WO2007080515A1 - Thrombosis preventing krill extract - Google Patents

Thrombosis preventing krill extract Download PDF

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Publication number
WO2007080515A1
WO2007080515A1 PCT/IB2007/000099 IB2007000099W WO2007080515A1 WO 2007080515 A1 WO2007080515 A1 WO 2007080515A1 IB 2007000099 W IB2007000099 W IB 2007000099W WO 2007080515 A1 WO2007080515 A1 WO 2007080515A1
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WIPO (PCT)
Prior art keywords
krill
krill oil
composition
patient
oil extract
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PCT/IB2007/000099
Other languages
French (fr)
Inventor
Peter Mose Larsen
Stephen John Fey
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Aker Biomarine Asa
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Publication of WO2007080515A1 publication Critical patent/WO2007080515A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/12—Production of fats or fatty oils from raw materials by melting out
    • C11B1/14—Production of fats or fatty oils from raw materials by melting out with hot water or aqueous solutions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides
    • A23D9/013—Other fatty acid esters, e.g. phosphatides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/02—Pretreatment
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • This invention relates to novel extracts derived from krill, which can prevent and/or treat thrombosis.
  • This invention also relates to a method for the extraction of lipid fractions from krill in order to obtain the novel extracts of the present invention. More specifically, the invention relates to an improved method of extracting lipid fractions without using high temperatures and/or organic solvents.
  • Krill is the common name for small, shrimp-like crustaceans that swarm in dense shoals, especially in Antarctic waters. It is one of the most important food sources (especially protein) for fish, some kind of birds and especially for baleen. Krill is also a good source of omega-3 fatty acids, which are well known for their beneficial effects on human health.
  • krill and/or marine enzymes for the treatment of a great variety of diseases in human and animals such as infections, inflammations, cancers, HIV/AIDS, pain, polyps, warts, hemorrhoids, plaque, wrinkles, thin hair, allergic itch, anti-adhesion, eye disease, acne, cystic fibrosis and immune disorders including autoimmune diseases and cancer.
  • krill and/or marine oils may be used for the treatment of autoimmune murine lupus and other autoimmune diseases and can also be used for treating cardiovascular diseases.
  • US Patent 6,800,299 discloses a method for extracting lipid fractions from marine and aquatic animal material by acetone extraction.
  • the resulting non-soluble and particulate fraction is preferably subjected to an additional solvent extraction with an alcohol, preferably ethanol, isopropanol or t-butanol or an ester of acetic acid, preferably ethyl acetate to achieve extraction of the remaining soluble lipid fraction from the marine and aquatic animal material.
  • an alcohol preferably ethanol, isopropanol or t-butanol or an ester of acetic acid, preferably ethyl acetate
  • the remaining non-soluble particulate content is also recovered since it is enriched in proteins and contains a useful amount of active enzymes.
  • a krill extract is also provided herein. It is reported that these marine and aquatic animal oils have anti-inflammatory properties. Marine and aquatic animal oils are also reported as helpful in reducing the incidence of cardiovascular disease. As a further example the patent mentions that krill may be used as a source of enzymes for debridement of ulcers and wounds or to facilitate food digestion.
  • WO02102394A2 discloses a process for the preparation of a krill oil extract, which process includes the steps of placing krill and/or marine material in a ketone solvent to achieve extraction of the soluble lipid fraction from the krill; then separating the liquid and solid contents; then recovering a first lipid rich fraction from the liquid contents by evaporation of the solvent present in the liquid contents; then placing the solid contents in an organic solvent to achieve extraction of the remaining soluble lipid fraction from the krill material; then separating the liquid and solid contents; then recovering a second lipid rich fraction by evaporation of the solvent from the liquid contents; and finally recovering the solid contents.
  • Diseases that can be treated and/or prevented by using the krill oil extract are inter alia cardiovascular diseases.
  • the Krill oil has been shown to decrease cholesterol in vivo, inhibit platelet adhesion and plaque formation and reduce vascular endothelial inflammation in a patient.
  • Canadian Patent 1 ,098,900 describes a method for extracting oils and producing proteins from krill comprising emulsification of lipids of krill in an aqueous medium, separation of the emulsion of lipids from the krill mass, alkaline extraction of proteins from the krill mass, separation of the protein extract produced from chitin integuments, and finally separation of protein from the protein extract.
  • krill is a prospective source of food and other practically useful products such as chitin and lipids which find wide application in different branches, such as food industry, textile, and medicine.
  • WO03011873A2 discloses a phospholipid extract from inter alia krill, with therapeutic properties, such as those essential for the maintenance of a healthy cardiovascular system.
  • the phospholipid extract comprises a variety of phospholipids, fatty acid, metals and a novel flavonoid.
  • the method for the preparation of this extract is generally carried out by a method similar to the one described in US Patent 6,800,299 (see above; includes organic solvents), which procedure produces two successive lipid fractions and a dry residue enriched in protein, including active enzymes.
  • WO8401715A1 and WO09533471A1 disclose various aspects of so-called krill enzymes, which are water-soluble. It is mentioned that in krill a mixture of different enzymes exists, such as e.g. proteinases (with acidic and neutral-to-alkaline pH-optima), peptidases (exo- and endopeptidases), lipases, phospholipases, amylases and other carbohydrate degrading enzymes, phosphatases nucleases, nucleotidases and esterases.
  • the proteolytic (trypsin-like) activity existing in a water extract from krill has been studied and described.
  • WO09533471A1 disclose the use of one or more krill enzymes for the manufacture of an intravasal pharmaceutical composition for thrombolysis in a mammal host.
  • krill oil prepared by a novel process, which is from a physical-chemical point of view very gentle to the krill material due to relatively low temperature and no use of organic solvents, comprises other therapeutically valuable components than known from conventional krill oil extracts as well as other known fish oil; such components include inter alia high molecular (MWt > 200 kDa) hydrophobic proteins.
  • a novel krill oil extract for the prevention and/or treatment of thrombosis.
  • the general extraction method of the present invention will now be described.
  • the starting material consisting of freshly harvested and preferably finely divided krill material, is subjected to extraction, for about two hours and preferably overnight.
  • extraction time is not critical to the yield of lipid extraction.
  • particles of less than 0.5 mm in diameter.
  • Extraction is preferably conducted under inert atmosphere and at a temperature in the order of about 5° C or less.
  • the inventors have also envisaged that the present invention may be carried out by applying supercritical CO2 extraction.
  • the beginning of the extraction will be conducted under agitation for about 10 to 40 minutes, preferably 20 minutes.
  • the solubilized lipid fractions are separated from the solid material by standard techniques including, for example, filtration, centrifugation or sedimentation. Filtration is preferably used.
  • a novel krill extract for prevention and/or treatment and/or therapy of thrombosis.
  • the novel oil extract is derived from krill found in any marine environment around the world, for example, the Antarctic ocean (euphasia superba), the Pacific ocean (euphasia pacifica), the Atlantic ocean, the Indian ocean, in particular coastal regions of Mauritius Island and/or Reunion Island of Madagascar, Canadian West Coast, Japanese Coast, St-Lawrence Gulf and Fundy Bay, and this oil extract is a lipid fraction.
  • a method for extracting lipid fractions from krill comprising the steps of:
  • a method for extracting lipid fractions from krill comprising the steps of:
  • a pharmaceutical composition for the treatment of thrombosis in a patient comprising an effective amount of a krill oil extract obtainable by a method according to the present invention.
  • omega-3 fatty acid refers to polyunsaturated fatty acids that have the final double bond in the hydrocarbon chain between the third and fourth carbon atoms from the methyl end of the molecule.
  • Non-limiting examples of omega-3 fatty acids include, but are not limited to 5,8,11 ,14,17-eicosapentaenoic acid (EPA), 4,7,10,13,16,19-docosahexanoic acid (DHA) and 7,10,13,16,19-docosapentanoic acid (DPA).
  • the method of preparation is a continuous flow process and so the times given represent the average time that the material is in each stage of the process and the temperatures are typical (and may vary by ⁇ 3°C).
  • the freshly captured krill are fed into the grinder together with process water and shredded at 2 0 C for 5 minutes.
  • the slurry is then passed into a heat exchanger and warmed gently up to a temperature about 35 0 C (max below 4O 0 C) (1-2 minutes) and then stored in a buffer tank for 5 to 10 minutes. All subsequent processes occur at temperatures below 4O 0 C.
  • a centrifugal decanter is then used to separate the solid material from the liquid (3 minutes).
  • the liquid fraction is then stored in a buffer tank for 5 to 10 minutes.
  • the temperature of the liquid is adjusted to 35 0 C using a countercurrent plate heat exchanger (1 minute).
  • the stock is diluted sequentially (1 :10), shaking for 6 minutes at each dilution.
  • Blood samples were taken from normal subjects. 3.8 mm plastic tubes containing 0.38 ml 0.129M sodium citrate buffer (CPD buffer, pH 5.5) were used to store the blood. The buffered blood was then mixed with the krill or fish oil to achieve a final oil concentration varying from 5x10 "2 to 5x10 "18 Vol%. The blood cells were treated with krill or fish oil for 60 minutes before aggregation tests were performed. Blood aggregation time
  • the thrombocyte aggregation tests were performed with a PFA 100 aggregometer (Dade Bering), which is a microprocessor controlled apparatus with single test vials.
  • the unit comprises a small reservoir, a capillary and a membrane, which is covered with 2 mg genuine, type 1 collagen and 50 mg adenosin-5 ' -diphosphate (ADP).
  • ADP adenosin-5 ' -diphosphate
  • the blood is pipetted directly into the reservoir and aspirated through a capillary with a diameter of 200 ⁇ m with a constant negative pressure resulting in high shear stress.
  • the capillary ends with a membrane having an aperture with a diameter of 150 ⁇ m.
  • the thrombocytes are then activated by collagen and ADP.
  • the test Upon aggregation the blood flow is stopped due to clogging, which is referred to as closing time.
  • the test automatically stops after 300 seconds.
  • the normal value is between 62.5
  • Dilute krill oil solutions were added to whole human blood samples and allowed to react in accordance with the following steps:
  • Figure 2 shows the effects of various oils on the rate of aggregation of human whole blood.
  • the fish oil can be diluted to a concentration of only about I x IO "4 before it looses its effect.
  • a commercially available krill oil can be diluted to about 5 x 10 '6 before it looses its effect (i.e. it is about 500 times more effective than fish oil).
  • Krill oil prepared in the manner described here can be diluted to a concentration of about 5 x 10 "12 before it looses its effect. This is a million times more effective than the existing krill oil preparations and five hundred million times better than fish oil (note that the abscissa is a logarithmic scale).
  • Figure 3 shows the inhibiting effect of krill oil on the aggregation of thrombocytes in blood samples from 6 subjects. It also appears that the effect varies from subject to subject; and furthermore blood from one of the subjects was not influenced at all by the presence of krill oil.
  • Krill oil A Krill caught in large nets and subjected to a long process time
  • Krill oil B Krill caught in smaller nets and subjected to a short process time
  • Fish oil B Pikasol (OTC registered natural pharmaceutical containing concentrated Omega-3 rich fish oil; contains 62% omega-3 fatty acids, mainly EPA and DHA; Pikasol is produced from highly refined fish oil from the cleanest oceans in the world)
  • the oils are dissolved in a 1 :1 mixture with glycerol and CPD (Gly/CPD-mixture). Every single dillution is performed with the Gly/CPD-mixture to ensure that the glycerol concentration remains constant about 5x10 "3 Vol%.
  • Krill oil prepared by the process according to the present invention has a strong inhibitory effect on human thrombocyte aggregation in blood samples
  • the difference between the intensity of the effect may possibly be ascribed to certain proteins of the krill oil, and
  • Phospholipids are to be extracted from the solid fraction obtained in example 1 (step 4) using ethanol. After removal of the ethanol, the phospholipids are to be mixed with the krill oil phase obtained from the liquid fraction in example 1 (step 7) into a krill oil composition.
  • the antithrombotic effects of this krill oil composition are to be compared with other krill oil products extracted with organic solvents by investigating the effect on the aggregation time of thrombocytes in-vitro.
  • the krill oil products (mixtures of krill triglycerides and krill phospholipids) for this comparison are to be extracted from krill or krill meal using organic solvents as described in US 6,800,299. It is to be observed that the anti-thrombotic effects of the krill oil composition obtained by the methods described herein are superior to any krill oil product extracted with organic solvents such as acetone.
  • the krill oil compositions tested in example 4 are to be administered in humans (in- vivo) for a period of 5 weeks. Diets are to contain approximately 38% of energy as fat excluding the lipid in the supplement. Around 2 g of each product are to be administered in a way that preserves the biological effect of the krill oil. Non-limiting examples of administration are oral, sublingual or transdermal. After termination of the experiment, ex vivo and in vitro platelet aggregation, and variables of coagulation, fibrinolysis, and hematology are to be evaluated.
  • Ex vivo platelet aggregation time are to be measured by filtragometry and in vitro platelet aggregation induced by collagen and ADP measured by PFA 100 aggregometer.
  • Variables of coagulation factor VII amidolytic activity and concentrations of fibrinogen and prothrombin fragment 1 and 2) and fibrinolysis [plasminogen activator inhibitor (PAI) activity and concentrations of tissue plasminogen activator (tPA)/PAI-1 complexes] are to be determined by standard methods.
  • PAI plasminogen activator inhibitor
  • tPA tissue plasminogen activator

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Abstract

In accordance with the present disclosure there is provided a novel marine lipid extract obtainable by a process wherein processing temperature below 60 0C; mechanical and physical disruption of the lipid cell membrane to facilitate low temperature extraction; processing takes place under inert gas to prevent oxidation or denaturation of fat and proteins; intermediate processing tanks kept at a minimum level to reduce residence time; and the oil is frozen immediately after recovery to stabilize it.

Description

THROMBOSIS PREVENTING KRILL EXTRACT
FIELD OF THE INVENTION
This invention relates to novel extracts derived from krill, which can prevent and/or treat thrombosis. This invention also relates to a method for the extraction of lipid fractions from krill in order to obtain the novel extracts of the present invention. More specifically, the invention relates to an improved method of extracting lipid fractions without using high temperatures and/or organic solvents.
BACKGROUND OF THE INVENTION
Krill is the common name for small, shrimp-like crustaceans that swarm in dense shoals, especially in Antarctic waters. It is one of the most important food sources (especially protein) for fish, some kind of birds and especially for baleen. Krill is also a good source of omega-3 fatty acids, which are well known for their beneficial effects on human health.
It is known in the art to use krill and/or marine enzymes for the treatment of a great variety of diseases in human and animals such as infections, inflammations, cancers, HIV/AIDS, pain, polyps, warts, hemorrhoids, plaque, wrinkles, thin hair, allergic itch, anti-adhesion, eye disease, acne, cystic fibrosis and immune disorders including autoimmune diseases and cancer.
It is also known in the art that krill and/or marine oils may be used for the treatment of autoimmune murine lupus and other autoimmune diseases and can also be used for treating cardiovascular diseases.
However, most of the krill oil extracts used for these treatments has only conserved its omega- 3 fatty acids as active ingredients, which is a very small part of all the active ingredients of the krill itself. This fact dramatically reduces the potential of the krill and/or marine oil as a treatment for these diseases.
There is an increasing demand for treatments using products derived from a natural source, therefore, it would be highly desirable to be provided with a krill and/or marine extract having an enhanced potential for prevention and/or treatment and/or management of disease. US Patent 6,800,299 discloses a method for extracting lipid fractions from marine and aquatic animal material by acetone extraction. The resulting non-soluble and particulate fraction is preferably subjected to an additional solvent extraction with an alcohol, preferably ethanol, isopropanol or t-butanol or an ester of acetic acid, preferably ethyl acetate to achieve extraction of the remaining soluble lipid fraction from the marine and aquatic animal material. The remaining non-soluble particulate content is also recovered since it is enriched in proteins and contains a useful amount of active enzymes. Also provided herein is a krill extract. It is reported that these marine and aquatic animal oils have anti-inflammatory properties. Marine and aquatic animal oils are also reported as helpful in reducing the incidence of cardiovascular disease. As a further example the patent mentions that krill may be used as a source of enzymes for debridement of ulcers and wounds or to facilitate food digestion.
WO02102394A2 discloses a process for the preparation of a krill oil extract, which process includes the steps of placing krill and/or marine material in a ketone solvent to achieve extraction of the soluble lipid fraction from the krill; then separating the liquid and solid contents; then recovering a first lipid rich fraction from the liquid contents by evaporation of the solvent present in the liquid contents; then placing the solid contents in an organic solvent to achieve extraction of the remaining soluble lipid fraction from the krill material; then separating the liquid and solid contents; then recovering a second lipid rich fraction by evaporation of the solvent from the liquid contents; and finally recovering the solid contents. Diseases that can be treated and/or prevented by using the krill oil extract are inter alia cardiovascular diseases. In this respect it is mentioned that the Krill oil has been shown to decrease cholesterol in vivo, inhibit platelet adhesion and plaque formation and reduce vascular endothelial inflammation in a patient.
Canadian Patent 1 ,098,900 describes a method for extracting oils and producing proteins from krill comprising emulsification of lipids of krill in an aqueous medium, separation of the emulsion of lipids from the krill mass, alkaline extraction of proteins from the krill mass, separation of the protein extract produced from chitin integuments, and finally separation of protein from the protein extract. The document mentions that krill is a prospective source of food and other practically useful products such as chitin and lipids which find wide application in different branches, such as food industry, textile, and medicine.
WO03011873A2 discloses a phospholipid extract from inter alia krill, with therapeutic properties, such as those essential for the maintenance of a healthy cardiovascular system. The phospholipid extract comprises a variety of phospholipids, fatty acid, metals and a novel flavonoid. The method for the preparation of this extract is generally carried out by a method similar to the one described in US Patent 6,800,299 (see above; includes organic solvents), which procedure produces two successive lipid fractions and a dry residue enriched in protein, including active enzymes.
WO8401715A1 and WO09533471A1 disclose various aspects of so-called krill enzymes, which are water-soluble. It is mentioned that in krill a mixture of different enzymes exists, such as e.g. proteinases (with acidic and neutral-to-alkaline pH-optima), peptidases (exo- and endopeptidases), lipases, phospholipases, amylases and other carbohydrate degrading enzymes, phosphatases nucleases, nucleotidases and esterases. The proteolytic (trypsin-like) activity existing in a water extract from krill has been studied and described. WO09533471A1 disclose the use of one or more krill enzymes for the manufacture of an intravasal pharmaceutical composition for thrombolysis in a mammal host.
The potential of krill oil to prevent thrombosis has been disclosed in the prior art; however such a preventive effect has so far only been ascribed to the presence of powerful antioxidants and the special composition of poly-unsaturated fatty acids. The present inventors have surprisingly found that krill oil prepared by a novel process, which is from a physical-chemical point of view very gentle to the krill material due to relatively low temperature and no use of organic solvents, comprises other therapeutically valuable components than known from conventional krill oil extracts as well as other known fish oil; such components include inter alia high molecular (MWt > 200 kDa) hydrophobic proteins.
SUMMARY OF THE INVENTION
In accordance with the present invention there is provided a novel krill oil extract for the prevention and/or treatment of thrombosis.
The general extraction method of the present invention will now be described. The starting material, consisting of freshly harvested and preferably finely divided krill material, is subjected to extraction, for about two hours and preferably overnight. However, extraction time is not critical to the yield of lipid extraction. To facilitate extraction, it is preferable to use particles of less than 0.5 mm in diameter. Extraction is preferably conducted under inert atmosphere and at a temperature in the order of about 5° C or less. The inventors have also envisaged that the present invention may be carried out by applying supercritical CO2 extraction.
Preferably, the beginning of the extraction will be conducted under agitation for about 10 to 40 minutes, preferably 20 minutes. The solubilized lipid fractions are separated from the solid material by standard techniques including, for example, filtration, centrifugation or sedimentation. Filtration is preferably used.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, there is provided a novel krill extract for prevention and/or treatment and/or therapy of thrombosis.
The novel oil extract is derived from krill found in any marine environment around the world, for example, the Antarctic ocean (euphasia superba), the Pacific ocean (euphasia pacifica), the Atlantic ocean, the Indian ocean, in particular coastal regions of Mauritius Island and/or Reunion Island of Madagascar, Canadian West Coast, Japanese Coast, St-Lawrence Gulf and Fundy Bay, and this oil extract is a lipid fraction.
According to a first aspect of the present invention there is provided a method for extracting lipid fractions from krill, said method comprising the steps of:
• placing the krill material in a blender to mechanically disrupt fat cell membranes;
• separating the liquid and solid components;
• recovering a lipid rich fraction from the liquid component; wherein the extraction is performed quickly at a temperature below 60 0C and does not involve the use of organic solvents.
According to another aspect of the present invention there is provided a method for extracting lipid fractions from krill, said method comprising the steps of:
• Feeding freshly captured krill into a grinder to produce a slurry
• Heating the slurry gently to a temperature below 900C for less than 45 minutes
• Separating the solid material from the liquid
• Separating the liquid into an aqueous phase and a krill oil phase
wherein the extraction does not involve the use of organic solvents. According to the invention there is also provided a pharmaceutical composition for the treatment of thrombosis in a patient comprising an effective amount of a krill oil extract obtainable by a method according to the present invention.
EXAMPLES
As used herein, the term omega-3 fatty acid refers to polyunsaturated fatty acids that have the final double bond in the hydrocarbon chain between the third and fourth carbon atoms from the methyl end of the molecule. Non-limiting examples of omega-3 fatty acids include, but are not limited to 5,8,11 ,14,17-eicosapentaenoic acid (EPA), 4,7,10,13,16,19-docosahexanoic acid (DHA) and 7,10,13,16,19-docosapentanoic acid (DPA).
Example 1
Preparation of the krill oil extract of the present invention (see also Fig 1 ).
Preparation of the krill oil
The method of preparation is a continuous flow process and so the times given represent the average time that the material is in each stage of the process and the temperatures are typical (and may vary by ±3°C).
1. The freshly captured krill are fed into the grinder together with process water and shredded at 20C for 5 minutes.
2. This is then fed into a refiner which separates the chitin shell from the slurry (1 minute, 3°C).
3. The slurry is then passed into a heat exchanger and warmed gently up to a temperature about 350C (max below 4O0C) (1-2 minutes) and then stored in a buffer tank for 5 to 10 minutes. All subsequent processes occur at temperatures below 4O0C.
4. A centrifugal decanter is then used to separate the solid material from the liquid (3 minutes).
5. The liquid fraction is then stored in a buffer tank for 5 to 10 minutes.
6. The temperature of the liquid is adjusted to 35 0C using a countercurrent plate heat exchanger (1 minute).
7. The liquid is then separated into an aqueous phase and a krill oil phase. Preparation of the stock solution ofkrill oil
1. Autoclave glycerol (analytical quality) and leave to cool to room temperature
2. Mix 100 μl_ of krill oil with 1000 μL autoclaved glycerol
3. Shake mixture for 6 min. on a minibead beater (Biospec. Products, USA) at room temperature
4. Add 900 μL diluted CPD solution (Compoflex®, Fresenius HemoCare 61348 Bad Hamburg, Germany containing: citric acid monohydrate 3.27g, Sodium citrate dihydrate 26.3g, sodium dihydrogen phosphate dehydrate 2.51 g, glucose monohydrate 25.3g made up to 1 L).
5. Shake mixture for 6 min. on minibead beater 4 times at room temperature
Preparation of the dilute solutions ofkrill oil
1. The stock is diluted sequentially (1 :10), shaking for 6 minutes at each dilution.
2. Immediately before use, shake mixture for 6 min. on a minibead beater at room temperature
Preparaf/on of the stock solution of CPD Glycerol control solution
1. Mix 100 μL of diluted CPD solution with 1000 μL autoclaved glycerol
2. Shake mixture for 6 min. on minibead beater at room temperature
3. Add 900 μL diluted CPD solution
4. Shake mixture for 6 min. on minibead beater 2 times at room temperature
5. Repeat 4. immediately before use
Example 2
Effect of the krill oil extract of the present invention on' the aggregation time of thrombocytes.
Preparation of human blood
Blood samples were taken from normal subjects. 3.8 mm plastic tubes containing 0.38 ml 0.129M sodium citrate buffer (CPD buffer, pH 5.5) were used to store the blood. The buffered blood was then mixed with the krill or fish oil to achieve a final oil concentration varying from 5x10"2 to 5x10"18 Vol%. The blood cells were treated with krill or fish oil for 60 minutes before aggregation tests were performed. Blood aggregation time
The thrombocyte aggregation tests were performed with a PFA 100 aggregometer (Dade Bering), which is a microprocessor controlled apparatus with single test vials. The unit comprises a small reservoir, a capillary and a membrane, which is covered with 2 mg genuine, type 1 collagen and 50 mg adenosin-5'-diphosphate (ADP). The blood is pipetted directly into the reservoir and aspirated through a capillary with a diameter of 200 μm with a constant negative pressure resulting in high shear stress. The capillary ends with a membrane having an aperture with a diameter of 150 μm. The thrombocytes are then activated by collagen and ADP. Upon aggregation the blood flow is stopped due to clogging, which is referred to as closing time. The test automatically stops after 300 seconds. The normal value is between 62.5 - 120.5 seconds for ADP.
Determination of anti-aggregation effect
Dilute krill oil solutions were added to whole human blood samples and allowed to react in accordance with the following steps:
1. Serial dilutions of the krill oil or other oils under investigation were added to the human blood samples and gently shaken for 1 hour at room temperature on a "HETO-blood turner" at a rotational speed of 10 rpm.
2. Exactly 800μL of the blood-oil sample were placed in the reaction cartridge (DADE PFA collagen/epitest cartridge containing 4μg epinephrine bitartrate and 2μg type 1 equine collagen). The blood was then allowed to clot at 37°C for up to 300 seconds (preset instrument maximum).
3. Measurements were read from the display and printed out recorded
Figure 2 shows the effects of various oils on the rate of aggregation of human whole blood.
Samples of human whole blood are aggregated at the start and end of every experiment ("Start blood" and "End blood" on the abscissa) to determine the rate of blood aggregation for the donor. As an additional control carried out just after and just before the start and end whole blood aggregation determinations, an aliquot of the vehicle is added and the aggregation determination is repeated ("Start Glycerol/CPD" and "End Glycerol/CPD"). These controls are performed to ensure that the ability of the blood to aggregate does not change during the experimentation (see the trend line for the glycerol/CPD points). Between these control experiments, the blood is treated (as described in the text) with various concentrations of the different oils for 1hr before its ability to aggregate is determined. Dotted line - fish oil; dashed line a commercially available krill oil; solid line krill oil prepared in the manner disclosed here. The graph shows typical data from a single patient.
As can be seen in Figure 2, the fish oil can be diluted to a concentration of only about I x IO"4 before it looses its effect. A commercially available krill oil can be diluted to about 5 x 10'6 before it looses its effect (i.e. it is about 500 times more effective than fish oil). Krill oil prepared in the manner described here can be diluted to a concentration of about 5 x 10"12 before it looses its effect. This is a million times more effective than the existing krill oil preparations and five hundred million times better than fish oil (note that the abscissa is a logarithmic scale).
Figure 3 (graph 1 ) shows the inhibiting effect of krill oil on the aggregation of thrombocytes in blood samples from 6 subjects. It also appears that the effect varies from subject to subject; and furthermore blood from one of the subjects was not influenced at all by the presence of krill oil.
In Figure 4 (graph 2) the effect of krill oil C on blood from the same subject was analysed twice with a 21 day interval. The effect of krill oil C on the aggregation of thrombocytes is significant; however it must be concluded that the difference in the concentration required to achieve a significant inhibition varies with more than 10"3 Vol%.
Example 3
Comparison of krill oils and fish oils with respect to the effect on blood aggregation
These experiments serve to demonstrate that the krill oil obtainable by the process of the present invention prevents formation of thrombosis (based on the same experimental procedure as laid down in Examples 1 and 2) to a higher degree than known krill oils and other fish oils. The experiments include 2 fish oils as well as 3 different krill oils:
Krill oil A: Krill caught in large nets and subjected to a long process time
Krill oil B: Krill caught in smaller nets and subjected to a short process time
Krill oil C: Krill sucked up and processed very rapidly (in accordance with the present invention)
Fish oil A: Newly cold pressed cod fish oil
Fish oil B: Pikasol (OTC registered natural pharmaceutical containing concentrated Omega-3 rich fish oil; contains 62% omega-3 fatty acids, mainly EPA and DHA; Pikasol is produced from highly refined fish oil from the cleanest oceans in the world)
The oils are dissolved in a 1 :1 mixture with glycerol and CPD (Gly/CPD-mixture). Every single dillution is performed with the Gly/CPD-mixture to ensure that the glycerol concentration remains constant about 5x10"3 Vol%.
It is known that the quality of krill oil may vary considerably due to the way the krill material has been "caught". As discussed above the prior envisages that the amount of phosholipids, omega-3 and omega-6 polyunsaturated fatty acids and various antioxidants is responsible for the therapeutic effects attributable to krill oil. As appears from Fig 5 (graph 3) the three krill oils have very different effects on the aggregation. Surprisingly, the different effects could not be ascribed to differences in the amount of e.g. polyunsaturated fatty acids. On the contrary it appeared (based on 2D gel electrophoresis) that 5 proteins were present in Krill oil C (according to the present invention) but only in minute amounts in krill oil B and not traceable in krill oil A. This observation stems with the fact that many proteins in krill are extremely sensible for proteolytic degradation, which starts right after the krill has been caught.
As already mentioned the therapeutic effect of antioxidants and polyunsaturated fatty acids from fish oil on cardiovascular diseases is well known. Accordingly, the present inventors have compared the effect the effect of Krill oil C and fish oils A and B with respect to their ability to prevent thrombose formation (verified with the above described aggregation test). Fig 6 (graph 4) demonstrates that Krill oil C (according to the present invention) has a far more pronounced inhibitory effect on the thrombocyte aggregation than is the case with the fish oils. Conclusions drawn from Examples 1-3 Based on the experimental evidence provided so far the following conclusions may be drawn:
• Krill oil prepared by the process according to the present invention has a strong inhibitory effect on human thrombocyte aggregation in blood samples,
• The difference between the intensity of the effect may possibly be ascribed to certain proteins of the krill oil, and
• There is a substantial difference between how blood from different subjects responds to the krill oil with respect to aggregation time, however it may validly said that the krill oil obtained with the process of the present invention is far more effective that krill oils and fish oils obtained by traditional high temperature/solvent extraction methods.
Example 4
Phospholipids are to be extracted from the solid fraction obtained in example 1 (step 4) using ethanol. After removal of the ethanol, the phospholipids are to be mixed with the krill oil phase obtained from the liquid fraction in example 1 (step 7) into a krill oil composition. The antithrombotic effects of this krill oil composition are to be compared with other krill oil products extracted with organic solvents by investigating the effect on the aggregation time of thrombocytes in-vitro. The krill oil products (mixtures of krill triglycerides and krill phospholipids) for this comparison are to be extracted from krill or krill meal using organic solvents as described in US 6,800,299. It is to be observed that the anti-thrombotic effects of the krill oil composition obtained by the methods described herein are superior to any krill oil product extracted with organic solvents such as acetone.
Example 5
The krill oil compositions tested in example 4, the krill oil extracted obtained in example 1 (step 7), krill oil obtained using organic solvents and a control are to be administered in humans (in- vivo) for a period of 5 weeks. Diets are to contain approximately 38% of energy as fat excluding the lipid in the supplement. Around 2 g of each product are to be administered in a way that preserves the biological effect of the krill oil. Non-limiting examples of administration are oral, sublingual or transdermal. After termination of the experiment, ex vivo and in vitro platelet aggregation, and variables of coagulation, fibrinolysis, and hematology are to be evaluated. Ex vivo platelet aggregation time are to be measured by filtragometry and in vitro platelet aggregation induced by collagen and ADP measured by PFA 100 aggregometer. Variables of coagulation (factor VII amidolytic activity and concentrations of fibrinogen and prothrombin fragment 1 and 2) and fibrinolysis [plasminogen activator inhibitor (PAI) activity and concentrations of tissue plasminogen activator (tPA)/PAI-1 complexes] are to be determined by standard methods. It is to be observed that the subjects treated with the krill oil composition described in example 4 and the krill lipid extract obtained in example 1 (step 7) show superior anti-thrombotic activity than subjects treated with krill oil compositions obtained using organic solvents and control. Prevention of thrombosis is linked to prevention of myocardial infarction and stroke. Hence, the krill oil composition described in example 4 and example 1 (Step 7) can be used to prevent these pathologies.

Claims

1. A method for extracting lipid fractions from krill, said method comprising the steps of:
• placing the krill material in a grinder or blender to mechanically disrupt cell membranes;
• separating the liquid and solid components;
• recovering a lipid rich fraction from the liquid component; wherein the extraction is performed at a temperature below 60 0C and does not involve the use of organic solvents.
2. A method as in claim 1 , wherein separating the liquid and solid components is effected by techniques selected from the group consisting of mechanical pressing, filtration, centrifugation and sedimentation.
3. A method as in claim 1 , wherein the extraction is performed at a temperature below 27°C, preferably below 150C, more preferably below 5°C.
4. A krill oil extract obtainable by a method according to any one of claims 1 to 3.
5. A krill oil extract according to claim 4 for use as a medicament.
6. A pharmaceutical composition comprising the krill oil extract of claim 4.
7. A pharmaceutical composition for the treatment of thrombosis in a patient comprising an effective amount of a krill oil extract obtainable by a method according to any one of claims 1 to 3 in association with a pharmaceutically acceptable carrier.
8. The composition of claim 7, further comprising at least one of compounds selected from the group consisting of glycerol, dimethyl-sulphoxide (DMSO), linoleic acid, alpha-linoleic acid, arachidonic acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid, cholesterol, triglycerides, monoglycerides, all-trans retinal, canthexanthin, carotene, zinc, selenium, sodium, potassium and calcium.
9. Use of the krill oil extract obtainable by the method of any one of claims 1-3 for the production of a medicament for decreasing development of thrombosis in a patient.
10. A composition for inhibiting platelet adhesion and plaque formation in arteries of a patient comprising an effective amount of krill oil extract in association with a pharmaceutically acceptable carrier, wherein said krill oil extract is obtainable from a method according to any one of claims 1 to 3.
11. A krill oil extract obtainable by a method comprising the steps of:
• Feeding freshly captured krill into a grinder to produce a slurry
• Heating the slurry gently to a temperature below 900C in less than 45 minutes
• Separating the solid material from the liquid
• Separating the liquid into an aqueous phase and a krill oil phase wherein the extraction does not involve the use of organic solvents.
12. A food product comprising the krill oil extract of claim 4.
13. An animal feed comprising the krill oil extract of claim 4.
14. A food supplement comprising the krill oil extract of claim 4.
15. A composition comprising the krill oil extract of claim 4 and phospholipids, said phospholipids having the following structure:
Figure imgf000015_0001
wherein R1 is a fatty acid, R2 is a fatty acid, and R3 is selected from the group consisting of H or choline, ethanolamine, inositol or serine.
16. The composition in claim 15, wherein at least 1% (w/w) of the said fatty acids are unsaturated fatty acids.
17. The composition in claim 15, wherein at least 1% (w/w) of the said fatty acids are omega-3 fatty acids.
18. A food product comprising the composition in any of the claims 15 to 17.
19. An animal feed comprising the composition in any of the claims 15 to 17
20. A food supplement comprising the composition in any of the claims 15 to 17.
21. A pharmaceutical comprising the composition in any of the claims 15 to 17.
22. A method of preventing platelet adhesion in a patient comprising administering to said patient a therapeutically effective amount of the composition in any of the claims 15 to 21.
23. A method for preventing stroke or heart attack in a patient comprising administering to said patient a therapeutically effective amount of the composition in any of the claims 15 to 21.
24. A method of preventing platelet adhesion and plaque formation in a patient comprising administering to said patient a therapeutically effective amount of krill oil, wherein said krill oil is obtained without organic solvent extraction.
25. A method of preventing platelet adhesion and plaque formation in a patient comprising administering to said patient a therapeutically effective amount of krill oil composition, wherein said krill oil composition comprises triglyceride, phospholipid and protein fractions.
26. The method of claim 25, wherein said protein fraction comprises high molecular weight hydrophobic proteins.
27. A composition comprising a krill oil extract isolated from krill comprising triglyceride, phospholipid and protein fractions.
28. A pharmaceutical comprising the composition of claim 27.
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