Classifications

• • 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
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/175—Amino acids
• • 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
  • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
  • A61K31/05—Phenols
• • 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
  • A61K31/12—Ketones
• • 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
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
  • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
  • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
  • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/401—Proline; Derivatives thereof, e.g. captopril
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/01—Hydrolysed proteins; Derivatives thereof
  • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
  • A61K38/014—Hydrolysed proteins; Derivatives thereof from animals from connective tissue peptides, e.g. gelatin, collagen
• • 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
  • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

• the present invention relates to a composition comprising curcuminoid with green tea polyphenol or with a combination of glycine, proline and hydroxyproline for use in preventing or treating osteoarthritis. It also relates to a method of preventing or treating osteoarthritis in mammals, the method comprising administering to said mammal a composition which comprises curcuminoid with green tea polyphenol or with a combination of glycine, proline and hydroxyproline.
• Cartilage deterioration can be caused by several reasons such as repeated exercise, instability of the joint, etc., which may result in inflammation of the joints. While a greater portion of humans with arthritis have rheumatoid arthritis, most of the arthritis occurring in companion animals is osteoarthritis.
• the first aspect of this invention relates to a composition comprising curcuminoid with green tea polyphenol or with a combination of glycine, proline and hydroxyproline for use in preventing or treating osteoarthritis. Treating osteoarthritis includes ameliorating osteoarthritis symptoms.
• the present invention relates, for all aspects, to any mammal, including a human.
• the present invention relates to a companion animal such as a dog, a cat or an equine animal (e.g. a horse) or any other such animal that suffers or is prone to suffer from osteoarthritis.
• composition of the present invention comprises curcuminoid.
• Curcuminoid is curcumin or a derivative of curcumin.
• the chemical structures of curcuminoids differ in their functional groups.
• Curcuminoid includes curcumin, demethoxycurcumin, b/ ' s-methoxycurcumin and/or tetrahydrocurcumin.
• Curcuminoids are natural phenols that are present, in particular, in the Indian spice turmeric. Turmeric is derived from the roots of the plant Curcuma longa. Curcuminoids have also been found in roots of other species in the plant family Zingiberaceae of the Curcuma genus. Curcuminoids have a distinctly earthy, bitter, peppery flavour and a mustardy smell.
• turmeric contains 60-80% curcumin, 15-30% demethoxycurcumin and 2-6% b/ ' s-demethoxycurcumin.
• the curcuminoid in the composition of the invention can be of any format, including a powder or lipid extract.
• curcuminoid can be mixed with phospholipids and/or cellulose, starch or derivatives thereof to form complexes. This may assist in stability and/or to further increase solubility and bioavailability of the curcuminoid.
• the curcuminoid can be mixed with essential oils, piperine or bromelain.
• the curcuminoid can be mixed with phosphatidycholine, for example lecithin.
• the curcuminoid of the present invention is curcumin, which is the most active curcuminoid.
• Curcumin according to the present invention includes demethoxycurcumin, b/ ' s-demethoxycurcumin and/or tetrahydrocurcumin.
• composition of the invention comprises curcuminoid and green tea polyphenol.
• Catechins in tea include epigallocatechin-3-gallate (EGCG), epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin (EGC), catechin, and gallocatechin (GC).
• EGCG epigallocatechin-3-gallate
• ECG epicatechin
• ECG epicatechin-3-gallate
• ECG epigallocatechin
• GC gallocatechin
• the green tea polyphenols include catechin.
• the catechin includes EGCG.
• Green tea extract usually contains at least about 25% polyphenols, about 12.5% of catechins and about 9.3% of EGCG.
• Epigallocatechin gallate is the ester of epigallocatechin and gallic acid. EGCG is the most abundant catechin in tea and is a potent antioxidant. It is particularly found in green tea. EGCG is a major polyphenol of green tea and exhibits anti-oxidant, anti-tumour and anti-mutagenic activities.
• composition of the invention comprises curcumin and a combination of glycine, proline and hydroxyproline.
• a combination of glycine, proline and hydroxyproline represents 50% of the total amino acid content of hydrolyzed collagen.
• a combination of glycine, proline and hydroxyproline is hydrolyzed collagen.
• the amino acid composition of hydrolyzed collagen is as set in the table below;
• Hydrolyzed collagen is obtained by the enzymatic hydrolysis of collagenous tissues found in the bones, skin, and connective tissue of animals such as cattle, fish, horses, pigs, and rabbits. Hydrolyzed collagen is well digested and is preferentially accumulated in cartilage.
• a preferred composition includes curcuminoid, green tea polyphenol and a combination of glycine, proline and hydroxyproline. Preferably, this composition includes curcumin, green tea polyphenol and hydrolyzed collagen.
• the invention is preferably a foodstuff. It can be any foodstuff, such as dry, semi moist or wet food product. In particular, the foodstuff may be a pet food product.
• the pet foodstuff is preferably a commercial pet food product.
• a product is preferably sold as a product for feeding to a pet animal, in particular a pet cat or a pet dog.
• a typical pet foodstuff contains about 20-30% crude protein and about 10-20% fat, the remainder being carbohydrate, including dietary fibre and ash.
• a typical wet or moist product contains (on a dry matter basis) about 40% fat, 50% protein and the remainder being fibre and ash.
• the foodstuff of the invention may be a dry product (with approximately 5 to approximately 15% moisture), a semi-moist product (with approximately 15 to approximately 70% moisture) or a wet product (with approximately 70 to approximately 90% moisture).
• the remaining components of the foodstuff are not essential to the invention and typical standard products can be included.
• the combined ingredients of the foodstuff according to the invention can provide all of the recommended vitamins and minerals for the particular animal in question (a complete and balanced food).
• the foodstuff according to the present invention encompasses any product which a pet consumes in its diet.
• the invention covers standard food products including liquids, as well as pet food snacks (for example, snack bars, pet chew, crunchy treat, cereal bars, snacks, biscuits and sweet products) and supplements.
• the foodstuff can be provided as a food supplement.
• the food supplement can be a powder, sauce, topping, biscuit, kibble, pocket or tablet that can be administered with or without an additional foodstuff. Where the food supplement is administered with an additional foodstuff, the food supplement can be administered sequentially simultaneously or separately.
• the food supplement may be mixed with the foodstuff, sprinkled over the foodstuff or served separately. Alternatively, the food supplement can be added to a liquid provided for drinking such as water or milk.
• the foodstuff is preferably a cooked product. It may incorporate meat or animal derived material (such as beef, chicken, turkey, lamb, fish, blood plasma, marrow bone etc. or one or more thereof).
• the product alternatively may be meat free (preferably including a meat substitute such as soya, maize gluten or a soya product) in order to provide a protein source.
• the foodstuff may contain additional protein sources such as soya protein concentrate, milk proteins, gluten etc.
• the foodstuff may also contain a starch source such as one or more grains (e.g. wheat, corn, rice, oats, barley etc.), or may be starch free.
• the foodstuff of the invention is preferably produced as a dry product containing from approximately 5% to approximately 15% moisture.
• the preferred dry food is more preferably presented as a small biscuit - like kibbles.
• composition in the first aspect of the invention may comprise curcuminoid at an amount ranging from about 0.005 to 1.1 % by weight of curcuminoid on an "as is" weight percent of the food.
• the amount of curcuminoid can be any amount from 0.005 to 1.1 % (as is).
• the amount of curcuminoid can be any amount from 0.1 to 1 % (as is).
• the amount of curcuminoid can be any amount from 0.1 to 0.6% (as is).
• the amount of curcuminoid can be any amount from 0.3 to 0.6% (as is).
• composition in the first aspect of the invention may comprise curcuminoid at an amount ranging from about 0.005 to 0.15% by weight of curcuminoid on an "as is" weight percent of the food.
• the amount of curcuminoid can be any amount from 0.005 to 0.15% (as is) (7 to 99 mg/400 kcal).
• the amount of curcuminoid in the composition ranges from about 0.01 to 0.07% (as is) (14 to 46 mg/400 kcal). Most preferably, the amount of curcuminoid is 0.035% (as is) (36 mg/400 kcal).
• the curcuminoid in the composition is curcumin at an amount ranging from about 0.005 to 0.15% by weight of curcumin on an "as is" weight percent of the food.
• the amount of curcumin can be any amount from 0.005 to 0.15% (as is) (7 to 99 mg/400 kcal).
• the amount of curcumin ranges from about 0.01 to 0.05% (as is) (14 to 32 mg/400 kcal).
• the amount of curcumin is 0.026% (as is) (27 mg/400 kcal).
• the composition in the first aspect of the invention may comprise green tea polyphenol in an amount ranging from about 0.01 to 1.1 % by weight of green tea polyphenol on an "as is" weight percent of the food.
• the amount of green tea polyphenol can be any amount from 0.01 to 1 .1 % (as is).
• the amount of green tea polyphenol can be any amount from 0.1 to 1 % (as is).
• the amount of green tea polyphenol can be any amount from 0.1 to 0.6% (as is).
• the amount of green tea polyphenol can be any amount from 0.3 to 0.6% (as is).
• the composition in the first aspect of the invention may comprise green tea polyphenol in an amount ranging from about 0.01 to 0.3 % by weight of green tea polyphenol on an "as is" weight percent of the food.
• the amount of green tea polyphenol can be any amount from 0.01 to 0.3 % (as is) (14 to 197 mg/400 kcal). Preferably, the amount of green tea polyphenol ranges from about 0.03 to 0.17% (as is) (43 to 1 13 mg/400 kcal). Most preferably, the amount of green tea polyphenol is 0.085% (as is) (87 mg/400 kcal).
• the green tea polyphenol is EGCG at an amount ranging from about 0.005 to 0.2% by weight of EGCG on an "as is" weight percent of the food (7 to 131 mg/400 kcal).
• the amount of EGCG can be any amount from 0.01 to 0.06% (as is) (14 to 39 mg/400 kcal). Most preferably, the amount of EGCG is 0.032% (as is) (33 mg/400 kcal).
• the composition in the first aspect of the invention may comprise a combination of glycine, proline and hydroxyproline in an amount ranging from about 0.5 to 10% by weight of combined glycine, proline and hydroxyproline on an "as is" weight percent of the food.
• the amount of combined glycine, proline and hydroxyproline can be any amount from 0.5 to 10% (as is) (720 to 6591 mg/400 kcal).
• the amount of combined glycine, proline and hydroxyproline ranges from about 1.2 to 5% (as is) (1736 to 3295 mg/400 kcal).
• the amount of combined glycine, proline and hydroxyproline is 2.7% (as is) (2780 mg/400 kcal).
• the combination of glycine, proline and hydroxyproline is hydrolyzed collagen in an amount ranging from about 0.5 to 5% by weight of combined glycine, proline and hydroxyproline on an "as is" weight percent of the food.
• the amount of hydrolyzed collagen can be any amount from 0.5 to 5% (as is) (720 to 3295 mg/400 kcal).
• the amount of hydrolyzed collagen ranges from about 0.7 to 3.2% (as is) (1016 to 2138 mg/400 kcal).
• the amount of hydrolyzed collagen is 1.7% (as is) (1750 mg/400 kcal).
• the composition may comprise curcumin in an amount of about 27 mg/400 kcal (35 mg/400 kcal of curcuminoids) with about 87 mg/400 kcal of green tea polyphenol and with about 2757 mg/400 kcal of combined glycine, proline and hydroxyproline, wherein the combination of glycine, proline and hydroxyproline is hydrolyzed collagen.
• the combination of glycine, proline and hydroxyproline is hydrolyzed collagen and is present in the composition at an amount of about 1747mg/400kcal.
• the composition may comprise curcumin in an amount of about 33 mg/400 kcal (43mg/400 kcal of curcuminoids) with about 106 mg/400 kcal of green tea polyphenol and with about 3295 mg/400 kcal of combined glycine, proline and hydroxyproline, wherein the combination of glycine, proline and hydroxyproline is hydrolyzed collagen.
• the combination of glycine, proline and hydroxyproline is hydrolyzed collagen and is present in the composition at an amount of about 2108mg/400kcal.
• the composition may comprise curcumin in an amount of about 15 mg/400 kcal (22mg/400 kcal of curcuminoids) with about 51 mg/400 kcal of green tea polyphenol and with about 1736 mg/400 kcal of combined glycine, proline and hydroxyproline, wherein the combination of glycine, proline and hydroxyproline is hydrolyzed collagen.
• the combination of glycine, proline and hydroxyproline is hydrolyzed collagen and is present in the composition at an amount of about 1016mg/400kcal.
• compositions for feeding to a mammal, in particular a companion animal apply to a composition for feeding to a mammal, in particular a companion animal.
• the second aspect of the invention relates to a method of preventing or treating osteoarthritis in mammals.
• Osteoarthritis is a degenerative and inflammatory condition that affects the joints in mammals. It is also known as degenerative arthritis or degenerative joint disease. Osteoarthritis is a group of abnormalities involving degradation of joints, including articular cartilage and sub-chondral bone.
• Osteoarthritis is the consequence of an imbalance of catabolism and anabolism, wherein catabolism is increased; anabolism is decreased causing the inflammation of chondrocytes.
• Chondrocytes are the only cells found in healthy cartilage. They produce and maintain the cartilaginous matrix, which consists mainly of collagen and proteoglycans.
• the composition of the invention has demonstrated to provide, inter alia, a decrease in inflammation, a decrease in catabolism and an increase in anabolism in in vitro inflammation-induced chondrocytes and in in vitro healthy chondrocytes.
• the composition of the invention prevents and/or treats osteoarthritis in animals.
• the present invention relates, for all aspects, to any mammal, including a human.
• the present invention relates to a companion animal such as a dog, a cat or an equine animal (e.g. a horse) or any other such animal that suffers or is prone to suffer from osteoarthritis.
• the second aspect of the invention provides a method for preventing and treating osteoarthritis in mammals, including ameliorating the symptoms of osteoarthritis, in particular companion animals.
• the method comprises administering to said animal a composition which comprises curcumin with green tea polyphenol or with a combination of glycine, proline and hydroxyproline.
• the animal may be in need thereof. Since a significant number of dogs suffer from osteoarthritis in their lifetime, all dogs can be considered as in need of prevention.
• the method comprises administering to said animal a composition comprising curcumin, green tea polyphenol and a combination of glycine, proline and hydroxyproline.
• a composition comprising curcumin, green tea polyphenol and a combination of glycine, proline and hydroxyproline.
• the combination of glycine, proline and hydroxyproline is hydrolysed collagen.
• the method is preferably administered to an animal, in particular a companion animal, that suffers from osteoarthritis and is in need of ameliorating the symptoms of osteoarthritis or in need of preventing further symptoms of osteoarthritis or in need of treatment of osteoarthritis.
• This may be to, for example a young pet animal, such as a puppy, or an older companion animal.
• the foodstuff may be administered in a dietary regime in accordance with the usual dietary regime of the companion animal.
• the foodstuff may comprise 100% of the diet of the companion animal or a lesser proportion, depending on the level of prevention or treatment required.
• the foodstuff allows the composition to be administered with ease thus avoiding a need to supplement the companion animal's food.
• the foodstuff can be administered by the animal's owner thus avoiding constant veterinary supervision.
• the foodstuff may be available at any outlet selling pet food products or may be available from a veterinarian.
• the foodstuff may be as described above according to the first aspect of the invention.
• administration also includes feeding or any other method of oral administration.
• Other means of administration may include tablets, capsules, injection, suppositories or any other suitable means.
• the present description includes a method for preparing the composition of the first aspect of the invention.
• the foodstuff can be made according to any method known in the art such as in Waltham Book of Dog and Cat Nutrition, Ed. ATB Edney, Chapter by A. Rainbird, entitled “A Balanced Diet” in pages 57 to 74 Pergamon Press Oxford.
• a process for the manufacture of a foodstuff as defined herein comprises mixing together ingredients with the composition comprising curcuminoid with green tea polyphenol or with a combination of glycine, proline and hydroxyproline and forming a foodstuff, in particular a pet foodstuff. Heating/cooking may be applied to any one or more of the ingredients prior to, during or following the mixing.
• composition can be sprayed onto the foodstuff, mixed in with the foodstuff or incorporated into the foodstuff in a matrix. Methods of inclusion of the composition are known in the art.
• the importance of the present invention is the beneficial properties of curcuminoid with either green tea polyphenol or with a combination of glycine, proline and hydroxyproline (optionally as hydrolyzed collagen). In particular, an effect which is more than the cumulative effect is seen.
• a further benefit is seen with the triple combination of ingredients of: curcuminoid, green tea polyphenol and glycine, proline and hydroxyproline (optionally as hydrolyzed collagen).
• the combination of the compounds of the composition of the present invention can provide a synergistic effect in terms of one or more of decreasing inflammation, decreasing catabolism and increasing anabolism.
• Example 1 Individual screening of compounds
• DMEM Dulbecco's Modified Eagle Medium
• HEPES N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)
• penicillin 100 U/ml
• streptomycin 0.1 mg/ml
• chondrocytes were released from cartilage by sequential enzymatic digestions with 0.5 mg/ml hyaluronidase type IV S (Sigma-Aldrich, Bornem, Belgium) for 30 min at 37 °C, 1 mg/ml pronase E (Merck, Leuven, Belgium) for 1 h at 37 °C and 0.5 mg/ml clostridial collagenase IA (Sigma-Aldrich, Bornem, Belgium) for 16 to 20 h at 37 °C.
• hyaluronidase type IV S Sigma-Aldrich, Bornem, Belgium
• pronase E Merck, Leuven, Belgium
• IA clostridial collagenase IA
• the enzymatically isolated cells were then filtered through a nylon mesh (70 ⁇ ), washed three times, counted and filled to the density of 0.25 x 106 cells/ml of DMEM (with phenol red and 4.5 g/L glucose) supplemented with 10 % foetal bovine serum, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine (all from Lonza, Verviers, Belgium) and 20 ⁇ g/ml proline (Sigma-Aldrich, Bornem, Belgium).
• chondrocytes were seeded in a 6-well plate at 0.5 X 106 cells/well by adding 2 ml of the previously described culture medium/well and cultured in monolayer for 5 days. Chondrocytes were then cultured in monolayer until confluence (for about 2 days) in DMEM (phenol red-free and containing only 1 g/L glucose) (Lonza, Verviers, Belgium) supplemented with 1 % fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine and 20 ⁇ g/ml proline. Only primary cultures were used to ensure the stability of chondrocyte phenotype.
• the culture medium was removed and replaced by fresh culture medium (DMEM phenol red-free and containing only 1 g/L glucose supplemented with 1 % fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine and 20 ⁇ g/ml proline) containing some nutraceuticals (12.5 ⁇ g/ml of each of them) and in the absence or in the presence of recombinant porcine I L-1 ⁇ (10 "11 M) (RD System, Abingdon, UK).
• fresh culture medium DMEM phenol red-free and containing only 1 g/L glucose supplemented with 1 % fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine and 20 ⁇ g/ml proline
• the anti-inflammatory power of the compounds was tested by measuring the chondrocyte viability and the production of PGE2 and NO.
• the compounds were added in the culture medium either before inflammation (prevention effect measurement), either simultaneously of the inflammation (treatment effect measurement).
• Green tea extract 25% polyphenols of which 12.5 % are catechines and 8% is EGCG: (Naturex)
• Curcuma powder 85% curcuminoids (Naturex)
• example 1 The methodology of example 1 was followed. Four different concentrations were tested to cover the range of concentrations corresponding to 10 "5 M, depending on the molecular weight: 0.5 ⁇ g ml, 2.5 ⁇ g ml, 12.5 ⁇ g ml and 62.5 ⁇ g ml.
• Example 3 Testing particular combinations of the compounds and the synergistic effects The method of example 1 was followed. Supplementation with compounds
• the culture medium was removed and replaced by fresh culture medium (DMEM phenol red-free and containing only 1 g/L glucose supplemented with 1 % fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine and 20 ⁇ g ml proline) containing some compounds (12.5 ⁇ g ml of each of them) and in the absence or in the presence of recombinant porcine I L-1 ⁇ (10 "11 M) (RD System, Abingdon, UK).
• fresh culture medium DMEM phenol red-free and containing only 1 g/L glucose supplemented with 1 % fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine and 20 ⁇ g ml proline
• curcuma extract (Naturex, Avumble, France), hydrolysate collagen (Gelita, Eberbach, Germany) and green tea extract (Naturex, Avumble, France).
• Curcuma extract was prepared as a 12.5 mg/ml solution in tetrahydrofuran (Merck, Leuven, Belgium) and then further diluted 1000 times in cell culture medium.
• Hydrolysate collagen and green tea extract were dissolved in water at the concentration of 12.5 mg/ml, filtered through a sterile mesh (0.20 ⁇ ) and then further diluted 1000 times in cell culture medium.
• the compounds were tested alone at the final concentration of 12.5 ⁇ g/ml or in combination (12.5 ⁇ g/ml curcuma extract + 12,5 ⁇ g/ml hydrolysate collagen; 12.5 ⁇ g/ml curcuma extract + 12.5 ⁇ g/ml green tea extract; 12.5 ⁇ g/ml curcuma extract + 12.5 ⁇ g/ml hydrolysate collagen + 12.5 ⁇ g/ml green tea extract) in the absence or in the presence of recombinant porcine I L-1 ⁇ (10 "11 M).
• the effects of the compounds were compared to controls: DMEM alone or DMEM + I L-1 ⁇ .
• conditioned culture medium of three wells of each condition was collected and stored at -20°C.
• the cells of these corresponding wells were scrapped, an RNA extraction was made using RNeasy mini kit (Qiagen, Venlo, Netherlands), a reverse transcriptase polymerase chain reaction was realised and then a quantitative real time polymerase chain reaction was realised, using the LightCycler 480 (Roche, Vilvoorde, Belgium) to analyse gene expression.
• conditioned culture medium of the remaining wells (3 of each condition) was collected (lactate dehydrogenase release assay) and stored at -20°C until analysis (nitrite and prostaglandin E2 assays).
• Cells were scrapped and homogenized in 500 ⁇ of Tris-HCI buffer by ultrasonic dissociation for 20 s at 4 °C, to measure DNA content.
• LDH lactate dehydrogenase
• Chondrocytes were homogenized in 500 ⁇ of Tris-HCI buffer by ultrasonic dissociation for 15 s at 4 °C. DNA content was measured in the cell extracts using the fluorimetric method of Hoechst.
• Nitric oxide (NO) production was determined by quantifying its derived product, nitrite, in the culture supernatant using a spectrophotometric method based upon the Griess reaction. Briefly, 100 ⁇ of the supernatant or sodium nitrite (NaN02) standard dilutions were mixed with 100 ⁇ of Griess reagent (0.5 % sulphanilamide, 0.05 % naphtyl ethylene diamine dihydrochloride, 2.5 % H3P04). The absorption was measured at 540 nm. The production of NO was expressed per microgram of DNA.
• PGE2 Prostaglandin E2 production was measured in the culture supernatant using the DetectX PGE2 High Sensitivity Immunoassay kit (Arbor Assays, Michigan, USA). Briefly, 100 ⁇ of the supernatant or PGE2 standard dilutions were pipetted into a clear microtiter plate coated with an antibody to capture mouse IgG. A PGE2-peroxidase conjugate (25 ⁇ ) is added to the standards and supernatants in the wells. The binding reaction is initiated by the addition of 25 ⁇ of a monoclonal antibody to PGE2. After an overnight incubation at 4°C, the plate is washed and 100 ⁇ of substrate is added. The substrate reacts with the bound PGE2-peroxidase conjugate. After a short incubation, the reaction is stopped and the intensity of the generated colour is detected at 450 nm wavelength. The production of PGE2 was expressed per microgram of DNA.
• RT PCR Quantitative real-time reverse transcriptase polymerase chain reaction
• RNA from cells from 3 wells of each condition was isolated using RNeasy mini kit (Qiagen, Venlo, Netherlands). Then, RNA was reverse transcribed. Quantitative real time Polymerase Chain Reaction (PCR) was performed by using the SYBR Premix Ex Taq (Tli RNaseH Plus) (Westburg, Leusden, Netherlands). The PCR template source was either first-strand cDNA or purified DNA standard.
• Primer sequences used to amplify the desired cDNA were as follows: bovine HPRT forward and reverse primers: 5'- AGTTTGGAAATACCTGGCG-3' and 5'-AGTCTTTAGGCTCGTAGTGC-3'; bovine interleukin (IL)-6 forward and reverse primers: 5'- TGGTGATGACTTCTGCTTTCC-3' and 5'- TGCCAGTGTCTCCTTGC-3'; bovine cyclooxygenase (COX)2 forward and reverse primers: 5'-GTCTGATGATGTATGCCACC-3' and 5'-ACGTAGTCTTCAATCACAATCT-3'; bovine induced nitric oxide synthase (iNOS) forward and reverse primers: 5'- GGCAAGCACCACATTGAGA-3' and 5'- TGCGGCTGGATTTCGGA-3'; bovine aggrecans (AGG) forward and reverse primers: 5'-TGCCTTTGACGTGAGC-3' and 5'- GCATTGTT
• Amplification was performed with a spectrofluorometric thermal cycler (LightCycler 480, Roche Diagnostics, Vilvoorde, Belgium).
• HPRT a housekeeping gene, as an internal control. Gene expression was normalized by calculating the ratio between the number of cDNA copies of IL-6, COX2, iNOS, AGG, COL2, MMP-3, ADAMTS4, ADAMTS5, and that of HPRT.
• Results were expressed as the mean percentage of increase compared to the control. Statistical significance was assessed using the t-test. Differences were considered statistically significant at p ⁇ 0.05. Table below details the results provided when combining the compounds and the synergistic effects observed. Table 4

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