WO2015142700A1 - Composition et procédé destinés à atténuer la douleur articulaire au moyen de phospholipides et d'astaxanthine - Google Patents

Composition et procédé destinés à atténuer la douleur articulaire au moyen de phospholipides et d'astaxanthine Download PDF

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Publication number
WO2015142700A1
WO2015142700A1 PCT/US2015/020671 US2015020671W WO2015142700A1 WO 2015142700 A1 WO2015142700 A1 WO 2015142700A1 US 2015020671 W US2015020671 W US 2015020671W WO 2015142700 A1 WO2015142700 A1 WO 2015142700A1
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Prior art keywords
astaxanthin
phospholipid
dietary supplement
oil
supplement composition
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PCT/US2015/020671
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English (en)
Inventor
John Minatelli
Rudi Moerck
W. Stephen Hill
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U.S. Nutraceuticals, Llc D/B/A Valensa International
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Priority claimed from US14/217,515 external-priority patent/US9238043B2/en
Priority claimed from US14/645,890 external-priority patent/US9913810B2/en
Application filed by U.S. Nutraceuticals, Llc D/B/A Valensa International filed Critical U.S. Nutraceuticals, Llc D/B/A Valensa International
Priority to KR1020167028853A priority Critical patent/KR20160134787A/ko
Priority to DE212015000073.7U priority patent/DE212015000073U1/de
Publication of WO2015142700A1 publication Critical patent/WO2015142700A1/fr

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    • 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
    • A61K35/56—Materials from animals other than mammals
    • A61K35/60—Fish, e.g. seahorses; Fish eggs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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 COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • 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
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • 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
    • A61K35/56—Materials from animals other than mammals
    • A61K35/612—Crustaceans, e.g. crabs, lobsters, shrimps, krill or crayfish; Barnacles
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • This invention relates to treating and alleviating joint pain and symptoms of osteoarthritis and/or rheumatoid arthritis.
  • krill oil is disclosed in U.S. Patent Publication Nos. 2004/0234587; 2004/0241249; and 2007/0098808, the disclosures which are hereby incorporated by reference in their entirety.
  • the use of krilf oil is also disclosed in a research paper published by L. Deutsch entitled, "Evaluation of the Effect of Neptune Krill Oil on Chronic infiammation and Arthritic Symptoms," published in the Journal of the American College of Nutrition, Volume 26, No. 1 , 39-49 (2007), the disclosure which is hereby incorporated by reference in its entirety.
  • this krill and/or marine oil can be obtained by the combination of detailed steps as taught in the '808 application, by placing krill and/or marine material in a ketone solvent, separating the liquid and solid contents, recovering a first lipid rich fraction from the liquid contents by evaporation, placing the solid contents and organic solvent in an organic solvent of the type as taught in the specification, separating the liquid and solid contents, recovering a second lipid rich fraction by evaporation of the solvent from the liquid contents and recovering the solid contents.
  • the resultant krill oil extract has also been used in an attempt to decrease lipid profiles in patients with hyperlipidemia.
  • the '808 publication gives details regarding this krill oil as derived using those general steps identified above.
  • a dietary supplement composition is formulated in a therapeutic amount to treat and alleviate symptoms of joint pain in a person having joint pain.
  • the composition includes astaxanthin and microbial fermented, low molecular weight hyaluronic acid or sodium hyaiuronate (hyaluronan).
  • the composition also includes at least one of a phospholipid, glycolipid, and sphingolipid, and is formulated into an oral dosage form.
  • the astaxanthin is 0.1 to 15 percent by weight of the at least one phospholipid, glycolipid, and sphingolipid.
  • the astaxanthin is derived from a natural or synthetic ester or synthetic dioi and may include a pharmaceutical or food grade diluent, fn another example, the phospholipid comprises at least one of Phosphatidylcholine,
  • the phospholipid may be derived from at least one of a plant, algae and animaf source or synthetic derivatives thereof.
  • the composition includes 0.5 to 12 mg of astaxanthin and includes 50 to 500 mg of the at least one of the phospholipid, g!ycolipid and
  • the dietary supplement composition is formulated into a single dosage capsule in an example.
  • the composition includes pro-inflammatory low molecular weight microbial fermented sodium hyaluronate fragments having a molecular weight of 0.5 to 300 kiiodaltons (kDa).
  • a method to treat and alleviate symptoms of joint pain includes administering to a person having joint pain a therapeutic amount of a dietary supplement composition, including astaxanthin and at least one of a phosphoiipid, glycolipid, and sphingoiipid, and formulated into an oral dosage form.
  • the astaxanthin is 0.1 to 15 percent by weight of the at least one phospholipid, glycolipid and sphingolipid.
  • the composition may be formed by dispersing the astaxanthin under high shear conditions into the at least one phosphoiipid, glycolipid and sphingoiipid.
  • FIG. 1 is a view showing a chemical structure of astaxanthin that can be used in accordance with a non-limiting example.
  • the composition as related to the krill oii includes EPA and DHA functionalized as marine phospholipids and acyltriglycerides derived from krill.
  • the krill, algae, roe extract and fish oil derived product and phospholipid compositions may include astaxanthin, such as esterified astaxanthin, and in one non-limiting example, low molecular weight polymers of hyaluronic acid or sodium hyaluronate (hyaluronan) in an oral dosage form.
  • pro-inflammatory low molecular weight microbial fermented sodium hyaluronate having a molecular weight of between 0.5 to 300 kDa, in another example between 0.5 to 230 kDa, and in yet another example, between 0.5 to 100 kDa.
  • DHA Docosahexaenoid Acid
  • Astaxanthin, Vitamin A, Vitamin E > 1.25 can vary depending on application and persons,
  • the composition includes pro-inflammatory microbial fermented sodium hyaiuronate fragments having a molecular weight of 0.5 to 300 kiioda!tons (kDa), in an example, and 0.5 to 230 kDa, and 0.5 to 100 kDa, all in an oral dosage form.
  • Natural high molecular weight hyaluronic acid is the major hydrodynamic component of synovia! fluid and importantly is known to be immuno-neutral to the innate immune system.
  • LMWtHA low molecular weight hyaluronic acid
  • LMWtHA fragments exhibit potent proinflammatory behavior. It therefore remains unclear why a pro-inflammatory component would elicit a favorable overall response in inflamed joint tissues. It is believed that such pro-inflammatory LMWtHA fragments promote site repair by simulation of the innate immune system repair mechanism and by simulating production of non-immunogenic high molecular weight hyaluronic acid bringing the joint back to homeostasis. A great deal of work by leading immunologists is still attempting to unravel all the aspects of the complicated signaling processes associated with the innate immune system. Studies using large animal models of osteoarthritis have shown that mild immunogenic
  • Hyaluronic Acids with molecular weights within the range of 0.5-1.0 x 10 s Da (Dalton) were generally more effective in reducing indices of synovial inflammation and restoring the rheological properties of SF (visco-induction) than non-immunogenic HA's with molecular weights > 2.3 x 10 6 Da.
  • pro-inflammatory low molecular weight hyaluronic acid is around 300 kDa to about 320 kDa or less, with many skilled in the art
  • hyaluronic acid fragments induce expression of inflammatory genes and they are low molecular weight kDa.
  • Clinical trials by the inventors and their assignee have shown the effectiveness of the composition when using krill oil, together with the low molecular weight hyaiuronic acid or hyaluronan and astaxanthin in accordance with a non-limiting example. In the clinical trials, no rescue medication was allowed as compared to the Deutsch study referenced above.
  • the low molecular weight hyaluronic acid had a molecular weight of about 40 kDa in the trial, but could range from 0.5 to 100 kDa in an example, or 0.5 to 230 kDa, or 0.5 to 300 kDa in yet other examples.
  • compositions and method used in the clinical trials of the current subject matter were directed to treating and alleviating joint pain.
  • the clinical subjects in the clinical trial did not have any confirmed osteoarthritis and/or rheumatoid arthritis.
  • the clinical study was directed to patients that have a non-disease state joint pain that is not associated with a disease state such as osteoarthritis and/or rheumatoid arthritis.
  • the composition was used as a supplement to treat and alleviate symptoms of joint pain of unknown etiology, including joint pain not associated with osteoarthritis and/or rheumatoid arthritis in this example.
  • Astaxanthin is a component of the composition.
  • the clinical trials of the joint care composition with the krill oil, low molecular weight hyaluronic acid and astaxanthin proved the effectiveness of the composition with surprising beneficial results.
  • Related scientific literature indicates that in a lipopoiysaccharide induced inflammatory rat model, astaxanthin at just 1 mg/kg in vitro and in vivo: (1) down regulates TNF-alpha production by 75%; (2) down regulates prostaglandin E-2 production (PGE-2) by 75%; (3) inhibits nitric oxide synthase (NOS) expression of nitric oxide by 58%; and (4) these effects on inflammatory markers were nearly as effective as prednisolone in this model.
  • PGE-2 prostaglandin E-2 production
  • NOS nitric oxide synthase
  • FIG. 1 shows an example of the astaxanthin as astaxanthin 3S, 3'S (3, 3'-dihydroxy-4, 4'-diketo-P-carotene).
  • the clinical trial of 15 mg astaxanthin alone is noted as beneficial.
  • Phospholipids may include plant based phospholipids such as from lecithin and lysophospholipids and/or giycophospholipids, including periila oil such as described in commonly assigned U.S. Patent No. 8,784,904, the disclosure which is hereby incorporated by reference in its entirety. Astaxanthin levels could very from 0.5-2 mg and 0.5-4 mg and in one embodiment is 2-4 mg or 2-6 mg and as broad as 0.5-12 mg and 7-12 mg.
  • astaxanthin In induced uveitis, astaxanthin also showed dose dependant ocular antiinflammatory activity by suppression of NO, PGE-2 and TNF-Alpha by directly blocking NO synthase activity. Astaxanthin is also known to reduce C-Reactive Protein (C-RP) blood levels in vivo. For example, in human subjects with high risk levels of C-RP three months of astaxanthin treatment resulted in 43% of patients serum C-RP levels to drop below the risk level. This may explain why C-RP levels dropped significantly in the Deutsch study identified above.
  • C-RP C-Reactive Protein
  • Astaxanthin is so powerful that it has been shown to negate the pro-oxidant activity of Vioxx, a COX-2 inhibitor belonging to the NSAIDS drug class which is known to cause cellular membrane lipid peroxidation leading to heart attack and stroke. For this reason Vioxx was removed from the US market. Astaxanthin is absorbed in vitro by iens epithelial cells where it suppresses UVB induced lipid peroxidative mediated ceil damage at umol/L concentrations. In human trials astaxanthin at 4mgs/day prevented post exercise joint fatigue following strenuous knee exercise when compared to untreated subjects. These results have been shown in:
  • a composition in one embodiment includes 300 mg of krill oil, 30 to 45 mg of low molecular weight hyaluronic acid, and 2 mg astaxanthin. It has now been found that 150 mg to 300 mg of krill oil is beneficial with one embodiment using 150 mg.
  • the astaxanthin can range from 0.5 to 2 mg, 2 to 4 mg, 0.5 to 6 mg, 0.5 to 8 mg, 0.5 to 10 mg, 0.5 to 12 mg, and 7 to 12 mg.
  • the use of added phospholipids and/or surfactants described below will aid in delivery of the astaxanthin.
  • the !ow molecular weight hyaluronic acid can vary from 10 to 70 mg, from 20 to 60 mg, from 25 to 50 mg, with one embodiment having 45 mg, and in another embodiment about 30 mg.
  • Astaxanthin has potent singlet oxygen quenching activity. Astaxanthin typically does not exhibit pro-oxidant activity unlike ⁇ -carotene, lutein, zeaxanthin and Vitamins A and E. Astaxanthin in some studies has been found to be about 50 times more powerful than Vitamin E, 1 times more powerful than ⁇ -carotene and three times more powerful than lutein in quenching of singlet oxygen. Astaxanthin is also well known for its ability to quench free radicals. Comparative studies have found astaxanthin to be 65 times more powerful than Vitamin C, 54 times more powerful than ⁇ -carotene, 47 times more powerful than lutein, and 14 times more powerful than Vitamin E in free radical quenching ability.
  • U.S. Patent No. 5,527,533 (the Tso patent), the disclosure which is hereby incorporated by reference in its entirety, discloses the benefits of astaxanthin for retarding and ameliorating central nervous system and eye damage. Astaxanthin crosses the blood-brain-retina barrier and this can be measured by direct measurement of retinal astaxanthin concentrations. Thus, Tso demonstrated protection from photon induced damage of photo-receptors, ganglion and neuronal cell damage.
  • HA binds to the surface of dendritic cells ("DCs") and stimulated T-ceils. Blockade of the CD44-HA interaction leads to impaired T-Cell activation both in vitro and in vivo. Studies have shown that in cancer cell lines,
  • LMWtHA fragments specifically induce nitric oxide synthase in dendritic cells.
  • DCs DCs
  • DCs are essential T-cell activators which function by presenting antigens to T-cells, thus apoptosis of DCs may short circuit the adaptive immune system response.
  • This effect was clearly CD44 dependent because pretreatment of DCs with anti-CD44 monoclonal antibodies blocked the NO mediated induction of DC apoptosis.
  • low molecular weight HA fragments interrupt the normal course of the well known T-cell mediated adaptive immune system response.
  • CD44 is a glycoprotein responsible in part for lymphocyte activation (also known as T-cell activation) and is known to specifically bind to HA.
  • low molecular weight HA fragments appear to up- regulate the innate immune response, particularly in chronic inflammatory conditions where the innate immune system may in some way be compromised.
  • krili oil is typically produced from Antarctic krill (euphausia superba), which is a zooplankton (base of food chain). It is one of the most abundant marine biomass of about 500 million tons according to some estimates. Antarctic krill breeds in the pure uncontaminated deep sea waters. It is a non-exploited marine biomass and the catch per year is less than or equal to about 0.02% according to some estimates. Because kri!l is harvested in large amounts and world supply of krili is being depleted, substitutes for krill such as other marine based oils, including algae based oils, are now being studied, developed and used.
  • krill oil and some other marine based and plant based oils have an oil based phospholipid bound EPA and DHA uptake into cellular membranes that is far more efficient than triacyigiyercide bound EPA and DHA, since liver conversion of triacylglycerides is itself inefficient and because phospholipid bound EPA and DHA can be transported into the blood stream via the lympathic system, thus, avoiding liver breakdown.
  • krill, algae and some marine and plant based oil consumption does not produce the burp-back observed with fish oil based products. Because of this burp-back feature of fish oils, it has been found that approximately 50% of all consumers who try fish oil never buy it again.
  • Some algae based oils have EPA conjugated with phospholipid and glycoiipid polar lipids, making the EPA uptake even more efficient.
  • Oral LD 50 600 mg/kg (rats);
  • NOAEL 465 mg/kg (rats); or
  • astaxanthin has three prime sources: 3 mg astaxanthin per 240 g serving of non-farmed raised salmon or a 1 % to 12% astaxanthin oleoresin or .5-2.5% beadlet derived from microalgae. Further verification is reflected in Lee et al., Molecules and Cells 6(1): 97-105, 2003; Ohgami et al., Investigative Ophthalmology and Visual Science 44(6): 2694-2701 , 2003; SpiHer et al., J. of the American College of Nutrition 21 (5): October 2002; and Fry et al., University of Memphis, Human Performance Laboratories, 2001 and 2004, Reports 1 and 2.
  • the current composition has krill, aigae, fish oil derived, roe, seed oil, or other phospholipid ingredients in combination with astaxanthin and low molecular weight polymers of hyaluronic acid or sodium hyaluronate in preferably an oral dosage form for the control of joint pain range of motion and stiffness. It should be understood that different proportions of the composition components and their percentages can be used depending on end use applications and other environmental and physiological factors when treating a patient.
  • the composition and method treats and alleviates symptoms of non-disease state joint pain and may be used to treat and alleviate symptoms of osteoarthritis and/or rheumatoid arthritis in a patient by administering a therapeutic amount of the composition, including the krill oil or other aigae based oil, fish oil derived product, roe, and other phospholipid materials in combination with astaxanthin and low molecular weight polymers of hyaluronic acid or sodium hyaluronate (hyaluronan) in an oral dosage form, preferably the low molecular weight polymers.
  • a therapeutic amount of the composition including the krill oil or other aigae based oil, fish oil derived product, roe, and other phospholipid materials in combination with astaxanthin and low molecular weight polymers of hyaluronic acid or sodium hyaluronate (hyaluronan) in an oral dosage form, preferably the low molecular weight polymers.
  • the krill oil alone is derived from Euphasia spp., comprising Eicosapentaenoic (EPA) and Docosahexaenoic (DHA) fatty acids in the form of triacylglycerides and phospholipids, although not less than 1% EPA and 5% DHA has been found advantageous.
  • EPA Eicosapentaenoic
  • DHA Docosahexaenoic
  • the kriil oil includes at least 15% EPA and 9% DHA, of which not less than 45% are in the form of phosphoiipids, and in another example 40%.
  • the composition can be delivered advantageously for therapeutic results with 1 -4000 mg of oil, such as krill or algae based oil, delivered per daily dose.
  • 500 mg is a preferred amount for a single capsule dosage, and in another example 1 ,000 mg.
  • 0.1-50 mg astaxanthin are supplemented to the oil per daily dose, but a preferred amount is about 2-4 mg and 0.5 to 12 mg.
  • phospholipids may be used.
  • the composition of the algae based oils and their fatty acid profile varies from the fatty acid profiles of krill oil as explained below and shown in the tables. It is possible to also use wax esters and omega-3 salts and ethyl esters.
  • the composition may also include an n-3 (omega-3) fatty acid rich oil derived from fish oil, algae oil, flax seed oil, or chia seed oil when the n-3 fatty acid comprises alpha-linolenic, stearidonic, eicosapentaenoic or docosapentaenoic acid.
  • the composition may include naturally-derived and synthetic antioxidants that are added to retard degradation of fatty acids and astaxanthin.
  • krill oil or algae based oil and other oils as described in synergistic combination with other ingredients. It has been determined that a fish oil derived, choline based, phospholipid bound omega-3 fatty acid mixture including phospholipid bound polyunsaturated EPA and DHA is advantageous for joint health when combined with the astaxanthin and low molecular weight hyaluronic acid or hyaluronate.
  • a mixture of fish oil derived, choline based, phospholipid bound fatty acid mixture including polyunsaturated EPA and DHA is Omega Choline 1520F as a phospholipid, omega-3 preparation, which is derived from natural fish oil and sold by Enzymotec Ltd.
  • the mixture of fish oil derived, choline based, phospholipid bound fatty acid mixture including polyunsaturated EPA and DHA in one example comprises
  • the omega choline includes at least 7% EPA and 12% DHA, of which not less than 15% are in the form of
  • the composition can be delivered advantageously for therapeutic results with 1-4000 mg of a mixture of fish oil and fish oil derived, choline based, phospholipid bound fatty acid mixture including polyunsaturated EPA and DHA delivered per daily dose.
  • about 150 mg to about 300 mg is used.
  • 2 to 4 mg astaxanthin are supplemented to the omega choline per daily dose, but may include a range of 0.5 to 4 mg, or 0.5 to 6 mg, 0.5 to 12 mg, or 7 to 12 mg, and other ranges as described before.
  • the composition may also include a natural or synthetic cyclooxygenase-1 or - 2 inhibitor comprising for example aspirin, acetaminophen, steroids, prednisone, or NSAIDs.
  • the composition may also include a gamma-linoleic acid rich oil comprising Borage (Borago officinalis L.) or Safflower (Carthamus tinctorius L), which delivers a metabolic precursor to PGEi synthesis.
  • the composition may also include an n-3 (omega-3) fatty acid rich oil derived from fish oil, algae oil, flax seed oil, chia seed oil or perilla seed oil wherein the n ⁇ 3 fatty acid source comprises a!pha-linolenic, stearidonic, eicosapentaenoic or
  • the composition may include naturally-derived and synthetic antioxidants that are added to retard degradation of fatty acids such as tocopherols, tocotrienols, carnosic acid or Carnoso! and/or astaxanthin.
  • herring roe extract as the source of phospholipids that may have some EPA and DHA, Synergistic results are obtained and vast improvements seen.
  • phospholipids from herring roe improved phospholipid and glucose tolerance in healthy, young adults as published by Bjorndal et ai., Lipids in Health Disease, 2014, 13:82.
  • the pure roe phospholipid may be formed using extraction techniques. It is a honey-like product that is thinned or diluted with fish oil and/or perilla oil or other seed or plant oil, in an example.
  • the herring roe extract is processed in one example using extraction by ethanol. Triacylglycerides are added and ethanol stripped out to have a robust solution. Seed oil, such as the perilla seed oil as described in the incorporated by reference '904 patent, may be added back to the ethanol extract before stripping to thin and form a high level phospholipid blend.
  • the roe oil extract may be mixed with fish oil and/or seed oil, such as the perilla, or any other marine oil.
  • the herring egg roe extract is mixed with perilla seed oil of at least 1 :1 and preferably as high as 6:1 ALA to LA with the concentrate as having at least 50%, and in another example 60%
  • phospholipids and in another example at least 30%, and in another example 40% triglycerides.
  • An example composition includes a combination of a roe extract from herring or a phospholipid rich roe extract with phospholipid bound EPA and DHA admixed with seed/fish oil and/or seed oil where the seed oil has a ratio of ALA to LA between 1 :1 and 1 :6, and optionally including astaxanthin in one example of about 2-4 mg or 0.5 to 12 mg or other ranges as noted above, and the low molecular weight hyaluronic acid, such as described above.
  • the amount of roe egg extract mixed with the seed oii such as perilla oii varies and is about 150 to 500 mg, or 300 to 500 mg, or up to 1 ,000 mg daily dose in one example and may include hyaluronic acid.
  • Other plant based phospholipids may be used, including commercially available lecithins and an egg yolk derivative, including lysophospholipids and glycophospholipids to act as surfactants. It is possible to use sunflower-based phospholipids and natural plant-based oils and natural surfactant extracts.
  • the astaxanthin is enhanced with fats, surfactants, or phospholipids and can be delivered more efficiently with phospholipids and sunflower based and/or the lipophilic perilla oil as described before.
  • the perilla oil is formed as a shelf stable, supercritical, C02 fluid extracted seed oil derived from a cracked biomass of perilla frutescens from 60 to 95 percent w/w of PUFAs in a ratio of from 4:1 to 6:1 alpha-linoienic acid (ALA) to linoleic acid (LA).
  • the perilla frutescens derived seed oil is made in an example by subjecting the perilla frutescens seed to supercritical fluid CO2 extraction to produce a seed oil extract; fractionating the resulting seed oil extract in separate pressure step-down stages for coliecting light and heavy fractions of seed oil extract; and separating the heavy fraction from the light fraction to form the final seed oil from the heavy fraction.
  • Selected antioxidants are included in another example and the perilla oil includes a mixture of selected lipophilic and hydrophilic antioxidants.
  • Lipophilic antioxidants can be used either alone or in combination with at least one of: a) phenolic antioxidants including at least one of sage, oregano, and rosemary; b) tocopherol; c) tocotrieno!(s); d) carotenoids including at least one of astaxanthin, lutein, and zeaxanthin; e) ascorbylacetate; f) ascorbylpalmitate; g) Butylated hydroxytoiuene (BHT); h)
  • a hydrophilic antioxidant or sequesterant may include hydrophilic phenolic antioxidants including at least one of grape seed extract, tea extracts, ascorbic acid, citric acid, tartaric acid, and malic acid.
  • a peroxide value of this perilla seed oil is under 10.0 meq/Km.
  • this perilla seed oil is from 85 to 95 percent w/w of PUFAs and the PUFAs are at least greater than 56 percent alpha-linolenic acid (ALA).
  • the perilla seed oil is shelf stable at room temperature up to 32 months.
  • this perilla seed oil is derived from a premilled or flake-rolled cracked biomass of perilla frutescens.
  • the mixture of selected antioxidants may include astaxanthin, phenolic antioxidants and natural tocopherols.
  • the perilla seed oil may also include at least one of dispersed nano- and micro-particles of rice or sugar cane based policosanol.
  • the composition is encapsulated into a single dosage capsule and referred to as a deep ocean caviar capsule.
  • the encapsulated composition includes herring caviar phospholipid extract (herring roe) perilla ⁇ perilla frutescens) seed extract, olive oil, Zanthin® astaxanthin (Haematococcus pluvialis algae extract), gelatin, spice extract, non-G O natural tocopherols, cholecalciferol, riboflavin, and methylcobalamin.
  • the composition includes fish as herring roe and tilapia gelatin. An example is set forth in the following chart. Properties:
  • Appearance Size 00 clear capsule with dark red oil
  • Vitamin B 2 (Riboflavin) 1.7 mg; 100% DV
  • the processing components may contain a mix of marine omega-3 phospholipids derived from herring caviar and peril!a seed oil. it may contain an 02BTM botanical peroxidation blocker, including spice extract, non-GMO tocopherols and ascorbyl palmitate. it can be packaged as a bulk product in sealed drums 45 and 190 kg net with inert headspace, complying with European and American standards for food products. It preferably stores at below room temperature. The product is protected against light and heat. If drums are opened for sampling, the headspace can be flushed with inert gas during sampling and prior to storing. Test Unit Acceptance Criterion Method
  • Heavy metals (sum of Pb, mg/kg 10 AM1015 Hg, Cd & In-organic As) 2 )
  • the astaxanthin is at least about 0.1 to about 15 percent by weight of the at least one phospholipid, glycolipid, and sphingolipid.
  • the astaxanthin in an example is derived from a natural or synthetic ester or synthetic dio!.
  • a pharmaceutical or food grade diluent may be added.
  • a dietary supplement composition When incorporated with a microbial fermented, low molecular weight hyaluronic acid or sodium hyaluronate (hyaluronan) as described before, a dietary supplement composition is formed and can be formulated in a therapeutic amount to treat and alleviate symptoms of joint pain in a person having joint pain.
  • hyaluronan sodium hyaluronate
  • the triglycerides have two types of molecules as a glycerol and three fatty acids, while the phospholipids contain glycerol and fatty acids, but have one glycerol molecule and two fatty acid molecules. In place of that third fatty acid, a polar group is instead attached to the glycerol molecule so that the
  • phospholipids are partly hydrophilic as compared to hydrophobic triglycerides.
  • Lysophospholipids may be used as a derivative of a phospholipid in which one or both acyl derivatives have been removed by hydrolysis.
  • Lecithin and its derivatives may be used as an emulsifier and surfactant as a wetting agent to reduce surface tension of liquids.
  • Other phospholipids may be used.
  • Different phospholipids include
  • phosphatidylinositol phosphatidic acid
  • lyso-phosphatidylchoiine phosphatidylchoiine
  • iyso- phosphatidylethanolamine phosphatidyiserine
  • phospholipids may include those products mentioned before, including phosphatidic acid.
  • compositions such as lecithin may be hydrolyzed
  • lysophospholipids that can be added to the roe extract as explained above.
  • One phospholipase is phospholipase A2 where the fatty acid is removed at the C2 position of glycerol. Fractionation may be used.
  • glycoiipids are primarily derivatives of ceramides where a fatty acid is bonded or connected to the amino alcohol sphingosine. It should be understood that the phospholipid sphingomyelin is also derived from a ceramide. Glycoiipids, however, contain no phosphates in comparison to the phospholipids. The fat is connected to a sugar molecule in a glycolipid and are fats bonded to sugars. Because it is built from a sphingosine, fat and sugar, some refer to it as a glycosphingolipid.
  • a sphingolipid is a lipid that contains a backbone of sphingoid basis and set of alphatic amino alcohols that include the sphingosine.
  • the phospholipid and other components may be derived from at least one of a plant, algae and animal source, or a synthetic derivative thereof.
  • the phospholipid and other components may be derived from at least one of soybean, sunflower, grapeseed, egg yolk, krili, fish body, fish roe, squid, and algae.
  • the phospholipid and other components may be formed as compound rich mono- or di-glcerides or fatty acids where the fatty acid contains between 2 and 20 carbon atoms.
  • the composition is formed by dispersing the astaxanthin and phospholipid and optionally a diluent under high shear conditions.
  • the diluent may be a pharmaceutical or food grade diluent as known to those skilled in the art.
  • the astaxanthin is about 2 to about 10 percent by weight of the phospholipid and glycolipid and derived from a natural or synthetic ester or synthetic diol. In yet another example, 50 to 500 mg of phospholipid, glycolipid, and sphingolipid may be used.
  • the dietary supplement composition may be formulated into a single dosage capsule.
  • the astaxanthin may be derived from Haematococcus pluvialis algae, Pfaffia, krill, or by synthetic routes, in the free or synthetic diol, monoester or diester form, both natural and synthetic, at a daily dose of 0.5-8 mg or 0.5-12 mg, in one example, and in another example, 1-2 mg, 2-4 mg, 1-6 mg, and other ranges, and up to 12 mg, including 7-12 mg.
  • the polymers of hyaluronic acid or sodium hyaluronate (hyaluronan) can be derived from microbial fermentation or animal tissue.
  • hyaluronan can be delivered per daily dose and preferably between 10 and 70 mgs/dose and at 20 to 60, 25 to 50, and 35 and 45 mg per dose.
  • the hyaluronan is micro- or nano- dispersed within the composition in one preferred example.
  • the hyaluronic acid is derived from a biofermenation process and has a molecular weight between 0.5 and 100 kilodaltons (kDa), and in another example, up to 300 kDa and preferably 0.5 to 300 kDa, and in another example, from 0.5 to 230 kDa as low molecular weight hyaluronic acid or hyaluronan.
  • a preferred range is 0.5 to 300 kDa.
  • the polymers of hyaluronic acid or sodium hyaluronate (hyaluronan) are derived from microbial fermentation or animal tissue.
  • the pure low molecular weight hyaluronic acid oligomers in an example are derived principally and practically from microbial fermentation, but couid also be derived from hydrolyzed animal tissues. This microbial fermentation process is known to produce extraordinarily pure low molecular sodium hyaluronate free from amino acid conjugation.
  • Human hyaluronic acid is typically synthesized in the body naturally or taken from the diet such as from chicken, beef, and other natural sources.
  • This natural hyaluronic acid has high molecular weight, i.e., greater than 300 kDa, as compared to microbial fermented sodium hyaluronate that is low molecular weight and defined in the literature as about 0.5 to 300 kDa.
  • the hyaiuronic acid naturally found in the body is a poiymer of acidified glucuronic acid and N-acetyl-giucosamine, which under physiological pH of about 7.4, exists as free acid, with partial sodium, potassium and ammonium salts.
  • Streptococcus in one example is used to ferment the sodium hyaluronate and is a mutant strain. Therefore, the resulting low molecular weight hyaluronic acid is obtained from a mutant strain of streptococcus bacteria.
  • the fermentation process is followed by isolation and denaturation of the organism and its proteins with ethanol and heat. This is followed by filtration.
  • the molecular weight is chemically modified with acid aqueous chemical hydrolysis as a chemical reaction.
  • the final product is isolated by ethanol precipitation of the sodium salt and drying to produce pro-inflammatory low molecular weight microbially fermented sodium hyaluronate fragments.
  • This low molecular weight sodium hyaluronate is a chemical reaction degradation product of a mutant strain streptococcus bacterial fermentation.
  • An example sodium hyaluronate is manufactured by fermentation using the bacterial strain streptococcus zooepidemicus.
  • the production strain is a non-hemo!ytic mutant of a parent strain, NCTC 7023.
  • the production strain is produced by nitroso-guanidine mutagenesis with a unique ribosomal genome sequence not naturally found in nature.
  • This manufacturing process has three main stages of 1) fermentation, 2) purification, and 3) refining.
  • the fermentation begins with a seed culture from the mutant production strain.
  • a starter culture inoculates the seed tank, which contains a broth medium that is grown out to become the seed broth.
  • the seed broth is transferred to a fermenter containing the sterilized culture medium and a culturing temperature of 33-37 degrees Celsius is maintained until fermentation is complete within 22-30 hours.
  • the filtrate containing the chemical hydrolysis derived low molecular weight hyaluronic acid produced during the chemical molecular weight modification step is then precipitated with ethanol, followed by washing or dehydrating. The precipitate is dried under vacuum to yield the final low molecular weight, microbial fermented sodium hyaluronate.
  • hyaluronic acid may be used. These include low molecular weight hyaluronic acid derived from chicken sterna! cartilage extract.
  • the hyaluronic acid may include elastin, elastin precursors, and collagen.
  • the hyaluronic acid may be contained in a matrix form with chondroitin sulfate and naturally occurring hydrolyzed collagen Type II nutraceutical ingredients and form lower weight molecules that the body may more readily absorb and deliver to different areas of the body as required.
  • Fresh chicken sternal cartilage could be cut and suspended in aqueous solution followed by treating the cartilage with a proteolytic enzyme to form a hydrolysate.
  • the proteolytic enzyme is capable of hydrolyzing collagen Type II to fragments having a lower molecular weight.
  • the hydrolysate is sterilized and filtered and concentrated and then dried to form powder enriched collagen Type il powder that is then isolated and includes a percentage of low molecular weight hyaluronic acid. Examples of manufacturing techniques can be found in U.S. Patent Nos. 6,780,841 and 6,025,327, the disclosures which are hereby incorporated by reference in their entirety.
  • the low molecular weight hyaluronic acid couid also be derived from the hydrolyzed collagen as derived from the bovine collagen Type I or the chicken sternal cartilage collagen Type II, or even a natural eggshell membrane that includes some hyaluronic acid, which can be extracted from the eggshell membrane.
  • the hyaluronic acid is processed to increase its molecular weight using cross-linking techniques as compared to using a low molecular weight hyaluronic acid.
  • the eggshell membrane can still be used to obtain the low molecular weight hyaluronic acid. It may be possible to use enzymatic degradation of eggshell membrane that undergoes manipulation to purify the hyaluronic acid.
  • the hyaluronic acid may be derived from dehydrated rooster combs such as disclosed in U.S. Patent No. 6,806,259 and U.S. Patent Publication No. 2006/0183709, which are incorporated herein by reference in their entirety, where the hyaluronic acid may be further processed. Often it is a higher molecular weight and will be processed to obtain a lower molecular weight of the desired 0.5 to 300 kDa. in many teachings, a certain molecular weight hyaluronic acid is processed to increase its molecular weight.
  • the hyaluronic acid may also be obtained from human umbilical cords or other techniques such as disclosed in U.S. Patent No. 4,141 ,973, the disclosure which is hereby incorporated by reference in its entirety, and further processed to obtain the desired molecular weight.
  • compositions that include about 50 mg of an active ingredient, for example, hyaluronic acid and a cartilage, such as a Type II collagen when astaxanthin is added.
  • an active ingredient for example, hyaluronic acid
  • a cartilage such as a Type II collagen when astaxanthin is added.
  • boron is used.
  • the composition includes 30-50 mg of collagen and about 4-6 mg of boron and 2-4 mg of hyaluronic acid with an average of each of the component ranges.
  • a cartilage blend as a mixture of cartilage and salt is about 40 mg with boron as 5 mg and hyaluronic acid as 3.3 mg.
  • the cartilage blend includes cartilage and potassium chloride to provide 10 mg of undenatured Type-2 collagen.
  • another composition to include the astaxanthin with the composition that is formed from glucosamine hydrochloride such as about .25 to 1.75 or about 1.5 grams and methylsulfonymethane ( SM) of about 500 to 1 ,000 and about 750 mg and including the addition of chondroitin sulfate of about 150 to 250 and about 200 mg.
  • glucosamine hydrochloride such as about .25 to 1.75 or about 1.5 grams and methylsulfonymethane ( SM) of about 500 to 1 ,000 and about 750 mg and including the addition of chondroitin sulfate of about 150 to 250 and about 200 mg.
  • SM methylsulfonymethane
  • the joint fluid may include the joint fluid as hyaluronic acid, such as 1 -5 mg and about 3.3 mg, and also vitamin D3 and other components such as antioxidants.
  • the astaxanthin can vary between 2 to 4 mg or 0.5 to 12 mg and other ranges as disclosed above. It should be understood that the astaxanthin and the at least one of phospholipid, glycolipid, and sphingolipid or other components as described above may be used for many different purposes and results. It may be used to aid in treating or improving b!ood lipid profiles and reducing LDL per-oxidation in humans. It may be used to counter or treat depression and other neurological disorders. It may be used for respiratory illnesses and skin ailments or diseases.
  • composition may include a natural or synthetic cyclooxygenase-1 or -2 inhibitor comprising for example aspirin, acetaminophen, steroids, prednisone, or NSAIDs.
  • the composition may also include a gamma-iinoleic acid rich oil comprising Borage (Borago officinalis L) or Safflower (Carthamus tinctorius L), which delivers a metabolic precursor to PGEi synthesis.
  • the composition may also include an n-3 (omega-3) fatty acid rich oil derived from fish oil, algae oil, flax seed oil, chia seed oil, or perilla seed oil.
  • the n-3 fatty acid comprises alpha-linolenic, stearidonic, eicosapentaenoic or docosapentaenoic acid.
  • an aigae based oil may be used instead of kril! oil. Hydroiyzed or unhydrolyzed collagen and elastin derived from eggshell membranes can also be advantageously added.
  • composition may also include anti-inflammatory and/or natural joint health promoting compounds comprising at least one of preparations of green lipped mussel (Perna canaliculus), Boswellia serrata, turmeric (Curcuma longa), stinging nettle (Urtica dioica), Andrographis, Cat's claw (Uncaria tomentosa), bromelain,
  • the composition may include naturally-derived and synthetic antioxidants that are added to retard degradation of fatty acids and astaxanthin.
  • compositions may use different ingredients in combination with the krill, algae or other oil, including the seed based oil, roe extract, and phospholipid and other surfactants.
  • the astaxanthin and hyaluronate may be combined with different ingredients and supplemental compositions for more specific purposes.
  • [0078JA pharmaceutically acceptable composition comprises a krill, fish, algae, roe extract or plant based oil and/or phospholipid and/or surfactant in combination with astaxanthin and hyaluronate optionally combined with one or more ingredients including but not limited to glucosamine sulfate, chondroitin sulfate, collagen,
  • methyisulfonmethane a gamma-linoieic acid or omega-3 fatty acid rich oil a
  • cyclooxgenase inhibitor or a lipogenase inhibitor for the treatment of symptoms related to non-disease joint pain and/or joint diseases, including but not limited to osteoarthritis and rheumatoid arthritis.
  • a dietary supplement acceptable composition comprises a krill, algae, fish, roe extract, or plant based oil and/or other phospholipid and/or surfactant in combination with astaxanthin and hyaluronate optionally combined one or more ingredients, inciuding but not limited to, glucosamine sulfate, chondroitin sulfate, collagen, methyisulfonmethane, a gamma-iinoleic acid or omega-3 fatty acid rich oil a cyclooxgenase inhibitor or a lipoxygenase inhibitor for the treatment of symptoms related to non-disease joint pain and/or joint diseases, including but not limited to osteoarthritis and rheumatoid arthritis.
  • a medical food acceptable composition comprises a krill, aigae, fish, roe extract, or plant based oil and/or other phospholipid and/or surfactant in combination with astaxanthin and hyaluronate and optionally combined with one or more ingredients including glucosamine sulfate, chondroitin sulfate, collagen, methyisulfonmethane, a gamma-linoleic acid or omega-3 fatty acid rich oil, a
  • cyclooxgenase inhibitor or a lipoxygenase inhibitor for the treatment of symptoms related to non-disease joint pain and/or joint diseases, including but not limited to osteoarthritis and rheumatoid arthritis.
  • a composition is formulated in a therapeutic amount to treat and alleviate symptoms of non-disease joint pain and/or joint diseases, including osteoarthritis and/or rheumatoid arthritis, wherein the composition includes a krill, aigae, fish, roe extract, or plant based oil and/or other phospholipid and/or surfactant in combination with astaxanthin and polymers of hyaluronic acid or sodium hyaluronate (hyaluronan) in an oral dosage form.
  • This composition includes other active
  • the composition oil is used with the HA, such as the low molecular weight HA, and astaxanthin to treat non-disease joint pain in one example, but can be used to treat osteoarthritis.
  • Osteoarthritis (OA) is the most prevalent form of arthritis and is a disease in which the cartilage that acts as a cushion between the bones in joints begins to wear away causing bone on bone joint swelling and joint pain. It is characterized by degeneration of articular cartilage along with peri-articular bone response, it affects both sexes, mainly in the fourth and fifth decades of life. The knee joint is most commonly affected joint. At present the management is by pharmacological and non-pharmacological therapy. Corrective surgical therapy and or joint replacement therapy in some cases may not be possible.
  • CAMs complimentary and alternative medicines
  • Glucosamine and chondroitin a!one or in combination are widely marketed as dietary suppfements to treat joint pain due to OA.
  • Two major clinical trials on glucosamine and chondroitin failed to show any significant improvement in WOMAC score over placebo except in the highest quartile of patients studied. Because of their limited effectiveness, the search for additional CAMs to treat OA continues (see for example Ruff et al., Eggshell Membrane in the Treatment of Pain and Stiffness from Osteoarthritis of the Knee: A Randomized, Multicenter, Double-Blind, Placebo- Controlled Clinical Study, Clin. Rheumatol (2009) 28:907-914).
  • a pure diol of the S, S'astaxanthin including a synthetic diol with a surfactant and/or the low molecular weight hyaluronic acid. It is possible to use that pure diol in combination with the EPA rich algae based oil or other fish, roe extract, or plant based oil and/or phospholipid and/or surfactant as described above, and which is admixed with either astaxanthin derived from Haematococcus pluviaiis or the free diol form in substantially pure S,S' enantiomer form. It is possible to add synthetically derived mixed enantiomers of the diol.
  • the diol of the S, S'astaxanthin is possible because in cases of kriil oil and possibly algae based oils and Hp derived and other types, there are principally diesters and monoesters respectively with very little diol, which is insoluble. Some research indicates that it may be many times more bioavailable than either the monoester or diester form. It is possible to synthesize asymmetrically the S,S' pure diol. Despite the pure diol's poor solubility in some examples, there may be an active transport mechanism related to its bioavailability, or conversely, that only in the diol form is the monoester or diester forms transferred from the intestines to the blood.
  • the phospholipid or glycolipid based product presenting EPA and/or DHA along with the added astaxanthin in its various forms and especially the S,S' enantiomeric form in principally monoester form from Haematococcus pluviaiis or pure diol form from asymmetric synthesis couid be viable.
  • it is possible to combine it with the algae derived glycol and phospholipid based EPA rich oil.
  • Astaxanthin (3,3'-dihydroxy- -p-carotene-4,4' ⁇ dione) is a xanthophyll carotenoid found in many marine species including crustaceans, saimonid fish and algae. Astaxanthin cannot be synthesized by mammals, but when consumed in the diet has shown effectiveness as an antioxidant, anti-inflammatory agent and with benefit to eye health, heart health, and the immune system.
  • Astaxanthin has a hydroxyl group on each ⁇ -ionone moiety, therefore it can be found in its free (diol) form as well as mono- or di-esterified.
  • astaxanthin is commonly found as a mixture: primarily mono-esters of C12-C18 fatty acids and lesser amounts of di-ester and free diol.
  • Synthetic astaxanthin is commonly provided in only the free diol form.
  • the astaxanthin molecule has two E/Z chiral centers and three optical R/S isomers.
  • Haematococcus pluvialis algae produces natural astaxanthin solely in the (3S,3'S) isomer. This is explained in the article from Renstrom B., G. Borch,
  • yeast Phaffia rhodozyma synthesizes only the 3R,3'R configuration. This is explained in the article from Andrewes A. and M. Starr entitled, "(SR.S'RJ-Astaxanthin from the Yeast Phaffia Rhodozyma," Phytochemistry,
  • Wild salmon predominately contain the (3S,3'S) form with a (3S,3'S), (3R,3'S , and (3R,3'R) isomer ratio of 22:1 :5.
  • 3S,3'S 3S,3'S
  • 3R,3'S 3R,3'R
  • 3R,3'R 3R,3'R
  • astaxanthin produced by traditional synthesis will contain a racemic mixture in a (3S,3'S), (3 3'S; meso), (3R,3'R) ratio of 1 :2:1. This ratio is also seen in many species of shrimp, which are able to racemize (3S,3'S) to the meso form. This is explained in the article from Schiedt, K., S. Bischof and E. Glinz entitled, "Metabolism of Carotenoids and in vivo Racemization of (3S,3'S)-Astaxanthin in the Crustacean Penaeus," Methods in Enzymology, 214:148-168, 1993, the disclosure which is hereby incorporated by reference in its entirety.
  • the uptake of free astaxanthin diol is about 4-5 times higher than that of esterified astaxanthin, likely due to the limitation of required enzymatic hydrolysis in the gut prior to absorption.
  • These intestinal enzymes may also be R/S selective on astaxanthin esters.
  • Coral-Hinostroza et al. found higher relative absorption of astaxanthin from (SR.S'R-astaxanthin dipalmitate compared to the other two isomers.
  • ingestion of racemic free diol astaxanthin does not show any stereospecific selection.
  • Astaxanthin for use in human food supplements is currently derived from the cultivated freshwater algae Haematococcus pluvialis. This algae produces 3S,3'S astaxanthin ester in a fatty acid matrix which can be isolated with solvent or carbon dioxide extraction. This oily extract can be used directly in edible formulations or further processed into solid powder or beadlet preparations. Many clinical studies have been conducted with H. pluvialis derived astaxanthin to demonstrate beneficial health effects and safety. Food additive approvals for astaxanthin-rich algae extracts have been approved for many suppliers in the US and EU.
  • Haematococcus algae cultivation for use in dietary supplements cannot always match demand for use of astaxanthin in dietary supplements.
  • Use of synthetic astaxanthin diol can also benefit applications which need a concentrated, standardized astaxanthin source.
  • Conventional racemic synthetic astaxanthin sources are used as a colorant in Salmonid aquaculture as a feed ingredient. This racemic mixture may have limited use since only one-quarter of the compound is the 3S,Z'S isomer commonly found in natural Salmon and has been studied in humans for efficacy and safety.
  • Astaxanthin may also be synthesized with in a stereospecific manner, so that the output is exclusively the generally accepted 3S,3'S isomer in a free dio! form.
  • the free diol crystals can be suspended in a vegetable oil or solid beadlet for use in edible preparations or pili, capsule, tablet form.
  • the 3S,3'S product has the advantage of greater consistency than aigai preparations and aiso with lower odor. Therefore algal- derived astaxanthin can be replaced with synthetic 3S,3'S astaxanthin diol in existing formulations with the same or increased effectiveness.
  • hyaluronic acid alone and/or in combination with astaxanthin is beneficial and synergistic.
  • low molecular weight hyaluronic acid in its different forms can be given to patients in an amount from 1-500 mg per day and preferably about 10-70 mg per day, and in another example, 20-60 mg, 25-50 mg, 35 mg, and 45 mg.
  • Astaxanthin of about 2-4 mg may be added in an example, but could range from 0.5 to 4 mg a day, and 7-12 mg range in another example, or 0.5 to 12 mg.
  • the hyaluronic acid may be given in the form of a pro-inflammatory low molecular weight sodium hyaluronate fragments that are about 0.5-300 kDa corresponding to the pro-inflammatory low moiecular weight fragments.
  • astaxanthin and phospholipids such as from krill oil, algae oil, roe, fish oil product, or plant based oils helps in delivering the hyaluronic acid, stiil the iow moiecular weight hyaluronic acid and in the form of the fragments preferably is still small enough to enter through the gut and be used in an oral administration.
  • astaxanthin with the low moiecular weight hyaluronic acid.
  • Different amounts can be used, and in one example, 2-4 mg per day, and in another example, 0.5-12 mg per day can be used with low molecular weight hyaluronic acid such as the amount of 1-500 mg and preferably about 10-70 mg and with 0.5-12 mg or 4-12 mg of astaxanthin.
  • About 40-120 mg of low molecular weight hyaluronic acid may be used in an example.
  • a dosage of astaxanthin may be about 6-8 mg and the low molecular weight hyaluronic acid could be in the range of about 60-80 mg.
  • hyaluronic acid fragments such as the pro-inflammatory low molecular weight sodium hyaluronate fragments are potent as innate immune system cell receptors signaling molecules associated with the
  • inflammatory cascade and the oral hyaluronic acid in the form of low molecular weight fragments can reach joints as compared to the higher molecular weight hyaluronic acid that is injected since it is not orally administered.
  • This algae based oil provides an algae sourced EPA or an EPA/DHA based oil in which oils are present in phospholipid and glycerolipid forms, as glycolipids.
  • Different aigae based oils derived from different microalgae may be used.
  • One preferred example algae based oil has the EPA titre higher than the DHA as compared to a class of omega-3's from fish oils that are triacylglycerides. These algae based oils are rich in EPA and in the phospholipid and g!ycoiipid forms.
  • An example marine based algae oil is produced by Parry Nutraceuticals as a division of EID Parry (India) Ltd. as an omega-3 (EPA) oil.
  • omega-3 fatty acids such as EPA and DHA. It is known that fish and krill do not produce omega-3 fatty acids but accumulate those fatty acids from the algae they consume. Omega-3 bioavailability varies and is made available at the site of physiological activity depending on what form it is contained. For example, fish oil contains omega-3 fatty acids in a triglyceride form that are insoluble in water and require emulsification by bile salts via the formation of micelles and subsequent digestion by enzymes and subsequent absorption.
  • omega-3 fatty acids that are bound to polar lipids such as phospholipids and giycolipids
  • polar lipids such as phospholipids and giycolipids
  • these omega-3 fatty acids have greater bioavailability for cell growth and functioning as compared to the omega-3 triglycerides of fish oil.
  • algae There are many varieties of algae that contain EPA conjugated with phospholipid and glycoiipid polar lipids or contain EPA and DHA conjugated with phospholipids and giycolipids.
  • algae or “microalgae” may be used interchangeably to each other with microalgae referring to photosynthetic organisms that are native to aquatic or marine habitats and are too small to be seen easily as individual organisms with the naked eye.
  • photoautotropic refers to growth with tight as the primary source of energy and carbon dioxide as the primary source of carbon.
  • biomass may refer to a living or recently dead biological cellular material derived from plants or animals.
  • polar may refer to the compound that has portions of negative and/or positive charges forming negative and/or positive poles.
  • oil may refer to a combination of fractionable lipid fractions of a biomass. As known to those skilled in the art, this may include the entire range of various hydrocarbon soluble in non-polar solvents and insoluble, or relatively insoluble in water as known to those skilled in the art.
  • the microalgae may also include any naturally occurring species or any genetically engineered microalgae to have improved lipid production.
  • the following first table shows the specification of an algae based oil as manufactured by Parry Nutraceutica!s identified above, followed by a second table for a fatty acid profile chart of that aigae based oil.
  • a third table is a comparative chart of the fatty acid profiles for non-algae based oils. These charts show that the algae based oil has a high EPA content of phospholipids and giycolipids.
  • nannochloropsis oculata as a source of EPA.
  • Another algae that may be used is thalassiosira weissftogii such as described in U.S. Patent No. 8,030,037 assigned to the above-mentioned Parry Nutraceuticals, a Division of EID Parry (India) Ltd., the disclosure which is hereby incorporated by reference in its entirety.
  • Other types of algae as disclosed include chaetoceros sp. or prymnesiophyta or green algae such as chlorophyta and other microalgae that are diamons tiatoms.
  • the chlorophyta could be tetraselmis sp. and include prymnesiophyta such as the class prymnesiophyceae and such as the order isochrysaies and more specifically, isochrysis sp. or pavlova sp.
  • prymnesiophyta such as the class prymnesiophyceae and such as the order isochrysaies and more specifically, isochrysis sp. or pavlova sp.
  • algae/fungi phospho!ipid/g!ycolipid sources include: grateloupia turuturu; porphyhdium omentum; monodus subterraneus; phaeodactylum tricomutum; isochrysis galbana; navicula sp.; pythium irregule; nannochloropsis sp.; and nitzschia sp.
  • Porphyridium cruentum is a red algae in the family porphyhdiophyceae and also termed rhodophyta and is used as a source for fatty acids, lipids, cell-wall polysaccharides and pigments. The polysaccharides of this species are sulphated. Some porphyridium cruentum biomass contains carbohydrates of up to 57%.
  • Phaeodactylum tricornutum is a diatom and unlike most diatoms, it can grow in the absence of silicon and the biogenesis of silicified frustules is facultative.
  • Isochrysis galbana is a microalgae and used in the bivalve aquaculture industry.
  • Navicula sp. is a boat-shaped algae and is a diatom. Pythium irregule is a soilborne pathogen found on plant hosts.
  • Nannochloropsis sp. occurs in a marine environment, but also occurs in fresh and brackish water.
  • the species are small, nonmotile spheres that do not express any distinct morphological feature.
  • These algae have chlorophyll A and lack chlorophyll B and C. They can build high concentrations of pigment such as astaxanthin, zeaxanthin and canthaxinthin. They are about 2-3 micrometers in diameter. They may accumulate high levels of polyunsaturated fatty acids.
  • Nitzschia sp. is a pinnate marine diatom and usually found in colder waters and associated with both Arctic and Antarctic polar sea ice where it is a dominant diatom. It produces a neurotoxin known as domoic acid which is responsible for amnesic shell fish poisoning. It may grow exponentially at temperatures between -4 and -6 degrees C. it may be processed to form and extrapolate the fatty acids.
  • microalgae As a source of polyunsaturated fatty acids, microalgae competes with other micro-organisms such as fungi and bacteria. There may be some bacterial strains that could be an EPA source, but microalgae has been found to be a more adequate and readily available source. Microalgae is a good source of oil and EPA when derived from phaeodactylum, isochrysis and monodus. The microalgae phaeodactylum tricornutum produces a high proportion of EPA. Other different strains and species of microalgae, fungi and possibly bacteria that can be used to source EPA include the following:
  • Different microalgae may be used to form the algae based oil comprising glycolipids and phospholipids and at least EPA and/or EPA/DHA. Examples include: Chlorophyta, Cyanophyta (Cyanobacteria), and Heteromonyphyta.
  • the microalgae may be from one of the following classes: Bacillariophyceae, Eustigmatophyceae, and Chrysophyceae.
  • the microalgae may be from one of the following genera:
  • Nannochloropsis Chlorella, Dunaliella, Scenedesmus, Selenastrum, Oscillatoria, Phormidium, Spirulina, Amphora, and Ochromonas.
  • microalgae species include: Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var. punctata, Amphora coffeiformis var. taylori, Amphora coffeiformis var. tenuis, Amphora
  • Ankistrodesmus Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri var.
  • Chetoceros sp. Chlamydomas perigranulata, Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsuiate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophlla, Chlorella kessleri, Chlorella lobophora, Chlorella luteoviridis, Chlorella luteoviridis var.
  • Chlorella salina Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris to. tertia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris to. tertia, Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella, Chlorella
  • Nephrochloris sp. Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, Nitzschia c!osterium, Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Osciltatoria subbrevis, Parachloretla kessleri, Pascheria acidophila, Pavlov
  • the microa!gae are autotrophic.
  • yeast that can be used include Cryptococcus curvatus, Cryptococcus terricolus, Lipomyces starkeyi, Lipomyces tipofer, Endomycopsis vernalis, Rhodotoru!a giutinis, Rhodotoru!a gracilis, Candida 107, Saccharomyces paradoxus, Saccharomyces mikatae,
  • Saccharomyces bayanus Saccharomyces cerevisiae, any Cryptococcus, C.
  • neoformans C. bogoriensis, Yarrowia lipolytica, Apiotrichum curvatum, T. bombicola, T. apicoia, T. petrophilum, C. tropicaiis, C. lipolytica, and Candida albicans. It is even possible to use a biomass as a wild type or genetically modified fungus.
  • Non-limiting examples of fungi include Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Penniclllium iilacinum, Hensenulo, Chaetomium, Ciadosporium, Malbranchea, Rhizopus, and Pythium.
  • bacteria may be used that includes lipids, proteins, and carbohydrates, whether naturally occurring or by genetic engineering.
  • Non-!imiting examples of bacteria include: Escherichia coli, Acinetobactersp. any actinomycete, Mycobacterium tuberculosis, any streptomycete, Acinetobacter calcoaceticus, P.
  • aeruginosa Pseudomonas sp., R. erythropolis, N. erthopolis, Mycobacterium sp., B., U. zeae, U, maydis, B. lichenformis, S. marcescens, P. fluorescens, B. subtilis, B. brevis, B. polmyma, C, lepus, N. erthropolis, T. thiooxidans, D. polymorphis, P. aeruginosa and Rhodococcus opacus.
  • Possible algae sourced, EPA/DHA based oils that are derived from an algae and contain glycol and phospholipid bound EPA and/or EPA/DHA and may include a significant amount of free fatty acids, triglycerides and phospholipids and giycolipids in the range of 35-40% or more of total lipids are disclosed in the treatise "Chemicals from Microalgae” as edited by Zvi Cohen, CRC Press, 1999.
  • the algae oil was provided at 1.5 grams of EPA and no DHA as compared to krill oil that was provided at 1.02 grams EPA and 0.54 grams DHA.
  • the participants consumed both oils in random order and separated by seven days and the blood samples were collected before breakfast and at several time points up to 10 hours after taking the oils.
  • the researchers determined that the algae based oil had a greater concentration of EPA and plasma than krill oil with the EPA concentration higher with the algae based oil at 5, 6, 8 and 10 hours (P ⁇ 0.05) intended to be higher at 4 hours (P 0.094).
  • the maximum concentration (CMAX) of EPA was higher with algae oil than with krill oil (P 0.010).
  • This difference may relate to the different chemical composition and possibly the presence of the glycolipids where the presence of DHA in krill oil limits the incorporation of EPA into plasma lipids.
  • the n-3 polyunsaturated fatty acids within glycolipids as found in the algae oil, but not in a krill oil may be an effective system for delivering EPA to humans.
  • Microalgae can be cultured photoautotrophically outdoors to prepare concentrated microalgae products containing Eicosapentaenotc acid (EPA) and
  • Docosahexaenoic acid which are the long-chain polyunsaturated fatty acids (PUFAs) found in fish oil. Both are very important for human and animal health.
  • the concentrated microalgae products as disclosed in the '037 patent may contain EPA and DHA and lipid products containing EPA and DHA purified from microalgae.
  • the concentrated microalgae composition may be prepared by cultivating microalgae photoautotrophically outdoors in open ponds under filtered sunlight in a continuous or batch mode and at a dilution rate of less than 35% per day. The microalgae may be harvested in the exponential phase when the cell number is increasing at a rate of at least 20% of maximal rate.
  • the microalgae is concentrated.
  • at least 40% by weight of lipids in the microalgae are in the form of glycodiacyiglycerides, phosphodiacylglycerides, or a combination thereof and at least 5% by weight of the fatty acids are DHA, EPA, or a combination thereof.
  • the microalgae are Tetraselmis sp. cultivated at above 20°C or in another example at above 30°C,
  • the EPA yield in the microalgae has been found to be at least 10 mg/liter culture.
  • the microalgae can be Isochrvsis sp. or Pavlova sp. in another example, or are Thalassiosira sp. or Chaetecoros sp.
  • the microalgae may be different diatoms and are cultivated photoautotrophically outdoors in open ponds for at least 14 days under filtered sunlight and at least 20% by weight of the fatty acids are EPA.
  • the use of this algae based oil overcomes the technical problems associated with the dwindling supplies of fish oil and/or Antarctic kri!l, which are now more difficult to harvest and obtain and use economically because these products are in high demand.
  • a major difference between fish oils and algae based oils is their structure.
  • Fish oils are storage lipids and are in the form of triacylglycerides.
  • the algae based oils as lipids are a mixture of storage lipids and membrane lipids.
  • the EPA and DHA present in algae based oils is mainly in the form of giycolipids and a small percentage is in the form of phospholipids. Giycolipids are primarily part of chloroplast membranes and phospholipids are part of ceil membranes.
  • the ⁇ 37 patent describes various methods for cuituring microalgae photoautotrophically outdoors to produce EPA and DHA.
  • One method used is filtering sunlight to reduce the light intensity on the photoautotrophic culture. Shade cloth or netting can be used for this purpose. It was determined that for most strains, the optimal solar intensity for growth, for maintaining a pure culture, and for omega-3 fatty acid accumulation was about 40,000 to 50,000 lux, approximately half of the 1 10,000 lux of full sunlight. Shade cloth or netting is suitable for filtering the sunlight to the desired intensity.
  • the dilution rate is 15-40% per day or 15-35% per day, and in yet other examples, the dilution rate is 10-30%, 10-35%, or 10-40% per day.
  • Another technique to successfully culture microalgae photoautotrophically outdoors and produce EPA and EPA/DHA is to harvest the microalgae in exponential phase rather than stationary phase.
  • Harvesting in exponential phase reduces the risk of contamination in outdoor photoautotrophic cultures and has surprisingly been found to give a good yield of EPA and DHA.
  • To drive fat accumulation in microbial cultures the cultures are harvested in stationary phase because cells in the stationary phase tend to accumulate storage lipids.
  • the '037 patent teaches that EPA and DHA accumulate in large amounts as membrane lipids in cultures harvested in the exponential phase.
  • the membrane lipids containing EPA and DHA are predominantly phosphodiacylglycerides and glycodiacylgiycerides, rather than the triaclyglycerides found in storage lipids.
  • cultures are harvested often when cell number is increasing at a rate at least 20% of the maximal rate, i.e., the maximal rate achieved at any stage during the outdoor photoautotrophic growth of the harvested culture.
  • the cultures are harvested in exponential phase when cell number is increasing at a rate of at least 30%, at least 40%, or at least 50% of maximal rate. It is also possible to use recombinant DNA techniques.
  • Example 1 The strain Thalassiosira sp. is a diatom and this strain used was isolated from Bay of Bengal, and it dominates during summer months. This example strain was isolated from seawater collected near Chemai, India, and the culture was maintained in open tubs. The particular strain was identified as
  • Thalassiosira weissflogii which is capable of growth at high temperatures (35-38°C).
  • the fatty acid profile was good even when the alga was grown at high temperature with 25-30% EPA (as a percentage of fatty acids).
  • Cutturinq The lab cultures were maintained in tubs in an artificial seawater medium, under fluorescent lights (3000-4000 lux) and the temperature was maintained at 25°C. initiai expansion of the culture was done under laboratory condition in tubs. The dilution rate was 15% to 30% of the total culture volume per day. Once the volume was 40-50 liters, it was transferred to an outdoor pond. The outdoor ponds were covered with netting to control the light (40,000 to 50,000 lux). The dilution continued until the culture reached 100,000 liters volume. The culture was held in 500 square meter ponds at this time with a culture depth of 20 cm. The culture was stirred with a paddle wheel and CO2 was mixed to keep the culture pH neutral.
  • the whole pond was harvested by filtration.
  • the filtered biomass was washed with saltwater (15 parts per thousand concentration) and then spray dried.
  • the mode of cuituring was batch mode.
  • the EPA productivity was 2-3 mg/lit/day.
  • the ponds can also be run continuously for several weeks by harvesting part of the culture, recycling the filtrate into the ponds and replenishing required nutrients.
  • Example 2 The strain Tetraselmis sp. is in the division Chlorophyta and the class Prosinophyceae or Micromanadophyceae. This strain was obtained from the Central Marine Fisheries Research Institute, India. It was isolated from the local marine habitats in India. The culture was maintained in flasks in artificial seawater medium, and expanded as described for Thalassiosira. With culture outdoors in open ponds as described for Thalassiosira, the strain gave a good lipid yield (200-300 mg/liter) and an EPA content of 6-7% of fatty acids.
  • Example 3 The strain Chaetoceros sp. is another diatom strain obtained from the Central Marine Fisheries Research Institute, India, and isolated from local marine habitats in India. Chaetoceros sp. was maintained in flasks and cultivated in outdoor ponds photoautotrophicafly as described in Example 1. It gave similar EPA productivity and EPA content as Thalassiosira as described in Example 1 .
  • Example 4 The strain Isochrysis sp. is in the Prymnesiophyta, class Prymnesiophyceae, order fsochrysidales. It was obtained from the Central Marine Fisheries Research Institute, India, and isolated from local marine habitats in India. It was maintained and grown as described in Example 1. It was expanded from
  • Example 5 Harvesting and Drying: The harvesting may be done by flocculation.
  • the commonly used floccufants include Alum with polymer and FeCI3 with or without polymer and chitosan.
  • the concentration of fiocculent will depend on the cell number in the culture before harvest. The range may vary from 100 ppm to 500 ppm.
  • harvesting is done by filtration using appropriate meshes. Removal of adhered chemicals (other than salt) is accomplished by washing the ceils in low salinity water.
  • the concentration of encapsulating agent may vary from 0.1 to 1.0% on a dry weight basis. Modified starch is a suitable encapsulating agent.
  • the spray dryer is usually an atomizer or nozzle type. The inlet temperature ranges from 160 to 190°C and the outlet temperature ranges from 60 to 90°C. The spray dried powder is used immediately for extraction. If storage is required, the powder is packed in aluminum laminated pouches and sealed after displacing the air by nitrogen. The packed powder is stored at ambient temperature until further use.
  • Example 6 Extraction of EPA/DHA is carried out using a wet slurry or dry powder and solvents, which include hexane, ethanol, methanol, acetone, ethyl acetate, isopropanol and cyciohexane and water, either alone or in combination of two solvents.
  • the solvent to biomass ratio depends on the starting material. If it is a slurry, the ratio is 1 :2 to 1:10. With a spray dried powder, on the other hand, the ratio is 1 :4 to 1 :30.
  • the extraction is carried out in an extraction vessel under inert atmosphere, with
  • the mixture is passed through a centrifuge or filtration system to remove the cell debris.
  • the lipid in the filtrate is concentrated by removing the solvent by distillation, which is carried out under vacuum.
  • the resulting product is a crude lipid extract, which contains approximately 10% omega-3 fatty acid (EPA/DHA).
  • the extract can be used as it is or purified further to enrich the omega-3 fatty acids. Further purification may involve removal of unsaponifiables such as pigments, sterols and their esters.
  • the aigae based oil composition may be used for different purposes as described.
  • the astaxanthin in the presence of a surfactant may be at below 4 mg/day and as noted before, optionally admixed with the low molecular weight hyaluronic acid or UC-li and/or as a chicken sternum collagen isolate.
  • the phospholipid may have little EPA and DHA.
  • a preferred astaxanthin concentration is about 2-4 mg and a chicken sternum collagen isolate can be about 40 mg and have a range of 30 to about 50 mg.
  • Other surfactants such as plant based phospholipids and commercially available lecithins that are modified and including egg yo!k compositions and/or sea based oils such as from perilla may be used.
  • Sea based phospholipids and lysolipid, also referred to as lysophospholipid, counterparts may be used.
  • a non-omega-3 platform may be used with the current invention.
  • the low molecular weight hyaluronic acid as described may vary from 1-500 mg, 0-70 mg, 35 mg, or 45 mg, and other ranges as described, and is a preferred low molecular weight microbial fermented product as described above.
  • Astaxanthin once a day during breakfast for 12 weeks A total of 70 subjects were recruited for the study, 35 in each group (Astaxanthin oleoresin complex and placebo- control) of both the sexes. Patients were explained the nature of the study and informed consent was obtained prior to the start of the study. Patient subjects were clinically examined by the Principal Investigator and team. X ray and blood samples were drawn at the commencement and at the end of study period. The case record forms were filled by the Principal Investigator and rechecked by the Clinical research associate. Sixty patient subjects completed the study. Ten were drop outs due to various reasons but not on account of intolerance to the astaxanthin oleoresin complex or placebo control. The results were tabulated by the expert data entry operators under supervision of Biometric expert. The results were subjected to Statistical analysis by an independent analyst.
  • Osteoarthritis symptoms were based on Western Ontario and Mc asters Universities (WOMAC) Osteoarthritis Index, VAS scale, Lequesne's functional scale as well as Sleep score as additional parameters besides radiological investigations. Further the assessment of Osteoarthritis symptoms based on haematological studies, specifically MP3 (Matrix metalloproteinase 3) in clinical parameters since Osteoarthritis patients show elevated levels of M P3 in blood as well as in synovial fluid. The elevated levels cause significant tissue damage through cartilage destruction.
  • WOMAC Western Ontario and Mc asters Universities
  • WOMAC Score The Western Ontario McMaster (WOMAC) is a validated instrument designed specifically for the assessment of lower extremity pain and function in Osteoarthritis (OA) of the knee. The patients were assessed on their pain, stiffness and difficulty in carrying out day-to-day activities.
  • the pain index was assessed for Activities - a) in walking on flat surface, going up or down on flat surface, at night while in bed, sitting or lying, standing upright; b) Stiffness - after first wakening in morning, after sitting/lying or resting later in the day; and c) difficulty in descending stairs, ascending stairs, standing up from a chair, while standing, bending to floor to pick up objects, walking on flat ground, getting in and out of autorickshaw/bus/car, going shopping, on rising from bed, while lying on bed, while sitting on chair, going on/off toilet, doing heavy domestic duties such as moving heavy boxes/scrubbing floor/lifting shopping bags, doing light domestic duties such as cleaning
  • VAS Visual Analog Scale
  • Pain parameters were assessed in Osteoarthritis patients taking astaxanthin oleoresin and the Placebo group using VAS. The assessment was carried out in a) Pain parameters - pain while using stairs, pain while walking on fiat ground, pain while standing upright, pain while sitting or lying down, pain at night in bed b) Physical functions - going downstairs, going upstairs, sitting, getting up from sitting, standing, bending to floor, walking on flat ground, getting into or out of automobiles, shopping, putting on socks/stockings, taking off
  • taquesne's index - Laquesne's index is the Functional index for
  • Osteoarthritis of the knee Assessment is carried out on a) Pain/discomfort - during nocturnal bed rest, morning stiffness or regressive pain after rising, after standing for 30 minutes; and b) Physical functions - maximum distance walked, activities of daily living like able to climb up a standard flight of stairs, able to climb down a standard flight of stairs, able to squat or bend on the knees, able to walk on uneven ground.
  • Sleep Scale - Sleep is an important element of functioning and well being. Sleep Scale was originally developed in the Medical Outcomes Study (MOS) intended to assess the extent of sleep problems.
  • the Medical Outcomes Study Sleep Scale includes 12 items assessing sleep disturbance, sleep adequacy, somnolence, quantity of sleep, snoring, and awakening short of breath or with a headache. A sleep problems index, grouping items from each of the former domains, is also available.
  • This assessment evaluated the psychometric properties of OS-Sleep Scale in Osteoarthritis patients taking Astaxanthin oleoresin compiex and Placebo group. The results on Sleep scale MOS is given in Table 7.
  • MMP3 Mestrix Metalloproteinase 3
  • WO AC INDEX exhibited significant differences (P ⁇ 0.001). This score is unique for the functional abilities in patients with chronic joint disorders such as
  • VAS Pain parameters Pain +Physical score: There were significant reductions in the mean scores at the end of treatment for patients taking astaxanthin oleoresin complex but not for Placebo P ( ⁇ 0.001). It is suggestive of improvement in the pain related aspects of Osteoarthritis.
  • Laquesne's index (Functional Index for OA of knee): There were significant reductions in the mean scores at the end of treatment for patients taking Astaxanthin oleoresin complex but not for Placebo (P ⁇ 0.05).
  • Astaxanthin oleoresin complex extracted through polar solvents from Haematococcus piuvialis alga may be suitable for the patients in the early stage of the Osteoarthritis to prevent the progression of the disorder. It may be useful to the patients with established Osteoarthritis to provide symptomatic relief from pain and improved quality of life. Astaxanthin oleoresin complex improves symptoms like pain as well as quality of physical activities of daily life in a significant manner. Osteoarthritis is seen to mark its presence at a younger age in India. It would be appropriate to initiate the treatment with Astaxanthin oleoresin complex right from the beginning as soon as the diagnosis is arrived at. Study with larger sample size at different centres is

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Abstract

La présente invention concerne une composition de complément alimentaire et un procédé d'utilisation associé, ladite composition étant formulée dans une quantité thérapeutique pour traiter et soulager les symptômes de la douleur articulaire chez une personne souffrant de douleur articulaire. Cette composition comprend de l'astaxanthine et de l'acide hyaluronique ou de l'hyaluronate de sodium (hyaluronane) fermenté microbien de faible poids moléculaire. La composition comprend également un phospholipide et/ou un glycolipide et/ou un sphingolipide. Ladite composition est formulée dans une forme posologique orale, et l'astaxanthine représente 0,1 à 15 pour cent en poids du phospholipide et/ou du glycolipide et/ou du sphingolipide.
PCT/US2015/020671 2014-03-18 2015-03-16 Composition et procédé destinés à atténuer la douleur articulaire au moyen de phospholipides et d'astaxanthine WO2015142700A1 (fr)

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