WO2005016372A1 - Compositions comprising polymetal-binding proteins and plant extracts for reducing free radicals - Google Patents

Compositions comprising polymetal-binding proteins and plant extracts for reducing free radicals Download PDF

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Publication number
WO2005016372A1
WO2005016372A1 PCT/CA2004/000445 CA2004000445W WO2005016372A1 WO 2005016372 A1 WO2005016372 A1 WO 2005016372A1 CA 2004000445 W CA2004000445 W CA 2004000445W WO 2005016372 A1 WO2005016372 A1 WO 2005016372A1
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composition
plant
free radicals
protein
lactoferrin
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PCT/CA2004/000445
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French (fr)
Inventor
Eric Lamiot
Najat Aattouri
Marta Santure
Sylvie Gauthier
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Advitech Solutions Inc.
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Publication of WO2005016372A1 publication Critical patent/WO2005016372A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A61K36/05—Chlorophycota or chlorophyta (green algae), e.g. Chlorella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/748—Cyanobacteria, i.e. blue-green bacteria or blue-green algae, e.g. spirulina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/82—Theaceae (Tea family), e.g. camellia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention relates to utraceutical products, with beneficial effect on health or prevention of physiological diseases.
  • the invention relates to a composition based on poly-metal binding proteins and added products obtained from plant sources having desired biological and biochemical properties.
  • Lactoferrin is a protein of 77 Da found in milk and in other malian secretion. ' This protein is an iron-binding protein, and many bioactivities were demonstrated in-vitro or in-vivo. This protein was related to a structural group of iron-binding protein containing transferrin and ovotransferrin, known for their biological activities. In its structure, a basic N-terminal motif is rich in tryptophane and arginine which make it possible to be responsible for some of the effects of the protein. Through pepsin hydrolysis, this protein release a peptide named lactoferricin which also have bioactivities.
  • lactoferrin can be carried out by affinity chromatography, chromatography with a silica-type support, or by some types of ion-exchange chromatography * . ' All these techniques allow the production of lactoferrin with a purity of between 70 to 95%, and some commercially available lactoferrin are already on the market. Because of the interest of human lactoferrin as a compound in infant formula, some methods exist to produce this human protein in different genetically engineered species. The antibacterial activity of lactoferrin is the most documented feature. This comes directly from antibacterial activity or can be a result, of iron depletion by said lactoferrin in the environment of the bacteria.
  • US Patent 6,172,040 proposes the use of lactoferrin immobilized in a . matrix substance for use as antimicrobial agent in food.
  • lactoferrin iron, copper, zinc or manganese
  • US Patent 5,296,464 for their different antibacterial activities. It is proposed that lactoferrin preparations be used in foods, on surfaces, or for topics applications. The use of lactoferrin is also proposed for therapy against acute or
  • lactoferrin can be used in combination with other antimicrobial agents, respectively lactoperoxidase and beta- lactams, as oral therapeutic agents.
  • lactoferrin can be used in combination with other antimicrobial agents, respectively lactoperoxidase and beta- lactams, as oral therapeutic agents.
  • lactoferrin many other bio-activities of lactoferrin are known, . and some of them have been patented. Examples include the anti-heparinic effect, the anti-toxin effect against endotoxins, the wound-healing effects in ophthalmic preparation, the prebiotic effect on bifidobacteria, the growth factor effect for use in infant formula, its activities in iron transport, antioxidant activities or use as metal chelator.
  • lactoferrin peptides can have some bioactivities, there are several possible applications of lactoferrin hydrolysates or lactoferrin peptides on different health targets.
  • ROS reactive oxygen species
  • enzymes such as superoxide dismutase, catalase, glutathione transferase, glutathione peroxidase and glutathione reductase
  • antioxidants such as tocopherols, ubiquinone, ubiquinol, ascorbic acid and dehydroascorbic acid.
  • One aim of the present invention is ' to provide a biocompatible composition for modulating physiological mechanisms in a human or an animal comprising ' a polymetal-binding protein and a plant or cyanobacterial extract having antioxidant activity.
  • the desired modulation may be increasing or reducing a metabolic or physiologic reaction.
  • the biocompatible composition of the present invention can be used as a . nutraceutical, a therapeutical or a cosmeceutical composition.
  • the polymetal-binding protein may be, but is not limited to, lactoferrin, or related proteins or derivatives or fragments thereof. Again, it will be recognized by someone skilled in the art that the polymetal-binding protein can be capable of modulating a metabolic or physiological reaction by binding at least one metal ion selected from the group consisting of iron, magnesium, copper, zinc, and manganese.
  • polymetal-binding protein is preferably at a concentration between 50 to 1500 mg per dose or in concentration allowing the composition to act on the targeted metabolic or physiologic reaction.
  • Another object of the present invention is to provide a nutraceutical or cosmeceutical composition for modulating or activating a metabolic body reaction in a human- or an animal.
  • This composition may comprise between about 50 and 1500 mg of lactoferrin or related iron binding proteins or their peptides per portion, and at least one plant extract with antioxidant properties which improve the action of lactoferrin or related iron binding proteins or their peptides on a metabolic reaction
  • Another object of the present invention is to provide a therapeutical composition for modulating a metabolic body reaction in a human or an animal.
  • This composition comprises: a) between 50 and 1500 mg of lactoferrin or related iron binding proteins or their peptides per day; and b) a plant extract with antioxidant properties which improve the action of lactoferrin or related iron binding proteins or their peptides on a metabolic reaction.
  • a further object of the present invention is to provide a topical composition for modulating a metabolic body reaction in a human or an animal.
  • This composition comprises: a) between 50 and 1500 mg of lactoferrin or related iron binding proteins or their peptides per portion; and b) a plant extract with antioxidant properties which improves the action of lactoferrin or related iron binding proteins or their peptides on a metabolic reaction
  • the modulation of .a metabolic reaction includes maintaining, restoring, reducing or increasing the rate of the metabolic reaction, the latter being an intestinal, an immune, an inflammatory, a skin, a neurological, a blood, a neuronal, a muscular, a hepatic, a cardiovascular, a renal, a gastric, a digestive, a pancreatic, an endocrine, a bone, a joint, or a respiratory reaction.
  • the composition of the present invention can be, for example but is not limited to, a food composition or a cosmeceutical composition.
  • a biocompatible composition for modulating free radicals quantity or related damages in a human or an animal cell, tissue, or biological fluid comprising a polymetal-binding protein and plant or cyanobacterial extract having antioxidant activity.
  • the polymetal-binding protein is preferably lactoferrin, or a related protein, or derivatives or fragments thereof.
  • the biocompatible composition of the present invention is characterized by, depending on needs, being capable to increase or reduce the quantity of free radicals and related damages into cells, tissues or body fluids in humans or animals.
  • the polymetal-binding protein is capable of modulating free radical quantity by binding at least one metal ion selected from the group consisting of iron, magnesium, copper, zinc, and manganese. Also, the polymetal-binding protein is preferably at a concentration between 50 to 1500 mg/dose or in concentration allowing the composition to act on the targeted metabolic or physiologic reaction..
  • the plant or cyanobacterial extract used in producing the composition of the present invention can be raw or refined, and can contain chromophores.
  • the biocompatible composition of the present invention can be used as a nutraceutical, a therapeutical, or a cosmeceutical composition.
  • Another aim of the present invention is to provide a method of alleviating or preventing inflammation in a human or animal comprising administering to said human or animal a combination of poly-metal binding protein and plant or cyanobacterial extract.
  • the combination that cause the reduction of free radicals quantity into a human or ⁇ animal body is completed by administering simultaneously or separately the poly-metal binding protein and the plant or cyanobacterial extract.
  • the inflammation can be partly or totally due to free radicals.
  • Another aim of the present invention is to provide the use of poly-metal protein combined with at least one of plant o cyanobacteria extract in the preparation of a composition for modulating free radical quantity or free radical related damages in body cells, tissues, or fluids. This use can be carried out for reducing the quantity of free radicals in or the damages induced by the free radicals in body cells, tissues, or fluids.
  • poly-metal protein combined with at least one of plant or cyanobacteria extract in the preparation of a composition for alleviating or treating inflammation in a human or an animal.
  • the lactoferrin is obtained from milk, whey or genetically modified organisms.
  • Lactoferrin peptide are obtained by hydrolysis of this protein with enzyme obtained ' from natural sources that include digestive, microbial, animal or plant source, or with genetically or chemically engineered enzymes.
  • Related iron-binding protein are obtained from eggs, blood, serum, plasma or tissues of vertebrates, or genetically modified organisms, peptides from iron-binding proteins are produced by the hydrolysis of these proteins with enzyme obtained from natural source that include digestive, microbial, animal or plant sources or with genetically or chemically engineered enzymes.
  • polymetal binding protein as used herein is intended to mean, for example, proteins which can bind metals, such as iron, zinc, copper or other metals, for example lactoferrin or related proteins.
  • related proteins as used herein is intended to mean, for example metal-binding proteins than lactoferrin such as transferrin obtained from blood or vertebrate tissues, or ovotransferrin obtained from eggs. Geneticaly engineered proteins using genes or genes fragments of these proteins are also included on the definition of this term.
  • peptides as used herein means fragments of these proteins obtained by hydrolysis of these proteins by enzymes obtained from natural sources that include digestive, microbial, animal, or plant sources, or with genetically engineered enzymes.
  • plant extracts as used herein is intended to mean plant extracts obtained from cells, leaves, stems or stalks of plants, algae, or cyanobacteria.
  • the plant extracts have antioxidant properties.
  • dose is intended to mean a sufficient amount of protein which must be used by human or animal each day to give the desired effect on the modulation of the metabolic reaction which is targeted.
  • an effective concentration of a free radical inhibitor or “concentration which is effective to inhibit free radical degradation” as used herein means a concentration of a free radical inhibitor compound that is capable of reducing the amount of free radical compounds as compared to the amount of free radical compounds obtained in the absence of the free radical inhibitor compound.
  • Methods of determining the ability of a compound to inhibit free radical formation or free radical-induced degradation are well known in the art. For example, a method for determining the free radical inhibitory capacity of a compound is based on the ability of such a compound to inhibit free radical polymerization of acrylic acid to polyacrylic acid.
  • a suitable assay for demonstrating an effective concentration of a selected free radical inhibitor compound is an assay which determines the ability of such a compound to inhibit the polymerization of a 30% aqueous solution of acrylic acid at 85°C. after initiation with 0.1% azobisisobutyronitrile.
  • the inhibitory capacity of such a. compound is determined by comparing the percentage of polyacrylic acid formed in the presence and absence of a predetermined concentration of the selected free radical inhibitor compound, with inhibition being demonstrated by a detectable decrease in the amount of polyacrylic acid formed in the reaction mixture containing the inhibitor as compared to the amount of polyacrylic acid formed in the reaction mixture to which an inhibitor was not added.
  • the amount of polyacrylic acid formed is inversely related to the amount of acrylic acid (i.e., monomer) remaining in the reaction mixture at the end of the assay.
  • the amount of acrylic acid may be routinely determined by measuring the iodine value of the reaction mixture or by other methods known in the art for determinating unsaturation.
  • a well-known free radical inhibitor compound such as hydroquinone mono methyl ether (HQMME) may be used as a standard inhibitor compound in the assay.
  • Other methods commonly used are the TBARS (ThioBarbituric Acid Reactive Substance) determination, which is the determination of Malondialdehyde (MDA) created when lipids are submitted to peroxidation by free radicals.
  • MDA Malondialdehyde
  • Inhibition of MDA formation when Fe3+ and ascorbic acids (which are able to generate free radical formation) are mixed with phosphatidyl choline (a lipid) is a good marker for free radical inhibition.
  • Another available method is the ability to neutralize the DPPH (2,2-Diphenyl-l-picrylhydrazyl), which is a stable free radical in solution.
  • a simple spectrometric test allows the determination of DPPH which is neutralized by a free radical inhibitor compound.
  • Cell culture tests are also available, which are related ' to the characterization of oxidative stress markers (Nitric Oxide (NO) or iNOS (inducible Nitric Oxide Synthase) for example) induced by pro-oxidative compounds added the cell culture media. Inhibition of the oxidative stress markers by a free radical inhibitor compound are commonly used to determine inhibition of the free radical degradation on cellular models.
  • Fig. 1 illustrates the effect of proinflammatory compounds on iNOS expression in Caco-2 cells
  • Fig. 2 illustrates the effect of. different concentrations .of lactoferrin on iNOS expression by Caco-2 cells inflamed with CM1;
  • . . Fig. 3. illustrates the effect of the algae extract and lactoferrin on iNOS expression by Caco-2 cells inflamed with CM1 ;
  • Fig. 4 illustrates the effect of green tea extract and lactoferrin on iNOS expression by Caco-2 cells inflamed with CM1.
  • composition of the invention comprises botanical extracts having antioxidant properties, combined VvTTH a polymetal-binding protein, such as,* but not limited to, lactoferrin related binding proteins or derivatives or fragments thereof. It has been surprisingly observed that the plant extracts have a . significant synergetic effect which enhances or completes the effect of a polymetal- binding protein or their derivatives or fragments thereof.
  • a new nutraceutical or cosmeceutical composition allowing the prevention, treatment or restoration of physiological functions in an human or an. animal, through . oral administration or topical application.
  • the composition is principally composed of two fractions, one being a polymetal-binding protein, such as lactoferrin or related binding proteins or peptides from lactoferrin or related binding proteins and another being a botanical or cyanobacterial extract with antioxidant properties.
  • lactoferrin is used in combination- with other agents, to completing its activities. It is the case of its i munostimulatory effect, which is completed, for example but not limited to, by the use of colostrum in the finished product.
  • plant extracts are synergistically used for health, or prevention or treatment of diseases.
  • some compounds may be used for examples for treatment of cancer, neurological diseases, immune problems, prostate diseases, hepatic disorder, or for their antiviral or antifungal activities.
  • plant extracts, raw or refined have antioxidant activities, and this characteristic has been exploited since a . long time for its positive effects on health. ' It is the case for quercetin and rutin, carotenoids, cysteine derivatives or polyphenols or procyanidins.
  • complex extracts of botanical sources have an antioxidant effect and can be used for their antioxidant properties in food, cosmetics, or neutraceutical formulations. These extracts can be used for examples as food preservation, for skin protection, or for use in food or nutraceutical compositions for example.
  • the combination of these two fractions, lactoferrin. and plant extract synergistically improves the activity of the polymetal-binding protein or their peptides, to modulate a body metabolism involved in a physiological reaction.
  • the physiological reaction can be a processing reaction in normal conditions, or a reaction following or being part of a disease or a disorder.
  • the disease or disorder can be acquired, such as for example but not . limited to, following an accident or intoxication, or naturally occurring as it is the case for genetically transmitted metabolic disorders.
  • Lactoferrin or its derivatives allow the binding or different metal ions, such as, but not limited to, iron, copper, zinc or manganese, which are ions involved in a high variety of physiological reactions. It is recognized for its antibacterial, antiviral and antifungal activities, as well ' as for . its immunomodulatory, anti-tumor or anti-inflammatory effects.
  • the combination of this polymetal-binding protein with a plant extract has proven to give body effects that were not believed possible to suspect before.
  • the plant or cyanobacterial extracts have antioxidant properties, and more preferably contains chromophores.
  • the extracts can be in form of flakes, or can be raw, crude, or refined at different levels into powder, for example.
  • the extract can be separated from impurities or other extraneous substances before being mixed with the poly-metal binding proteins.
  • the plant or cyanobacterial extract can be processed before being mixed with the poly-metal binding protein.
  • the extract portion of the composition of the invention can be administered at the same time of shortly before or after administration of the poly-metal binding proteins.
  • the combination plant or cyanobacterial extract and poly-metal binding protein is preferably orally administered, but can also be topically applied onto the human or animal body.
  • the present invention entails a body cells or tissues treatment composition or capable of inhibiting, removing or neutralizing free radical degradation of the body cells or tissues, which may comprises a nutraceutical base composition or a topical cosmetic base composition, which contains a concentration of a plant or cyanobacterial extract combined with a poly- metal binding protein.
  • the invention also relates to the use of lactoferrin or related metal- binding proteins or fragments thereof.
  • These polymetal-binding proteins can be produced from different sources, preferably from milk, whey, egg, serum, plasma or blood, but can also be originated from genetically modified organisms.
  • the protein itself can be of hen, bovine, human, porcine, ovine, caprine, or other vertebrate origin, preferably hen, bovine or human, or can be a chimeric protein obtained by molecular biology.
  • the peptides can be produced by chemical or enzymatic hydrolysis of lactoferrin or related metal-binding proteins, preferably by enzymatic hydrolysis, preferably with digestive enzyme.
  • the peptides can be genetically engineered organisms by using lactoferrin or related metal-binding proteins, or gene fragments and expression of these fragments in an appropriate organism, followed by purification of the peptides.
  • the composition comprising poly-metal binding protein mixed with plant extract is . used to neutralise the effect of free radicals into the body of a human or an animal.
  • the composition is useful in reducing the flow or circulation of free radicals involved, directly or indirectly, into a metabolic reaction , such as in body inflammation processes.
  • Inflammation which can be induced by several factors like for examples chemical compounds, physical agents (like UN), infections or a ⁇ toimmune disorders, and which is part of many diseases, like atherosclerosis, rheumatic disease, arthritis, intestinal bowel disease, or skin disorder, induce the activation of several oxidant-generating enzymes such as ⁇ ADPH (reduced, form of nicotinamide adenine dinucleotide phosphate) oxidase, i ⁇ OS, or some peroxidases. .
  • ⁇ ADPH reduced, form of nicotinamide adenine dinucleotide phosphate
  • i ⁇ OS oxidase
  • peroxidases oxidase
  • These enzyme produce high concentration of diverse free radicals including nitric oxide, superoxide anion, hydrogen peroxide, which are able to react with each other to generate more and more reactive oxygen and nitrogen species.
  • Lactoferrin or related metal-binding proteins or their peptides can be used for their antiviral, antifungal or antimicrobial activities, for their antioxidant properties, for their effects on immuno-stimulation, for their anti-inflammatory, metal-binding or anti- aging effects.
  • the composition can be used for its positive synergistic effect on the physiology of the liver, its anti-cancer properties, its effect on neurological disorders, its effect on healing stimulation or its growth factor activity.
  • the botanical extract can be obtained from various types of plants such as algae, chlorella, or from cyanobacteria. Leaves, stems or stalks or other part of the botanical source can be used for the production of the extract.
  • the extract used may contain at least one of the chromophore used by the plant or cyanobacteria in the photosynthetic pathway.
  • the extract used in the composition has antioxidant effects. This extract is used for its synergetic effect on polymetal-binding proteins, lactoferrin or related metal- binding proteins or derivatives or fragments thereof, which can improve or complete the action of metal-binding proteins.
  • Formulation of the present invention can be found in the form of or integrated to different supports.
  • the nutraceutical products may be presented in various forms, i.e. pills, caplets, tablets, powders or granules within a daily dose where the component of the formulation is active on the physiological function.
  • compositions can be added.
  • the daily dose is calculated to provide the required activity for the ingredients in the final formula.
  • ingredients of the composition in a formulation for topical application, such as creams, pomades or ointments. Also in that case, concentrations of the ingredients are chosen to obtain the desired effect on the physiological targets.
  • Caco-2 cells are widely used to simulate intestinal cells in model culture. These cells spontaneously differentiate after confluence. This model can be also used as a good model for studying inflammation. In this specific test, inflammation is induced by addition of inflammatory compounds, such as H 2 O 2 or some proinflammatory cytokines. Examination of the inflammation can be carried out by evaluation of the synthesis of some inflammatory markers by the Caco-2 cells. One of this marker is iNOS (inducible Nitric Oxide Synthase), which can be followed by the Western-blot test, with detection by specific antibodies. Different inflammatory agents were tested on Caco-2 cells in order to determine the best one to use in further experiments. Fig.
  • iNOS inducible Nitric Oxide Synthase
  • CMl Cytomix 1 (IFN- ⁇ , 1000 U/ml; IL-l ⁇ , 17 nanograms/ml and TNF- ⁇ , 10 ng ml)
  • CM2 Cytomix 2 (IFN- ⁇ , 600 U/ml; IL-l ⁇ , 10 nanograms/ml and TNF- ⁇ , 50 ng/ml)
  • CM3 Cytomix 3 (IFN- ⁇ , 200 U/ml; IL-l ⁇ , 5 nanograms/ml and TNF- ⁇ , 100 ng/ml), H 2 O 2 /CMl, CMl with H 2 0 2 /ml).
  • CMl cytomix 1
  • IFN-gamma 1000 U/ml
  • IL-1 beta 17 nanograms/ml
  • TNF-alpha 10 ng/ml
  • Caco-2 cells, inflamed with CMl were treated with different concentrations of a commercial lactoferrin.
  • Results of western-blot and respective density of the iNOS spots are shown in Fig. 2(Effect of different concentrations of lactoferrin on iNOS expression by Caco-2 cells inflammed with CMl. Lactoferrin concentrations are indicated in micrograms per ml). Lactoferrin decreases the synthesis of iNOS at 250, 500 and 750 micrograms per ml. A dose of 750 micrograms per ml was chosen for the other experiments.
  • Caco-2 cells, inflamed with CMl were treated with a commercial algae extract (chlorella), lactoferrin, and a mixture of both at the same concentrations.
  • chlorella commercial algae extract
  • lactoferrin lactoferrin
  • Caco-2 cells, inflamed with CMl were treated with a commercial green . tea extract at different concentrations, lactoferrin, and a mixture of both at 5 concentrations where green tea has an anti-inflammatory effect.
  • Results shown in Fig. 4 (Effect of the green tea extract (TV) at different concentrations, lactoferrin at 750 micrograms per ml and mix of lactoferrin at 750 micrograms per ml and green tea extract at 100 micrograms per ml on iNOS expression by Caco-2 cells inflamed with CMl), demonstrate that the effect of green tea at 100 micrograms per ml and l o lactoferrin together decrease iNOS in a higher level than the individual compounds at the same concentrations, and that the algebrical sum of the separate effects of green tea at 100 micrograms per ml and lactoferrin is lower than the effect of the compounds together, indicating a synergistic effect between lactoferrin and green tea extract.

Abstract

The present invention relates to a composition comprising a polymetal­binding protein or derivatives of fragments thereof, as physiologically active ingredients acting synergistically with a plant or cyanobacteria extract having antioxidant activity.

Description

COMPOSITIONS COMPRISING POLYMETAL-BINDING PROTEINS AND PLANT EXTRACTS FOR REDUCING FREE RADICALS
TECHNICAL FIELD The present invention relates to utraceutical products, with beneficial effect on health or prevention of physiological diseases. Particularly, the invention relates to a composition based on poly-metal binding proteins and added products obtained from plant sources having desired biological and biochemical properties.
BACKGROUND ART Lactoferrin is a protein of 77 Da found in milk and in other mamalian secretion.' This protein is an iron-binding protein, and many bioactivities were demonstrated in-vitro or in-vivo. This protein was related to a structural group of iron-binding protein containing transferrin and ovotransferrin, known for their biological activities. In its structure, a basic N-terminal motif is rich in tryptophane and arginine which make it possible to be responsible for some of the effects of the protein. Through pepsin hydrolysis, this protein release a peptide named lactoferricin which also have bioactivities.
The production of lactoferrin can be carried out by affinity chromatography, chromatography with a silica-type support, or by some types of ion-exchange chromatography*. ' All these techniques allow the production of lactoferrin with a purity of between 70 to 95%, and some commercially available lactoferrin are already on the market. Because of the interest of human lactoferrin as a compound in infant formula, some methods exist to produce this human protein in different genetically engineered species. The antibacterial activity of lactoferrin is the most documented feature. This comes directly from antibacterial activity or can be a result, of iron depletion by said lactoferrin in the environment of the bacteria.
US Patent 6,172,040 proposes the use of lactoferrin immobilized in a . matrix substance for use as antimicrobial agent in food.
The use of different metal binding forms of lactoferrin (iron, copper, zinc or manganese) is proposed in US Patent 5,296,464 for their different antibacterial activities. It is proposed that lactoferrin preparations be used in foods, on surfaces, or for topics applications. The use of lactoferrin is also proposed for therapy against acute or
• chronic infections caused by Streptococcus pyogenes and Staphylococc' us aureus. Alternatively, it has been described in the art that the lactoferrin can be used in combination with other antimicrobial agents, respectively lactoperoxidase and beta- lactams, as oral therapeutic agents. Many other bio-activities of lactoferrin are known, . and some of them have been patented. Examples include the anti-heparinic effect, the anti-toxin effect against endotoxins, the wound-healing effects in ophthalmic preparation, the prebiotic effect on bifidobacteria, the growth factor effect for use in infant formula, its activities in iron transport, antioxidant activities or use as metal chelator. As lactoferrin peptides can have some bioactivities, there are several possible applications of lactoferrin hydrolysates or lactoferrin peptides on different health targets.
According to the free radical theory of premature aging of the body cells, different environmental products can produce reactive oxygen species (ROS) that damage the cells. ROS are a collection of reactive free radicals produced by the oxygen molecule, and include singlet oxygen, the superoxide radical, hydrogen peroxide, and the hydroxyl radical, as well as the reaction products produced by these free radicals. Due to their reactivity, ROS relatively indiscriminately react with other molecules, and generate a cascade of harmful free radical reactions on the skin.
The body cells, tissues or fluids possess certain defense mechanisms against the generation of ROS. These defenses include the presence of enzymes such as superoxide dismutase, catalase, glutathione transferase, glutathione peroxidase and glutathione reductase, as well as antioxidants such as tocopherols, ubiquinone, ubiquinol, ascorbic acid and dehydroascorbic acid. Unfortunately, some products entering the body can easily overwhelm these defense systems, such that the amount of superoxide dismutase and glutathione transferase in the body declines significantly upon irradiation with these products. Simultaneously with the loss of these reducing enzymes, there is a dramatic increase in conjugated double bonds formed in the body cells from the linoleates present in cell membranes. There is also an increase in thiobarbituric acid reactive substances present in certain body tissues, which represent a collection of molecules that are formed from ROS.
In spite of advances in recent years in the protection of body tissues and cells from harmful products, the epidemic of inflammation, cancers and tissue damages due to the effects of these products has continued unabated. The loss of portions of the control from environmental pollution for example is believed to have contributed to an increase in ambient harmful product dissemination that reaches exposed body cells, tissues, or fluids. There is a significant need for commercially acceptable or improved preparations that can be administered to human and animals, to offset the harmful effects of the products described above. From the state of the art described above, it is clear that it is still highly desirable to be provided with new synergistic compositions in the benefit of health of humans and animals.
SUMMARY OF THE INVENTION
One aim of the present invention is' to provide a biocompatible composition for modulating physiological mechanisms in a human or an animal comprising ' a polymetal-binding protein and a plant or cyanobacterial extract having antioxidant activity. The desired modulation may be increasing or reducing a metabolic or physiologic reaction. k
The biocompatible composition of the present invention can be used as a . nutraceutical, a therapeutical or a cosmeceutical composition.
The polymetal-binding protein may be, but is not limited to, lactoferrin, or related proteins or derivatives or fragments thereof. Again, it will be recognized by someone skilled in the art that the polymetal-binding protein can be capable of modulating a metabolic or physiological reaction by binding at least one metal ion selected from the group consisting of iron, magnesium, copper, zinc, and manganese.
In the above biocompatible composition, polymetal-binding protein is preferably at a concentration between 50 to 1500 mg per dose or in concentration allowing the composition to act on the targeted metabolic or physiologic reaction..
Another object of the present invention is to provide a nutraceutical or cosmeceutical composition for modulating or activating a metabolic body reaction in a human- or an animal. This composition may comprise between about 50 and 1500 mg of lactoferrin or related iron binding proteins or their peptides per portion, and at least one plant extract with antioxidant properties which improve the action of lactoferrin or related iron binding proteins or their peptides on a metabolic reaction
Another object of the present invention is to provide a therapeutical composition for modulating a metabolic body reaction in a human or an animal. This composition comprises: a) between 50 and 1500 mg of lactoferrin or related iron binding proteins or their peptides per day; and b) a plant extract with antioxidant properties which improve the action of lactoferrin or related iron binding proteins or their peptides on a metabolic reaction.
A further object of the present invention is to provide a topical composition for modulating a metabolic body reaction in a human or an animal. This composition comprises: a) between 50 and 1500 mg of lactoferrin or related iron binding proteins or their peptides per portion; and b) a plant extract with antioxidant properties which improves the action of lactoferrin or related iron binding proteins or their peptides on a metabolic reaction The modulation of .a metabolic reaction includes maintaining, restoring, reducing or increasing the rate of the metabolic reaction, the latter being an intestinal, an immune, an inflammatory, a skin, a neurological, a blood, a neuronal, a muscular, a hepatic, a cardiovascular, a renal, a gastric, a digestive, a pancreatic, an endocrine, a bone, a joint, or a respiratory reaction. It will be understood by someone skilled in the art that the composition of the present invention can be, for example but is not limited to, a food composition or a cosmeceutical composition.
In accordance with another aspect of the present invention, there is provided a biocompatible composition for modulating free radicals quantity or related damages in a human or an animal cell, tissue, or biological fluid comprising a polymetal-binding protein and plant or cyanobacterial extract having antioxidant activity. The polymetal-binding protein is preferably lactoferrin, or a related protein, or derivatives or fragments thereof.
The biocompatible composition of the present invention is characterized by, depending on needs, being capable to increase or reduce the quantity of free radicals and related damages into cells, tissues or body fluids in humans or animals.
The polymetal-binding protein is capable of modulating free radical quantity by binding at least one metal ion selected from the group consisting of iron, magnesium, copper, zinc, and manganese. Also, the polymetal-binding protein is preferably at a concentration between 50 to 1500 mg/dose or in concentration allowing the composition to act on the targeted metabolic or physiologic reaction..
The plant or cyanobacterial extract used in producing the composition of the present invention can be raw or refined, and can contain chromophores. The biocompatible composition of the present invention can be used as a nutraceutical, a therapeutical, or a cosmeceutical composition.
Another aim of the present invention is to provide a method of alleviating or preventing inflammation in a human or animal comprising administering to said human or animal a combination of poly-metal binding protein and plant or cyanobacterial extract.
Preferably, the combination that cause the reduction of free radicals quantity into a human or ■ animal body is completed by administering simultaneously or separately the poly-metal binding protein and the plant or cyanobacterial extract..
The inflammation can be partly or totally due to free radicals.
Another aim of the present invention is to provide the use of poly-metal protein combined with at least one of plant o cyanobacteria extract in the preparation of a composition for modulating free radical quantity or free radical related damages in body cells, tissues, or fluids. This use can be carried out for reducing the quantity of free radicals in or the damages induced by the free radicals in body cells, tissues, or fluids.
Also, there is provide the use of poly-metal protein combined with at least one of plant or cyanobacteria extract in the preparation of a composition for alleviating or treating inflammation in a human or an animal.
In a further embodiment of the present invention, the lactoferrin is obtained from milk, whey or genetically modified organisms. Lactoferrin peptide are obtained by hydrolysis of this protein with enzyme obtained ' from natural sources that include digestive, microbial, animal or plant source, or with genetically or chemically engineered enzymes. Related iron-binding protein are obtained from eggs, blood, serum, plasma or tissues of vertebrates, or genetically modified organisms, peptides from iron-binding proteins are produced by the hydrolysis of these proteins with enzyme obtained from natural source that include digestive, microbial, animal or plant sources or with genetically or chemically engineered enzymes.
For the purpose of the present invention the following terms are defined below. The term "polymetal binding protein" as used herein is intended to mean, for example, proteins which can bind metals, such as iron, zinc, copper or other metals, for example lactoferrin or related proteins. The term "related proteins" as used herein is intended to mean, for example metal-binding proteins than lactoferrin such as transferrin obtained from blood or vertebrate tissues, or ovotransferrin obtained from eggs. Geneticaly engineered proteins using genes or genes fragments of these proteins are also included on the definition of this term. The term "peptides" as used herein means fragments of these proteins obtained by hydrolysis of these proteins by enzymes obtained from natural sources that include digestive, microbial, animal, or plant sources, or with genetically engineered enzymes.
The expression "plant extracts" as used herein is intended to mean plant extracts obtained from cells, leaves, stems or stalks of plants, algae, or cyanobacteria. The plant extracts have antioxidant properties.
The term "dose" as used herein is intended to mean a sufficient amount of protein which must be used by human or animal each day to give the desired effect on the modulation of the metabolic reaction which is targeted.
The phrase "an effective concentration of a free radical inhibitor" or "concentration which is effective to inhibit free radical degradation" as used herein means a concentration of a free radical inhibitor compound that is capable of reducing the amount of free radical compounds as compared to the amount of free radical compounds obtained in the absence of the free radical inhibitor compound. Methods of determining the ability of a compound to inhibit free radical formation or free radical-induced degradation are well known in the art. For example, a method for determining the free radical inhibitory capacity of a compound is based on the ability of such a compound to inhibit free radical polymerization of acrylic acid to polyacrylic acid. A suitable assay for demonstrating an effective concentration of a selected free radical inhibitor compound is an assay which determines the ability of such a compound to inhibit the polymerization of a 30% aqueous solution of acrylic acid at 85°C. after initiation with 0.1% azobisisobutyronitrile. The inhibitory capacity of such a. compound is determined by comparing the percentage of polyacrylic acid formed in the presence and absence of a predetermined concentration of the selected free radical inhibitor compound, with inhibition being demonstrated by a detectable decrease in the amount of polyacrylic acid formed in the reaction mixture containing the inhibitor as compared to the amount of polyacrylic acid formed in the reaction mixture to which an inhibitor was not added. As. will be appreciated, the amount of polyacrylic acid formed is inversely related to the amount of acrylic acid (i.e., monomer) remaining in the reaction mixture at the end of the assay. The amount of acrylic acid may be routinely determined by measuring the iodine value of the reaction mixture or by other methods known in the art for determinating unsaturation. A well-known free radical inhibitor compound such as hydroquinone mono methyl ether (HQMME) may be used as a standard inhibitor compound in the assay. Other methods commonly used are the TBARS (ThioBarbituric Acid Reactive Substance) determination, which is the determination of Malondialdehyde (MDA) created when lipids are submitted to peroxidation by free radicals. Inhibition of MDA formation when Fe3+ and ascorbic acids (which are able to generate free radical formation) are mixed with phosphatidyl choline (a lipid) is a good marker for free radical inhibition. Another available method is the ability to neutralize the DPPH (2,2-Diphenyl-l-picrylhydrazyl), which is a stable free radical in solution. A simple spectrometric test allows the determination of DPPH which is neutralized by a free radical inhibitor compound. Cell culture tests are also available, which are related ' to the characterization of oxidative stress markers (Nitric Oxide (NO) or iNOS (inducible Nitric Oxide Synthase) for example) induced by pro-oxidative compounds added the cell culture media. Inhibition of the oxidative stress markers by a free radical inhibitor compound are commonly used to determine inhibition of the free radical degradation on cellular models.
DESCRIPTION OF DRAWINGS Fig. 1 illustrates the effect of proinflammatory compounds on iNOS expression in Caco-2 cells;
Fig. 2 illustrates the effect of. different concentrations .of lactoferrin on iNOS expression by Caco-2 cells inflamed with CM1;
. . Fig. 3. illustrates the effect of the algae extract and lactoferrin on iNOS expression by Caco-2 cells inflamed with CM1 ; and
Fig. 4 illustrates the effect of green tea extract and lactoferrin on iNOS expression by Caco-2 cells inflamed with CM1.
DETAILED DESCRIPTION OF THE INVENTION The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention, may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The inventors of the present invention have developed a composition having new synergistic characteristics on the physiology of a human or an animal. More particularly, the composition of the invention comprises botanical extracts having antioxidant properties, combined VvTTH a polymetal-binding protein, such as,* but not limited to, lactoferrin related binding proteins or derivatives or fragments thereof. It has been surprisingly observed that the plant extracts have a . significant synergetic effect which enhances or completes the effect of a polymetal- binding protein or their derivatives or fragments thereof.
In accordance with the present invention, there is provided a new nutraceutical or cosmeceutical composition allowing the prevention, treatment or restoration of physiological functions in an human or an. animal, through . oral administration or topical application. The composition is principally composed of two fractions, one being a polymetal-binding protein, such as lactoferrin or related binding proteins or peptides from lactoferrin or related binding proteins and another being a botanical or cyanobacterial extract with antioxidant properties.
In one embodiment of .the present invention, lactoferrin is used in combination- with other agents, to completing its activities. It is the case of its i munostimulatory effect, which is completed, for example but not limited to, by the use of colostrum in the finished product.
Another embodiment of the invention, plant extracts are synergistically used for health, or prevention or treatment of diseases. When presents in a botanical material, some compounds may be used for examples for treatment of cancer, neurological diseases, immune problems, prostate diseases, hepatic disorder, or for their antiviral or antifungal activities. According to one particular aspect of the present invention, plant extracts, raw or refined, have antioxidant activities, and this characteristic has been exploited since a. long time for its positive effects on health.' It is the case for quercetin and rutin, carotenoids, cysteine derivatives or polyphenols or procyanidins.
More than the use of purified products, complex extracts of botanical sources have an antioxidant effect and can be used for their antioxidant properties in food, cosmetics, or neutraceutical formulations. These extracts can be used for examples as food preservation, for skin protection, or for use in food or nutraceutical compositions for example.
The combination of these two fractions, lactoferrin. and plant extract, synergistically improves the activity of the polymetal-binding protein or their peptides, to modulate a body metabolism involved in a physiological reaction. The physiological reaction can be a processing reaction in normal conditions, or a reaction following or being part of a disease or a disorder. One skilled in the art will recognize that the disease or disorder can be acquired, such as for example but not . limited to, following an accident or intoxication, or naturally occurring as it is the case for genetically transmitted metabolic disorders.
Lactoferrin or its derivatives allow the binding or different metal ions, such as, but not limited to, iron, copper, zinc or manganese, which are ions involved in a high variety of physiological reactions. It is recognized for its antibacterial, antiviral and antifungal activities, as well ' as for . its immunomodulatory, anti-tumor or anti-inflammatory effects. The combination of this polymetal-binding protein with a plant extract has proven to give body effects that were not believed possible to suspect before. Preferably, the plant or cyanobacterial extracts have antioxidant properties, and more preferably contains chromophores. It will be recognized by a person skilled in the art to which the invention pertains that the extracts can be in form of flakes, or can be raw, crude, or refined at different levels into powder, for example. The extract can be separated from impurities or other extraneous substances before being mixed with the poly-metal binding proteins. In different specific conditions, as for example for rendering some compounds more available to, or more stable into, the organisms after absorption, the plant or cyanobacterial extract can be processed before being mixed with the poly-metal binding protein. The extract portion of the composition of the invention can be administered at the same time of shortly before or after administration of the poly-metal binding proteins. It will be recognized that the combination plant or cyanobacterial extract and poly-metal binding protein is preferably orally administered, but can also be topically applied onto the human or animal body. In another of its aspects, the present invention entails a body cells or tissues treatment composition or capable of inhibiting, removing or neutralizing free radical degradation of the body cells or tissues, which may comprises a nutraceutical base composition or a topical cosmetic base composition, which contains a concentration of a plant or cyanobacterial extract combined with a poly- metal binding protein.
The invention also relates to the use of lactoferrin or related metal- binding proteins or fragments thereof. These polymetal-binding proteins can be produced from different sources, preferably from milk, whey, egg, serum, plasma or blood, but can also be originated from genetically modified organisms. The protein itself can be of hen, bovine, human, porcine, ovine, caprine, or other vertebrate origin, preferably hen, bovine or human, or can be a chimeric protein obtained by molecular biology. The peptides can be produced by chemical or enzymatic hydrolysis of lactoferrin or related metal-binding proteins, preferably by enzymatic hydrolysis, preferably with digestive enzyme. In another method to produce lactoferrin or related metal-binding proteins, the peptides can be genetically engineered organisms by using lactoferrin or related metal-binding proteins, or gene fragments and expression of these fragments in an appropriate organism, followed by purification of the peptides. ■
In one preferred embodiment of the present invention, the composition comprising poly-metal binding protein mixed with plant extract, is . used to neutralise the effect of free radicals into the body of a human or an animal. Most particularly, the composition is useful in reducing the flow or circulation of free radicals involved, directly or indirectly, into a metabolic reaction , such as in body inflammation processes. Inflammation, which can be induced by several factors like for examples chemical compounds, physical agents (like UN), infections or aμtoimmune disorders, and which is part of many diseases, like atherosclerosis, rheumatic disease, arthritis, intestinal bowel disease, or skin disorder, induce the activation of several oxidant-generating enzymes such as ΝADPH (reduced, form of nicotinamide adenine dinucleotide phosphate) oxidase, iΝOS, or some peroxidases. . These enzyme produce high concentration of diverse free radicals including nitric oxide, superoxide anion, hydrogen peroxide, which are able to react with each other to generate more and more reactive oxygen and nitrogen species. All of these free radicals are able to induce DΝA (Desoxiribo-Νucleic Acid) or RΝA (Ribo-Νucleic ' Acid) damages, which can induced carcinogenesis, modification of proteins (for example oxidation, nitrosation, nitration) which induced alteration of protein structure and function, and membrane lipid peroxidation, which is related to cellular dysfunction. It is clear that antioxidant compounds are able to decrease inflammatory reactions. In the present invention, lactoferrin or related metal-binding proteins or their peptides are used for their biological activities for health benefits. Lactoferrin or related metal-binding proteins or their peptides can be used for their antiviral, antifungal or antimicrobial activities, for their antioxidant properties, for their effects on immuno-stimulation, for their anti-inflammatory, metal-binding or anti- aging effects. The composition can be used for its positive synergistic effect on the physiology of the liver, its anti-cancer properties, its effect on neurological disorders, its effect on healing stimulation or its growth factor activity.
In one embodiment of the present invention, the botanical extract, can be obtained from various types of plants such as algae, chlorella, or from cyanobacteria. Leaves, stems or stalks or other part of the botanical source can be used for the production of the extract. The extract used may contain at least one of the chromophore used by the plant or cyanobacteria in the photosynthetic pathway. The extract used in the composition has antioxidant effects. This extract is used for its synergetic effect on polymetal-binding proteins, lactoferrin or related metal- binding proteins or derivatives or fragments thereof, which can improve or complete the action of metal-binding proteins.
Formulation of the present invention can be found in the form of or integrated to different supports. The nutraceutical products may be presented in various forms, i.e. pills, caplets, tablets, powders or granules within a daily dose where the component of the formulation is active on the physiological function.
Other possible supports are functional foods, such as drinks, bars, mixed powders for drinks or puddings, or any form where ingredients of the composition can be added. In that case, the daily dose is calculated to provide the required activity for the ingredients in the final formula. It is possible to use ingredients of the composition in a formulation for topical application, such as creams, pomades or ointments. Also in that case, concentrations of the ingredients are chosen to obtain the desired effect on the physiological targets.
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
EXAMPLE I Assessment of the composition against inflammation Caco-2 cells are widely used to simulate intestinal cells in model culture. These cells spontaneously differentiate after confluence. This model can be also used as a good model for studying inflammation. In this specific test, inflammation is induced by addition of inflammatory compounds, such as H2O2 or some proinflammatory cytokines. Examination of the inflammation can be carried out by evaluation of the synthesis of some inflammatory markers by the Caco-2 cells. One of this marker is iNOS (inducible Nitric Oxide Synthase), which can be followed by the Western-blot test, with detection by specific antibodies. Different inflammatory agents were tested on Caco-2 cells in order to determine the best one to use in further experiments. Fig. 1 shows the results of iNOS western blot of samples with different inflammatory agents (treatments are: no compounds, IFN-γ : Interferon gamma 1000 U/ml, CMl: Cytomix 1 (IFN-γ, 1000 U/ml; IL-lβ, 17 nanograms/ml and TNF-α, 10 ng ml), CM2: Cytomix 2 (IFN-γ, 600 U/ml; IL-lβ, 10 nanograms/ml and TNF-α, 50 ng/ml), CM3: Cytomix 3 (IFN-γ, 200 U/ml; IL-lβ, 5 nanograms/ml and TNF-α, 100 ng/ml), H2O2/CMl, CMl with H202/ml). The highest production of iNOS is obtained with inflammation with cytomix 1 (CMl), a mixture of different cytokines (IFN-gamma, 1000 U/ml; IL-1 beta, 17 nanograms/ml and TNF-alpha, 10 ng/ml). EXAMPLE H Evaluation of different concentrations of lactoferrin
Caco-2 cells, inflamed with CMl were treated with different concentrations of a commercial lactoferrin. Results of western-blot and respective density of the iNOS spots are shown in Fig. 2(Effect of different concentrations of lactoferrin on iNOS expression by Caco-2 cells inflammed with CMl. Lactoferrin concentrations are indicated in micrograms per ml). Lactoferrin decreases the synthesis of iNOS at 250, 500 and 750 micrograms per ml. A dose of 750 micrograms per ml was chosen for the other experiments.
EXAMPLE IH Combination of lactoferrin with chlorella
Caco-2 cells, inflamed with CMl were treated with a commercial algae extract (chlorella), lactoferrin, and a mixture of both at the same concentrations.
• The results shown in Fig. 3 (Effect of the algae extract (chlorella) at 750 micrograms per ml, lactoferrin at 750 micrograms per ml and mix of both at the same concentrations . on iNOS expression by Caco-2 cells inflamed with CMl), demonstrate that the combined effect of chlorella and lactoferrin together significantly decreases iNOS in a higher level than if compounds are separately tested, and that the sum of the separate effects of chlorella and lactoferrin is lower than the effect of the compounds as a whole, indicating a synergistic effect between lactoferrin and chlorella. EXAMPLE IV Combination of lactoferrin with green tea extract
Caco-2 cells, inflamed with CMl were treated with a commercial green . tea extract at different concentrations, lactoferrin, and a mixture of both at 5 concentrations where green tea has an anti-inflammatory effect. Results shown in Fig. 4 (Effect of the green tea extract (TV) at different concentrations, lactoferrin at 750 micrograms per ml and mix of lactoferrin at 750 micrograms per ml and green tea extract at 100 micrograms per ml on iNOS expression by Caco-2 cells inflamed with CMl), demonstrate that the effect of green tea at 100 micrograms per ml and l o lactoferrin together decrease iNOS in a higher level than the individual compounds at the same concentrations, and that the algebrical sum of the separate effects of green tea at 100 micrograms per ml and lactoferrin is lower than the effect of the compounds together, indicating a synergistic effect between lactoferrin and green tea extract.
15
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention 20 and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth,' and as follows in the scope of the appended claims.

Claims

WE CLAIM:
1. A biocompatible composition for modulating free radicals quantity or related damages in a human or an animal cell, tissue, or biological fluid comprising a polymetal-binding protein and plant or cyanobacterial extract having antioxidant activity.
2. The biocompatible composition of claim 1, wherein said polymetalbinding protein is lactoferrin, or a related protein, or derivatives or fragments thereof,
3. The biocompatible composition of claim l,which increases or reduces the quantity of free radicals.
4. The biocompatible composition of claim 1, wherein said modulating is increasing or reducing damages induced by said free radicals.
5. The biocompatible composition, of claim 1, wherein said polymetalbinding protein is capable of modulating .free radical quantity by binding at least one metal ion selected from the group consisting of iron, magnesium, copper, zinc, and manganese.
6. The biocompatible composition of claim 1, wherein said polymetalbinding protein is at a concentration between 50 to 1500 mg/dose or in concentration allowing the composition to act on the targeted metabolic or physiologic reaction..
7. The biocompatible composition of claim 1, wherein said plant or cyanobacterial extract is raw or refined,
8. The biocompatible composition of claim 1, which is a nutraceutical, a therapeutical, or a cosmeceutical composition.
9. The biocompatible composition of claim 1, wherein said plant or cyanobacterial extracts contain chromophores.
5 10. A method of alleviating or preventing inflammation in a human or animal comprising administering to said human or animal a combination of poly- metal binding protein and plant or cyanobacteria extract.
11. • A method as defined in claim 10, wherein said combination* causes reduction of free radicals quantity.
10.
12. A method as defined in claim 10, wherein said inflammation is partly or totally, due to free radicals.
13. Use of poly-metal protein combined with at least one of plant or cyanobacteria extract in the preparation of a composition for modulating free radical quantity or free radical related damages in body cells, tissues, or fluids.
15 14. The use of claim 13, wherein said modulation is reducing the quantity of free radicals in body cells, tissues, or fluids.
15. The use of claim 13, wherein said modulating is reducing the damages induced by said free radicals iii body cells, tissues, or fluids.
16. The use of claim 13, wherein said body is human or animal body.
17. A composition for modulating a metabolic body reaction in a human or an animal comprising a polymetal-binding protein and plant or cyanobacterial extract having antioxidant activity.
18. The composition of claim 17, wherein said metabolic reaction involves action of free radicals.
19. Use of poly-metal protein combined with ' at least one of plant or cyanobacteria extract in the preparation of a composition for alleviating or treating inflammation in a human or an animal.
PCT/CA2004/000445 2003-03-25 2004-03-24 Compositions comprising polymetal-binding proteins and plant extracts for reducing free radicals WO2005016372A1 (en)

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