DE212015000073U1 - Composition for relieving joint pain with phospholipids and astaxanthin - Google Patents

Abstract

A nutritional supplement composition comprising astaxanthin, proinflammatory, low molecular weight, microbially fermented sodium hyaluronate fragments having a molecular weight of 0.5 to 300 kilodaltons (kDa) and a carrier having a lipid selected from the group consisting of a phospholipid, glycolipid and sphingolipid an oral dosage form, wherein the astaxanthin constitutes 0.1 to 15% by weight of the carrier and the lipid selected from the group consisting of a phospholipid, glycolipid and sphingolipid for use as adjunct to treating and relieving joint pain ,

Description

Related Application (s)

The present application claims priority to U.S. Patent Application Serial No. 14 / 645,890, filed March 12, 2015, and U.S. Patent Application Serial No. 14 / 217,515, filed March 18, 2014.

Field of the invention

The present invention relates to a composition for the treatment and alleviation of joint pain and symptoms of osteoarthritis and / or rheumatoid arthritis.

Background of the invention

The use of krill oil is in the U.S. Patent Publication No. 2004/0234587 ; 2004/0241249 and 2007/0098808 , the disclosures of which are hereby fully incorporated by reference. The use of krill oil has also been published in a research paper by L. Deutsch, entitled "Evaluation of the Effect of Neptune Krill Oil on Chronic Inflammatory and Arthritic Symptoms," Journal of the American College of Nutrition, Vol. 26, No. 1, 39-49 (2007). has been published and the disclosure of which is hereby incorporated by reference in its entirety.

Published applications' 587, '249 and' 808 discuss the beneficial aspects of using krill oil in conjunction with pharmaceutically acceptable carriers. By way of example, this krill oil and / or marine oil can be obtained by the combination of detailed steps taught in the '808 application by adding krill and / or a marine material in a ketone solvent, separating the liquid and solid components. a first lipid-rich fraction is recovered from the liquid constituents by evaporation, the solid constituents and the organic solvent are added to an organic solvent of the type taught in the specification, the liquid and the solid constituents are separated, a second lipid-rich fraction by evaporation of the Solvent is recovered from the liquid components and the solid components are recovered. The resulting krill oil extract has also been used in an attempt to lower lipid profiles in patients with hyperlipidemia. Document '808 reports in detail on this krill oil obtained by means of these general steps described above.

Summary of the invention

The jointly transmitted great-grandparents and Grandparent US Pat. Nos. 8,481,072 and 8,945,608 and the jointly assigned U.S. Patent No. 8,557,275 claiming the priority of the '072 patent, the disclosures of which are hereby fully incorporated by reference, discuss the beneficial use of oils derived from krill and / or fish. These patents disclose the beneficial and synergistic effects of mitigating joint pain when an oil derived from krill and / or fish is used in combination with other agents such as low molecular weight hyaluronic acid and astaxanthin. The use of krill oil was a major point of the patents '072 and '608 , An oil derived from krill or fish, such as in the patent '275 describes phospholipid and glycolipid-bound EPA (eicosapentaenoic acid) as compared to fish oils which are triacylglycerides.

Further development has been achieved with different species of algae producing only EPA or EPA and DHA (docosahexaenoic acid). A further development by means of rouge extract and / or phospholipid sources and / or other surface-active substances has also been carried out. Further development has also been made using low molecular weight hyaluronic acid from various sources and with improvements in the use of astaxanthin and its improved bioavailability, such as incorporation of a phospholipid or other components.

A nutritional supplement composition is formulated in a therapeutic amount to treat and alleviate the symptoms of joint pain in a person suffering from joint pain. The composition comprises astaxanthin and microbially fermented low molecular weight hyaluronic acid or sodium hyaluronate (hyaluronan). The composition also includes at least one of the following: phospholipid, glycolipid and sphingolipid, and is formulated into an oral dosage form. The Astaxanthin is 0.1 to 15% by weight of at least one of the following: phospholipid, glycolipid and sphingolipid.

In one example, the astaxanthin is derived from a natural or synthetic ester or synthetic diol and may contain a pharmaceutical grade or food grade diluent. In another example, the phospholipid comprises at least one of the following: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, lyso-phosphatidylcholine, lyso-phosphatidylethanolamine and lyso-phosphatidylserine. The phospholipid may be derived from at least one of: a vegetable source, an algae source or an animal source or synthetic derivatives thereof.

In yet another example, the composition contains 0.5 to 12 mg astaxanthin and contains 50 to 500 mg of at least one of the following: phospholipid, glycolipid and sphingolipid. The nutritional supplement composition is formulated into a single dose capsule in one example. The composition comprises proinflammatory, low molecular weight, microbially fermented sodium hyaluronate fragments having a molecular weight of 0.5 to 300 kilodaltons (kDa).

Treating and mitigating the symptoms of joint pain involves administering to a person suffering from joint pain a therapeutic amount of a nutritional supplement comprising astaxanthin and at least one of the following: phospholipid, glycolipid and sphingolipid, which is formulated into an oral dosage form. The astaxanthin constitutes 0.1 to 15% by weight of at least one of the following: phospholipid, glycolipid and sphingolipid. The composition may be formed by dispersing the astaxanthin under high shear conditions in at least one of the following: phospholipid, glycolipid and sphingolipid.

Brief description of the drawings

Other objects, features and advantages of the present invention will become apparent from the detailed description of the invention which follows, with reference to the accompanying drawings, in which:

1 is a view showing a chemical structure of astaxanthin, which can be used in accordance with a non-limiting example.

Detailed Description of the Preferred Embodiments

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. However, the invention may be embodied in many different forms and is not to be considered as limited to the embodiments disclosed herein. Rather, these embodiments are presented so that the present disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Below is a description of the joint health composition and related method disclosed in the patents '072 . '608 and '275 relating to krill oil and / or fish-derived oil, and containing novel details relating to an algae-based oil, fish oil-derived, roe and / or vegetable oils, including phospholipids further away from the platform base of the Omega-3. Novel details and new uses and compositions of different sources of hyaluronic acid and phospholipids and astaxanthin are described.

The krill oil related composition contains EPA and DHA functionalized as marine phospholipids and krill derived acyl triglycerides. The product derived from krill, algae, rye extract and fish oil and the phospholipid compositions may contain astaxanthin, such as esterified astaxanthin and, by way of nonlimiting example, low molecular weight polymers of hyaluronic acid or sodium hyaluronate (hyaluronan) in an oral dosage form. In one example, it comprises proinflammatory, low molecular weight, microbially fermented sodium hyaluronate having a molecular weight of 0.5 to 300 kDa, in another example, 0.5 to 230 kDa, and in yet another example, 0.5 to 100 kDa. Some of these components of krill oil in one example are illustrated in the following table: components percentage (%) PHOSPHOLIPIDS PC, PE, PI, PS, SM, CL > 40 OMEGA-3 (functionalized on PL) > 30 Eicosapentaenoic Acid (EPA) * > 17 (15% in one example and 10% in another) Docosahexaenoic acid (DHA) + > 11 (9% in one example and 5% in another) ANTIOXIDANTS (mg / 100g) Astaxanthin, Vitamin A, Vitamin E > 1.25 *> 55% PL-EPA / total EPA
+> 55% PL-DHA / total DHA

These quantities may vary depending on the application and the individuals.

The composition contains in one example proinflammatory, microbially fermented sodium hyaluronate fragments having a molecular weight of 0.5 to 300 kilodaltons (kDa), and 0.5 to 230 kDa and 0.5 to 100 kDa, all in oral dosage form. Natural high molecular weight hyaluronic acid is the major hydrodynamic component of synovial fluid and, importantly, is known to be immune-neutral to the innate immune system. These are nature's shock-absorbing agents and lubricants of the bone joint. It has been found that oral bioavailability for fragments of low molecular weight hyaluronic acid (LMWtHA) is especially excellent for the connective tissue, maximizing interaction with synovial fluid-producing target cells. Thus, in a preferred composition containing krill oil, algae-based oil, fish oil-derived product, roe and phospholipids or other compositions, astaxanthin and LMWtHA, two anti-inflammatory components, are combined with a highly flammable component.

The scientific literature indicates that LMWtHA fragments show potent proinflammatory behavior. It therefore remains unclear why a proinflammatory component causes a favorable overall response in inflamed joint tissues. It is believed that such pro-inflammatory LMWtHA fragments promote local repair by simulating the innate immune repair mechanism, and by simulating production of non-immunogenic high molecular weight hyaluronic acid, the joint is returned to homeostasis. Much of the work done by leading immunologists is still exposing all aspects of the complicated signaling processes associated with the innate immune system. Studies on extensive animal models of osteoarthritis have shown that mild immunogenic hyaluronic acids with molecular weights in the range of 0.5-1.0 x 10 ⁶ Da (Dalton) are generally more effective in reducing the signs of synovial inflammation and restoring the rheological properties of SF (viscoinduction) were non-immunogenic HAs with molecular weights> 2.3 × 10 ⁶ Da.

It will be appreciated by those skilled in the art that a pro-inflammatory, low molecular weight hyaluronic acid will be around 300 kDa to about 320 kDa or less, with many experts using 300 kDa as the upper limit. It is well known that low molecular weight hyaluronic acids and sodium hyaluronates act as proinflammatory agents and are believed to be upregulators of the inflammatory cascade with respect to the innate immune system. Some reports indicate that hyaluronic acid fragments cause the expression of inflammatory genes and are low molecular weight kDa. Clinical trials conducted by the inventors and their assignee have demonstrated the efficacy of the composition when krill oil is used together with the low molecular weight hyaluronic acid or hyaluronan and astaxanthin according to a non-limiting example. In clinical trials, no rescue medication was approved compared to the German study referred to above. The low molecular weight hyaluronic acid had a molecular weight of about 40 kDa in the experiment, but in one example could be in the range of 0.5 to 100 kDa or in still other examples in the range of 0.5 to 230 kDa or 0.5 to 300 kDa.

The composition used in the clinical trials of the present subject was directed to the treatment and alleviation of joint pain. The subjects of the clinical study had no confirmed osteoarthritis and / or rheumatoid arthritis. The abridged exclusion criteria specifically listed the subjects as having no autoimmune disease or similar disease and the study excluded those subjects who knew that their joint pain was due to osteoarthritis and / or rheumatoid arthritis. The clinical study was directed to patients who had joint pain in a non-disease state unrelated to a disease state, such as osteoarthritis and / or rheumatoid arthritis. The composition has been used as a supplement to treat and alleviate joint pain symptoms of unknown etiology, including joint pain, which are not associated with osteoarthritis and / or rheumatoid arthritis in this example.

Astaxanthin is a component of the composition. The clinical trials relating to the joint care composition with krill oil, low molecular weight hyaluronic acid and astaxanthin demonstrated the effectiveness of the composition with surprisingly beneficial results. The related scientific literature indicates that astaxanthin with only 1 mg / kg in vitro and in vivo: (1) down-regulates TNF-alpha production by 75% in a lipopolysaccharide-induced inflammatory rat model; (2) downregulated prostaglandin E-2 production (PGE-2) by 75%; (3) nitric oxide synthase (NOS) inhibits nitric oxide expression by 58%; and (4) these effects on inflammatory markers in this model were almost as effective as prednisolone. Such information suggests, but does not prove, that astaxanthin may be an effective single product for reducing OA and / or RH pain or other symptoms associated with OA and / or RH. 1 shows an example of astaxanthin as astaxanthin 3S, 3'S- (3,3'-dihydroxy-4,4'-diketo-β-carotene). The clinical trial with only 15 mg astaxanthin is considered to be beneficial.

The patents incorporated by reference '072 and '608 describe the clinical trial with astaxanthin alone, in which one dose of a softgel containing 15 mg of formulated and described astaxanthin was administered once daily during breakfast for 12 weeks. This large astaxanthin single dose was effective to treat osteoarthritis and joint pain. It has now been found that lower astaxantine dosages, instead of these much higher dosages, such as 15 mg, can be used in clinical trials when at least one of the following is added: a phospholipid, glycolipid and sphingolipid, or used alone with the low molecular weight hyaluronic acid becomes. A pharmaceutical grade or food grade diluent or other surfactant may be added. Other beneficial and often synergistic results are obtained when astaxanthin is used in the presence of low molecular weight hyaluronic acid as described above or UC-II. Phospholipids may be plant-based phospholipids, such as lecithin and lyso-phospholipids and / or glycophospholipids, including perilla oil as described in the commonly assigned US Pat U.S. Patent No. 8,784,904 is described, the disclosure of which is fully incorporated by reference herein. The astaxanthin levels could vary from 0.5-2 mg and 0.5-4 mg, and in one embodiment would be 2-4 mg or 2-6 and even 0.5-12 mg and 7-12 mg.

• 1) Lee u. a., Molecules and Cells, 16(1): 97–105; 2003 ;
• 2) Ohgami u. a., Investigative Ophthalmology and Visual Science 44(6): 2694–2701, 2003 ;
• 3) Spiller u. a., J. of the Amer. College of Nutrition, 21(5): Oktober 2002 ; und
• 4) Fry u. a., Univ. of Memphis Human Performance Laboratories, 2001 und 2004. Berichte 1 & 2 .

In induced uveitis, astaxanthin also demonstrated dose-dependent ocular anti-inflammatory activity by suppressing NO, PGE-2, and TNF-alpha by directly blocking NO synthase activity. It is also known that astaxanthin reduces the C-reactive protein (C-RP) levels in the blood in vivo. For example, in human subjects with high risk levels of C-RP, a three-month astaxanthin treatment in 43% of patients resulted in serum C-RP levels falling below the risk level. This may explain why the C-RP levels in the above-mentioned German study fell significantly. Astaxanthin is so potent that it has been shown to defeat the pro-oxidative effect of Vioxx, a COX-2 inhibitor, that belongs to the class of NSAIDS medications known to cause cellular membrane lipid peroxidation leading to heart attack and heart attack Stroke leads. That's why Vioxx was taken off the market in the US. Astaxanthin is absorbed by lens epithelial cells in vitro, where it suppresses UVB-induced lipid-peroxidative mediated cell damage in umol / L concentrations. In human trials, astaxanthin at 4 mg / day prevented post-workout joint fatigue following strenuous knee training compared to untreated subjects. These results were shown in:

• 1) Lee et al., Molecules and Cells, 16 (1): 97-105; 2003 ;
• 2) Ohgami et al., Investigative Ophthalmology and Visual Science 44 (6): 2694-2701, 2003 ;
• 3) Spiller et al., J. of the Amer. College of Nutrition, 21 (5): October 2002 ; and
• 4) Fry et al., Univ. of Memphis Human Performance Laboratories, 2001 and 2004. Reports 1 & 2 ,

In one embodiment, a composition contains 300 mg of krill oil, 30 to 45 mg of low molecular weight hyaluronic acid, and 2 mg of astaxanthin. It has now been found that 150 mg to 300 mg Krill oil is advantageous in an 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 lie. The use of the added phospholipids and / or surfactants described below aids in the delivery of astaxanthin. The low 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 another embodiment having about 30 mg.

Astaxanthin has a potent singlet oxygen scavenging effect. Astaxanthin typically has no pro-oxidative effect, unlike β-carotene, lutein, zeaxanthin, and vitamins A and E. In some studies, astaxanthin was found to be about 50 times more potent than vitamin E. 11 times more potent than β-carotene and is three times stronger than lutein in capturing singlet oxygen. Astaxanthin is also well known for its ability to trap free radicals. Comparative studies have found that astaxanthin is 65 times more potent than vitamin C, 54 times stronger than beta-carotene, 47 times more potent than lutein, and 14 times more potent than vitamin E in its ability to capture free radicals.

The U.S. Patent 5,527,533 (the Tso patent), the disclosure of which is fully incorporated herein by reference, discloses the benefits of astaxanthin for delaying and ameliorating damage to the central nervous system and the eyes. Astaxanthin traverses the blood-brain-retina barrier, and this can be measured by direct measurement of the retinal astaxanthin levels. Therefore, Tso showed protection against photon-induced photoreceptor damage, ganglion and neuronal cell damage.

Studies have shown that HA binds to the surface of DCs and stimulated T cells. Blockade of the CD44-HA interaction results in 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. In DCs, NO expression caused apoptosis (cell death) of dendritic cells. DCs are essential T-cell activators that act by presenting antigens to T-cells so that apoptosis of DCs can 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. It appears that low molecular weight HA fragments disrupt the normal course of the well-known T cell mediated adaptive immune system response. CD44 is a glycoprotein which, in part, is responsible for lymphocyte activation (also known as T cell activation) and is known for specific binding to HA. In contrast, as previously discussed, low molecular weight HA fragments appear to up-regulate the innate immune response, especially in chronic inflammatory conditions where the innate immune system may be somewhat compromised.

• 1) Mummert u. a., J. of Immunol. 169, 4322–4331 ;
• 2) Termeer u. a., Trends in Immunology, Bd. 24, März 2003 ;
• 3) Yang u. a., Cancer Res. 62, 2583–2591 ; und
• 4) McKee u. a., J. Biol. Chem. 272, 8013–8018 .

Support for these teachings can be found in:

• 1) Mummert et al., J. of Immunol. 169, 4322-4331 ;
• 2) Termeer et al., Trends in Immunology, Vol. 24, March 2003 ;
• 3) Yang et al., Cancer Res. 62, 2583-2591 ; and
• 4) McKee et al., J. Biol. Chem. 272, 8013-8018 ,

Additional information can be found in the following publications: Ghosh P. Guidolin D. Semin Arthritis Rheum., 2002 Aug; 32 (1): 10-37 ; and P. Rooney, M. Wang, P. Kumar and S. Kumar, Journal of Cell Science 105, 213-218 (1993) ,

As noted above, krill oil is typically produced from Antarctic krill (Euphausia superba), which is a zooplankton (lower end of the food chain). It is one of the most common marine biomass with about 500 million tons according to some estimates. Antarctic krill reproduces in clean, uncontaminated deep-sea waters. It is an unused marine biomass and the catch per year is estimated to be about 0.02% per year. As krill is harvested in large quantities and the world's supply of krill is wasted, substitutes for krill, such as other marine-based oils, including algae-based oils, are now being studied, developed and used.

It is believed that krill oil and some other marine based and plant based oils have oil-based phospholipid-bound EPA and DHA uptake in cell membranes, which is much more efficient than triacylglyceride-bound EPA and DHA since the conversion of triacylglycerides in the As such, liver is not efficient and phospholipid-bound EPA and DHA can be transported via the lymphatic system into the bloodstream, thereby avoiding liver failure. Furthermore Consuming oil based on krill, algae and some marine and plant matter does not produce any burp observed with fish oil based products. It has been found that because of this barking feature of fish oils, about 50% of all consumers who taste fish oil will never buy it again. Some algae-based oils contain EPA conjugated to phospholipid- and glycolipid-polar lipids, which makes EPA uptake even more effective.

• Orales LD 50: 600 mg/kg (Ratten);
• NOAEL: 465 mg/kg (Ratten); oder
• Serumpharmakokinetik: Stewart u. a. 2008
• 1) T_1/2: 16 Stunden;
• 2) T_max: 8 Stunden;
• 3) C_max: 65 μg/l.

Astaxanthin has an excellent safety protocol. In one study, the following results were obtained:

• Oral LD 50: 600 mg / kg (rats);
• NOAEL: 465 mg / kg (rats); or
• Serumpharmakokinetik: Stewart and others 2008
• 1) T _1/2 : 16 hours;
• 2) T _max : 8 hours;
• 3) C _max : 65 μg / l.

There was no negative effect on healthy adults in eight weeks of 6 mg daily supplementation. Spiller et al. 2003 ,

By way of non-limiting example, astaxanthin has three major sources: 3 mg astaxanthin per 240 g portion of unbred salmon or 1% to 12% astaxanthinoleoresin or 1.5-2.5% globules derived from microalgae. Another verification takes place in Lee et al., Molecules and Cells 16 (1): 97-105, 2003 ; Ohgami et al., Investigative Ophthalmology and Visual Science 44 (6): 2694-2701, 2003 ; Spiller 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 ,

It now reports beneficial and synergistic effects which have been observed when using a krill, algae and fish oil-derived product, roe extract and seed oil and other phospholipid-based compositions in combination with other active ingredients. In particular, the present composition contains as ingredients krill, algae, fish oil, rye, seed oil or other phospholipids in combination with astaxanthin and low molecular weight polymers of hyaluronic acid or sodium hyaluronate, preferably in an oral dosage form, for controlling the range of joint pain in terms of movement and stiffness. It is anticipated that different proportions of the compositional components and their percentages may be used to treat a patient, depending on the end-uses and other environmental and physiological factors.

By way of non-limiting example, the composition treats and ameliorates symptoms of joint pain in a non-disease state and may be used to treat and ameliorate symptoms of osteoarthritis and / or rheumatoid arthritis in a patient by administering a therapeutic amount of a composition with the krill oil or other algae-based oil, a 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. In one example, the krill oil is derived only from Euphasia spp., Which comprises fatty acids of eicosapentaen (EPA) and docosahexaen (DHA) in the form of triacylglycerides and phospholipids, and it has been found that not less than 1% EPA and 5% DHA are beneficial.

In another example, the krill oil contains at least 15% EPA and 9% DHA, of which not less than 45% are in the form of phospholipids, and 40% in another example. The composition can be advantageously delivered for therapeutic results with 1-4000 mg of oil, such as krill or algae-based oil, which is delivered at a daily dose. In another example, 500 mg is a preferred amount for a single capsule dose and in another example 1,000 mg. In another example, 0.1-50 mg of astaxanthin is added to the oil supplementally in daily dosage, but a preferred amount is about 2-4 mg and 0.5 to 12 mg. The oils on algae and other marine basis and the roe extract with phospholipid and plant-based oils and phospholipids can be used. The composition of algae-based oils and their fatty acid profile differ from the fatty acid profiles of krill oil, as explained below and shown in the Tables. It is also possible to use wax esters and omega-3 salts as well as ethyl esters.

The composition may also comprise an n-3 (omega-3) fatty acid rich oil derived from fish oil, algae oil, linseed oil or chia seed oil when the n-3 fatty acid comprises alpha-linolenic acid, stearidonic acid, eicosapentaenoic or docosapentaenoic acid. The composition may have naturally-derived and synthetic antioxidants added to retard the degradation of fatty acids and astaxanthin.

Details of one type of CO2 extraction and processing technique (such as supercritical CO2 extraction) and peroxidation blocker technique that may be used are described in the commonly assigned US Pat U.S. Patent No. 8,652,544 ; U.S. Patent No. 8,586,104 ; U.S. Patent No. 8,784,904 and the U.S. Patent Publication No. 2009/0181114 , the disclosures of which are hereby fully incorporated by reference.

As indicated above, there are favorable aspects for using krill oil or algae-based oil and other described oils in synergistic combination with other ingredients. It has been found that a choline-based phospholipid-bound omega-3 fatty acid mixture derived from fish oil and containing phospholipid-linked polyunsaturated EPA and DHA is beneficial to joint health when combined with astaxanthin and low molecular weight hyaluronic acid or Hyaluronate is combined. A commercially available example of a mixture of a choline-based phospholipid-linked fatty acid mixture derived from fish oil containing polyunsaturated EPA and DHA is Omega Choline 1520F as a phospholipid, an omega-3 derived from natural fish oil from Enzymotec Ltd. is sold. An example of such a composition is described below: Ingredients (g / 100 g): Pure marine phospholipids not less than 15 DHA * not less than 12 EPA ** not less than 7 * Docosahexaenoic acid ** eicosapentaenoic acid Omega 3 not less than 22 Omega-6 <3 Analytical data: Peroxide value (meq / kg) not more than 5 Drying loss (g / 100 g) not more than 2 Physical Properties: consistency viscous liquid

The mixture of a fish oil-derived, choline-based, phospholipid-bound fatty acid mixture containing polyunsaturated EPA and DHA in one example comprises the fatty acids of eicosapentaen (EPA) and docosahexaen (DHA) in the form of triacylglycerides and phospholipids. In another example, the omegacholine contains at least 7% EPA and 12% DHA, of which no less than 15% are in the form of phospholipids. The composition can advantageously be delivered for therapeutic results with 1-4000 mg of a mixture of fish oil and a fish oil-derived, choline-based, phospholipid-bound fatty acid mixture containing polyunsaturated EPA and DHA, which is delivered in a daily dose. In one example about 150 mg to about 300 mg are used. In another example, 2 to 4 mg of astaxanthin per omega-choline is added per daily dose, but ranges from 0.5 to 4 mg or 0.5 to 6 mg, 0.5 to 12 mg or 7 to 12 mg and other ranges may be as described above.

It is also possible to use a mixture of a fish oil-derived, choline-based, phospholipid-bound omega-3 fatty acid mixture (containing polyunsaturated EPA and DHA) mixed with astaxanthin and the low molecular weight hyaluronic acid. It is also of it it can be assumed that an enriched version of a mixture of a fish oil-derived, choline-based, phospholipid-bound fatty acid mixture containing polyunsaturated EPA and DHA can be used, whereby the proportion of fishblend diluents added has been lowered and the proportion of fish oil-derived phospholipids increased has been. This can be accomplished by using supercritical CO2 and / or solvent extractions to selectively remove triacylglycerides of phospholipids using, for example, the techniques disclosed in the incorporated patents. The composition may also contain a natural or synthetic cyclooxygenase-1 or -2 inhibitor comprising, for example, aspirin, acetaminophen, steroids, prednisone or NSAIDs. The composition may also contain an oil rich in gamma linoleic acid, including borage (Borago officinalis L.) or safflower (Carthamus tinctorius L.) which releases a metabolic precursor to PGE ₁ synthesis.

The composition may also contain an oil rich in n-3 (omega-3) fatty acid derived from fish oil, algae oil, linseed oil, chia seed oil or perilla seed oil, the n-3 fatty acid source being alpha-linolen, steridone, eicosapentaen or docosapentaenoic acid. The composition may contain naturally-derived and synthetic antioxidants added to delay degradation of fatty acids such as tocopherols, tocotrienols, carnosic acid or carnosol and / or astaxanthin.

It has been found advantageous to use herring gene extract as a source of phospholipids which may have some EPA and DHA. Synergistic results are obtained and there are big improvements to be seen. A study has indicated that herbal phospholipids improved phospholipid and glucose tolerance in healthy, young adults, such as Bjorndal et al., Lipids in Health Disease, 2014, 13:82 released. The pure rouge phospholipid can be formed by extraction techniques. It is a honey-like product thinned or diluted in one example with fish oil and / or perilla oil or other seed or vegetable oil.

The description before dilution with fish oil and / or perilla oil is as follows: Percentage of phospholipids 60 Phospholipid mg / g 600 Phosphatidylcholine amount mg / g 520 Cholinäquivalente 83 Total EPA mg / g (TG & PL bound) 75 Total DHA mg / g (TG & PL bound) 195 EPA mg / g bound to phospholipid 67 DHA mg / g bound to phospholipid 175 EPA + DHA mg / g bound to phospholipid 242

In one example, the herring gene extract is processed by extraction with ethanol. Triacylglycerides are added and ethanol is stripped off to obtain the stable solution. Seed oil, such as perilla seed oil, disclosed in the incorporated patent '904 may be added back to the ethanol extract before stripping to dilute and formulate a mixture with a high content of phospholipid.

The rye oil extract may be mixed with fish oil and / or seed oil, such as perilla, or other marine oil. In one example, the herring seed extract is mixed with perilla seed oil at least 1: 1 and preferably up to 6: 1 ALA to LA, the concentrate being at least 50% and in another example 60% phospholipids and in another example at least 30% and in another Example 40% triglycerides.

An exemplary composition contains a combination of a herring gene extract or a phospholipid-rich Rogen extract with phospholipid-bound EPA and DHA mixed with seed / fish oil and / or seed oil, the seed oil having a ratio of ALA to LA between 1: 1 and 1: 6, and optionally contains astaxanthin in an example of about 2-4 mg or 0.5 to 12 mg or other ranges as indicated above and the low molecular weight hyaluronic acid described above. The The amount of roe egg extract mixed with the seed oil, such as the perilla oil, varies and amounts to 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 can be used, including commercially available lecithins and an egg yolk derivative containing lysophospholipids and glycophospholipids to act as surfactants. It is possible to use sunflower-based phospholipids and plant-based natural oils as well as extracts of natural surfactants. The astaxanthin is enhanced with fats, surfactants or phospholipids and can be delivered more effectively with phospholipids and sunflower-based oil and / or lipophilic perilla oil, as previously described.

In one example, the perilla oil is formed as a durable, supercritical, CO2 fluid-extracted seed oil derived from a broken biomass of Perilla frutescens of 60 to 95 percent w / w PUFAs in a ratio of 4: 1 to 6: 1 alpha-linolenic acid (ALA ) to linoleic acid (LA). The seed oil derived from Perilla frutescens is prepared in one example by subjecting the perilla frutescens seeds to supercritical fluid CO2 extraction to produce a seed oil extract; the resulting seed oil extract is fractionated in separate pressure reduction stages to collect light and heavy fractions of the seed oil extract; and separating the heavy fraction from the light fraction to form the final seed oil from the heavy fraction.

The selected antioxidants are included in another example and the perilla oil comprises a mixture of selected lipophilic and hydrophilic antioxidants. Lipophilic antioxidants may be used either alone or in combination with at least one of the following: a) phenolic antioxidants having at least one of the following: sage, oregano and rosemary; b) tocopherol; c) tocotrienol / e; d) carotenoids having at least one of the following: astaxanthin, lutein and zeaxanthin; e) ascorbyl acetate; f) ascorbyl palmitate; g) butylated hydroxytoluene (BHT); h) docosapentaenoic acid (BHA); or i) tertiary butylhydroquinone (TBHQ). A hydrophilic antioxidant or complex may comprise hydrophilic phenolic antioxidants with at least one of: grapeseed extract, tea extracts, ascorbic acid, citric acid, tartaric acid and malic acid.

In one example, a peroxide value of this perilla seed oil is below 10.0 meq / km. In another example, the perilla seed oil is 85 to 95 percent w / w PUFAs and the PUFAs are at least greater than 56 percent alpha-linolenic acid (ALA). The perilla seed oil is stable at room temperature for up to 32 months. In another example, this perilla seed oil is derived from a pre-milled or flaked crushed biomass of Perilla frutescens. The mixture of selected anti-oxidants may include astaxanthin, phenolic antioxidants and natural tocopherols. The perilla seed oil may also have at least one of the following: dispersed nano- and microparticles of rice or sugarcane-based policosanol.

In one example, the composition is enclosed in a single dose capsule and referred to as a deep sea avian capsule. In a specific example, the entrapped composition Heringskaviarphospholipidextrakt comprises (herring roe) Perilla (Perilla frutescens) -Samenextrakt, olive oil, Zanthin ^® astaxanthin (Haematococcus pluvialis alga extract), gelatin, spice extract, non-genetically modified (non-GMO) natural tocopherols, cholecalciferol, Riboflavin and methylcobalamin. The composition contains fish as herring roe and tilapia gelatin. An example is given in the following table. Properties: appearance clear capsule size 00 with dark red oily filling fatty acids ALA minute 140 mg EPA minute 18 mg DHA minute 50 mg Ges. Omega-3 minute 210 mg phospholipids 195 mg astaxanthin 500 μg Vitamin D ₃ 1000 IU; 250% DV Vitamin B ₂ (riboflavin) 1.7 mg; 100% DV Vitamin B ₁₂ 6 μg; 100% DV microbiological USP <61> / FDA BAM Total plate count <1000 cfu / g Yeast & mold <100 cfu / g E. coli missing in 10 g Salmonella missing in 10 g S. aureus missing in 10 g storage conditions tightly closed containers, 15- 30 ° C, 30-50% relative humidity Shelf life 24 months minimum packaging HDPE or PET bottle (number to be determined) All ingredients are BSE-free and not genetically modified

The processing components may contain a mixture of marine omega-3 phospholipids derived from herring caviar and perilla seed oil. An O2B ^™ botanical peroxidation blocker with spice extract, non-genetically modified tocopherols and ascorbyl palmitate may be included. Packing may be bulk in sealed drums with 45 and 190 kg net and inert headspace, which comply with European and American standards for food products. Storage preferably takes place below room temperature. The product is protected from light and heat. When drums are opened for sampling, the headspace may be purged with inert gas during sampling and prior to storage. test unit acceptance criteria method appearance Yellow viscous oil AM2020 solubility Soluble in oil and dispersible in water AM2021 minimum maximum ALA (C18: 3 n-3) mg / g as TG ³⁾ 230 AM1044 EPA (C20: 5n-3) mg / g as TG ³⁾ 30 AM1001 DHA (C22: 6n-3) mg / g as TG ³⁾ 85 AM1001 Total omega-3 ¹⁾ mg / g as TG ³⁾ 370 AM1001 ALA (C18: 3 n-3) mg / g as FFA ⁴⁾ 215 AM1044 EPA (C20: 5n-3) mg / g as FFA ⁴⁾ 28 AM1001 DHA (C22: 6n-3) mg / g as FFA ⁴⁾ 80 AM1001 Total omega-3 ¹⁾ mg / g as FFA ⁴⁾ 335 AM1001 Ges. PC mg / g 250 AM1002 Ges. PL mg / g 300 AM1002 Ges. Neutral lipids mg / g 700 AM1003 Water content of Karl Fisher % 3.0 AM1004 peroxide meq / kg 10.0 AM1005 Heavy metals (sum of Pb, Hg, Cd & inorganic As) ²⁾ mg / kg 10 AM1015 1) Total n-3: ALA, EPA, DHA, 18: 4, 20: 4, 21: 5, 22: 5
2) Frequency analysis
3) All ALA, EPA, DHA or ges. Omega-3 expressed as triglycerides
4) All ALA, EPA, DHA or ges. Omega-3 expressed as free fatty acids

It has surprisingly been found that the bioavailability of astaxanthin can be increased when incorporated or used with at least one of the following: phospholipid, glycolipid and sphingolipid and optionally with food grade and / or pharmaceutical grade diluents. Lower dosages compared to the 15 mg used in previous clinical trials may be used. The astaxanthin is at least about 0.1 to about 15 weight percent of at least one of the following: phospholipid, glycolipid, and sphingolipid. In one example, the astaxanthin is derived from a natural or synthetic ester or synthetic diol. A pharmaceutical grade or food grade diluent may be added. When incorporated with a microbial fermented low molecular weight hyaluronic acid or sodium hyaluronate (hyaluronan) as described above, a nutritional supplement composition is formed which can be formulated in a therapeutic amount to treat and ameliorate symptoms of joint pain in a person with joint pain.

It can be assumed that the triglycerides have two types of molecules as glycerine and three fatty acids, while the phospholipids contain glycerol and fatty acids, but have one glycerol molecule and two fatty acid molecules. Instead of the third fatty acid, a polar group is instead attached to the glycerol molecule, so that the phospholipids are partially hydrophilic compared to hydrophobic triglycerides. Lysophospholipids can be used as a derivative of a phospholipid in which one or both acyl derivatives have been removed by hydrolysis. Lecithin and its derivatives can be used as an emulsifier and the surfactant as a wetting agent to reduce the surface tension of liquids. Other phospholipids can be used. The various phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, lyso-phosphatidylcholine, lyso-phosphatidylethanolamine and lyso-phosphatidylserine. Some may come from egg yolk and may also be chemically extracted using hexane, ethanol, acetone, petroleum ether or benzene, as well as being mechanically extracted, including from various sources such as soybeans, eggs, milk, marine sources and sunflowers. When phospholipids are derived from soy and sunflower, they may contain the aforementioned products, including phosphatidic acid. Various compositions, such as lecithin, can be enzymatically hydrolyzed and have a fatty acid that is removed by phospholipase to form the lyso-phospholipids that can be added to the rye extract, as discussed above. A phospholipase is the phospholipase A2, which removes the fatty acid in the C2 position of glycerol. A fractionation can be used.

The glycolipids are mainly derivatives of ceramides in which a fatty acid is linked or linked to the aminoalcohol sphingosine. It should be noted that the phospholipid sphingomyelin also comes from a ceramide. However, glycolipids contain no phosphates compared to the phospholipids. The fat is linked to a sugar molecule in a glycolipid and these are fats that are attached to sugar. As it is composed of a sphingosine, fat and sugar, it is sometimes referred to as glycosphingolipid. A sphingolipid is a lipid containing a sphingoid-based scaffold and a series of aliphatic amino alcohols having sphingosine. As already mentioned, the phospholipid and other components may be derived from at least one of the following: a plant, an alga and an animal source or a synthetic derivative thereof. The phospholipid and other components may be derived from at least one of soybeans, sunflowers, grape seeds, egg yolks, krill, fish carcasses, fries, squid and algae. The phospholipid and other components may be formed as a compound rich in mono- or diglycerides or fatty acids, the fatty acid containing between 2 and 20 carbon atoms. During processing, the composition is formed by dispersing 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.

In another example, the astaxanthin is about 2 to about 10 weight percent phospholipid and glycolipid and is 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 nutritional supplement composition can be formulated into a single dose capsule.

The astaxanthin may be derived from Haematococcus pluvialis algae, Pfaffia, krill or obtained 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 with 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) may be derived from a microbial fermentation or animal tissue. About 1-500 mg of hyaluronan may be given per daily dose, and preferably between 10 and 70 mg / dose and 20 to 60, 25 to 50 and 35 and 45 mg per dose. The hyaluronan is microdispersed or nanodispersed in the composition in a preferred example. In another example, the hyaluronic acid is from a biofermentation 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 0.5 up to 230 kDa as low molecular weight hyaluronic acid or hyaluronan. A preferred range is 0.5 to 300 kDa. In another example, the hyaluronic acid or sodium hyaluronate (hyaluronan) polymers are derived from a microbial fermentation or animal tissue.

In one example, the pure low molecular weight hyaluronic acid oligomers are principally and practically derived from microbial fermentation, but could also be derived from hydrolyzed animal tissues. It is known that this microbial fermentation process produces an exceptionally pure low molecular weight sodium hyaluronate that is free of amino acid conjugation.

Human hyaluronic acid is typically synthesized naturally in the body or taken from the diet, such as chicken, beef and other natural sources. This natural hyaluronic acid has a high molecular weight, d. H. over 300 kDa, compared to microbial fermented sodium hyaluronate, which has a low molecular weight and is defined in the literature at about 0.5 to 300 kDa. The hyaluronic acid found naturally in the body is a polymer of acidified glucuronic acid and N-acetylglucosamine and exists at a physiological pH of about 7.4 as the free acid, with partial sodium, potassium and ammonium salts. Streptococcus is used in one example 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 acidic aqueous chemical hydrolysis as a chemical reaction. The final product is isolated by ethanol precipitation of the sodium salt and drying to produce proinflammatory, low molecular weight, microbially fermented sodium hyaluronate fragments.

This low molecular weight sodium hyaluronate is the degradation product of a chemical reaction from a bacterial fermentation of a Streptococcus mutant strain. An example of sodium hyaluronate is produced by fermentation using the bacterial strain Streptococcus zooepidemicus. The production strain is a non-hemolytic mutant of the parent strain NCTC 7023. The production strain is produced by nitroso-guanidine mutagenesis with a single ribosomal genome sequence that is not naturally occurring in nature.

This manufacturing process has three main stages 1) fermentation, 2) purification and 3) further processing. The fermentation starts with a seed culture from the mutant production strain. A starter culture inoculates the seed tank containing a culture solution medium that grows and becomes the seed culture solution. The seed culture solution is transferred to a fermenter containing the sterilized culture medium and a culture temperature of 33-37 ° C is maintained until the fermentation is complete within 22-30 hours.

This fermentation culture solution is mixed with ethanol to obtain precipitated crude sodium hyaluronate. The 50-70% ethanol concentration used during the purification inactivates the Streptococcus organism. The crude product is dissolved in purified water and filtered to remove both impurities and inactivated microbial fragments. This gives a clear filtrate. The water has a temperature of 50-70 ° C when used in the dissolution step and inactivates any remaining Streptococcus organism. The sodium hyaluronate with the target molecular weight is then obtained by controlling the pH, temperature and hold time in the dissolution step. The higher the pH and the temperature in the specific range and the longer the retention time in the specific range, the lower the resulting molecular weight of the sodium hyaluronate. The filtrate containing the low molecular weight hyaluronic acid from the chemical hydrolysis generated during the chemical molecular weight modification step is then precipitated with ethanol, followed by washing and dehydration. The precipitate is dried in vacuo to give the final low molecular weight microbial fermented sodium hyaluronate.

Other sources of low molecular weight hyaluronic acid may be used. These include low molecular weight hyaluronic acid derived from chicken breast cartilage extract. The hyaluronic acid may contain elastin, elastin precursors and collagen. The hyaluronic acid may be contained in a matrix form with chondroitin sulfate and naturally occurring hydrolyzed type II collagen as dietary supplement ingredients and form lower molecular weight molecules that the body is more readily able to absorb and deliver to different areas of the body as needed. Fresh chicken breast bone cartilage could be cut and suspended in aqueous solution, after which the cartilage is treated with a proteolytic enzyme to form a hydrolyzate. The proteolytic enzyme can hydrolyze Type II collagen into lower molecular weight fragments. The hydrolyzate is sterilized and filtered and concentrated and then dried to form an enriched collagen type II powder, which is then isolated and has a percentage of low molecular weight hyaluronic acid. Examples of manufacturing methods can be found in the U.S. Pat. Nos. 6,780,841 and 6,025,327 are found, the disclosures of which are fully incorporated herein by reference.

It is possible that the low molecular weight hyaluronic acid could also be derived from the hydrolyzed collagen derived from bovine type I collagen or type II chicken breast cartilage collagen, or even from a natural egg shell membrane having some hyaluronic acid which can be extracted from the egg shell membrane. Although some teachings use hyaluronic acid derived from eggshell membrane, as for example in the patents incorporated by reference, the hyaluronic acid is processed to increase its molecular weight via crosslinking techniques as compared to the use of 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 the enzymatic degradation of the eggshell membrane, which is subjected to processing to purify the hyaluronic acid.

The hyaluronic acid can come from dehydrated cockscomb, such as in the U.S. Patent No. 6,806,259 and in the U.S. Patent Publication No. 2006/0183709 which are fully incorporated herein by reference where the hyaluronic acid can be further processed. Often, it has a higher molecular weight and is processed to obtain a lower molecular weight of the desired 0.5 to 300 kDa. In many teachings, a hyaluronic acid of a certain molecular weight is processed to increase its molecular weight. Hyaluronic acid can also be obtained from human umbilical cords or other techniques, such as U.S. Patent No. 4,141,973 the disclosure of which is fully incorporated herein by reference and further processed to obtain the desired molecular weight.

It has been determined that synergistic or advantageous improvements may be made with respect to some commercially available compositions comprising about 50 mg of an active agent, for example hyaluronic acid, and a cartilage, such as type II collagen, when astaxanthin is added. Sometimes boron is used. For example, the composition comprises 30-50 mg of collagen and about 4-6 mg of boron and 2-4 mg of hyaluronic acid at an average of each of the component regions. It has been found that an effective and synergistic result is obtained when astaxanthin alone and / or low molecular weight hyaluronic acid is added, such as 0.5 to 4 mg or 0.5 to 12 mg astaxanthin plus 30-45 mg low molecular weight hyaluronic acid, although even lower levels could be used, such as 1-5 mg. This composition could include type II collagen with added astaxanthin and low molecular weight hyaluronic acid with the optional addition of boron. One (1) to 500 mg of hyaluronic acid could be used.

In one example, a cartilage mixture as a mixture of cartilage and salt is about 40 mg, with boron having 5 mg and hyaluronic acid having 3.3 mg. The cartilage mixture has cartilage and potassium chloride to provide 10 mg of non-denatured type-2 collagen. It is possible for another composition to include the astaxanthin in the composition consisting of glucosamine hydrochloride, such as about 1.25 to 1.75 or about 1.5 grams, and methylsulfonylmethane (MSM) at about 500 to 1,000 and about 750 mg is formed and to include the addition of chondroitin sulfate at about 150 to 250 and about 200 mg. It may also contain the synovial fluid as hyaluronic acid, such as 1-5 mg and about 3.3 mg, as well as vitamin D3 and other components such as antioxidants. The astaxanthin may vary between 2 and 4 mg or 0.5 and 12 mg and other ranges as disclosed above. It should be noted that the astaxanthin and at least one of the following: phospholipid, glycolipid and sphingolipid or other components as described above can be used for many different purposes and results. It can be used to help treat or improve blood lipid profiles and reduce LDL peroxidation in humans. It can be used to combat or treat depression or other neurological disorders. It can be used for respiratory diseases and skin disorders or diseases.

It has been found to be beneficial and synergistic to use astaxanthin with low molecular weight hyaluronic acid. It can optionally be incorporated with the UC-II in the areas described above. Astaxanthin beads could be added to the UC-II. This type of composition is advantageous over glucosamine chondroitin pills because in the latter two, much larger pills are needed daily to support the joint and cartilage. The composition may comprise a natural or synthetic cyclooxygenase-1 or -2 inhibitor comprising, for example, aspirin, acetaminophen, steroids, prednisone or NSAIDs. The composition may also contain an oil which is rich in gamma-linolenic acid and the borage (Borago officinalis L.) or safflower (Carthamus tinctorius L.) comprises providing a metabolic precursor for _PGE1 synthesis.

The composition may also comprise an oil rich in an n-3 (omega-3) fatty acid derived from fish oil, algae oil, linseed oil, chia seed oil or perilla seed oil. In one example, the n-3 fatty acid comprises alpha-linolenic acid, stearidonic acid, eicosapentaenoic acid or docosapentaenoic acid. In an exemplary composition given above, it has been found that an algae-based oil can be used instead of krill oil. Hydrolyzed or unhydrolyzed collagen and elastin derived from eggshell membranes may also be advantageously added. The composition may also contain anti-inflammatory and / or natural joint health promoting compounds comprising at least one of the following preparations: green-lipped mussel (Perna canaliculus), Boswellia serrata, turmeric (Curcuma longa), stinging nettle (Urtica dioica), Kalmegh, cat's claw (Uncaria tomentosa), bromelain, methylsulfonylmethane (MSM), chondroitin sulfate, glucosamine sulfate, s-adenosylmethionine, proanthocyanidins, procyanidins or flavonoids. The composition may contain naturally-derived and synthetic antioxidants added to delay degradation of fatty acids and astaxanthin.

Different compositions may use different ingredients in combination with krill, algae or other oil, including the seed-based oil, rouge extract and phospholipid and other surfactants. The astaxanthin and hyaluronate may be combined for more specific purposes with different ingredients and supplemental compositions.

A pharmaceutically acceptable composition comprises a krill, fish, algae, rye extract or vegetable oil, and / or phosphoplipid and / or surfactant in combination with astaxanthin and hyaluronate, optionally combined with one or more ingredients, including but not limited to exclusively - glucosamine sulphate, chondroitin sulphate, collagen, methylsulphonmethane, a gamma-linoleic acid or omega-3 fatty acid rich oil, a cyclooxygenase inhibitor or a lipogenase inhibitor for the treatment of symptoms related to non-disease joint pain and / or joint disease, including but not limited to Osteoarthritis and rheumatoid arthritis.

In yet another example, a nutritional supplement acceptable composition comprises a krill, algae, fish, rye extract or plant based oil and / or another 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, methylsulfone methane, gamma-linoleic acid or omega-3 fatty acid rich oil Cyclooxygenase inhibitor or a lipoxygenase inhibitor for the treatment of symptoms associated with non-disease joint pain and / or joint disease including, but not limited to, osteoarthritis and rheumatoid arthritis.

In yet another example, a composition acceptable for medicinal nutrition comprises a krill, algae, fish, rotoxtract 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 sulphate, chondroitin sulphate, collagen, methylsulphonmethane, a gamma-linoleic acid-rich or omega-3 fatty acid rich oil, a cyclooxygenase inhibitor or a lipoxygenase inhibitor for the treatment of symptoms associated with non-arthritic joint pain and / or joint disease, including but not limited to osteoarthritis and rheumatoid arthritis.

In yet another example, a composition is formulated in a therapeutic amount to treat and alleviate symptoms of non-disease joint pain and / or joint disease, including osteoarthritis and / or rheumatoid arthritis, the composition comprising a krill, algae, fish , Rogenxtrakt- or plant-based oil and / or another 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 ingredients as illustrated and illustrated above in the context of the method and composition.

The composition oil, whether it be a krill, algae, fish, rogeneous extract or plant based oil, and / or another phospholipid and / or surfactant is used with the HA, such as the low molecular weight HA, and astaxanthin, However, to treat non-disease joint pain in one example, it may also be used to treat osteoarthritis. Osteoarthritis (OA) is the most prevalent form of arthritis and is a disease in which the cartilage, which acts as a cushion between the bones in joints, begins to wear, causing bone-on-bone joint swelling and joint pain. It is accompanied by degeneration of articular cartilage along with periarticular bone reaction. It affects both sexes, mainly in the fourth and fifth decades of life. The knee joint is the most commonly affected joint. At present, treatment is by pharmacological and non-pharmacological therapy. Corrective surgical therapy and / or joint replacement therapy may not be feasible in some cases.

The traditional treatments for osteoarthritis include the use of analgesics, non-steroidal anti-inflammatory drugs (NSAIDs) or cyyclooxygenase-2 specific (COX-2) NSAIDs alone or in combination. Advances in recombinant protein synthesis also provide relief in the symptoms of OA and RH. Injections with steroids or high molecular weight hyaluronic acid are also used with some success, however, these therapies have sufficiently known harmful side effects.

Many of these treatments alone have shown limited efficacy in clinical trials. In order to avoid the cardiac risks and gastrointestinal problems associated with traditional OA treatments (especially long-term use), many patients have turned to complementary and alternative medicines (CAMs), such as nutritional supplements. Glucosamine and chondroitin, alone or in combination, are widely marketed as nutritional supplements to treat joint pain due to OA. Two important clinical trials regarding glucosamine and chondroitin (the GAIT study) did not show any significant improvement in the WOMAC score over placebo, except in the highest quartile of the patients studied. Due to their limited efficacy, the search for additional CAMs to treat OA will continue (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 ).

It is also possible to use a pure diol of S, S'-astaxanthin, including a synthetic diol with a surfactant and / or the low molecular weight hyaluronic acid. It is possible to use this pure diol in combination with the algae-based EPA-rich oil or other fish, rye extract or plant-based oil and / or phospholipid and / or surfactant as described above, with admixture either with astaxanthin derived from Haematococcus pluvialis 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 S, S'-astaxanthin is feasible because the krill oil and possibly the algae-based oils and those derived from Hp and other species are predominantly diesters and monoesters each containing very little diol which is insoluble. Some research suggests that it is many times more bioavailable than either the monoester or diester form. It is possible to asymmetrically synthesize the pure S, S'-diol. Despite the poor solubility of pure diol found in some examples, it may be an active transport mechanism in the Give context with its bioavailability, or vice versa, so that only in the diol form the monoester or diester forms are transferred from the intestine into the blood. The phospholipid or glycolipid-based product, which represents EPA and / or DHA, together with the added astaxanthin in its various forms and especially the S, S'-enantiomeric form in mainly the monoester form of Heamatococcus pluvialis or the pure diol form from an asymmetric synthesis could be feasible. Therefore, it is possible to combine it with the algae-derived glycol and phospholipid-based, EPA-rich oil.

As noted above, astaxanthin (3,3'-dihydroxy-β-β-carotene-4,4'-dione) is a xanthophyll carotenoid found in many marine species, including crustaceans, salmonid fish and algae. Astaxanthin can not be synthesized by mammals, but when ingested, it shows antioxidant activity, anti-inflammatory and promotes eye health, heart health, and the immune system.

Astaxanthin has one hydroxyl group on each β-ionone part, so it can be found in its free (diol) form as well as mono- or di-esterified. In natural products, astaxanthin is commonly found as a mixture: mainly monoesters of C12-C18 fatty acids and minor amounts of diester and free diol. Synthetic astaxanthin is usually provided only in the free diol form.

The astaxanthin molecule has two E / Z chiral centers and three optical R / S isomers. The Haematococcus pluvialis alga produces natural astaxanthin only in the (3S, 3'S) isomer. This is in the article of Renstrøm B., G. Borch, O. Skulberg and S. Liaane-Jensen, "Optical Purity of (3S, 3'S) Astaxanthin From Haematococcus Pluvialis", Phytochemistry, 20 (11): 2561-2564, 1981 , the disclosure of which is fully incorporated herein by reference.

Alternatively, the yeast Phaffia rhodozyma synthesizes only the 3R, 3'R configuration. This is in the article of Andrewes A. and M. Starr, entitled "(3R, 3'R) -Astaxanthin from the Yeast Phaffia Rhodozyma", Phytochemistry, 15: 1009-1011, 1976 , the disclosure of which is fully incorporated herein by reference.

Wild salmon contains predominantly the (3S, 3'S) -form with a (3S, 3'S), (3R, 3'S) and (3R, 3'R) -isomer ratio of 22: 1: 5. This is in the article of Turujman, S, W. Wamer, R. Wei and R. Albert, entitled "Rapid Liquid Chromatographic Method to Distinguish Wild Salmon From Aquacultured Salmon Fed Synthetic Astaxanthin", J. AOAC Int., 80 (3): 622-632, 1997 , the disclosure of which is fully incorporated herein by reference.

However, astaxanthin prepared by traditional synthesis will contain a racemic mixture in a (3S, 3'S), (3R, 3'S; meso) (3R, 3'R) ratio of 1: 2: 1. This relationship is also evident in many shrimp species that can (3S, 3'S) racemize to meso form. This is in the article of Schiedt, K., S. Bischof and E. Glinz, entitled "Metabolism of Carotenoids and In Vivo Racemization of (3S, 3'S) -Astaxanthin in the Crustaecean Penaeus", Methods in Enzymology, 214: 148-168, 1993 , the disclosure of which is fully incorporated herein by reference.

However, most of the astaxanthin in shrimp is in the carapace (shell), so limited amounts of meso-isomer are consumed in the human diet.

Nutritional studies with free diol or fatty acid esters of astaxanthin have been shown to increase the amount of astaxanthin in human plasma. This is in the article of Liesterlie, M., B. Bjerkeng and S. Liaan-Jensen entitled "Plasma Appearance and Distribution of Astaxanthin E / Z and R / S Isomers in Plasma Lipoproteins of Men After Single Dose Administration of Astaxanthin", J. Nutr. Biochem, 11: 482-490, 2000 ; and the article of Coral-Hinostroza, G., T. Ytestøyl, B. Ruyter and B. Bjerkeng, entitled "Plasma Appearance of Unesterified Astaxanthin Geometric E / Z and Optical / S Isomers in Men Given Single Doses of a Mixture of Optical 3 and 3 ' R / S Isomers of Astaxanthin Fatty Acyl Diesters ", Comp. Biochem Phys. C., 139: 99-110, 2004 , the disclosures of which are fully incorporated by reference herein.

The intake of free astaxanthinediol is about 4-5 times higher than that of esterified astaxanthin, probably due to the limitation of the required enzymatic hydrolysis in the intestine before absorption. These intestinal enzymes may also be R / S selective with respect to astaxanthin esters. Coral-Hinostroza et al. (2004) have a higher relative absorption of astaxanthin from (3R, 3'R) -Astaxanthindipalmitat in Comparison to the other two isomers found. However, uptake of racemic free diol astaxanthin does not show stereospecific selection.

Astaxanthin for use in human nutritional supplements currently comes from the cultured freshwater algae Haematococus pluvialis. This alga produces 3S, 3'S-astaxanthin esters in a fatty acid matrix that can be isolated with a solvent or carbon dioxide extraction. This oily extract can be used directly in edible formulations or further processed to a solid powder or bead preparations. Many clinical studies have been conducted with H. pluvialis-derived astaxanthin to demonstrate beneficial health effects and safety. Dietary supplements for astaxanthin-rich algae extracts have been approved for many US and EU suppliers.

Heamatococcus algae cultivation for use in nutritional supplements may not always meet the need for the use of astaxanthin in nutritional supplements. The use of synthetic astaxanthinediol may also be useful in applications requiring a concentrated, standardized source of astaxanthin. Conventional racemic synthetic astaxanthin sources are used as a dye in a salmon aquaculture feed component. This racemic mixture may be of limited utility since only one quarter of the compound is the 3S, 3'S isomer commonly found in natural salmon and has been tested for efficacy and safety in humans.

Astaxanthin can also be synthesized stereospecifically so that the yield is only the generally accepted 3S, 3'S isomer in free diol form. The free diol crystals may be suspended in a vegetable oil or solid beads for use in edible preparations or pill, capsule, tablet form. The 3S, 3S product has the advantage that it has a higher consistency than algae preparations and also less odor. Thus, in existing formulations, the alax-derived astaxanthin may be replaced with synthetic 3S, 3'S-astaxanthinediol of the same or increased potency.

As indicated above, it has also been surprisingly found that the use of hyaluronic acid alone and / or in combination with astaxanthin is beneficial and synergistic. For example, a low molecular weight hyoluronic acid in its various forms can be administered to patients in an amount of 1-500 mg daily and preferably about 10-70 mg daily and in another example 20-60 mg, 25-50 mg, 35 mg and 45 mg , Astaxanthin at about 2-4 mg may be added in one example, but in another example the range may also range from 0.5 to 4 mg daily and 7-12 mg or 0.5 to 12 mg. The hyaluronic acid may be administered in the form of proinflammatory low molecular weight sodium hyaluronate fragments having about 0.5-300 kDa, which corresponds to the proinflammatory low molecular weight fragments. Although the use of astaxanthin and phospholipids such as krill oil, algae oil, roe, fish oil product or plant-based oils aids in the release of hyaluronic acid, the low molecular weight hyaluronic acid, even in the form of the fragments, is preferably still small enough to pass through the intestine so that it is used in an oral administration.

It is also advantageous to use astaxanthin with the low molecular weight hyaluronic acid. Different amounts can be used; in one example 2-4 mg daily and in another example 0.5-12 mg daily with low molecular weight hyaluronic acid may be used, such as the amount of 1-500 mg and preferably about 10-70 mg and 0.5-12 mg or 4-12 mg astaxanthin. About 40-120 mg of low molecular weight hyaluronic acid can be used in one example. An astaxanthin dose may be about 6-8 mg and the low molecular weight hyaluronic acid may be in the range of about 60-80 mg. Although the larger amounts of astaxanthin can be used alone with low molecular weight hyaluronic acid alone, it is possible to use 2 mg astaxanthin and minor amounts of low molecular weight hyaluronic acid such as 20 mg and up to 40 mg as non-limiting examples. It is believed that hyaluronic acid fragments, such as the proinflammatory low molecular weight sodium hyaluronate fragments, are innate immune system cell receptor signaling molecules associated with the inflammatory cascade and the oral hyaluronic acid in the form of low molecular weight fragments can reach the joints, unlike the higher molecular weight hyaluronic acid that is injected as they can not be administered orally.

As stated above, the algae-based oil has been found to be advantageous in one example. This algae-based oil provides an EPA-based oil or an EPA / DHA from an algae source containing oils in phospholipid and glycerolipid forms as glycolipids. Oils based on different algae derived from different microalgae can be used. An oil based on a preferred exemplary alga has a higher EPA titer than the DHA compared to the omega-3 class of fish oils. which are triacylglycerides. These algae-based oils are rich in EPA and phospholipid and glycolipid forms. An exemplary marine-based algae oil is manufactured by Parry Nutraceuticals, EID Parry (India) Ltd. produced as omega-3 (EPA) oil.

Algae are known to be an important source of 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 these fatty acids from the algae they consume. The bioavailability of omega-3 varies and is provided at the site of physiological activity, depending on the form in which 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 through the formation of micelles and subsequent digestion by enzymes and subsequent absorption. However, these omega-3 fatty acids bound to polar lipids, such as phospholipids and glycolipids, do not depend on bile digestion and undergo a simpler digestion process prior to absorption. Therefore, these omega-3 fatty acids, for example from an algae-based oil, have greater bioavailability for cell growth and function compared to the omega-3 triglycerides of fish oil. There are many types of algae containing EPA conjugated to phospholipid and glycolipid polar lipids or containing EPA and DHA conjugated to phospholipids and glycolipids.

In this specification, the term "algae" or "microalgae" may be used interchangeably with each other, where microalgae refer to photosynthetic organisms that reside in the aquatic or marine habitat and are too small to be considered simply as individual organisms visible to the naked eye. When the term "photoautotrophic" is used, it refers to growth by light as the main energy source and carbon dioxide as the main carbon source. Other forms of biomass, which may include algae or microalgae, may be used, and the term "biomass" may refer to a living or recently dead biological cellular material derived from plants or animals. The term "polar" may refer to the compound having portions of negative and / or positive charges that form negative and / or positive poles. The term "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 full range of various hydrocarbons which are soluble in nonpolar solvents and which are insoluble or relatively insoluble in water, as known to those skilled in the art. The microalgae may also include any naturally occurring or genetically modified microalgae that has improved lipid production.

The following first table shows the details of an algae-based oil as produced by the above-mentioned "Parry Nutraceuticals" and is supplemented by a second table for a fatty acid profile plot of this algae-based oil. A third table is a comparative graph of fatty acid profiles for non-algae based oils. These graphs show that the algae-based oil has a high EPA content of phospholipids and glycolipids. DETAILED INFORMATION: OIL BASED ON ALGAE PARAMETER DETAILED INFORMATION SOP. NO TEST METHOD / REFERENCE Physical Properties appearance viscous oil QA-88 internally colour brownish-black QA-88 internally odor characteristic QA-88 internally taste characteristic QA-88 internally in general. composition Drying loss (%) 2.0-3.0 QA 038 USP <731> drying loss Ash (%) 0.5-1.0 QA 080 AOAC official procedure 942.05, 16th edition Protein (%) 1.0-2.0 QA-021 AOAC official procedure 978.04, 16th edition Carbohydrate (%) 1.0-2.0 AOAC 18th issue 2006 / by difference Rem. Solvent (ppm) (as ethyl acetate) (as acetone) NMT 100 NMT 30 QA-074 GC headspace USP <467> lipid composition Total lipid (%) 92.0 to 95.0 QA-86 AOAC official procedure 933.08 Chlorophyll (%) NMT 1.50 QA 078 Jeffrey & Humphrey (1975 - Photosynthetic pigments of Algae (1989) Total carotenoids (%) NMT 1.50 QA-85 By JHFA method - 1986 Ges. Unsaponifiable (%) NMT 12.0 QA 086 AOAC official procedure 933.08 Omega-3 [EPA + DHA] -% w / w NLT 15.00 QA 087 Company procedure Total omega-3 (% w / w) NLT 17.00 Total omega-6 (% w / w) NMT 5.00 Total EFA (% w / w) NLT 20 Percentage of lipids triglycerides 15-20% phospholipids 5-10% glycolipids 35-40% Free fatty acids 15-20% Microbial parameters QA-039 AOAC, 1995, chapter 17 Standard plate counting (cfu / lg) NMT 1,000 Yeast and mold (cfu / 1 g) NMT 100 Coli forms (/ 10 g) negative E. coli (/ 10 g) negative Staphylococcus (/ 10 g) negative Salmonella (/ 10g) negative Fatty acid profile (range%) Myristic acid [14] NLT 4.0 QA-086 & 087 In-house GC process Palmitic acid [16: 0] NLT 16.0 Palmitoic Acid [16: 1, n-9] NLT 12.0 Hexadecadienoic acid [16: 2, n-4] NLT 4.0 Hexadecatrienoic acid [16: 3, n-4] NLT 12.0 Stearic acid [18: 0] NLT 0.10 Oleic acid [18: 1] NLT 1.0 Linoleic acid [18: 2, n-6] - LA NLT 1.0 Alpha-linolenic acid [18: 3, n-3] - ALA NLT 0.50 Stearidonic acid [18: 4, n-3] - SA NLT 0.10 Arachidonic acid [20: 4, n-6] - AA NLT 0.25 Eicosapentaenoic acid [20: 5, n-3] NLT 15.0 Decosahexaenoic acid [20: 6, n-3] NLT 1.5 heavy metals Lead (ppm) NMT 1.0 Arsenic (ppm) NMT 0.5 External lab reports AOAC 18th Issue 2006 by ICPMS Cadmium (ppm) NMT 0.05 Mercury (ppm) NMT 0.05 Safety: Safety for the intended purpose Shelf life: 24 months from the date of manufacture Stability: stable under non-open conditions Storage: I in a cool, dry place without sunlight storage, after using container with nitrogen flush Documentation: each shipment carries COA Packaging: 1 kg, 5 kg and 20 kg food-grade containers FATTY ACID PROFILE DIAGRAM OIL BASED ON ALGAE FATTY ACID OMEGA-3 (EPA) OIL BASED ON ALGAE Total fatty acid, g / 100 g oil 75 g Fatty acid [% sat. Fatty acid] Myristic acid [14: 0] 6.87 Pentadecanoic acid [15: 0] N / A Palmitic acid [16: 0] 20.12 Palmitoic acid [16: 1, ω-9] 18.75 Hexadecadienoic acid [16: 2, ω-4] 6.84 Hexadecatrienoic acid [16: 4, ω-4] 12.54 Heptadecanoic acid [17: 0] N / A Stearic acid [18: 0] 0.68 Oleic acid [18: 1, ω-9] 3.56 Linoleic acid [18: 2, ω-6] 2.68 Alpha-linolenic acid [18: 3, ω-3] 3.73 Gamma-linolenic acid [18: 3, ω-6] N / A Stearidonic acid [18: 4, ω-3] 0.33 Arachidonic acid [20: 4, ω-6] 0.97 Eicosapentaenoic acid [20: 5, ω-3] EPA 23.00 Docosapentaenoic acid [22: 5, ω-3] DHA N / A Docosahexaenoic acid [22: 6, ω-3] DHA 3.26 Other 3.54 EPA / DHA [g / 100 g of oil] 15.75 Total ω-3 fatty acids [g / 100 g of oil] 18.20 LIPID CLASS DETAILS [g / 100 g of oil] Unsaponifiable [carotenoids, chlorophyll, sterol, fatty alcohol, etc.] 12 Free fatty acids 20 triglycerides 20 phospholipids 10 glycolipids 38 total 100 STABILITY (months) 24 FATTY ACID COMPARATIVE DIAGRAM OILS ON NON-ALGAE BASIS FATTY ACID FISH OIL MAX EPA KRILL OIL MARTEK OIL Total fatty acid, g / 100 g oil 95 g 70-80 g 95 g Fatty acid [% sat. Fatty acid] Myristic acid [14: 0] 8.68 11.09 11.47 Pentadecanoic acid [15: 0] N / A N / A N / A Palmitic acid [16: 0] 20.35 22.95 26.36 Palmitoic acid [16: 1, ω-9] 11.25 6.63 N / A Hexadecadienoic acid [16: 2, ω-4] N / A N / A N / A Hexadecatrienoic acid [16: 4, ω-4] N / A N / A N / A Heptadecanoic acid [17: 0] N / A N / A N / A Stearic acid [18: 0] 4.67 1.02 0.50 Oleic acid [18: 1, ω-9] 13.07 17.93 1.50 Linoleic acid [18: 2, ω-6] 1.28 0.14 0.61 Alpha-linolenic acid [18: 3, ω-3 0.33 2.11 0.40 Gamma-linolenic acid [18: 3, ω-6] N / A N / A N / A Stearidonic acid [18: 4, ω-3] 1.69 7.01 0.33 Arachidonic acid [20: 4, ω-6] 0.50 N / A N / A Eicosapentaenoic acid [20: 5, ω-3] EPA 20.31 19.04 1.0 Docosapentaenoic acid [22: 5, ω-3] DHA N / A N / A 15.21 Docosahexaenoic acid [22: 6, ω-3] DHA 13.34 11.94 42.65 Other 4.53 0.14 N / A EPA / DHA [g / 100 g of oil] 31,96 21.68 41.46 Total ω-3 fatty acids [g / 100 g of oil] 33.85 28,00 41,60 LIPID CLASS DETAILS [g / 100 g of oil] Unsaponifiable [carotenoids, chlorophyll, sterol, fatty alcohol, etc.] 5 5 5 Free fatty acids 0.5 30 0.5 triglycerides 94.5 25 94.5 phospholipids zero 40 zero glycolipids zero zero zero total 100 100 100 STABILITY [months] 12 24 6

Different types of marine based algal oils can be used, including Nannochloropsis oculata as the EPA source. Another alga that can be used is Thalassiosira weissflogii, as for example in the U.S. Patent No. 8,030,037 described in the above-mentioned Parry Neutraceuticals, Plant EID Parry (India) Ltd. has been overwritten, the disclosure of which is fully incorporated herein by reference. Other types of algae disclosed include Chaetoceros sp. or Prymnesiophyta or green algae, such as Chlorophyta and other microalgae, which are diamonds tiatoms. The Chlorophyta could be Tetraselmis sp. and Prymnesiophyta include, such as the class Prymnesiophyceae and z. B. the order Isochrysales and in particular Isochrysis sp. or Pavlova sp.

There are many other species of algae that can be used to produce EPA and DHA as an algae-based oil, whether marine based or not, used in accordance with a non-limiting example. In some cases, isolation of the phospholipid and glycolipid-bound EPA and DHA-based oils may require manipulation of the algal species growth cycle.

Other algae / fungi Phospholipid / glycolipid sources include: Grateloupia turuturu; Porphyridium cruentum; Monodus subterraneus; Phaeodactylum tricomutum; Isochrysis galbana; Navicula sp. Pythium irregule; Nannochloropsis sp .; and Nitzschia sp.

Details regarding Grateloupia turuturu are titled in the article "Grateloupia Turuturu (Halymeniaceae, Rhodophyta) is the Correct Name of the Non-Native Species in the Atlantic Known as Grateloupia Doryphora", Eur. J. Phycol. (2002) 37: 349-359 , with the authors Brigitte Gavio and Suzanne Fredericq, the disclosure of which is fully incorporated herein by reference.

Porphyridium cruentum is a red algae of the family Porphyridiophyceae and is also called Rhodophyta and used as a source of fatty acids, lipids, cell wall polysaccharides and pigments. The polysaccharides of this type are sulfated. Part of the Porphyridium cruentum biomass contains up to 57% carbohydrates.

Monodus subterraneus is titled in an article "Biosynthesis of Eicosapentaenoic Acid (EPA) in the Fresh Water Eustigmatophyte Monodus Subterraneus (Eustigmatophyceae)", J. Phycol, 38, 745-756 (2002), with the authors Goldberg, Shayakhmetova and Cohen described, the disclosure of which is fully incorporated herein by reference. The biosynthesis of PUFAs from algae is complicated and the biosynthesis from this alga is described in the article.

Phaeodactylum tricomutum is a diatom and unlike most diatoms it can grow in the absence of silicon and the biogenesis of silicified frustules is optional.

Isochrysis galbana is a microalgae and is used in the bivalve aquaculture industry.

Navicula sp. is a boat-shaped alga and is a diatom. Pythium irregule is a ground bearded pathogen found on plant hosts.

Nannochloropsis sp. occurs in a marine environment, but also occurs in fresh and brackish water. The species are small, non-motile spheres that show no clear morphological feature. These algae have chlorophyll A and lack chlorophyll B and C. They can build up high levels of pigment such as astaxanthin, zeaxanthin and canthaxinthine. They are about 2-3 microns in diameter. They can accumulate high levels of polyunsaturated fatty acids.

Nitzschia sp. is a pinnate marine diatom commonly found in colder waters and associated with both Arctic and Antarctic polar ice, where it is a dominant diatom. It produces a neurotoxin known as domoic acid, which is responsible for amnestic haddock poisoning. It can grow exponentially at temperatures between -4 and -6 ° C. it can be processed to form and extrapolate the fatty acids.

• I. Kieselalgen Asterionella japonica Bidulphia sinensis Chaetoceros septentrionale Lauderia borealis Navicula biskanteri Navicula laevis (heterotrof.) Navicula laevis Navicula incerta Stauroneis amphioxys Navicula pellicuolsa Bidulphia aurtia Nitzschia alba Nitzschia chosterium Phaeodactylum tricornutum Phaeodactylum tricornutum Skeletonema costatum
• II. Chrysophyceae Pseudopedinella sp. Cricosphaera elongate
• III. Eustigmatophyceae Monodus subterraneus Nannochloropsis
• IV. Prymnesiophyceae Rodela violacea 115.79 Porphyry. Cruentum 1380.Id
• V. Prasinophyceae Pavlova salina
• VI. Dinophyceae Cochlodinium heteroloblatum Cryptecodinium cohnii Gonyaulax catenella Gyrodinium cohnii Prorocentrum minimum
• VII. Andere Mikroalgen Chlorella minutissima Isochrysis galbana ALII4 Phaeodactylum tricornutum WT Porphyridium cruentum Monodus subterraneus
• VIII. Pilze Mortierella alpine Mortierella alpine IS-4 Pythium irregulare
• IX. Bakterien SCRC-2738

As a source of polyunsaturated fatty acids, the microalgae competes with other microorganisms such as fungi and bacteria. There may be some bacterial strains that could be an EPA source, but it has been found that the microalgae are a more suitable and readily available source. The microalgae is a good source of oil and EPA when it comes from Phaeodactylum, Isochrysis and Monodus. The microalgae Phaeodactylum tricornutum produces a high EPA content. Other different ones Strains and species of microalgae, fungi and possibly bacteria that can be used as the EPA source include:

• I. diatoms Asterionella japonica Bidulphia sinensis Chaetoceros septentrionale Lauderia borealis Navicula biskanteri Navicula laevis (heterotrof.) Navicula laevis Navicula incerta Stauroneis amphioxys Navicula pellicuolsa Bidulphia aurtia Nitzschia alba Nitzschia chosterium Phaeodactylum tricornutum Phaeodactylum tricornutum Skeletonema costatum
• II. Chrysophyceae Pseudopedinella sp. Cricosphaera elongate
• III. Eustigmatophyceae Monodus subterraneus Nannochloropsis
• IV. Prymnesiophyceae Rodela violacea 115.79 Porphyry. Cruentum 1380.Id
• V. Prasinophyceae Pavlova salina
• VI. Dinophyceae Cochlodinium heteroloblatum Cryptecodinium cohnii Gonyaulax catenella Gyrodinium cohnii Prorocentrum minimum
• VII. Other microalgae Chlorella minutissima Isochrysis galbana ALII4 Phaeodactylum tricornutum WT Porphyridium cruentum Monodus subterraneus
• VIII. Mushrooms Mortierella alpine Mortierella alpine IS-4 Pythium Irregulare
• IX. Bacteria SCRC-2738

Various microalgae can 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 Heterocontophyta. The microalgae can be from one of the following classes: Bacillariophyceae, Eustigmatophyceae and Chrysophyceae. The microalgae can be from one of the following genera: Nannochloropsis, Chlorella, Dunaliella, Scenedesmus, Selenastrum, Osillatoria, Phormidium, Spirulina, Amphora and Ochromonas.

Other nonlimiting examples of microalgae species that may be used include: Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphora coffeiforrnis, Amphora coffeiformis var. Linea, Amphora coffeiformis var. Punctata, Amphora coffeiformis var. Taylori Amphora coffeiformis var. Tenuis, Amphora delicatissima, Amphora delicatissima var. Capitata, Amphora spp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracllis, Chaetoceros muelleri, Chaeloceros muelleri var. subsalsum, Chaetoceros sp., Chlamydomas perigranulata, Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. Vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella lobophora, Chlorella luteoviridis, Chlorella luteoviridis var. Aureoviridis, Chlorella luteoviridis var. Lutescens , Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella protothecoides var. Acidicola, Chlorella regularis, Chlorella regularis var. Minima, Chlorella regularis var ata, chlorella reisiglii, chlorella saccharophila, chlorella
saccharophila var. ellipsoidea, chlorella salina, chlorella simplex, chlorella sorokiniana, chlorella sp., chlorella sphaerica, chlorella stigmatophora, chlorella vanniellii, chlorella vulgaris, chlorella vulgaris fo. tertia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris fo. Tertia, Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella pelrcel, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis, Eremosphaera sp , Effipsoidon sp., Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnion sp., Haematococcus pluvialis, Hymenomonas sp., Isochrysis aff. galbana, Isochrysis galbana, Lepocinclis, Micractinium, Micractinium, Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula saprophila, Navicula sp.
Nephrochloris spp., Nephroselmis spp., Nitschia communis, Nitzschia alexandrina, Nitzschia closterium, 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., Oscillatoria subbrevis, Parachlorella kessleri, Pascheria acidophila, Pavlova sp., Phaeodactylum tricomutum, Phagus, Phormidium, Platymonas sp. , Pleurochrysis carterae, Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamil, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis , Stichococcus sp., Synechococcus sp., Synechocystist, Tagetes erecta, Tagetes patula, Tetraedron,
Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii and Viridiella fridericiana. Preferably, the microalgae are autotrophic.

It is also possible to form the oil comprising glycolipids and phospholipids and at least EPA of genetically modified yeast. Non-limiting examples of yeasts that may be used include Cryptococcus curvatus, Cryptococcus terricolus, Lipomyces Stäryl Lipomyces lipofer, Endomycis pseudis, Rhodotorula glutinis, Rhodotorula gracilis, Candida 107, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces bayanus, Saccharomyces carevisiae, any Cryptococcus, C. neoformans, C. bogoriensis, Yarrowia lipolytica, Apiotrichum curvatum, T. bombicola, T. apicola, T. petrophilum, C. tropicalis, C. lipolytica and candida albicans. It is even possible to use a biomass as wild type or genetically modified fungus. Non-limiting examples of fungi that may be used include: Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum, Hensenulo, Chaetomium, Cladosporium, Malbranchea, Rhizopus and Pythium.

It is also possible that bacteria can be used that have lipids, proteins and carbohydrates, whether naturally occurring or obtained by genetic manipulation. Non-limiting examples of bacteria are: Escherichia coli, Acinetobacter sp., Each Actinomycet, Mycobacterium tuberculosis, each Streptomycet, 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. polyma, C. lepus, N. erthropolis, T. thiooxidans, D. polymorphis, P. aeruginosa and Rhodococcus opacus.

Possible sources of algae based on EPA / DHA derived from algae containing glycol and phospholipid-bound EPA and / or EPA / DHA and a significant amount of free fatty acids, triglycerides and phospholipids and glycolipids in the range of 35% -40% or more Whole lipids may have are in the paper "Chemicals from Microalgae", as by Zvi Cohen, CRC Press 1999 issued, revealed. Reference is also made to a study in men given a single dose of oil from a polar lipid-rich oil from the alga Nannochloropis oculata as a source of EPA and in the article entitled "Acute Appearance of Fatty Acids in Human Plasma - A Comparative Study Between Polar Lipid Rich Oil from the Microalgae Nannochloropis Oculata in Krill Oil in Healthy Young Males", published in Lipids in Health and Disease, 2013, 12: 102 by Kagan et al , is described. The EPA in the algae oil was higher by about 25.05 to 13.63 for the fatty acid composition as a percentage of the oil than in the krill oil. The algae oil was provided with 1.5 grams of EPA and no DHA compared to the krill oil provided with 1.02 grams of EPA and 0.54 grams of DHA. Participants consumed both oils in random order, seven days apart, and the blood samples were collected before breakfast and at several times up to 10 hours after ingestion of the oils.

The researchers found that the algae-based oil had a higher concentration of EPA and plasma than krill oil, with the EPA concentration being higher for algae-based oil at 5, 6, 8 and 10 hours (P <0.05), at 4 hours (P = 0.094) should be higher. The maximum concentration (CMAX) of EPA was higher with algal oil than with krill oil (P = 0.010). The maximum change in the concentration of EPA from its fasting concentration was higher than with krill oil (P = 0.006). The area under the concentration curve (AUC) and the incremental AUC (IAUC) were larger (P = 0.020 and P = 0.006). This difference may be related 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. Furthermore, the n-3 polyunsaturated fatty acids in glycolipids as found in algal oil but not in krill oil can be an effective system for delivering EPA to humans.

The microalgae can be photoautotrophically cultured outdoors to produce concentrated microalgae products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are the long chain polyunsaturated fatty acids (PUFAs) found in fish oil. Both are very important for the health of humans and animals. The concentrated microalgae products mentioned in the patent '037 EPA and DHA and lipid products containing EPA and DHA purified from microalgae may be included. The concentrated microalgae composition can be prepared by cultivating the microalgae photoautotroph outdoors in open basins under filtered sunlight in continuous or batch mode and at a dilution rate of less than 35% per day. The microalgae can be harvested in the exponential phase as the cell count increases at a rate of at least 20% of the maximum rate. In one example, the microalgae is concentrated. In another example, at least 40% by weight of the lipids in the microalgae are in the form of glycodiacylglycerides, phosphodiacylglycerides or a combination thereof, and at least 5% by weight of the fatty acids are DHA, EPA or a combination thereof.

In one example, the microalgae is Tetraselmis sp., Which is cultured at over 20 ° C or in another example above 30 ° C. It has been found that the EPA yield in the microalgae is at least 10 mg / liter of culture. In another example, the microalgae Isochrvsis sp. or Pavlova sp. be or are Thalassiosira sp. or Chaetecoros sp. The microalgae can be different diatoms and are cultivated photoautotrophically outdoors for at least 14 days in open basins 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 dwindling supplies of fish oil and / or Antarctic krill, which are now harder to harvest and economically obtainable and use because of the high demand for these products. One 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 are mainly in the form of glycolipids, and a small percentage are in the form of phospholipids. Glycolipids are mainly part of the chloroplast membranes, and phospholipids are part of the cell membranes.

The patent '037 describes various methods of cultivating microalgae photoautotrophically outdoors to produce EPA and DHA. One method that is used is filtering sunlight to reduce the light intensity on the photoautotrophic culture. For this purpose a shading cloth or net can be used. It has been determined that for most strains, the optimal sun intensity for growing, for maintaining a pure culture, and for omega-3 fatty acid enrichment is about 40,000 up to 50,000 lux, that is about half of the 110,000 lux of full sunlight, was. The shading or shading mesh is suitable for filtering the sunlight to the desired intensity.

It is also possible to cultivate the microalgae photoautotrophically in the open and EPA and DHA by means of small dilutions and a slow dilution rate or less than 40% per day, preferably less than 35% per day, more preferably from about 15% to about 30% per Day to produce. In other examples, the dilution rate is 15-40% per day or 15-35% per day and in still other examples the dilution rate is 10-30%, 10-35% or 10-40% per day. These smaller dilutions and lower dilution rates than commonly used will help prevent outdoor photoautotrophic contamination. This also promotes the strong culture growth, which leads to a good DHA or EPA yield.

Another technique for successfully cultivating microalgae photoautotrophically outdoors and producing EPA and EPA / DHA is to harvest the microalgae in an exponential phase rather than in a stationary phase. Harvesting in an exponential phase reduces the risk of contamination of photoautotrophic crops outdoors and surprisingly has led to a good yield of EPA and DHA. In order to promote fat accumulation in microbial cultures, the cultures are harvested in a stationary phase because cells in the stationary phase tend to accumulate storage lipids. The patent '037 teaches that EPA and DHA accumulate in large quantities as membrane lipids in cultures harvested in the exponential phase. The membrane lipids containing EPA and DHA are mainly phosphodiacylglycerides and glycodiacylglycerides and not the triacylglycerides found in memory lipids. These cultures are often harvested as the cell count increases at a rate of at least 20% of the maximum rate, ie, the maximum rate achieved at each stage during the photoautotrophic growth of the harvested outdoor culture. In specific examples, the cultures are harvested in the exponential phase as the cell count increases at a rate of at least 30%, at least 40%, or at least 50% of the maximum rate. It is also possible to use the recombinant DNA techniques.

The patent '037 contains several examples to which the reader is referred for descriptive and educational purposes.

Example 1: The strain Thalassiosira sp. is a diatom and this strain was isolated from the Bay of Bengal and dominates during the summer months. This exemplary strain was isolated from seawater collected near Chemai, India, and the culture was kept in open pans. The special strain was identified as Thalassiosira weissflogii, capable of growth at high temperatures (35-38 ° C). The fatty acid profile was good with 25-30% EPA (as a percentage of fatty acids), even when the algae were grown at high temperature.

Cultivation: The laboratory cultures were kept in wells in an artificial seawater medium under the fluorescent lights (3000-4000 lux) and the temperature was 25 ° C. The initial expansion of the culture was done under laboratory conditions in tubs. The dilution rate was 15% to 30% of the total culture volume per day. Once the volume reached 40-50 liters, it was transferred to an outdoor basin. The outdoor pools were covered with nets to control the light (40,000 to 50,000 lux). The dilution was continued until the culture reached a volume of 100,000 liters. The culture was held in basins of 500 square meters with a cultural depth of 20 cm. The culture was stirred with a paddle wheel and CO2 mixed in to keep the culture pH neutral. When the EPA levels in the basin reached a desired level (10-15 mg / L), the entire basin was harvested by filtration. The filtered biomass was washed with brine (15 parts per thousand concentration) and then spray dried. The kind of cultivating was the batch operation. The EPA productivity was 2-3 mg / liter / day. The basins can also be run continuously for several weeks by harvesting part of the culture, returning the filtrate to the basins, and replenishing the required nutrients.

Example 2: The strain Tetraselmis sp. belongs to the department Chlorophyta and the class Prosinophyceae or Micromanadophyceae. This strain was obtained from the Central Marine Fisheries Research Institute in India. He was isolated from the local marine habitats in India. The culture was kept in flasks in artificial seawater medium, and expanded as described for Thalassiosira. With the outdoor culture in open basins, as described for Thalassiosira, the strain gave a good lipid yield (200-300 mg / liter) and an EPA content of 6-7% fatty acids.

Example 3: The strain Chaetoceros sp. is another diatom strain obtained from the Central Marine Fisheries Research Institute of India and isolated from local marine habitats in India. Chaetoceros sp. was held in flasks and photoautotrophically cultured in outdoor pools as described in Example 1. He achieved similar EPA productivity and EPA content as Thalassiosira as described in Example 1.

Example 4: The strain Isochrysis sp. belongs to Prymnesiophyta, class Prymnesiophyceae, order Isochrysidales. It was obtained from the Central Marine Fisheries Research Institute of India and isolated from local marine habitats in India. It was maintained and grown as described in Example 1. It was expanded in 14-15 days from the laboratory culture to a 50,000 liter outdoor basin culture with a dilution rate of 15-30% per day. The lipid content at harvest was 100-150 mg lipids / liter. The rate of lipid production was 25-50 mg / liter / day. DHA made up 10-12% of total fatty acids.

Example 5 Harvesting and Drying: Harvesting may be by flocculation. The commonly used flocculants include alum with polymer and FeCl3 with or without polymer and chitosan. The concentration of the flocculant depends on the number of cells in the pre-harvest culture. The range can vary from 100 ppm to 500 ppm. Alternatively, the harvesting takes place by filtration by means of suitable nets. The removal of attached chemicals (which are not salt) is achieved by washing the cells in water of low salinity.

The harvested thin pulp is then spray dried. The thin slurry is sometimes encapsulated to prevent oxidation. The concentration of the encapsulant may vary from 0.1 to 1.0% on a dry weight basis. The modified starch is a suitable encapsulant. The spray dryer usually belongs to the type nebulizer or nozzle. The inlet temperature is in the range of 160 to 190 ° C and the outlet temperature is in the range of 60 to 90 ° C. The spray-dried powder is used directly for extraction. If storage is required, the powder is packed in aluminum-lined bags and sealed with nitrogen after displacing the air. The packaged powder is stored at ambient temperature until further use.

Example 6: The extraction of EPA / DHA is carried out by means of a wet slurry or a dry powder and solvents containing hexane, ethanol, methanol, acetone, ethyl acetate, isopropanol and cyclohexane and water, either alone or in combination with two solvents , The ratio of solvent to biomass depends on the starting material. If there is a thin slurry, the ratio is 1: 2 to 1:10. In contrast, with a spray-dried powder, the ratio is 1: 4 to 1:30. The extraction is carried out in an extraction vessel in an inert atmosphere at temperature ranges of 25 to 60 ° C and for a time varying from one hour to 10 hours. The solvent is added once or in part based on the lipid level in the cells.

After extraction of the crude lipid, the mixture is passed through a centrifuge or filtration system to remove cell debris. The lipid in the filtrate is concentrated by removing the solvent by distillation, which is carried out in vacuo. The resulting product is a crude lipid extract containing approximately 10% omega-3 fatty acid (EPA / DHA). The extract may be used as is or may be further purified to enrich the omega-3 fatty acids. Further purification may involve the removal of unsaponifiable substances such as pigments, sterols and their esters. The algae-based oil composition can be used for various purposes as described above.

In the patents '072 and '608 , which are incorporated by reference, a clinical trial is described only by astaxanthin, in which a dose of a softgel containing 15 milligrams of astaxanthin was administered once daily during breakfast for 12 weeks and 70 subjects were recruited for the study. This was a comparative single-blind clinical study and a total of 70 subjects recruited for the study, with 35 in each group, corresponding to an astaxanthinoleukine complex and a placebo control. The results of the clinical trial are presented below and demonstrate efficacy in using high doses of astaxanthin at levels of 15 mg. However, it has been found that surprisingly effective results are obtained when using 2 to 4 mg or 0.5 to 12 mg or other described ranges of astaxanthin alone in the presence of a suitable surfactant, such as a sunflower or perilla based phospholipid becomes. This could include the roe extract with phospholipid as described above. It could also be a plant-based phospholip and a lecithin alone or modified as a lysophospholipid source. It is possible to use glycophospholipids. An exemplary Perillaöl is described and in in commonly assigned, incorporated by reference '904. The astaxanthin and the surfactant may optionally be mixed with a low molecular weight hyaluronic acid as described above or UC-II. The astaxanthin may be present in the presence of a surfactant of less than 4 mg / day and, as indicated above, optionally admixed with the low molecular weight hyaluronic acid or the UC-II and / or as a chicken breast cartilage collagen isolate. The phospholipid may have some EPA and DHA. In one example, a preferred astaxanthin concentration is about 2-4 mg and a chicken breast cartilage colocal isolate may be about 40 mg and have a range of from about 30 to about 50 mg. Other surfactants such as plant-based phospholipids and commercially available lecithins which are modified and egg yolk compositions and / or marine-based oils such as perilla may be used. Sea-based phospholipids and lysolipid, also known as lysophospholipid, can be used as counterparts. A non-omega-3 platform can be used with the present invention. The described low molecular weight hyaluronic acid can vary from 1-500 mg, 10-70 mg, 35 mg or 45 mg and in other ranges described, and is a preferred low molecular weight microbial fermented product as described above.

The clinical trial described in the patents '072 and '608 is now described.

Clinical trial to evaluate the efficacy of Haematococcus pluvialis astaxanthin-oleoresin complex in osteoarthritis patients: The study was conducted as a comparative single-blind clinical trial with astaxanthinoleoin complex in 60 osteoarthritis patients compared to placebo control for a period of 12 weeks n = 60 (30A + 30P). The dosage consisted of a softgel containing 15 mg astaxanthin once daily during breakfast for 12 weeks. A total of 70 subjects were recruited for the study, 35 in each group (astaxanthinoleukine complex and placebo control) of both sexes. Patients were explained the nature of the study and informed consent was obtained prior to the start of the study. Patient test persons were clinically examined by the principal investigator and the team. Radiographs and blood samples were taken at the beginning and at the end of the study period. The case record forms were completed by the principal investigator and re-examined by the clinical research associate. Sixty patient test persons completed the study. Ten were eliminated for a variety of reasons, but not due to intolerance to the astaxanthinoleukine complex or placebo control. The results were tabulated by the expert data entry staff under the guidance of the biometrics expert. The results were subjected to a static analysis by an independent analyst.

The evaluation of osteoarthritic symptoms was based on the Western Ontario and McMaster Universities (WOMAC) osteoarthritis index, the VAS scale, the Lequesne functional scale and the sleep scale as additional parameters in addition to radiological examinations. Further, the evaluation of osteoarthritic symptoms was based on hematological studies, particularly MMP3 (matrix metalloproteinase 3) in clinical parameters, as osteoarthritis patients show elevated levels of MMP3 in the blood as well as in synovial fluid. The elevated levels cause significant tissue damage through cartilage destruction.

Results of the clinical trial and discussions:

Overall Health Assessment Points - The overall health assessment of osteoarthritis patients referred to the difficulty to A) donning - turn off buttons, wash and hair; b) getting up - get up straight from the chair, go to bed and get out of bed, sit cross-legged on the floor and get up; c) food - cut vegetables, bring a full cup / glass to the mouth; d) walking - going outdoors in the flat terrain, climbing five steps; and e) hygiene - taking a bath, washing and drying the body, going to the bathroom and back down; f) Stretching - after a 2 kg object stretch directly over the head and lay it down, bending down to pick up clothing from the ground; g) open a previously opened bottle, open and close the taps, open the door locks; h) Activities - work in the office / house, do errands, get in the car / car and get off again. The summary of the results is given in Table 3.

At the end of 3 months, there were significant reductions in the mean score of patients taking astaxanthinoleukine complex, but not for the placebo group. There were no significant differences between the astaxanthin and placebo groups at baseline or baseline levels. There were significant differences between the astaxanthin and placebo groups at 3 months.

The WOMAC Score - Western Ontario McMaster (WOMAC) is a validated instrument designed specifically for the assessment of lower extremity pain and the function of the knee in osteoarthritis (OA). Patients were assessed for pain, stiffness, and severity in performing daily activities. The pain index was evaluated for activity - a) when walking on a flat surface, walking up and down on a flat surface, sitting or lying in bed at night, standing upright; b) stiffness - after first awakening in the morning, after sitting / lying down or resting later during the day; and c) difficulties in walking down stairs, climbing stairs, getting up from the chair, bending down to the floor while standing, picking up objects, walking on flat ground, getting in and out of tuk-tuks / bus / car, going shopping, getting up from bed, lying in bed, sitting in a chair, going to the bathroom and downstairs, doing heavy household work, such as clearing heavy boxes / scrubbing the floor / picking up shopping bags, doing light household chores, such as cleaning Room / Table / Cooking / Dusting while sitting cross-legged, stand up from the position with your legs crossed while crouching on the floor. The summary of the results is given in Table 4.

There were significant reductions at the end of 3 months in the mean values for patients taking the astaxanthinoleukine complex but not for the placebo group. There were no significant differences in baseline values between patients taking astaxanthinoleukine complex and the placebo group. There were significant differences between the astaxanthin and placebo groups at 3 months.

VAS (visual analogue scale) for pain parameters - Pain parameters were evaluated by VAS in osteoarthritis patients taking astaxanthinoleoresin and the placebo group. The assessment was performed with respect to a) pain parameters - pain during use of the stairs, pain during walking on flat ground, pain during standing, pain during sitting or lying down, pain at night in bed b) physical functions - the Going down stairs, going up stairs, sitting, getting up from sitting, standing, bending to the floor, walking on flat ground, getting into or out of vehicles, shopping, putting on socks / socks, taking off socks / stockings, going to bed, off the bed get up, get into the bathtub or get out of the bathtub, sit on the toilet seat or get up from it, go into the lotus position during heavy housework while doing light housework. The summary of the results of pain parameters (pain + movement) values are given in Table 5.

There were significant reductions in the mean values at the end of the 3 months for patients taking astaxanthinoleukine complex, but not for the placebo group. There were no significant differences between baseline astaxanthinoleoin complex and placebo groups. There were significant differences between astaxanthinoleukine complex and placebo at 3 months.

Laquesne Index - The Laquesne Index is the functional index for osteoarthritis of the knee. The assessment will be made in terms of a) pain / discomfort - during nocturnal bed rest, morning stiffness or regressive pain after getting up, standing for 30 minutes; and b) Movement functions - maximum walking distance, activities of daily life such as walking up a standard staircase, going down a standard staircase, squatting down or leaning on the knees, walking on uneven ground. The results of the Laquesne Index are given in Table 6.

At the end of 3 months, there were significant reductions in mean scores for the patients taking astaxanthinoleukine complex but not for the placebo group. There were no significant differences between baseline astaxanthinoleoin complex and placebo groups. There were significant differences between astaxanthinoleukine complex and placebo at 3 months.

Sleep scale - Sleep is an important element of functioning and well-being. The sleep scale was originally developed in the Medical Outcomes Study (MOS), which aims to assess the extent of sleep problems. The Sleep Outline of the Medical Outcomes Study includes 12 points that assess sleep disorder, sleep affinity, somnolence, sleep, snoring, and respiratory or headache recovery. A sleep problem index grouping scores from each of the earlier scopes is also available. This assessment assessed the psychometric properties of the MOS sleep scale in osteoarthritis patients who took astaxanthinoleukine complex and the placebo group. The results with respect to the MOS sleep scale are given in Table 7.

There were significant reductions in mean values at the end of the 3 months for patients taking astaxanthinoleukine complex but not for the placebo group. There were no significant differences between the astaxanthinoleukine complex group and the placebo group at baseline. There were significant differences between the astaxanthinoleukine complex group and the placebo group for most sizes.

MMP3 (Matrix Metalloproteinase 3) Assay - Evaluation of osteoarthritic symptoms based on hematological studies, in particular MMP3 (matrix metalloproteinase 3) has been performed in clinical parameters as osteoarthritis patients show elevated levels of MMP3 in the blood as well as in synovial fluid. The elevated levels cause significant tissue damage through cartilage destruction. The results of MMP3 analysis in osteoarthritis patients before and after 3 months of astaxanthinoleukine complex administration are in 2 specified. The results of MMP3 analysis in osteoarthritis patients before and after 3 months of placebo administration are in 3 specified. The MMP3 levels did not show any significant change, but the trend is towards a reduction.

In total, 70 subjects were randomized to the study. Patients were explained the nature of the study and the active (astaxanthinoleukine complex gels with astaxanthin 15 mg) and placebo treatments. A written informed consent was obtained from the subjects prior to the start of the study. At the beginning of the study, the subjects were clinically evaluated and blood samples were taken for the CBC / ESR & MMP3 study. Special orthopedic and radiological examinations were performed. Patients were randomized to placebo and active treatment for a 12-week period. The patient test subjects were instructed to continue their other routine treatments, if any. At the end of 4 weeks, the subjects were asked for a second visit to refill the samples. The same procedure was used on the third visit and the procedure of the first visit was repeated on the fourth visit. The results were tabulated by registration staff and a detailed statistical analysis was made using these results. At baseline, the groups were similar and comparable.

Advantages of the invention: The total health score (getting up, dressing, eating, walking, hygiene, gripping, stretching, daily activities) showed significant changes between the astaxanthinoleukine complex and the placebo group (P <0.001). The improvement was evident in all parameters of daily activities.

The WOMAC INDEX showed significant differences (P <0.001). This evaluation is unique to the functional abilities of patients with chronic joint complaints, such as osteoarthritis.

VAS Pain Parameters (Pain + Exercise) Evaluation: There were significant reductions in mean end-of-treatment values for patients taking the astaxanthinoleukine complex, but not for placebo P (<0.001). It shows the improvement in the pain aspects of osteoarthritis.

Laquesne Index: (functional index of OA of the knee): There were significant reductions in mean end-of-treatment values for patients taking astaxanthinoleoin complex but not for placebo (P <0.05).

Sleep scale from the medical outcome study: There were significant reductions in mean end-of-treatment values for patients taking astaxanthinoleoin complex but not for placebo (P <0.001).

There were significant differences between the average sleep every night (h). Patients who took astaxanthinoleukine complexions slept longer than the placebo group (P <0.01).

The improvement in sleep time clearly demonstrates the effectiveness of treatment with the astaxanthinoleukine complex. Astaxanthin helps to a better sleep, as can be seen from the sleep evaluation. This is due to a reduction in pain, and other symptoms of MMP3 symptoms did not show any significant change, but the trend is towards reduction. The reduction in MMP3 levels indicates an improvement in cartilaginous health due to a reduction in cartilage destruction process in a positive way, although there is no direct evidence of this effect or any statistically significant effect in the present study. There was no change in the radiological image. No significant side effect / intolerance was noted during the study period. The astaxanthinoleukine complex seems to be safe for general use.

The astaxanthinoleukine complex extracted by Haematococcus pluvialis alga polar solvents may be suitable for patients in the early stages of osteoarthritis to prevent the progression of the disorder. It may be useful for patients with established osteoarthritis to obtain symptomatic relief in terms of pain and improved quality of life. Astaxanthinoleukine complex significantly improves symptoms such as pain and the quality of exercise in daily life. In India, osteoarthritis occurs at an earlier age. It would be useful to initiate treatment with astaxanthinoleukine complex right from the beginning as soon as a diagnosis is made. A larger study in different centers is recommended to further investigate the mechanism of action of the astaxanthinoleukine complex in osteoarthritis. Table 1: Carotenoid profile of Haematococcus pluvialis cell powder and astaxanthinoleukine complex carotenoids Zell powder Astaxanthinoleukine complex 5% Beta-carotene 0.62 ± 0.01 0.62 ± 0.01 canthaxanthin 1.21 ± 0.03 1.20 ± 0.03 astacin 3.09 ± 0.06 3.09 ± 0.06 semiastacin 1.35 ± 0.03 1.35 ± 0.03 Dicis astaxanthin 1.07 ± 0.02 1.03 ± 0.05 Trans astaxanthin 75.70 ± 1.53 75.75 ± 1.51 9-cis-astaxanthin 9.20 ± 0.77 9.19 ± 0.77 13-cis-astaxanthin 6.10 ± 0.94 6.08 ± 0.93 lutein 1.66 ± 0.03 1.65 ± 0.03 Table 2: Approximation analysis, carotenoid profile and fatty acid profile of astaxanthinoleukine complex PARAMETER Astaxanthinoleukine complex 5% Physically appearance Free flowing colour dark red approximation Protein% 0.95 ± 0.03 Carbohydrate% 0.11 ± 0.01 Lipid% 94.89 ± 0.12 Ash 3.82 ± 0.08 Humidity % 0.23 ± 0.02 carotenoids 5.14 ± 0.04 CAROTINOID% Ges. Carotenoids 5 Ges. Astaxanthin 4.68 [All-trans-astaxanthin [3, .90 9-cis-astaxanthin 0.47 13-cis-astaxanthin 0.31 15-cis-astaxanthin 0 Dicis astaxanthin] 0.05] Beta carotene 0.03 canthaxanthin 0.06 lutein 0.08 Fatty Acid Profile, range% C14: 0 myristic acid 0.23 C15: 0 pentadecanoic acid 0.1 C16: 0 palmitic acid 24.57 C16: 1 palmitoleic acid 0.57 C16: 2 hexadecadienoic acid 0.45 C16: 3 hexadecatrienoic acid 0.14 C16: 4 hexadecatetraenoic acid 1.15 C17: 0 heptadecanoic acid 2.14 C18: 0 stearic acid 1.61 C18: 1 oleic acid 38.93 C18: 2 linoleic acid 17.22 C18: 3, n-6 gamma-linolenic acid 0.84 C18: 3, n-3 alpha-linolenic acid 8.14 C18: 4 octadecatetraenoic acid 1.3 C20: 2 eicosadienoic acid 0.81 C20: 4 arachidonic acid 0.85 C22: 0 behenic acid 0.5 Table 3: Overall Health Score Total health evaluation value treatments basal duration Signifiianzniveau 1 month 2 months 3 months astaxanthin 18 14.68 13.19 12.13 S, P <0.001 placebo 20.25 19.8 19.48 19.51 NS, P = 0.4 S = significant, NS = not significant, P = probability Table 4: WOMAC Score WOMAC treatments basal duration level of significance 1 month 2 months 3 months astaxanthin 36.39 31.87 28.42 26.52 S, P <0.001 placebo 38.07 36.62 36.59 36.1 NS, P = 0.6 S = significant, NS = not significant, P = probability Table 5: VAS pain parameter result pain parameters treatments basal duration level of significance 1 month 2 months 3 months astaxanthin 891.94 828.71 772.58 748.39 S, P <0.001 placebo 945.86 923.28 916.21 915.17 NS, P = 0.1 S = significant, NS = not significant, P = probability Table 6: Laquesne Index parameter astaxanthin placebo level of significance 1. During the nightly bed rest basal 0.6 +/- 0.7 0.6 +/- 0.7 NS, P = 1.0 3 months 0.8 +/- 0.7 0.5 +/- 0.7 S, P = 0.05 2. Morning stiffness or regressive pain after getting up basal 0.9 +/- 0.6 0.6 +/- 0.7 NS, P = 0.9 3 months 0.6 +/- 0.6 0.6 +/- 0.5 NS, P = 0.9 3. After standing for 30 minutes basal 0.4 +/- 0.5 0.6 +/- 0.7 NS, P = 0.9 3 months 0.3 +/- 0.6 0.5 +/- 0.7 S, P = 0.05 4. Go maximum distance basal 1.3 +/- 0.7 1.7 +/- 1.3 NS, P = 0.9 3 months 0.6 +/- 0.5 1.7 +/- 1.3 S, P = 0.001 5. Activities of daily life a) can climb up a standard staircase basal 0.8 +/- 0.5 0.9 +/- 0.3 NS, P = 0.9 3 months 0.7 +/- 0.5 1.0 +/- 0.4 S, P = 0.03 b) can go down a standard staircase basal 1.3 +/- 0.3 1.6 +/- 0.9 NS, P = 0.9 3 months 0.9 +/- 0.6 1.6 +/- 0.9 S, P = 0.03 c) crouch or bend the knees basal 1.3 +/- 0.3 1.6 +/- 0.9 NS, P = 0.9 3 months 0.9 +/- 0.6 1.6 +/- 0.9 S, P = 0.03 d) can walk on uneven ground basal 1.3 +/- 0.3 1.6 +/- 0.9 NS, P = 0.9 3 months 0.9 +/- 0.6 1.6 +/- 0.9 S, P = 0.03 S = signifiiant, NS = not significant, P = probability Table 7: Sleep scale MOS sleep parameters astaxanthin placebo level of significance 1. Time to fall asleep (min) in the last four weeks basal 2.3 +/- 1.3 2, .6 +/- 1.3 NS, P = 0, .9 3 months 1.6 +/- 1.1 2.5 +/- 1.3 S, P <0.001 2nd average Sleep every night (hours during the last 4 weeks) 6, +/- 1.3 5, +/- 1.8 S, P <0.001 3. Was not sleep emotionally quiet? basal 3, +/- 1.9 3, +/- 1.9 NS, P = 0.9 S, P = 0.02 3 months 2.6 +/- 2.1 3.8 +/- 2.1 4. Get enough sleep to feel rested? basal 3.8 +/- 1.9 3.8 +/- 1.9 NS, P = 1.0 3 months 2.9 +/- 2.1 3.6 +/- 2.1 S, P = 0.03 5. A short breath or a headache? basal 5.6 +/- 1.2 4.6 +/- 2.2 NS, P = 0.6 3 months 5.6 +/- 1.2 4.5 +/- 2.8 NS, P = 0.6 6. Do you feel sleepy ^{or tired} during the day ^{? basal} 5, +/- 1.2 4, +/- 2.2 NS, P = 0.6 3 months 5.7 +/- 1.8 4.5 +/- 2.8 NS, P = 0.6 7. Do you have problems falling asleep? basal 3.6 +/- 2.7 4.6 +/- 2.2 NS, P = 0.3 3 months 4.4 +/- 2.1 4.5 +/- 2.8 NS, P = 0.9 NS, P = 0.7 8. Do you wake up during your sleep and are you having trouble falling asleep? basal 4.1 +/- 2.9 4.6 +/- 2.2 3 months 4.9 +/- 2.3 4.5 +/- 2.8 NS, P = 0.7 9. Are you having trouble staying awake during the day? basal 4.7 +/- 1.8 4.6 +/- 2.2 NS, P = 0.9 3 months 5.4 +/- 1.8 4.5 +/- 2.8 S, P = 0.05 10. Do you snore in your sleep? basal 5.5 +/- 1.1 4.4 +/- 1.5 NS, P = 0.2 3 months 5.7 +/- 0.8 4.8 +/- 1.3 S, P = 0.05 11. Do you take a nap during the day (5 minutes or more)? basal 4.1 +/- 1.5 4.4 +/- 1.5 NS, P = 0.6 3 months 3.7 +/- 1.5 4.8 +/- 1.3 S, P = 0.05 12. Do you get the necessary amount of sleep? basal 3.2 +/- 1.8 4.5 +/- 1.5 NS, P = 0.6 3 months 3.7 +/- 1.8 4.8 +/- 1.3 S, P = 0.05 S = significant, NS = not significant, P = probability

Many modifications and other embodiments of the invention will be apparent to those skilled in the art having the benefit of the teachings presented in the foregoing description and the accompanying drawings. It is therefore to be understood that the invention is not limited to the particular embodiments disclosed and that modifications and embodiments are intended to be included within the scope of the appended claims.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

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Claims (16)

A nutritional supplement composition comprising astaxanthin, proinflammatory, low molecular weight, microbially fermented sodium hyaluronate fragments having a molecular weight of 0.5 to 300 kilodaltons (kDa) and a carrier having a lipid selected from the group consisting of a phospholipid, glycolipid and sphingolipid an oral dosage form, wherein the astaxanthin constitutes 0.1 to 15% by weight of the carrier and the lipid selected from the group consisting of a phospholipid, glycolipid and sphingolipid for use as adjunct to treating and relieving joint pain , The dietary supplement composition of claim 1, wherein the astaxanthin comprises a natural or synthetic ester or a synthetic diol derivative. The nutritional supplement composition of claim 1, further comprising a pharmaceutical grade or food grade diluent. The dietary supplement composition of claim 1, wherein the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, lyso-phosphatidylcholine, lyso-phosphatidylethanolamine and lyso-phosphatidylserine. The nutritional supplement composition of claim 1, wherein the phospholipid comprises a vegetable source, algae source, animal source or a synthetic derivative. The dietary supplement composition of claim 1, wherein the composition contains from 0.5 to 12 mg astaxanthin. The dietary supplement composition of claim 1, wherein the composition contains from 50 to 500 mg of the carrier with the lipid selected from the group consisting of a phospholipid, glycolipid and sphingolipid. The nutritional supplement composition of claim 1, wherein the nutritional supplement composition is formulated into a single dose capsule. A nutritional supplement composition comprising astaxanthin, proinflammatory, low molecular weight, microbially fermented sodium hyaluronate fragments having a molecular weight of 0.5 to 300 kilodaltons (kDa) and a carrier having a lipid comprising a phospholipid and formulated into an oral dosage form, wherein the astaxanthin is 0 , 1 to 15% by weight of the carrier and the lipid with the phospholipid, for use as adjunct to treating and alleviating the symptoms of joint pain. The dietary supplement composition of claim 9, wherein the astaxanthin comprises a natural or synthetic ester or a synthetic diol derivative. A nutritional supplement composition according to claim 9, further comprising a pharmaceutical grade or food grade diluent. The dietary supplement composition of claim 9, wherein the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, lyso-phosphatidylcholine, lyso-phosphatidylethanolamine and lyso-phosphatidylserine. The nutritional supplement composition of claim 9, wherein the phospholipid comprises a vegetable source, algae source, animal source or a synthetic derivative. The nutritional supplement composition of claim 9, wherein the composition comprises from 0.5 to 12 mg astaxanthin. The dietary supplement composition of claim 9, wherein the composition contains from 50 to 500 mg of the carrier having the lipid with the phospholipid. The nutritional supplement composition of claim 9, wherein the nutritional supplement composition is formulated into a single dose capsule.

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