole blood, increases the coagulation time. Blood coagulation first accelerates, then slows down, changes in general occur towards hypocoagulation. The bleeding time increases slightly.
  • the composition of the present invention may inhibit platelet aggregation. Normalizes the process of fibrin formation, which improves tissue trophism. The probability of thrombosis interferes. Having fibrinolytic activity, normalizes fibrinolysis.
  • the composition of the present invention may have antimutagenic activity both in vitro and in vivo. It has strong antioxidant activity.
  • the composition of the present invention may prevent lipid peroxidation, stabilize cell membranes, including lysosome membranes. It captures free radicals, primarily “OH”, which is believed to be largely the basis of the anti-inflammatory effect of the drug. To achieve this effect, its concentration in the blood from 0.5 to 10 millimoles per liter is sufficient.
  • the composition of the present invention may enhance the synthesis of superoxide dismutase (SOD) in cells.
  • SOD superoxide dismutase
  • the composition of the present invention may also be useful as an antistress. It has anti-stress and sedative effects. Eliminates the reactions of anxiety and fear.
  • the composition of the present invention may have a strong oxidizing agent, and act as an antiseptic itself.
  • the composition of the present invention may have the ability to inhibit spore germination.
  • the composition of the present invention may inhibit the growth of all microorganisms saprophying in the blood of cancer patients. When exposed to the body, the composition of the present invention may block glycolytic metabolic pathways. Reduces the accumulation of lactate in the blood, thereby reducing lactocytosis.
  • the composition of the present invention may quickly relieve swelling of brain tissue and improves cerebral blood flow, normalizes cerebral hemodynamics, and improves the clinical condition of the animal.
  • the composition of the present invention may reduce the heart rate, increase the voltage of the teeth on the ECG.
  • the described effects are dose dependent. If small doses of the drug enhance the activity of the heart, then very large doses can have a toxic effect on the heart, up to a stop. It has a hypotensive effect. Lowers blood pressure. Normalizes the reactivity of the autonomic nervous system.
  • the introduction of the composition of the present invention may be accompanied by a diuretic effect.
  • Increasing diuresis by 50% acts as an osmotic diuretic.
  • diuresis can increase by an order of magnitude.
  • the composition of the present invention may be a mild immunosuppressant, which has an immunosuppressive effect against B -lymphocytes, reduces the level of immune complexes circulating in the blood, at the same time it stimulates nonspecific reactivity, and increases the body's resistance to adverse effects. Induces interferon synthesis. Does not affect the phagocytic activity of polymorphonuclear leukocytes (PMNL). In general, it increases the functional state of the macrophage system. Increases the cytolytic activity of blood serum. It has a resolving effect in the focus of inflammation. Inhibits macrophage activity in the focus of inflammation.
  • a mild immunosuppressant which has an immunosuppressive effect against B -lymphocytes, reduces the level of immune complexes circulating in the blood, at the same time it stimulates nonspecific reactivity, and increases the body's resistance to adverse effects. Induces interferon synthesis. Does not affect the phagocytic activity of polymorphonu
  • the composition of the present invention may be distributed completely over the cells at a concentration of 10-15 millimoles per liter. It is a cellular protector of the intracellular type. Penetrates (penetrates cell membranes). It has a protective effect on cells (celluloprotective effect).
  • the composition of the present invention may act as a cryoprotectant. Stabilizes lysosome membranes and other cell membranes. Passes through intact cell membranes.
  • the composition of the present invention may be oxidized to dimethyl sulfone, which is excreted in urine and feces.
  • the composition of the present invention may have a sedative effect, tranquilizing activity.
  • the composition of the present invention may be used for the treatment of strokes and injuries of the brain and spinal cord, due to the ability to normalize central and systemic hemodynamics.
  • the composition of the present invention may have a pronounced decongestant effect on brain tissue of the animal.
  • the composition of the present invention may be used for treatment of trigeminal neuritis (trigemini tis) is long, from 1 to 6 months.
  • the composition of the present invention may also have an antiviral effect. It may be used to treat shingles as an independent drug, and together with antiviral compounds.
  • the composition of the present invention may be effective in treating animals with chronic inflammatory lung diseases.
  • a good effect may be obtained from administration of the composition of the present invention, optionally, with the addition of proteolytic enzymes and antibiotics.
  • the composition of the present invention may also have an antiulcer activity, as it may inhibit the secretory function of the stomach, reducing the secretion of gastric juice. Increasing exocrine liver function by 50% and enhancing bile secretion.
  • the composition of the present invention may be used to treat rheumatoid arthritis. Gout treatment. Treatment of bursitis, arthritis, tenosynovitis.
  • the composition of the present invention may modify the metabolism of connective tissue, in particular collagen. Reduces destructive changes in the joints. It has a basic effect on the course of chronic arthritis.
  • the composition of the present invention may be used for Amyloidosis treatment.
  • the composition of the present invention may dissolve amyloid fibrils.
  • Patients within 3-5 years are prescribed oral administration of a 3-5% DMSO solution in water of 30 ml 3 times a day while taking peppermint preparations.
  • the oral route of administration of the composition of the present invention in the form of a solution may be used to prevent the formation of urinary stones.
  • the composition may be rapidly adsorbed through the mucosa of the gastrointestinal tract.
  • the composition of the present invention may be used in the treatment of interstitial cystitis.
  • the composition of the present invention may be instilled through a catheter into the bladder.
  • the composition of the present invention may be used independently in the treatment of bruises, sprains, hemorrhages, edema, purulent wounds, chronic osteomyelitis, erysipelas, phlebitis, and thrombophlebitis.
  • the composition of the present invention may be effective in preventing and correcting septic complications. Especially effective is the treatment of sepsis by intravenous administration of antibiotics together with the composition of the present invention may.
  • the composition of the present invention may be effective in the treatment of purulent wounds.
  • a solution of the composition of the present invention may, after which the cavity is drained. Reduces the amount of purulent discharge from the wounds. Stimulates the formation and growth of granulations.
  • the composition of the present invention may be used in the treatment of open fractures, osteomyelitis, contractures, tendons, pains, and the like.
  • an immuno-boosting formulation to be mixed with the components of the present composition.
  • the immuno-boosting formulation may comprise Copper containing nanoparticles, butyl ether, preferably, polyalkoxylated butyl ether and a hydrophobic substance.
  • the combination of the composition of the present invention together with the immuno-boosting formulation may be referred to herein as "AR- DEO”.
  • the combination of the composition of the present invention together with the immuno-boosting formulation may synergistically boost the immune system of the animal.
  • the term "enhancing the immunological system of an infant” may include, but not limited to, shortening of disease and/or outbreak periods, diminishment of likelihood of becoming ill, reduced number and/or severity of symptoms associated with diseases and the like.
  • animal as used herein may refer to any member of the kingdom Animalia, including for example, domesticated livestock and animal husbandry, such as, chickens, cattle, sheep, pigs; marine (oceanic) or freshwater fish and the like.
  • the composition of the present invention may possess bacteriocidic and/or bacteriostatic characteristics.
  • the copper containing composition of the present invention as potent nonspecific antiseptic substance is effective against a wide variety of harmful economically important pathogens, including E.coli, Salmonella, Staphylococcus, Clostridium, Mycoplasma species etc.
  • composition of the present invention may be useful to treat or prevent a wide variety of diseases, including for example, bacterial or viral infections.
  • the composition of the present invention may be useful to treat or prevent a wide variety of diseases, including for example, Acute hepatopancreatic necrosis disease, African swine fever, Akabane, Anthrax, Australian bat lyssavirus, Avian influenza, Avian paramyxovirus, Blackhead, Bluetongue, Bovine ephemeral fever, Bovine viral diarrhoea virus, Brucellosis, Caprine arthritis encephalitis, Cat- scratch disease, Cattle tick, Coccidiosis, Copper deficiency in ruminants, Cryptococcosis, Enzootic bovine leucosis, Epizootic ulcerative syndrome, Equine herpesvirus type 1 (EHV-1), Equine infectious anemia, Equine influenza, Equine viral arteritis, External parasites in poultry, Foot-and-mouth disease in cattle, Fowl cholera, Fowl pox, Giardiasis, Hendra virus, Hydatid disease
  • diseases including for example,
  • the unique combination of the composition of the present invention with the immune boosting formulation including Copper containing nanoparticles, butyl ether and a hydrophobic substance allows for the effective impact of the copper on the pathogen organisms such as bacteria, viruses, and other pathogens.
  • a raw material for formation of copper containing nanoparticles of the present invention may include a substance selected from the group: Copper acetate, copper acetate mono hydrate, basic copper carbonate monohydrate, copper chloride dihydrate, copper oxide, copper sulphate pentahydrate, cupric chelate of amino acids, cupric chelate of glycine hydrate.
  • the copper salt is preferably an organic salt of metal with variable valence and ability to dissociate at high temperature
  • examples of copper salts may include copper bismuth acetate, acetate of cuprum and the like.
  • the copper of the present invention is preferably Cuprum acetate, more preferably, Cuprum acetate monohydrate.
  • the hydrophobic substance may include any suitable fat selected from the group including fats, fatty acids, fatty acid esters, fatty acid triesters, salts of fatty acids, fatty alcohols, phospholipids, solid lipids, waxes, lauric acid, stearic acid, alkenes, waxes, alcohol esters of fatty acids, long chain alcohols and glucoles, and combinations thereof; optionally wherein said salt of fatty acids comprises one or more of aluminum, sodium, potassium and magnesium salts of fatty acids; optionally wherein said fatty coating layer comprises one or more of paraffin wax composed of a chain of alkenes, normal paraffins; natural waxes, synthetic waxes, vegetable oil such as sesame oil, hydrogenated vegetable oil, hydrogenated castor oil; fatty acids, such as lauric acid, myristic acid, palmitic acid, palmitate, palmitoleate, hydroxypalmitate, stearic acid, arachidic acid, oleic acid,
  • the hydrophobic substance may preferably be a vegetable oil, more preferably, a natural vegetable oil with high content of non-saturated fatty acids especially with conjugated bonds (dien bonds) such as linoleic acid including for example: olive oil, sesame oil, linseed oil and the like.
  • the vegetable oil is most preferably sesame oil.
  • the immune-boosting formulation may include Copper in a concentration of 0.1 - 4.5% w/v, preferably 0.5 - 3.5% w/v, more preferably 0.7 - 3.5% w/v; and a hydrophobic substance in a concentration of 30- 95% v/v, preferably 40-60% v/v, more preferably, 45-55% v/v.
  • the immune-boosting formulation may include Copper acetate monohydrate in an amount of 35gr, 250 ml of surfactant, e.g., polyalkoxylated butyl ether and 250 ml of Sesame oil, for the preparation of 500 ml of the composition.
  • surfactant e.g., polyalkoxylated butyl ether and 250 ml of Sesame oil
  • the composition of the present invention may further include another known immuno-boosting formulation mixed with the components of the present composition.
  • the immuno-boosting formulation may include Dimethyl sulfoxide (DMSO), a vegetable oil, a fatty acid, Chlorine dioxide and a surfactant.
  • DMSO Dimethyl sulfoxide
  • the vegetable oil of the immuno-boosting formulation may include sesame oil, cocoa oil, castor oil and the like. According to some embodiments, the vegetable oil of the immuno-boosting formulation is preferably sesame oil.
  • the hydrophobic substance of the immuno-boosting formulation may include any suitable fatty acid selected from the group including heptanoic acid, lauric acid, myristic acid, palmitic acid, palmitate, palmitoleate, hydroxypalmitate, arachidic acid, oleic acid, stearic acid, behenic acid, sodium stearate, calcium stearate, magnesium stearate, hydroxyoctacosanyl hydroxystearate, oleate esters of short-chain, esters of fatty acids, fatty alcohols, esterified fatty diols, hydroxylated fatty acid, hydrogenated fatty acid, triesters of fatty acids, triglycerides received from fatty acids and fatty acid eutectics.
  • suitable fatty acid selected from the group including heptanoic acid, lauric acid, myristic acid, palmitic acid, palmitate, palmitoleate, hydroxypalmitate, arachidic acid, oleic acid,
  • the hydrophobic substance of the immuno-boosting formulation is preferably a short chain hydrophobic acid such as heptanoic acid.
  • the surfactant of the immuno-boosting formulation is preferably Polysorbate 20, Tween 80 and/or Span 80 and their analogs from Arlacel or BRIJ detergent lines or their mixtures that provide adjustment of predetermined specific HLB values.
  • the composition of the present invention may be mixed with the immune-boosting formulation to provide a final mixture, for example, to be provided to the animals, e.g., in their drinking waters.
  • composition of the present invention may be mixed with the immune-boosting formulation in equal amounts and/or volumes.
  • the composition of the present invention may be referred to herein as solution B, and the immune-boosting formulation may be referred to herein as solution A, and final mixture (solution A and solution B) may be referred to herein as AR-DEO.
  • the composition of the present invention may be in a liquid form or a dry powder form.
  • the composition of the present invention is mixed in the drinking water of an animal.
  • the final mixture (AR-DEO) may be in a liquid form or a dry powder form.
  • the final mixture disclosed herein may be mixed in the drinking water of an animal.
  • composition of the present invention for boosting the immune system of an animal.
  • the use may include adding the composition of the present invention (solution B) to the drinking water of the animals using generally the following regimen:
  • Week 6 1.5 liters of solution A to a 1000 liters of drinking water for 3-5 days.
  • the use may include adding a mixture of the composition of the present invention (solution B) with the immune boosting formulation (Solution A) to the drinking water of the animals using generally the following regimen:
  • the desired dosage may be provided using generally the following dosage: 2ml of solution B + 2ml of solution A per 10 Kg body weight of the animal.
  • Copper containing material in nanoparticle form has two unique features:
  • one of the benefits of the process of the present invention is the incorporation of the metal atoms , e.g., the copper atoms, into the chain of organic molecules.
  • the second process takes place. It is partial polymerization of oxidized fatty acids.
  • the nanoparticles including shielded atoms of heavy metals may provide fast, effective, and safe action of the composition of the present invention.
  • FIG. 1 illustrates a laboratory analysis of particle size, as measured by a Malvren Zetasizer device.
  • Figure 1 illustrates the results from a sample of 70mg/L of AR-DEO
  • Figure 2 illustrates the results from a sample of 125 mg/L of AR-DEO
  • Figure 3 illustrates the results from a sample of AR-DEO + con + vitkanol
  • Figure 4 illustrates the results from a sample of AR-DEO concentrate
  • Example 2 An exemplary preparation of solution A and solution B:
  • Sesame Oil 200ml Copper Acetate 10 gr Ethyl an NS 500 LQ - 200ml Solution B Sesame oil 100 ml Heptanoic acid 100ml Polysorbate 20 - 200 ml Dimethyl sulfoxide - 5ml Chlorine dioxide - 1 gr
  • AR-DEO is a copper feed additive for enrichment and balancing of nursing and weaned pigs’ diets.
  • AR-DEO contains the active ingredient - an organic compound of copper, based on naturally occurring oils (component “A”) and auxiliary substances (component “B”) - carboxylic acid, emulsifier, nonionic surfactant, sodium chloride. Concentrate of the "A” and “B” mixture components in a ratio of 1: 1 contains at least 12.5 grams of an organic copper compound in 1 liter.
  • Components "A” and “B” are available in separate packages in the form of 100% concentrate. In appearance, they constitute opaque oily liquid from light brown to dark brown color and have a characteristic odor. They are packed in 1,5,10 - liter plastic bottles and cans, sealed with tamper-evident cup. Each package should be marked listing the manufacturer, its address and trademark, the name of the drug, its specifications, its purpose and method of application, its name and the content of the active substances, the shipping measurement, the date of manufacture, the shelf life, the storage conditions, the precautions and provide it with direction for use. 4.
  • the product is to be stored in original unopened packaging in covered ventilated storage rooms, protected from light, out of direct sunlight and away from heat sources, in a dry place inaccessible to unauthorized persons or animals, at a temperature from + 3 °C to + 35 °C.
  • the shelf life is 3 years from the manufacturing date.
  • the feed additive AR-DEO is a source of copper to meet the nursing and weaned pigs’ needs.
  • An organic copper compound based on natural oils in combination with auxiliary substances has higher bioavailability compared with inorganic compounds.
  • copper is involved in skeletal, immune system, reproductive function formation, and helps to increase growth rate and feed efficiency of animals and improve their security.
  • This additive in the doses recommended for use is bioavailable, harmless and areactogenic. According to the toxic effect, it belongs to the substance hazard category 4. It does not accumulate in tissues and organs. Within 72 hours, it is eliminated from the body.
  • This additive is active in a wide temperature range in the water of any hardness, it is resistant to pH fluctuations.
  • AR-DEO is a copper feed additive for enrichment and balancing of nursing and weaned pigs’ diets and it is intended for internal use by drinking with water or feeding with food. Introduction to animal feed and premixes of the feed and premix plants is not required.
  • the daily dosage for nursing and weaned pigs is a mixture of 1 ml of the components "B" and "A" concentrate mixture in the ratio of 1: 1 for 1 kg of live weight. It is given in the drinking water or in the feed in the form of oil in water emulsion with 5.1% concentration.
  • the master emulsion is prepared in a plastic container, convenient for mixing the components by active shaking.
  • the container is filled with 10 parts of potable water, heated to 60-70 °C, and 1 part "B" component is added.
  • the component "A” in an amount equal to the amount of component "B” is added until a white emulsion, and the master emulsion is further stirred by shaking for the same time until homogenous state.
  • the result is a 20% emulsion, which is used to prepare working concentrations of 5.1% oil in water emulsions.
  • the emulsion remains active for 7 days.
  • Working emulsion remains active for 3 days.
  • weaned piglets drink it with water, or by eating with dry or wet feed for 5 consecutive days.
  • the daily dose in the form of oil in water emulsion of 1% concentration is fed using medicator.
  • the daily dose in the form of oil in water emulsion of 5% concentration is mixed with the daily amount of feed. The course is repeated in both cases after 3 weeks.
  • the product is compatible with other feed additives and drugs provided the interval between the application should be at least 3-4 hours.
  • Method of digestion a. Heat sample for temperature 200°C within 15 min. b. Hold temperature at 200°C for 15 min. . c. Cool to room temp., transfer vessel content to a 50 ml volumetric flask. Wash vessel 3-5 times with demineralized water (3-5 ml for each time). d. Fill up to the mark with DI water. Mix well.
  • Digestion a. Transfer digestion vessel contents to a 50 ml volumetric flask. b. Wash vessel 3-5 times with demineralized water (3-5 ml for each time). c. Fill up to the mark with DI water. Mix well.
  • Cu (oil) Cu(tot) — Cu(W)
  • FFA Free Fatty Acids
  • TAG triacylglycerides
  • Standard solution contains Oleic Acid Methyl Ester (C18: 1 FAME) and Linoleic Acid Methyl Ester(C18:2 FAME)) with concentrations more than 1%
  • the solution can be stored in dry place not more than 24h 4.
  • reaction vial Remove reaction vial from water bath, cool to room temperature (no water!), open reaction vial, add 8ml of demineralized water close the vial and mix well. Reopen the vial, add 5ml of hexane, reclosed the vial and shake for 1 min.
  • Retention Times guide may vary on other equipment.
  • FFA Free Fatty Acids
  • TAG triacylglycerides
  • SA Stearic Acid Cone
  • Retention Times guide may vary on other equipment.
  • FFA Free Fatty Acids
  • TAG triacylglycerides
  • Standard solution contains Palmitic Acid Methyl Ester with concentrations more than 1%
  • the solution can be stored in dry place not more than 24h
  • reaction vial Remove reaction vial from water bath, cool to room temperature (no water!), open reaction vial, add 8ml of demineralized water close the vial and mix well. Reopen the vial, add 5ml of hexane, reclosed the vial and shake for 1 min.
  • Palmitic Acid FAME- detected area of peak corresponded Palmitic Acid FAME ⁇ All peaks- summary area of detected peaks (excluding peaks detected for Solvent blank sample (see #7). 8.3 Calculation of Palmitic Acid concentration in the sample
  • Area Palmitic Acid st - area of peak corresponded Palmitic Acid FAME for external standard solution
  • Retention Times guide may vary on other equipment
  • FFA Free Fatty Acids
  • TAG triacylglycerides
  • Standard solution contains Arachidonic Acid Methyl Ester with concentrations more than 1%
  • the solution can be stored in dry place not more than 24h
  • reaction vial Remove reaction vial from water bath, cool to room temperature (no water!), open reaction vial, add 8ml of demineralized water close the vial and mix well. Reopen the vial, add 5ml of hexane, reclosed the vial and shake for 1 min.
  • Retention Times guide may vary on other equipment
  • Example 9 Analyses of natural oils, fats and lipids.
  • Titrate sample and 2 blank solutions (25ml of solvent for sample) with titrant. If more than one endpoint is detected choose the one closest to pH 8.2.
  • Potassium Hyroxide put in 1000ml volumetric flask 30g Potassium Hyroxide, add 20ml of Water and up to mark by 95% Ethanol. Stand for 24h, and filter.
  • Potassium Iodide Solution Dissolve 100g of Potassium Iodide in 1000ml water. Store in a brown-glass bottle 1.4 Sodium Thiosulphate solution. 24.8g of Sodium Thiosulphate dissolve in 500- 700ml of water, add 2-3 drops of Chloroform. Add water to 1000ml. Wait overnight and filter solution and store in a brown bottle.
  • Standard solution of FAMEs (Fatty acids methyl esters) contain C14, C16.0, C16.1, C18.0, C18.1, C18.2, C20.0 FAMEs with concentration enough for FID detection (not less than 1% )
  • Trans-esterification process can be performed with alkaline or acid catalysis.
  • Integrated hexane layer washed 3 times by 50ml of 10% Ethanol solution, dried by Sodium Sulfate and evaporated in weighed vial.

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Abstract

There is provided herein a composition for boosting the immune system of an animal comprising Dimethyl sulfoxide (DMSO), a vegetable oil, a fatty acid, Chlorine dioxide and a surfactant.

Description

A COPPER BASED COMPOSITION FOR ANIMAL CONSUMPTION
TECHNICAL FIELD
The present invention relates to the field of special feed additives, specifically to the field of supporting and enhancing the immune system in animals.
BACKGROUND
Animals, much like humans, are prone to get infected with diseases. Zoonoses (also known as zoonosis and as zoonotic diseases) are infectious diseases caused by bacteria, viruses and parasites that spread between animals (usually vertebrates). The term "Animal disease" usually relates to an impairment of the normal state of an animal that interrupts or modifies its vital functions.
Concern with diseases that afflict animals dates from the earliest human contacts with animals and is reflected in early views of religion and magic. Diseases of animals remain a concern principally because of the economic losses they cause and the possible transmission of the causative agents to humans.
The branch of medicine called veterinary medicine deals with the study, prevention, and treatment of diseases not only in domesticated animals but also in wild animals and in animals used in scientific research. The prevention, control, and eradication of diseases of economically important animals are agricultural concerns.
The diseases of animals are of increasing importance, for a primary public-health problem throughout the world is animal -protein deficiency in the diet of humans. Indeed, both the United Nations Food and Agricultural Organization (FAO) and the World Health Organization (WHO) have been attempting to solve the problem of protein deficits in a world whose human population is rapidly expanding.
There is a long felt need for a solution for reducing animal disease and morbidity in a safe and effective manner.
SUMMARY
According to some demonstrative embodiments, there is provided herein a composition for boosting the immune system of an animal comprising Dimethyl sulfoxide (DMSO), a vegetable oil, a fatty acid, Chlorine dioxide and a surfactant.
According to some embodiments, the vegetable oil may be sesame oil, the fatty acid may be heptanoic acid and the surfactant may be Tween 80.
According to some embodiments, the composition may further include an immune- boosting formulation including Copper containing nanoparticles and a hydrophobic substance.
According to some embodiments, the hydrophobic substance may be selected from a group containing vegetable oil, vegetable oil with unsaturated connections, vegetable oil with dienic unsaturated connections.
According to some preferred embodiments, the vegetable oil may be sesame oil.
According to some embodiments, the immune-boosting formulation may further include polyalkoxylated butyl ether.
According to some embodiments, the vegetable oil of the immune-boosting formulation may be sesame oil, the fatty acid of the immune-boosting formulation may be heptanoic acid and the surfactant of thee immune-boosting formulation may be Tween 80.
According to some embodiments, the composition may preferably also include polyalkoxylated butyl ether.
According to some embodiments, there is provided herein a use of the composition of the present invention for boosting the immune system of farm animals and poultry.
According to some embodiments, the use of the present invention may include adding the above composition to the drinking water of said animals and poultry.
According to some embodiments, the use of the present invention may include adding the composition to the drinking water according to the following regimen:
Weeks 1-2 - 0.5 liters of said composition to a 1000 liters of drinking water for 3-5 days;
Weeks 3-5 - 1 liter of said composition to a 1000 liters of drinking water for 3-5 days;
Week 6 - 1.5 liters of said composition to a 1000 liters of drinking water for 3-5 days.
BRIEF DESCRIPTION OF FIGURES
Non-limiting examples of embodiments of the invention are described below with reference to figures attached hereto that are listed following this paragraph.
Reference is made to figures 1-4 which illustrates a laboratory analysis of particle size, as measured by a Malvren Zetasizer device.
All tests were conducted in three measurements (same sample was repeated to verify the stability of the particles in a suspension, whilst the following parameters have been measured:
Figure 1 illustrates laboratory analysis results from a sample of 70mg/L of AR-DEO, as measured by a Malvren Zetasizer device in accordance with some demonstrative embodiments.
Figure 2 illustrates laboratory analysis results from a sample of 125 mg/L of AR-DEO, as measured by a Malvren Zetasizer device in accordance with some demonstrative embodiments.
Figure 3 illustrates laboratory analysis results from a sample of AR-DEO + con + vitkanol 1 , as measured by a Malvren Zetasizer device in accordance with some demonstrative embodiments.
Figure 4 illustrates laboratory analysis results from a sample of AR-DEO concentrate, as measured by a Malvren Zetasizer device in accordance with some demonstrative embodiments.
DETAILED DESCRIPTION
According to some demonstrative embodiments there is provided herein a composition for oral consumption by an animal including Dimethyl sulfoxide (DMSO), a vegetable oil, a fatty acid, Chlorine dioxide and a surfactant.
Chlorine dioxide tends to quickly decompose upon exposure to air. According to some demonstrative embodiments, mixing the chlorine dioxide with DMSO preserves the Chlorine dioxide and diminishes the decomposition and/or absorbance of humidity, e.g., upon exposure to air.
According to some demonstrative embodiments, the composition of the present invention allows for the formation of chlorinic acid, e.g., upon ingestion of the composition. According to these embodiments, the chlorinic acid allows for the effective neutralization of harmful bacteria and/or viruses within a living organism.
According to some demonstrative embodiments, the unique combination of components of the composition of the present invention allows for a stable composition, which does not readily decompose upon exposure to the environmental conditions (such as air, humidity, elevated temperature etc.), while exhibiting antibacterial and antiviral properties.
According to some embodiments, the vegetable oil may include sesame oil, cocoa oil, castor oil and the like. According to some embodiments, the vegetable oil may preferably be sesame oil.
According to some embodiments, the fatty acid may be selected from the group including heptanoic acid, lauric acid, myristic acid, palmitic acid, palmitate, palmitoleate, hydroxypalmitate, arachidic acid, oleic acid, stearic acid, behenic acid, sodium stearate, calcium stearate, magnesium stearate, hydroxyoctacosanyl hydroxystearate, oleate esters of short-chain, esters of fatty acids, fatty alcohols, esterified fatty diols, hydroxylated fatty acid, hydrogenated fatty acid, triesters of fatty acids, triglycerides received from fatty acids and fatty acid eutectics.
According to some embodiments, the fatty acid of the composition is preferably a short chain hydrophobic acid such as heptanoic acid.
According to some demonstrative embodiments, the surfactant may preferably be Polysorbate 80 (Tween 80) Polysorbate 20 and/or Sorbitan monooleate (Span 80) and their analogs from Arlacel or BRIJ detergent lines or their mixtures that provide adjustment of predetermined specific HLB values.
According to some embodiments, the composition of the present invention may exhibit various useful characteristics including, for example, where it improves microcirculation in brain tissue and other areas of the body of the animal; improves microcirculation in the tissues of the joints; prevents the development of DIC syndrome, especially its local manifestations.
According to some embodiments, the composition of the present invention may also reduce coagulation time, improving microcirculation in the lesion. Reduces the tendency of red blood cells to aggregate. Normalizes the process of fibrin formation, which improves tissue trophism. Improves the nutritional conditions of the transplanted skin flap by expanding the capillary network, increasing the total number of functioning vessels. It creates the most favorable conditions for the healing of skin flaps and preventing the development of necrosis in them.
In addition, the composition may have a vasodilating effect, has a moderately pronounced hypotensive effect. Reduces peripheral vascular resistance and improves the rheological properties of blood, reduces the adhesion and aggregation of blood cells.
According to some embodiments, the composition may increase the permeability of the walls of venules. It easily penetrates the skin and cell membranes without damaging them. Penetrating through intact skin, it carries with it into the cells and intercellular tissue various medicinal substances.
According to some embodiments, the composition of the present invention may also increase the permeability of the histohematological barriers of the lungs and liver, however, without affecting the permeability of the histohematic barriers of the kidneys. Increases the permeability of the blood-brain and blood-brain barrier. Deposits medicinal substances in tissues for 1-2 weeks. It has a unique ability to conduct (transport) the substances dissolved in it through the skin and mucous membranes without damaging them. It breaks the hydrophobic bonds and lipoprotein complexes of cell membranes, which underlies the increase in their permeability under the influence of the composition. Promotes prolonged deposition of drugs in the tissues.
According to some embodiments, the composition of the present invention may also possess anti-inflammatory activity which may be comparable to non-steroidal anti inflammatory drugs. It has not only local, but also systemic anti-inflammatory effects.
According to some embodiments, the composition of the present invention may have an antiexudative effect, inhibits the activity of proteases, the synthesis of prostaglandins. At the same time, it increases the secretion of cortisol, strengthens the membrane of lysosomes. Reduces cellular infiltration in the focus of inflammation.
According to some embodiments, the composition of the present invention may have a direct antiproteolytic effect, prevents the development of edema and necrosis, inhibits platelet aggregation. It has an antipyretic effect, suppresses exudation processes in the focus of inflammation. The composition of the present invention including at least 33% DMSO may quickly lead to a decrease in soft tissue edema. Stimulates reparative processes in tissues, proliferative processes in the focus of inflammation.
According to some embodiments, the composition of the present invention may also quickly eliminate the phenomena of inflammation of the soft tissues. It accelerates the development of granulation tissue, slowing down the processes of scar formation. Gentle scars form. Promotes resorption of inflammatory infiltrates, including in the stroma of the lungs and pleura. It resolves rough scars, playing the role of a “chemical orderly” in the area of inflammation, a biochemical cleaner.
According to some embodiments, the composition of the present invention may interact with protein breakdown products, forming intermolecular (hydrogen and hydrophobic) bonds with functional groups of polypeptides - OH, NH, SH, NH2. Promotes their removal from the focus of inflammation. The composition of the present invention may inhibit the proliferation of fibroblasts, and in combination with corticosteroids completely suppresses it.
According to some embodiments, the composition of the present invention may inhibit collagen synthesis. Reliably and for a long time reduces the level of prostaglandins PgE b, PgF 2a by suppressing the intensity of the synthesis of the latter. According to some embodiments, the composition of the present invention may also reduce and slow down the development of radiation and drug pneumonitis.
According to some embodiments, the composition of the present invention may not have an allergenic effect. The composition of the present invention may not cause contact sensitization with cutaneous application. On the contrary, it is characterized by desensitizing and antiallergic activity. Inhibits, according to various sources, anaphylactic reactions by 41 - 72%.
According to some embodiments, the antiallergic activity of the composition of the present invention may be proportional to the concentration of the drug used. The basis of antiallergic activity is the ability to suppress B-lymphocytes and plasma cells, the blockade of histaminergic serotonergic receptors, antihistamine action, the ability to inactivate histamine released from mast cells, and deplete the pool of histamine-producing cells.
According to some embodiments, the composition of the present invention may be a long-acting anesthetic with a pronounced local analgesic effect. It has a long analgesic effect. Selectively blocks nerve fibers conducting a pain impulse. It can be considered as a symptomatic analgesic for pain syndromes of various origins.
According to some embodiments, a prolonged blockade of nerve endings and trunks may be achieved by the introduction of the composition of the present invention with an anesthetic, and the final concentration of the solvent should be 30-50%.
According to some embodiments, the composition of the present invention may potentiate the effect of antibiotics.
According to some embodiments, the composition of the present invention may also inhibit bacterial lactamases. The phenomenon of a selective increase in the concentration of antibiotics in the affected tissue with the combined administration of antibiotics with dimexide, their selective accumulation was discovered. The concentration of the antibiotic in the lesion increases by 2.5-3 times; thus, the composition of the present invention may enhance the antibacterial effect of antibiotics.
According to some embodiments, the introduction of antibiotics together with the composition of the present invention may intravenously and/or orally open up new paths in antibiotic therapy and anti-inflammatory therapy.
According to some embodiments, the composition of the present invention may have an anticoagulant effect. For example, after intravenous administration of the drug in the form of a 10-20% solution, the plasma volume decreases, its viscosity, as well as the viscosity of whole blood, increases the coagulation time. Blood coagulation first accelerates, then slows down, changes in general occur towards hypocoagulation. The bleeding time increases slightly. The composition of the present invention may inhibit platelet aggregation. Normalizes the process of fibrin formation, which improves tissue trophism. The probability of thrombosis interferes. Having fibrinolytic activity, normalizes fibrinolysis.
According to some embodiments, the composition of the present invention may have antimutagenic activity both in vitro and in vivo. It has strong antioxidant activity. The composition of the present invention may prevent lipid peroxidation, stabilize cell membranes, including lysosome membranes. It captures free radicals, primarily “OH”, which is believed to be largely the basis of the anti-inflammatory effect of the drug. To achieve this effect, its concentration in the blood from 0.5 to 10 millimoles per liter is sufficient. At the same time, the composition of the present invention may enhance the synthesis of superoxide dismutase (SOD) in cells.
According to some embodiments, the composition of the present invention may also be useful as an antistress. It has anti-stress and sedative effects. Eliminates the reactions of anxiety and fear.
According to some embodiments, the composition of the present invention may have a strong oxidizing agent, and act as an antiseptic itself.
According to some embodiments, the composition of the present invention may have the ability to inhibit spore germination.
According to some embodiments, the composition of the present invention may inhibit the growth of all microorganisms saprophying in the blood of cancer patients. When exposed to the body, the composition of the present invention may block glycolytic metabolic pathways. Reduces the accumulation of lactate in the blood, thereby reducing lactocytosis.
According to some embodiments, the composition of the present invention may quickly relieve swelling of brain tissue and improves cerebral blood flow, normalizes cerebral hemodynamics, and improves the clinical condition of the animal.
According to some embodiments, the composition of the present invention may reduce the heart rate, increase the voltage of the teeth on the ECG. The described effects are dose dependent. If small doses of the drug enhance the activity of the heart, then very large doses can have a toxic effect on the heart, up to a stop. It has a hypotensive effect. Lowers blood pressure. Normalizes the reactivity of the autonomic nervous system.
According to some embodiments, by inhibiting the secretion of the antidiuretic hormone of the pituitary gland, the introduction of the composition of the present invention may be accompanied by a diuretic effect. Increasing diuresis by 50%, acts as an osmotic diuretic. According to some authors, under the influence of the drug, diuresis can increase by an order of magnitude.
According to some embodiments, the composition of the present invention may be a mild immunosuppressant, which has an immunosuppressive effect against B -lymphocytes, reduces the level of immune complexes circulating in the blood, at the same time it stimulates nonspecific reactivity, and increases the body's resistance to adverse effects. Induces interferon synthesis. Does not affect the phagocytic activity of polymorphonuclear leukocytes (PMNL). In general, it increases the functional state of the macrophage system. Increases the cytolytic activity of blood serum. It has a resolving effect in the focus of inflammation. Inhibits macrophage activity in the focus of inflammation.
According to some embodiments, the composition of the present invention may be distributed completely over the cells at a concentration of 10-15 millimoles per liter. It is a cellular protector of the intracellular type. Penetrates (penetrates cell membranes). It has a protective effect on cells (celluloprotective effect).
According to some embodiments, the composition of the present invention may act as a cryoprotectant. Stabilizes lysosome membranes and other cell membranes. Passes through intact cell membranes.
According to some embodiments, in liver microsomes, the composition of the present invention may be oxidized to dimethyl sulfone, which is excreted in urine and feces.
According to some embodiments, the composition of the present invention may have a sedative effect, tranquilizing activity.
According to some embodiments, the composition of the present invention may be used for the treatment of strokes and injuries of the brain and spinal cord, due to the ability to normalize central and systemic hemodynamics. The composition of the present invention may have a pronounced decongestant effect on brain tissue of the animal.
According to some embodiments, the composition of the present invention may be used for treatment of trigeminal neuritis (trigemini tis) is long, from 1 to 6 months.
According to some embodiments, the composition of the present invention may also have an antiviral effect. It may be used to treat shingles as an independent drug, and together with antiviral compounds.
According to some embodiments, the composition of the present invention may be effective in treating animals with chronic inflammatory lung diseases.
According to some embodiments, In the treatment of animals with chronic inflammatory lung diseases, a good effect may be obtained from administration of the composition of the present invention, optionally, with the addition of proteolytic enzymes and antibiotics.
According to some embodiments, the composition of the present invention may also have an antiulcer activity, as it may inhibit the secretory function of the stomach, reducing the secretion of gastric juice. Increasing exocrine liver function by 50% and enhancing bile secretion.
According to some embodiments, the composition of the present invention may be used to treat rheumatoid arthritis. Gout treatment. Treatment of bursitis, arthritis, tenosynovitis. The composition of the present invention may modify the metabolism of connective tissue, in particular collagen. Reduces destructive changes in the joints. It has a basic effect on the course of chronic arthritis.
According to some embodiments, the composition of the present invention may be used for Amyloidosis treatment. The composition of the present invention may dissolve amyloid fibrils. Patients within 3-5 years are prescribed oral administration of a 3-5% DMSO solution in water of 30 ml 3 times a day while taking peppermint preparations.
According to some embodiments, the oral route of administration of the composition of the present invention in the form of a solution may be used to prevent the formation of urinary stones. The composition may be rapidly adsorbed through the mucosa of the gastrointestinal tract.
According to some embodiments, the composition of the present invention may be used in the treatment of interstitial cystitis. The composition of the present invention may be instilled through a catheter into the bladder.
According to some embodiments, the composition of the present invention may be used independently in the treatment of bruises, sprains, hemorrhages, edema, purulent wounds, chronic osteomyelitis, erysipelas, phlebitis, and thrombophlebitis. According to some embodiments, the composition of the present invention may be effective in preventing and correcting septic complications. Especially effective is the treatment of sepsis by intravenous administration of antibiotics together with the composition of the present invention may.
According to some embodiments, the composition of the present invention may be effective in the treatment of purulent wounds. For washing purulent sagging and cavities using a solution of the composition of the present invention may, after which the cavity is drained. Reduces the amount of purulent discharge from the wounds. Stimulates the formation and growth of granulations.
According to some embodiments, the composition of the present invention may be used in the treatment of open fractures, osteomyelitis, contractures, tendons, pains, and the like. According to some demonstrative embodiments, there is provided herein an immuno-boosting formulation to be mixed with the components of the present composition.
According to some embodiments, the immuno-boosting formulation may comprise Copper containing nanoparticles, butyl ether, preferably, polyalkoxylated butyl ether and a hydrophobic substance.
According to some embodiments, the combination of the composition of the present invention together with the immuno-boosting formulation may be referred to herein as "AR- DEO".
According to some embodiments, the combination of the composition of the present invention together with the immuno-boosting formulation may synergistically boost the immune system of the animal.
According to some embodiments, the term "enhancing the immunological system of an infant" (also referred to herein as "boosting the immunological system", "immune-stimulating effect", " immuno-stimulating" or "enhance immuno-stimulation"), may include, but not limited to, shortening of disease and/or outbreak periods, diminishment of likelihood of becoming ill, reduced number and/or severity of symptoms associated with diseases and the like.
According to some demonstrative embodiments, the term "animal" as used herein may refer to any member of the kingdom Animalia, including for example, domesticated livestock and animal husbandry, such as, chickens, cattle, sheep, pigs; marine (oceanic) or freshwater fish and the like.
According to some embodiments, the composition of the present invention may possess bacteriocidic and/or bacteriostatic characteristics. According to some embodiments, the copper containing composition of the present invention as potent nonspecific antiseptic substance is effective against a wide variety of harmful economically important pathogens, including E.coli, Salmonella, Staphylococcus, Clostridium, Mycoplasma species etc.
According to some demonstrative embodiments, the composition of the present invention may be useful to treat or prevent a wide variety of diseases, including for example, bacterial or viral infections.
According to some demonstrative embodiments, the composition of the present invention may be useful to treat or prevent a wide variety of diseases, including for example, Acute hepatopancreatic necrosis disease, African swine fever, Akabane, Anthrax, Australian bat lyssavirus, Avian influenza, Avian paramyxovirus, Blackhead, Bluetongue, Bovine ephemeral fever, Bovine viral diarrhoea virus, Brucellosis, Caprine arthritis encephalitis, Cat- scratch disease, Cattle tick, Coccidiosis, Copper deficiency in ruminants, Cryptococcosis, Enzootic bovine leucosis, Epizootic ulcerative syndrome, Equine herpesvirus type 1 (EHV-1), Equine infectious anemia, Equine influenza, Equine viral arteritis, External parasites in poultry, Foot-and-mouth disease in cattle, Fowl cholera, Fowl pox, Giardiasis, Hendra virus, Hydatid disease, Infectious laryngotracheitis, Intestinal torsion, Japanese encephalitis, Johne's disease, Leptospirosis, Listeriosis, Lumpy jaw, Marek's disease, Melioidosis, Neosporosis, Neurological diseases, Newcastle disease, Nipah virus, Ovine brucellosis, Piglet scours, Porcine sarcoptic mange, Psittacosis, Q fever, Rabies, Ringworm, Salmonella, Spotty liver poultry, Strangles, Swine brucellosis, Swine influenza, Tetanus, Transit tetany, Vibriosis (Campylobacteriosis), Warts on cattle, White-nose syndrome, microchiroptera, Wooden tongue, Worm parasites and the like.
According to some demonstrative embodiments, the unique combination of the composition of the present invention with the immune boosting formulation including Copper containing nanoparticles, butyl ether and a hydrophobic substance allows for the effective impact of the copper on the pathogen organisms such as bacteria, viruses, and other pathogens.
According to some embodiments, a raw material for formation of copper containing nanoparticles of the present invention may include a substance selected from the group: Copper acetate, copper acetate mono hydrate, basic copper carbonate monohydrate, copper chloride dihydrate, copper oxide, copper sulphate pentahydrate, cupric chelate of amino acids, cupric chelate of glycine hydrate.
According to some embodiments, the copper salt is preferably an organic salt of metal with variable valence and ability to dissociate at high temperature, examples of copper salts may include copper bismuth acetate, acetate of cuprum and the like.
According to some embodiments, the copper of the present invention is preferably Cuprum acetate, more preferably, Cuprum acetate monohydrate.
According to some embodiments, the hydrophobic substance may include any suitable fat selected from the group including fats, fatty acids, fatty acid esters, fatty acid triesters, salts of fatty acids, fatty alcohols, phospholipids, solid lipids, waxes, lauric acid, stearic acid, alkenes, waxes, alcohol esters of fatty acids, long chain alcohols and glucoles, and combinations thereof; optionally wherein said salt of fatty acids comprises one or more of aluminum, sodium, potassium and magnesium salts of fatty acids; optionally wherein said fatty coating layer comprises one or more of paraffin wax composed of a chain of alkenes, normal paraffins; natural waxes, synthetic waxes, vegetable oil such as sesame oil, hydrogenated vegetable oil, hydrogenated castor oil; fatty acids, such as lauric acid, myristic acid, palmitic acid, palmitate, palmitoleate, hydroxypalmitate, stearic acid, arachidic acid, oleic acid, stearic acid, sodium stearate, calcium stearate, magnesium stearate, hydroxyoctacosanyl hydroxystearate, oleate esters of long-chain, esters of fatty acids, fatty alcohols, esterified fatty diols, hydroxylated fatty acid, hydrogenated fatty acid (saturated or partially saturated fatty acids), partially hydrogenated soybean, partially hydrogenated cottonseed oil, aliphatic alcohols, phospholipids, lecithin, phosphathydil choline, triesters of fatty acids, coconut oil, hydrogenated coconut oil, cacao butter; palm oil; fatty acid eutectics; mono and diglycerides, poloxamers, block-co-polymers of polyethylene glycol and polyesters, and a combination thereof; optionally wherein said wax comprises one or more of beeswax, carnauba wax, Japan wax, bone wax, paraffin wax, Chinese wax, lanolin (wool wax), shellac wax, spermaceti, bayberry wax, candelilla wax, castor wax, esparto wax, jojoba oil, ouricury wax, rice bran wax, soy wax, ceresin waxes, Montan wax, ozocerite, peat waxes, microcrystalline wax, petroleum jelly, polyethylene waxes, Fischer-Tropsch waxes, chemically modified waxes, substituted amide waxes; polymerized a-olefms, or a combination thereof.
According to some embodiments, the hydrophobic substance may preferably be a vegetable oil, more preferably, a natural vegetable oil with high content of non-saturated fatty acids especially with conjugated bonds (dien bonds) such as linoleic acid including for example: olive oil, sesame oil, linseed oil and the like.
According to some embodiments, the vegetable oil is most preferably sesame oil.
According to some demonstrative embodiments, the immune-boosting formulation may include Copper in a concentration of 0.1 - 4.5% w/v, preferably 0.5 - 3.5% w/v, more preferably 0.7 - 3.5% w/v; and a hydrophobic substance in a concentration of 30- 95% v/v, preferably 40-60% v/v, more preferably, 45-55% v/v.
For example, the immune-boosting formulation may include Copper acetate monohydrate in an amount of 35gr, 250 ml of surfactant, e.g., polyalkoxylated butyl ether and 250 ml of Sesame oil, for the preparation of 500 ml of the composition.
According to some demonstrative embodiments, the composition of the present invention may further include another known immuno-boosting formulation mixed with the components of the present composition.
According to some embodiments, the immuno-boosting formulation may include Dimethyl sulfoxide (DMSO), a vegetable oil, a fatty acid, Chlorine dioxide and a surfactant.
According to some embodiments, the vegetable oil of the immuno-boosting formulation may include sesame oil, cocoa oil, castor oil and the like. According to some embodiments, the vegetable oil of the immuno-boosting formulation is preferably sesame oil.
According to some embodiments, the hydrophobic substance of the immuno-boosting formulation may include any suitable fatty acid selected from the group including heptanoic acid, lauric acid, myristic acid, palmitic acid, palmitate, palmitoleate, hydroxypalmitate, arachidic acid, oleic acid, stearic acid, behenic acid, sodium stearate, calcium stearate, magnesium stearate, hydroxyoctacosanyl hydroxystearate, oleate esters of short-chain, esters of fatty acids, fatty alcohols, esterified fatty diols, hydroxylated fatty acid, hydrogenated fatty acid, triesters of fatty acids, triglycerides received from fatty acids and fatty acid eutectics.
According to some embodiments, the hydrophobic substance of the immuno-boosting formulation is preferably a short chain hydrophobic acid such as heptanoic acid.
According to some demonstrative embodiments, the surfactant of the immuno-boosting formulation is preferably Polysorbate 20, Tween 80 and/or Span 80 and their analogs from Arlacel or BRIJ detergent lines or their mixtures that provide adjustment of predetermined specific HLB values.
According to some embodiments, the composition of the present invention may be mixed with the immune-boosting formulation to provide a final mixture, for example, to be provided to the animals, e.g., in their drinking waters.
According to some embodiments, the composition of the present invention may be mixed with the immune-boosting formulation in equal amounts and/or volumes.
According to some demonstrative embodiments, the composition of the present invention may be referred to herein as solution B, and the immune-boosting formulation may be referred to herein as solution A, and final mixture (solution A and solution B) may be referred to herein as AR-DEO.
According to some demonstrative embodiments, the composition of the present invention may be in a liquid form or a dry powder form.
According to some preferred embodiments, the composition of the present invention is mixed in the drinking water of an animal.
According to some demonstrative embodiments, the final mixture (AR-DEO) may be in a liquid form or a dry powder form.
According to some preferred embodiments, the final mixture disclosed herein may be mixed in the drinking water of an animal.
According to some embodiments, there is provided herein a use of the composition of the present invention for boosting the immune system of an animal.
According to some demonstrative embodiments, the use may include adding the composition of the present invention (solution B) to the drinking water of the animals using generally the following regimen:
Weeks 1-2 - 0.5 liters of solution A to a 1000 liters of drinking water for 3-5 days;
Weeks 3-5 - 1 liter of solution A to a 1000 liters of drinking water for 3-5 days;
Week 6 - 1.5 liters of solution A to a 1000 liters of drinking water for 3-5 days.
According to some demonstrative embodiments, the use may include adding a mixture of the composition of the present invention (solution B) with the immune boosting formulation (Solution A) to the drinking water of the animals using generally the following regimen:
Weeks 1-2 - 0.5 liters of solution B and 0.5 liters of solution A to a 1000 liters of drinking water for 3-5 days;
Weeks 3-5 - 1 liter of solution B and 1 liter of solution A to a 1000 liters of drinking water for 3-5 days;
Week 6 - 1.5 liters of solution B and 1.5 liters of solution A to a 1000 liters of drinking water for 3-5 days.
According to some embodiments, for cattle, swine and fish the desired dosage may be provided using generally the following dosage: 2ml of solution B + 2ml of solution A per 10 Kg body weight of the animal.
Copper containing material in nanoparticle form has two unique features:
Huge surface area provides abnormally high antiseptic effect of nutritionally and regulatory accepted dosage, and
Its insolubility in physiological liquids provides timely removal of copper from the body without excessive accumulation in farm products.
According to some embodiments, one of the benefits of the process of the present invention is the incorporation of the metal atoms ,e.g., the copper atoms, into the chain of organic molecules. At the same time, the second process takes place. It is partial polymerization of oxidized fatty acids. As a result, we have a mixture of nanoparticles with specific dimensions. According to some embodiments, the nanoparticles including shielded atoms of heavy metals may provide fast, effective, and safe action of the composition of the present invention.
Reference is made to figures 1-4 which illustrate a laboratory analysis of particle size, as measured by a Malvren Zetasizer device.
All tests were conducted in three measurements (same sample was repeated to verify the stability of the particles in a suspension, whilst the following parameters have been measured:
Figure imgf000017_0001
Figure 1 illustrates the results from a sample of 70mg/L of AR-DEO Figure 2 illustrates the results from a sample of 125 mg/L of AR-DEO Figure 3 illustrates the results from a sample of AR-DEO + con + vitkanol 1 Figure 4 illustrates the results from a sample of AR-DEO concentrate
Example 1 - preparation of solution A
Reactant, reagents, and materials used in reaction:
Figure imgf000017_0002
Figure imgf000018_0001
Example 2: An exemplary preparation of solution A and solution B:
Solution A
Sesame Oil 200ml Copper Acetate 10 gr Ethyl an NS 500 LQ - 200ml Solution B Sesame oil 100 ml Heptanoic acid 100ml Polysorbate 20 - 200 ml Dimethyl sulfoxide - 5ml Chlorine dioxide - 1 gr
Example 3 - Directions of use for AR-DEO
I. General
1. AR-DEO is a copper feed additive for enrichment and balancing of nursing and weaned pigs’ diets.
2. AR-DEO contains the active ingredient - an organic compound of copper, based on naturally occurring oils (component "A") and auxiliary substances (component "B") - carboxylic acid, emulsifier, nonionic surfactant, sodium chloride. Concentrate of the "A" and "B" mixture components in a ratio of 1: 1 contains at least 12.5 grams of an organic copper compound in 1 liter.
3. Components "A" and "B" are available in separate packages in the form of 100% concentrate. In appearance, they constitute opaque oily liquid from light brown to dark brown color and have a characteristic odor. They are packed in 1,5,10 - liter plastic bottles and cans, sealed with tamper-evident cup. Each package should be marked listing the manufacturer, its address and trademark, the name of the drug, its specifications, its purpose and method of application, its name and the content of the active substances, the shipping measurement, the date of manufacture, the shelf life, the storage conditions, the precautions and provide it with direction for use. 4. The product is to be stored in original unopened packaging in covered ventilated storage rooms, protected from light, out of direct sunlight and away from heat sources, in a dry place inaccessible to unauthorized persons or animals, at a temperature from + 3 °C to + 35 °C. The shelf life is 3 years from the manufacturing date.
5. It does not contain hormones, genetically modified products.
6. Content of harmful impurities does not exceed the maximum allowable limits that are in force.
II. Biological properties
1. The feed additive AR-DEO is a source of copper to meet the nursing and weaned pigs’ needs.
2. An organic copper compound based on natural oils in combination with auxiliary substances has higher bioavailability compared with inorganic compounds. In the animal organism copper is involved in skeletal, immune system, reproductive function formation, and helps to increase growth rate and feed efficiency of animals and improve their security.
3. This additive in the doses recommended for use is bioavailable, harmless and areactogenic. According to the toxic effect, it belongs to the substance hazard category 4. It does not accumulate in tissues and organs. Within 72 hours, it is eliminated from the body.
4. This additive is active in a wide temperature range in the water of any hardness, it is resistant to pH fluctuations.
II. Application procedure
1. AR-DEO is a copper feed additive for enrichment and balancing of nursing and weaned pigs’ diets and it is intended for internal use by drinking with water or feeding with food. Introduction to animal feed and premixes of the feed and premix plants is not required.
2. The daily dosage for nursing and weaned pigs is a mixture of 1 ml of the components "B" and "A" concentrate mixture in the ratio of 1: 1 for 1 kg of live weight. It is given in the drinking water or in the feed in the form of oil in water emulsion with 5.1% concentration.
3. Before using the "B" and "A" components are shaken in their original packaging to an even consistency and are used for preparation of a master oil in water emulsion. The master emulsion is prepared in a plastic container, convenient for mixing the components by active shaking. The container is filled with 10 parts of potable water, heated to 60-70 °C, and 1 part "B" component is added. After 3 minutes of shaking, the component "A" in an amount equal to the amount of component "B" is added until a white emulsion, and the master emulsion is further stirred by shaking for the same time until homogenous state. The result is a 20% emulsion, which is used to prepare working concentrations of 5.1% oil in water emulsions. The emulsion remains active for 7 days. Working emulsion remains active for 3 days.
4. Nursing pigs 1-10 days of age drink daily dose in two divided doses in the morning and in the evening for 5 consecutive days in the form of an emulsion of 1% concentration by individual forced drinking. The course is repeated starting with the 1st day after weaning by drinking with water in the form of an oil in water emulsion concentration of 1% by medicator of Dosatron type. While medicator operation a mixing of the master emulsion by shaking 1 time every 30 minutes is required.
5. Nursing pigs older than 10 days, drink water, or are fed with dry or wet food for 5 consecutive days. When drinking a daily dose is given in the form of oil in water emulsion of 1% concentration. When feeding a daily dose in the form of an emulsion of 5% concentration is mixed with the daily amount of feed. The course is repeated starting with the 1st day after weaning by drinking a water in the form of an emulsion of 1% concentration using medicator.
6. Weaned piglets drink it with water, or by eating with dry or wet feed for 5 consecutive days. When drinking a water, the daily dose in the form of oil in water emulsion of 1% concentration is fed using medicator. When feeding with the feed the daily dose in the form of oil in water emulsion of 5% concentration is mixed with the daily amount of feed. The course is repeated in both cases after 3 weeks.
7. The product is compatible with other feed additives and drugs provided the interval between the application should be at least 3-4 hours.
8. Side effects and complications of the drug are not registered, contraindications are not established.
9. When side effects and complications the use of the drug must be canceled.
10. Slaughtering of animals and poultry for meat is permitted not earlier than 96 hours after discontinuation of the drug. In the case of compelled slaughtering the meat is used for animal feed.
Example 4 - Determination of Cu (water soluble and oil immobilized).
1. Pretreatment of sample for total Cu.
1.1 Sample digestion
Weigh 0.5g of sample in a Teflon microwave digestion vessel. Add 9ml concentrated Nitric Acid and 1ml of 30% H2O2 solution.
Method of digestion a. Heat sample for temperature 200°C within 15 min. b. Hold temperature at 200°C for 15 min. . c. Cool to room temp., transfer vessel content to a 50 ml volumetric flask. Wash vessel 3-5 times with demineralized water (3-5 ml for each time). d. Fill up to the mark with DI water. Mix well.
1.2 Preparation of solutions for analysis
1.2.1. Digestion: a. Transfer digestion vessel contents to a 50 ml volumetric flask. b. Wash vessel 3-5 times with demineralized water (3-5 ml for each time). c. Fill up to the mark with DI water. Mix well.
1.2.2 Preparation of solution 1:100 dilution
Transfer 1 ml of digested solution (1.2.1.c) into a 100ml volumetric flask, add demineralized water to the mark. Mix well.
1.2.2 Preparation of solution 1:2,000 dilution
Transfer 5ml of solution (see 1.2.2) to a 100ml volumetric flask. Add demineralized water to the mark. Mix well.
2. Pretreatment of sample to water soluble Cu in the sample
2.1 Isolation of water-soluble Cu
To a 50ml conical flask transfer 15ml of sample. Add 20 ml water and 0.5 g of NaiSCri ( anhydrous). Mix well and separate water (lower) layer by separation funnel.
Add to water layer 10-15 ml of hexane and separate water (lower) layer with a separation funnel.
2.2. Digestion of water-soluble Cu sample
Put 10ml of water layer (see p2.1) in 20ml volumetric flask, add 2ml concentrated nitric acid, and place on the heat plate (t=95°C in the solution) Evaporate to approx. 2 ml, add 2 ml concentrated hydrochloric acid, and heat to elimination brown gas, and evaporate to approx. 1.5-2 ml. Remove from heat and cool. Replace in 10ml volumetric flask and add 2% nitric acid to the mark.
2.3 Preparation of diluted solution for analysis
2.3.1 Preparation of solution 1:100 dilution
1 ml Solution after digestion (see p 2.2) put in to the 100ml volumetric flask, add demineralized water to the mark. Mix well.
2.3.2 Preparation of solution 1:2000 dilution
5ml of solution 1:100 dilution (see p 2.3.1) add to 100ml volumetric flask. Add demineralized water to the mark. Mix well.
3. Measurement condition Wavelength- 324.754 nm (recommended)
Calibration - 0.0mg/1- 50mg/l (recommended)
Plasma power- 1400W
Gas- Argon HP (99.985%)
Coolant Flow -14.50 1/min (recommended)
Auxiliary Flow - 1.001/min (recommended)
Nebulizer Flow- 0.801/min (recommended)
3.1 Calibration curve requirement Concentration range- Up to 40mg/l (recommended)
Number of calibration points- usually blank + 6 points.
R2 not less than 0.999
4. Results calculation
4.1 Calculation of total Cu concentration
4.1.1 For solution 1:100 dilution (see. 2.3.1)
Figure imgf000022_0001
Where Cudet - measured Cu concentration Ml - mass of sample for analysis (see p1.1.1)
4.1.2 For solution 1:2,000 (see p1.2.2)
Figure imgf000022_0002
Where Cudet- measured Cu concentration Ml - mass of sample for analysis (see p1.1.1)
5.1 Calculation of water-soluble Cu concentration
5.1.1 For solution 1:100 dilution (see. p 2.3.1)
Figure imgf000022_0003
Where Cudet- detected Cu concentration M2- mass of sample for analysis (see p2.1)
5.1.2 For solution 1:2,000 (see p2.3.2)
Figure imgf000023_0001
Where Cudet- detected Cu concentration
M2- mass of sample for analysis (see p2.1)
Note:
In all case of calculation only Cudet in the calibration curve interval can be used for calculation.
5.2 Calculation of organic Cu concentration
Calculated according to equation:
Cu (oil) = Cu(tot) — Cu(W)
Where:
Cu(tot)- Total Cu concentration (see p 4.1)
Cu(W)- water soluble Cu concentration (see p 5.1)
Example 5 - Determination of % Linoleic Acid in Fatty Acid profile
1. Analysis principle
Free Fatty Acids (FFA) and triacylglycerides (TAG) transform in fatty acid methyl esters (FAME) by esterification (for FFA) and transesterification (for TAG). The FAMEs are isolated from the reaction mixture by extraction and analyzed by GC with FID detection.
2. Used analytes and marker.
2.1- Fatty Acid Methyl Esters Standard solution contains Oleic Acid Methyl Ester (C18: 1 FAME) and Linoleic Acid Methyl Ester(C18:2 FAME)) with concentrations more than 1%
Sigma: Cat. No. CRM4780
2.2 Methanol dry (for analysis, for GC)
2.4 Hexane (for analysis, for GC)
2.5 Sulfuric acid (for analysis)
2.6 Sodium sulfate anhydride (for analysis)
2.7 Demineralized water (conductivity less than 0.08 mS).
3. Acid transesterification solution preparation
In a 50 ml conical flask put 40ml of dry Methanol. Cool by ice bath and add 10 ml of sulfuric acid by drops with gentle mixing. Maintain a maximum temperature of not more than 50°C. Close the flask.
The solution can be stored in dry place not more than 24h 4. Transesterification reaction
Weigh 0.2 ml of sample oil in a 20ml vial add 2 ml of acid transesterification solution (see #3) and close by screw cap . Place on water bath (t=80°C) for 1-2 h with occasional ( every 5-7min) shaking.
Remove reaction vial from water bath, cool to room temperature (no water!), open reaction vial, add 8ml of demineralized water close the vial and mix well. Reopen the vial, add 5ml of hexane, reclosed the vial and shake for 1 min.
Open the vial and transfer the organic (upper) layer to a 2 ml vial.
Add 0.1g of sodium sulfate anhydride. Close the vial, mix wall.
Filter the dried organic layer by 0.45μm Nylon filter into a 250μl glass insert vial, and place in a 2ml GC vial and immediately closed by screw cap with septum. Sample solution is ready for GC analysis.
5. Preparation of analytical external standard solution of FAMEs
Place 1.5 ml of hexane in a 2ml GC, add 0.1ml of accurately weighed Fatty Acid Methyl Esters Standard solution, close by screw cap with septa and mix well for 1 min.
The solution is ready for use.
6. Requirements for GC analytical system
Column- DB-FFAP, L=30m, D=0.320mm, Film=0.25μm
Injector temp=250°C Split ratio=5
Flow- 1.7 ml/min, Carrier gas- Helium
Column Oven temperature- 120°C (hold 5min)/ rate 4°C/min to 240°C/ hold lOmin
Detector- Temperature 240°C, Flow: Make up flow (helium)-30ml/min Hydrogen flow- 35ml/min, air flow-300ml/min.
Injection volume- 1 μl
7. Sample of sequence
1. Solvent blank (Hexane)
2. Sample#1 (prepared according to #4)
3. Sample#2* (prepared according to #4)
4. Sample#3* (prepared according to #4)
5 -
6. Sample #N* (prepared according to #4)
7. External standard solution of FAMES (prepared according to #5)
*- If more than 1 sample of oil analyzed 8. Calculation of results
8.1 Determination of retention times of FAMEs
See Appendix 1 for times observed.
8.2 Calculation of Linoleic Acid (%) in Fatty Acid profile
Calculate the Linoleic Acid in Fatty Acid content:
Where:
Figure imgf000025_0001
%LA- % Linoleic Acid in Faty Acid profile
Area Linoleic Acid FAME- detected area of peak corresponded Oleic Acid FAME Σ All peaks- summary area of detected peaks (excluding peaks detected for Solvent blank sample (see #7).
8.3 Calculation of Linoleic Acid concentration in the sample
Calculate the Linoleic Acid content:
Figure imgf000025_0002
Where:
Cone (OA) - concentration of Oleic Acid in the sample Area Linoleic Acid FAME - area of Oleic Acid FAME peak of sample C st - concentration of Linoleic Acid FAME in the standard M st - mass of standard for preparation external standard solution Area Linoleic Acid st - area of peak corresponded Oleic Acid FAME for external standard solution.
M sample- mass of sample used for analysis (see #4)
9. Acceptance criterion
No significant peaks in RT corresponded to Linoleic Acid FAME ±0.2 min. for Hexane (Solvent blank) injection. Improve solvent quality if the Solvent blank injection has peaks within 0.2min. of the analyte peaks.
10. Appendix 1
Retention Times guide: may vary on other equipment.
Figure imgf000025_0003
Figure imgf000026_0001
Example 6 - Determination of % Srearic in Fatty Acid profile
1. Analysis principle
Free Fatty Acids (FFA) and triacylglycerides (TAG) transform in fatty acid methyl esters (FAME) by esterification (for FFA) and transesterification (for TAG). The FAMEs are isolated from the reaction mixture by extraction and analyzed by GC with FID detection.
2. Used analytes and marker.
2.1- Fatty Acid Methyl Esters Standard solution contains Stearic Acid Methyl Ester with concentrations more than 1% Sigma: Cat. No. CRM4780
2.2 Methanol dry (for analysis, for GC)
2.4 Hexane (for analysis, for GC)
2.5 Sulfuric acid (for analysis)
2.6 Sodium sulfate anhydride (for analysis) 2.7 Demineralized water (conductivity less than 0.08 mS).
3. Acid transesterification solution preparation
In a 50 ml conical flask put 40ml of dry Methanol. Cool by ice bath and add 10 ml of sulfuric acid by drops with gentle mixing. Maintain a maximum temperature of not more than 50°C. Close the flask. The solution can be stored in dry place not more than 24h
4. Transesterification reaction
Weigh 0.2 ml of sample oil in a 20ml vial add 2 ml of acid transesterification solution (see #3) and close by screw cap . Place on water bath (t=80°C) for 1-2 h with occasional ( every 5-7min) shaking. Remove reaction vial from water bath, cool to room temperature (no water!), open reaction vial, add 8ml of demineralized water close the vial and mix well. Reopen the vial, add 5ml of hexane, reclosed the vial and shake for 1 min.
Open the vial and transfer the organic (upper) layer to a 2 ml vial.
Add 0.1g of sodium sulfate anhydride. Close the vial, mix wall.
Filter the dried organic layer by 0.45μm Nylon filter into a 250μl glass insert vial, and place in a 2ml GC vial and immediately closed by screw cap with septum. Sample solution is ready for GC analysis.
5. Preparation of analytical external standard solution of FAMEs
Place 1.5 ml of hexane in a 2ml GC, add 0.1ml of accurately weighed Fatty Acid Methyl Esters Standard solution, close by screw cap with septa and mix well for 1 min.
The solution is ready for use.
6. Requirements for GC analytical system Column- DB-FFAP, L=30m, D=0.320mm, Film=0.25μm Injector temp=250°C Split ratio=5
Flow- 1.7 ml/min, Carrier gas- Helium
Column Oven temperature- 120°C (hold 5min)/ rate 4°C/min to 240°C/ hold 10min Detector- Temperature 240°C, Flow: Make up flow (helium)-30ml/min Hydrogen flow- 35ml/min, air flow-300ml/min.
Injection volume- 1 μl
7. Sample of sequence
1. Solvent blank (Hexane)
2. Sample#1 (prepared according to #4)
3. Sample#2* (prepared according to #4)
4. Sample#3* (prepared according to #4)
5 -
6. Sample #N* (prepared according to #4)
7. External standard solution of FAMES (prepared according to #5)
*- If more than 1 sample of oil analyzed
8. Calculation of results
8.1 Determination of retention times of FAMEs
See Appendix 2 for times observed.
8.2 Calculation of Stearic Acid (%) in Fatty Acid profile
Calculate the Srearic Acid in Fatty Acid content:
Figure imgf000028_0001
Where:
%SA- % Srearic Acid in Fatty Acid profile
Area Srearic Acid FAME- detected area of peak corresponded Srearic Acid FAME Σ All peaks- summary area of detected peaks (excluding peaks detected for Solvent blank sample (see #7).
8.3 Calculation of Stearic Acid concentration in the sample
Calculate the Stearic Acid content:
Figure imgf000028_0002
Where:
Cone (SA) - concentration of Stearic Acid in the sample Area Stearic Acid FAME - area of Stearic Acid FAME peak of sample C st - concentration of Stearic Acid FAME in the standard M st - mass of standard for preparation external standard solution Area Srearic Acid st - area of peak corresponded Srearic Acid FAME for external standard solution
M sample- mass of sample used for analysis (see #4)
9. Acceptance criterion
No significant peaks in RT corresponded to Stearic Acid FAME ±0.2 min. for Hexane (Solvent blank) injection. Improve solvent quality if the Solvent blank injection has peaks within 0.2min. of the analyte peaks.
10. Appendix 2
Retention Times guide: may vary on other equipment.
Figure imgf000028_0003
Figure imgf000029_0001
Example 7 - Determination of % Palmitic in Fatty Acid profile
1. Analysis principle
Free Fatty Acids (FFA) and triacylglycerides (TAG) transform in fatty acid methyl esters (FAME) by esterification (for FFA) and transesterification (for TAG). The FAMEs are isolated from the reaction mixture by extraction and analyzed by GC with FID detection.
2. Used analytes and marker.
2.1- Fatty Acid Methyl Esters Standard solution contains Palmitic Acid Methyl Ester with concentrations more than 1%
Sigma: Cat. No. CRM4780
2.2 Methanol dry (for analysis, for GC)
2.4 Hexane (for analysis, for GC)
2.5 Sulfuric acid (for analysis)
2.6 Sodium sulfate anhydride (for analysis)
2.7 Demineralized water (conductivity less than 0.08 mS).
3. Acid transesterification solution preparation
In a 50 ml conical flask put 40ml of dry Methanol. Cool by ice bath and add 10 ml of sulfuric acid by drops with gentle mixing. Maintain a maximum temperature of not more than 50°C. Close the flask.
The solution can be stored in dry place not more than 24h
4. Transesterification reaction
Weigh 0.2 ml of sample oil in a 20ml vial add 2 ml of acid transesterification solution (see #3) and close by screw cap . Place on water bath (t=80°C) for 1-2 h with occasional ( every 5-7min) shaking.
Remove reaction vial from water bath, cool to room temperature (no water!), open reaction vial, add 8ml of demineralized water close the vial and mix well. Reopen the vial, add 5ml of hexane, reclosed the vial and shake for 1 min.
Open the vial and transfer the organic (upper) layer to a 2 ml vial.
Add 0.1g of sodium sulfate anhydride. Close the vial, mix wall.
Filter the dried organic layer by 0.45μm Nylon filter into a 250pl glass insert vial, and place in a 2ml GC vial and immediately closed by screw cap with septum. Sample solution is ready for GC analysis.
5. Preparation of analytical external standard solution of FAMEs
Place 1.5 ml of hexane in a 2ml GC, add 0.1ml of accurately weighed Fatty Acid Methyl Esters Standard solution, close by screw cap with septa and mix well for 1 min.
The solution is ready for use.
6. Requirements for GC analytical system Column- DB-FFAP, L=30m, D=0.320mm, Film=0.25μm Injector temp=250°C Split ratio=5
Flow- 1.7 ml/min, Carrier gas- Helium
Column Oven temperature- 120°C (hold 5min)/ rate 4°C/min to 240°C/ hold lOmin Detector- Temperature 240°C, Flow: Make up flow (helium)-30ml/min Hydrogen flow- 35ml/min, air flow-300ml/min.
Injection volume- 1 μl
7. Sample of sequence
1. Solvent blank (Hexane)
2. Sample#1 (prepared according to #4)
3. Sample#2* (prepared according to #4)
4. Sample#3* (prepared according to #4)
5 -
6. Sample #N* (prepared according to #4)
7. External standard solution of FAMES (prepared according to #5)
*- If more than 1 sample of oil analyzed
8. Calculation of results
8.1 Determination of retention times of FAMEs
See Appendix 3 for times observed.
8.2 Calculation of Palmitic Acid (%) in Fatty Acid profile
Calculate the Palmitic Acid in Fatty Acid content:
Figure imgf000030_0001
Where
%PA- % Palmitic Acid in Fatty Acid profile
Area Palmitic Acid FAME- detected area of peak corresponded Palmitic Acid FAME Σ All peaks- summary area of detected peaks (excluding peaks detected for Solvent blank sample (see #7). 8.3 Calculation of Palmitic Acid concentration in the sample
Calculate the Palmitic Acid content:
Figure imgf000031_0001
Where Cone (PA) - concentration of Palmitic Acid in the sample
Area Palmitic Acid FAME - area of Palmitic Acid FAME peak of sample C st - concentration of Palmitic Acid FAME in the standard M st - mass of standard for preparation external standard solution Area Palmitic Acid st - area of peak corresponded Palmitic Acid FAME for external standard solution
M sample- mass of sample used for analysis (see #4)
9. Acceptance criterion
No significant peaks in RT corresponded to Palmitic Acid FAME ±0.2 min. for Hexane (Solvent blank) injection. Improve solvent quality if the Solvent blank injection has peaks within 0.2min. of the analyte peaks.
10. Appendix 3
Retention Times guide: may vary on other equipment
Figure imgf000031_0002
Example 8 - Determination of % Arachidonic in Fatty Acid profile 1. Analysis principle
Free Fatty Acids (FFA) and triacylglycerides (TAG) transform in fatty acid methyl esters (FAME) by esterification (for FFA) and transesterification (for TAG). The FAMEs are isolated from the reaction mixture by extraction, and analyzed by GC with FID detection.
2. Used analytes and marker.
2.1- Fatty Acid Methyl Esters Standard solution contains Arachidonic Acid Methyl Ester with concentrations more than 1%
Sigma: Cat. No. CRM4780
2.2 Methanol dry (for analysis, for GC)
2.4 Hexane (for analysis, for GC)
2.5 Sulfuric acid (for analysis)
2.6 Sodium sulfate anhydride (for analysis)
2.7 Demineralized water (conductivity less than 0.08 mS).
3. Acid transesterification solution preparation
In a 50 ml conical flask put 40ml of dry Methanol. Cool by ice bath, and add 10 ml of sulfuric acid by drops with gentle mixing. Maintain a maximum temperature of not more than 50°C. Close the flask.
The solution can be stored in dry place not more than 24h
4. Transesterification reaction
Weigh 0.2 ml of sample oil in a 20ml vial add 2 ml of acid transesterification solution (see #3) and close by screw cap . Place on water bath (t=80°C) for 1-2 h with occasional ( every 5-7min) shaking.
Remove reaction vial from water bath, cool to room temperature (no water!), open reaction vial, add 8ml of demineralized water close the vial and mix well. Reopen the vial, add 5ml of hexane, reclosed the vial and shake for 1 min.
Open the vial and transfer the organic (upper) layer to a 2 ml vial.
Add 0.1g of sodium sulfate anhydride. Close the vial, mix wall.
Filter the dried organic layer by 0.45μm Nylon filter into a 250pl glass insert vial, and place in a 2ml GC vial and immediately closed by screw cap with septum. Sample solution is ready for GC analysis
5. Preparation of analytical external standard solution of FAMEs
Place 1.5 ml of hexane in a 2ml GC, add 0.1ml of accurately weighed Fatty Acid Methyl Esters Standard solution, close by screw cap with septa and mix well for 1 min.
The solution is ready for use.
6. Requirements for GC analytical system
Column- DB-FFAP, L=30m, D=0.320mm, Film=0.25μm Injector temp=250°C Split ratio=5 Flow- 1.7 ml/min, Carrier gas- Helium
Column Oven temperature- 120°C (hold 5min)/ rate 4°C/min to 240°C/ hold lOmin Detector- Temperature 240°C, Flow: Make up flow (helium)-30ml/min Hydrogen flow- 35ml/min, air flow-300ml/min.
Injection volume- 1 μl
7. Sample of sequence
10. Solvent blank (Hexane)
11. Sample# 1 (prepared according to #4)
12. Sample#2* (prepared according to #4)
13. Sample#3 * (prepared according to #4)
14. -
15. Sample #N* (prepared according to #4)
16. External standard solution of FAMES (prepared according to #5)
*- If more than 1 sample of oil analyzed
8. Calculation of results
8.1 Determination of retention times of FAMEs
See Appendix 4 for times observed.
8.2 Calculation of Arachidonic Acid (%) in Fatty Acid profile
Calculate the Arachidonic Acid in Fatty Acid content:
Figure imgf000033_0001
Where
%AA- % Arachidonic Acid in Fatty Acid profile
Area Arachidonic Acid FAME- detected area of peak corresponded Arachidonic Acid
FAME Σ All peaks- summary area of detected peaks (excluding peaks detected for Solvent blank sample (see #7).
8.3 Calculation of Arachidonic Acid concentration in the sample
Calculate the Arachidonic Acid content:
Figure imgf000033_0002
Where Cone (AA) - concentration of Arachidonic Acid in the sample Area Arachidonic Acid FAME - area of Arachidonic Acid FAME peak of sample C st - concentration of Arachidonic Acid FAME in the standard M st - mass of standard for preparation external standard solution Area Arachidonic Acid st - area of peak corresponded Arachidonic Acid FAME for external standard solution
M sample- mass of sample used for analysis (see #4)
9. Acceptance criterion
No significant peaks in RT corresponded to Arachidonic Acid FAME ±0.2 min. for Hexane (Solvent blank) injection. Improve solvent quality if the Solvent blank injection has peaks within 0.2min. of the analyte peaks.
10. Appendix 4
Retention Times guide: may vary on other equipment
Figure imgf000034_0001
Example 9 - Analyses of natural oils, fats and lipids.
A. Acid Value (Number) determination 1. Reagents used:
1.1 Solvent for sample preparation: Into a 500ml volumetric flask transfer 125ml of dry Chloroform and make up to the mark with Methanol. Stir well. 1.2 Solution for titration (Factored 0.1N solution of methanolic KOH or NaOH.
Put in 1000ml volumetric flask approx 800ml of Methanol. Add 5.6g of KOH or 4.0 g NaOH and stir to complete dissolution (overnight), add Methanol to the mark and stir well. 2. Sample preparation.
Place 5 g of the sample in a dry glass beaker. Add 25ml of Solvent (1.1).
3. Titration
Titrate sample and 2 blank solutions (25ml of solvent for sample) with titrant. If more than one endpoint is detected choose the one closest to pH=8.2.
3. Calculation
Figure imgf000035_0001
B. Saponification Number
1. Reagents
1.1 0.5 N ethanolic Potassium Hydroxide Solution
Put in 1000ml volumetric flask 30g Potassium Hyroxide, add 20ml of Water and up to mark by 95% Ethanol. Stand for 24h, and filter.
1.2 0.5N Hydrochloric Acid (factored).
2. Sample pretreatment
Weight approx 2g of oil into 250ml conical flask Add 25 ml of ethanolic Potassium Hydroxide Solution.
Attach a reflux condenser and heat (not less than 100oC during lh with stirring). Titrate hot solution with 0.5N Hydrochloric Acid.
Do same above procedure for blank sample (without oil)
3. Calculation
Figure imgf000035_0002
C. Iodine Number (Value)
1. Reagents
1.1 Wijs Solution 0.1M (Merck cat #1091631000 or equiv).
1.2 Solvent for analysis- Cyclohexane + Glacial Acetic Acid (1/1 V)
1.3 Potassium Iodide Solution. Dissolve 100g of Potassium Iodide in 1000ml water. Store in a brown-glass bottle 1.4 Sodium Thiosulphate solution. 24.8g of Sodium Thiosulphate dissolve in 500- 700ml of water, add 2-3 drops of Chloroform. Add water to 1000ml. Wait overnight and filter solution and store in a brown bottle.
2. Sample treatment
2.1 Weigh 0.2-0.3g of oil. Add 20ml of Solvent and 20ml of Wijs solution. Close the beaker. Leave for reaction for 2h in dark place. Add 20ml of Potassium Iodide solution and 100- 150ml of water.
2.2 For blank, prepare a solution in the same way but without oil.
2.3 Titration with Sodium Thiosulphate solution with good stirring NOTE-According to reaction, for titration of Blank theoretically need about 40ml of solution.
3. Calculation of result
Figure imgf000036_0001
D. Fatty acids profile determination
1. Establish retention times for each component:
1.1. Reagents
1.1.1. Standard solution of FAMEs (Fatty acids methyl esters) contain C14, C16.0, C16.1, C18.0, C18.1, C18.2, C20.0 FAMEs with concentration enough for FID detection (not less than 1% )
1.1.1. Solvent (Hexane)
1. 2. GC condition and equipment Requirements.
GC column- not polar, L- not less than 30m.
T inj - 250°C T det - 250°C
Column oven temp (t =80oC, hold 2min, to 250°C at 10°C/min, hold 3 min.
Note: Column oven can be corrected for the best separation of C18.0, C18.1 and C18.2 FAMEs.
2. Transesterification process.
Trans-esterification process can be performed with alkaline or acid catalysis.
2.1 Alkaline catalyzed transesterification of oils (used if % free fatty acids less then
1.5% )
2.1.1 Reagents 2.1.1.1 Alkaline solution 2g of KOH put in approx 80ml of Methanol in 100ml Volumetric Flask. After dissolution up by Methanol to the mark.
2.1.1.2 Hexane (solvent).
2.1.2 Alkaline transesterification process.
Put approx 0.2ml of Oil in 20ml Volumetric Flask< add 10ml of Alcaline solution and mix well during 20-25 min. Add approx 10 ml of deminiralized water, mix, add 2 ml of hexane, mix, and still to phase separation. Replace organic (upper) phase in 2ml vial, replace residual water by sodium sulfate anhydride, and put filter dried sample for vial for analysis.
2.2 Acid transesterification (used if free fatty acids content more than 5%).
2.2.1 Used analytes
To prepare acid solution: 40 ml of Methanol put in in 50 ml conical flask, put in ice water, add 10ml of sulfuric acid (by drops).
2.2.2 Acid trans-esterification process
Put 0.2 ml of oil in 40-45ml vials and add 2 ml of acid solution. Put in water bath (t=80C) for 1-2 h with periodic shaking. Transfer to a 10 ml vial, adding approx 8ml water and extract with 2ml of hexane. Replace organic (upper) phase in 2ml vial, replace residual water by sodium sulfate anhydride, and put filter dried sample for vial for analysis.
E. Peroxide Value
1. Reagents
1.1 Sodium thiosulfate solution 0.01 or 0.00 IN
Prepared from 0.1N solution by dilution
Preparation 0.1N solution of thiosulfate: 15.8g of thiosulfate dissolve in 1L water. Store in dark bottle.
1.2 Solvent for analysis
Into a 1L volumetric flask add 400 ml Chloroform and add to the mark with Glacial Acetic Acid.
1.3 Potassium iodide solution
The solution should be freshly prepared before using. Mix 1.0 g potassium iodine and
1.3 ml distilled water.
2. Titration
Weigh 1 g or more of the sample (exactly on 0,0001 g) in an Erlenmeyer volumetric flask. Add 30 mL of the solvent mixture to dissolve the sample. Add 0,5 mL of the potassium iodine solution, close the Erlenmeyer flask with a stopper and stir well for 60 sec. Add 30 mL of distilled water, place the electrode and titration tip in the sample and start the method (stir very well). Carry out a blank titration without the sample in the same matter before. Calculation (in miliequvqlent O per Kg)
Figure imgf000038_0001
Where N-Normality of Na2S203
F. Alkaline impurities
In a test-tube mix 10 ml of neutralized acetone and 0.3 ml of water and add 0.05 ml of a 0.4 g/1 solution of bromophenol blue R in alcohol R. Neutralise the solution if necessary with 0.01 M sodium hydroxide. Add 10 ml of the oil to be examined, shake and allow to stand. Not more than 0.1 ml of 0.01 M hydrochloric acid is required to change the color of the upper layer
G. Unsaponifiable matter
1. Reagents
1.1 Ethanol
1.2 50% (m/w) solution of NaOH in water
1.3 10% (v/v) solution of Ethanol in water
1.4 Hexane
1.5 Sodium Sulfate Anhydrous.
2 Saponification process
5g of sample add to 75ml of Ethanol, add 50ml 50% solution of NaOH. Mix< heat to 60°C-80°C with stirring during 1.5-2h. and transferred to the separatory funnel. Reaction weasel wash by 100ml of Ethanol, 50 ml of cold water, 50 ml of hot water and 15ml of Hexane in separatory funnel. Unsaponificated matter extracted 5 times with 100ml of hexane.
3. Analysis
Integrated hexane layer washed 3 times by 50ml of 10% Ethanol solution, dried by Sodium Sulfate and evaporated in weighed vial.
Use the same process for blanks but without oil.
4. Calculation:
Figure imgf000039_0001
H. Active Chlorine in oil
I. Reagents and analytes
1.1 HNO3:H2O solution (1:1)
1.2 Potassium iodide (KI)
1.3 Na2S2O3 0.01N solution for titration.
2. Analysis
2.1 Weight 10ml of oil sample in 50ml volumetric flask. Add DI Water to the mark. If two layers are formed use the water layer.
2.2 Put all the water (or well-mixed mixture) in a 250ml glass beaker and add 70-100ml ofHNO3:H2O 1 : 1 solution and 2g KI. Put in a dark place for lOmin.
2.3 Titrate with Na2S2O3 0.01N.
3. Calculation:
Figure imgf000039_0002
Where:
V titr= titration volume F = factor of solution M = mass of sample
I. Elements (for example Cu) in vegetable oil
1. Analytes
1.1 Hexane 1.2 K2SO4 anhydrous
1.3 HNO3 cone.
1.4 H2O2 cone, solution
2. Pretreatment of sample
Weight 5-10 ml of oil in a 50- ml volumetric flask. Add 5-7 g of MgSO4 and water to the mark. Mix well and separate the oil layer.
Dry oil layer by MgSO4. Filter. Weigh 0.5 ml of separated dried layer in the reaction vessel (SI)
Weigh 0.5 ml of sample in reaction vessel (S2) Digest both samples with HNO3 (9ml) and H2O2 (1ml) solution in microwave oven. Condition T1 to T2 in 15 mins., T2=200°C, P=1800W, t=15min.
Dilution 1:100 and 1:2000.
3. Concentration of element:
Figure imgf000040_0001
Descriptions of embodiments of the invention in the present application are provided by way of example and are not intended to limit the scope of the invention. The described embodiments comprise different features, not all of which are required in all embodiments of the invention. Some embodiments utilize only some of the features or possible combinations of the features. Variations of embodiments of the invention that are described, and embodiments of the invention including different combinations of features noted in the described embodiments, will occur to persons of the art. The scope of the invention is limited only by the claims.

Claims

Claims
1. A composition for boosting the immune system of an animal comprising
Dimethyl sulfoxide (DMSO), a vegetable oil, a fatty acid, Chlorine dioxide and a surfactant.
2. The composition of claim 1, wherein said vegetable oil is sesame oil, said fatty acid is heptanoic acid and said surfactant is Polysorbate 80.
3. The composition of claim 1, further comprising an immune-boosting formulation comprising copper containing nanoparticles and a hydrophobic substance.
4. The composition of claim 3, wherein said hydrophobic substance is selected from a group containing vegetable oil, vegetable oil with unsaturated connections, vegetable oil with dienic unsaturated connections.
5. The composition of claim 4, wherein said vegetable oil is sesame oil.
6. The composition of claim 5, further comprising polyalkoxylated butyl ether.
7. A use of a composition according to any one of the above claims for boosting the immune system of farm animals and poultry.
8. The use of claim 7, comprising adding the composition of claim 1 to the drinking water of said animals and poultry.
9. The use of claim 8, comprising adding the composition to the drinking water according to the following regimen:
Weeks 1-2 - 0.5 liters of said composition to a 1000 liters of drinking water for 3-5 days
Weeks 3-5 - 1 liter of said composition to a 1000 liters of drinking water for 3-5 days
Week 6 - 1.5 liters of said composition to a 1000 liters of drinking water for 3-5 days
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