(19) Japan Patent Office (JP)
`
`(1 1) Japanese Unexamined Patent
`Application Publication Number
`
`(12) Japanese Unexamined Patent
`Application Publication (A)
`
`H2-215351
`
`(51) Int. Cl.5
`A23 J 7/00
`A23 L 1/30
`1/33
`
`A
`B
`
`Identification codes
`
`JPO file numbers
`6712-4B
`8ll4-4B
`21 l4-4B*
`
`(43) Publication date: August 28, 1990
`
`Request for examination: Not yet requested Number of claims: 3 (Total of6 pages)
`
`(54) Title of the invention
`
`METHOD FOR EXTRACTING KRILL PHOSPHOLIPIDS, FUNCTIONAL FOOD
`PRODUCT HAVING BRAIN FUNCTION ENHANCING EFFECT, AND BRAIN
`FUNCTION ENHANCING AGENT
`
`(21) Japanese Patent Application
`
`Hl-34846
`
`(72) Inventor
`
`(22) Date of Application
`Kazuki Maiuyama
`
`(72) Inventor
`
`Masami Nishikawa
`
`(72) Inventor
`
`Shoji Kimura
`
`(71) Applicant
`(74) Agent
`Con't on last page
`
`Taiyo Fishery Co., Ltd.
`Hiroo Otsu, patent attorney
`
`February 14, 1989
`% Taiyo Fishery Co., Ltd.
`3-2-9 Tsukijima, Cl1uo-ku, Tokyo-to
`“/0 Taiyo Fishery Co., Ltd.
`3-2-9 Tsukijima, Cl1uo-ku, Tokyo-to
`
`% Taiyo Fishery Co., Ltd.
`3-2-9 Tsukijima, Chuo-ku, Tokyo-to
`l-l-2 Otemachi, Chiyoda-ku, Tokyo-to
`
`SPECIFICATION
`
`l. TITLE OF THE INVENTION
`
`Method for Extracting Krill Phospholipids,
`Functional Food Product Having Brain Function
`Enhancing Effect, and Brain Function Enhancing
`Agent
`2. SCOPE or PATENT CLAIMS
`
`(1) A method for extracting krill phospholipids
`wherein raw krill
`is dewatered using a vacuum
`freezing drying method, all
`the lipids therein are
`extracted using ethanol, an elution of the total lipids
`thus obtained is made with an ethanol solvent, an
`acetone solvent, or a hexane solvent, the phosphatidyl
`choline
`and,
`using
`adsorption
`column
`a
`chromatography with a
`silica gel
`as
`filler,
`phosphatidyl ethanol amine are fractionated and then
`isolated using a faction collector.
`(2) A functional food product having a brain function
`enhancing effect, wherein at
`least one or more of
`phosphatidyl choline or phosphatidyl ethanol amine
`
`isolated from krill or a derivative of these is mixed
`
`into a base food ingredient as an active ingredient.
`(3) A brain function enhancing agent, wherein at least
`one or more of phosphatidyl choline or phosphatidyl
`ethanol amine isolated from krill or a derivative of
`
`these is contained as an active ingredient.
`
`3. DETAILED DESCRIPTION OF THE INVENTION
`
`[Industrial Field of Use]
`a method for
`invention is
`The present
`separating and extracting phospholipids from krill
`and is more particularly a method for
`isolating
`phosphatidyl choline and phosphatidyl ethanol amine
`which show important bioactivity in living bodies,
`relating to
`a
`technology characterized in that
`phosphatidyl choline and phosphatidyl ethanol amine
`thus extracted can be used as a food product or
`pharmaceutical product.
`
`[Prior Art]
`We have begun entering an era of the aging
`society in recent years, and as this has happened
`senile dementia has become a major societal issue.
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`
`Japanese Unexamined Patent Application Publication H2-215351 (2)
`
`divided into
`can broadly be
`Senile dementia
`caused by
`Alzheimer's-type dementia which is
`dysfunction
`of
`the
`nervous
`system and
`cerebrovascular
`dementia which
`is
`caused
`by
`dysfunction of the cerebral blood vessels. With
`Alzheimer's-type
`dementia,
`it
`is
`known
`that
`production of acetyl choline, a neurotransmitter,
`drops significantly as a neurochemical change in the
`brain. To prevent and treat this ailment, an attempt is
`made to restore biological function by supplementing
`metabolism of the reduced choline. Examples include
`"Method and Composition for Treating an Illness by
`Administering Lecithin" in PCT Application S56-
`500374
`A,
`"Brain
`Hyperfunction
`Agent
`Composition" in JP S59-167514 A, and "Therapeutic
`Composition and Treatment Method for Neurological
`Damage and Chemotaxis" in S60-214734 A.
`In other words, by ingesting phosphatidyl
`choline, which is a choline-containing phospholipid,
`acetyl choline is supplied to the brain, which is
`expected to prevent and treat Alzheimer's-type
`dementia and other neurological disorders.
`is
`Phosphatidyl
`ethanol
`amine, which
`type of phospholipid,
`is converted into
`another
`phosphatidyl
`choline
`through
`a methyl
`group
`transition
`reaction
`from S-adenosylmethionine.
`Consequently, phosphatidyl ethanol amine is also
`expected to be usable in treatment of Alzheimer's -
`type dementia and other neurological disorders and as
`a therapeutic agent therefor.
`The inventors focused particularly on the
`phospholipids phosphatidyl choline and phosphatidyl
`ethanol amine, which are glycerophospholipids in
`researching and developing a method for industrially
`extracting these in a form usable as a raw ingredient
`in food and pharmaceutical products.
`Conventionally, soy beans have typically
`been used as
`a
`raw material when refining
`phospholipids from a natural substance for industrial
`use. Soy bean phospholipids have mainly been
`commercialized as health food products and the like.
`Conventional
`soy
`bean
`phospholipid
`refining
`processes involve extracting the total phospholipids
`from raw soy beans with a chloroform ethanol
`solvent. The total phospholipids are then fractionated
`with acetone, thereby separating them into a soluble
`section and an insoluble section.
`In the acetone-
`
`fieed
`lipids, cholesterol,
`soluble section, neutral
`lipids, and so on are fractionated. In the acetone-
`insoluble section, phospholipids are fractionated.
`Next, the acetone-insoluble section is treated with a
`90% ethanol solution, thereby obtaining phosphatidyl
`choline, which is soluble in alcohol, and phosphatidyl
`ethanol amine, which is not soluble.
`
`[Problem to be Solved by the Invention]
`
`However, when refining phospholipids from
`soy beans, the phosphatidyl choline and phosphatidyl
`ethanol amine which are obtained have a purity of
`around 70% to 80% and it is very difficult to obtain a
`refined product with a purity of 90% or more. The
`method using chloroform ethanol described above
`also entails the fear that harmful substances might
`remain, no matter how the soy beans are refined and
`fractionated, making it difficult to use this in food
`products, which is a problem.
`The inventors noticed that while krill has
`
`it
`gotten attention as a rich source of protein,
`nevertheless rots easily and has a high water content,
`making it expensive to store and transport, which is
`why no effective method of use has been established.
`The inventors also noticed that while krill has a high
`phospholipid content, there has been no technological
`development
`which
`would
`allow use
`of
`phospholipids, which the inventors focused on and
`which are an active ingredient, in high-value-added
`and economically valuable
`functional
`food
`or
`pharmaceutical products.
`The inventors
`
`therefore realized that
`
`if
`
`useful phospholipids could be obtained from krill,
`which is an untapped marine resource, at high
`purities,
`this would be extremely profitable as an
`effective method for utilizing krill. The inventors
`therefore proceeded with R&D of such a refinement
`method, and arrived at the present invention.
`Specifically,
`the present
`invention is an
`extraction method, wherein krill
`is used as a raw
`ingredient,
`total phospholipids are fractionated, and
`high-purity phosphatidyl choline and phosphatidyl
`ethanol amine are refined and isolated from the total
`
`phospholipids thus obtained, being a technology
`whereby the bioactive substances thus obtained are
`used as a functional food product having a brain
`function enhancing effect and a brain function
`enhancing agent.
`[Means for Solving the Problem]
`The present
`invention uses the following
`means to solve this problem.
`a method for
`The present
`invention is
`extracting krill phospholipids wherein raw krill
`is
`dewatered using a vacuum freezing drying method,
`all the lipids therein are extracted using ethanol, an
`elution of the total lipids thus obtained is made with
`an ethanol solvent, an acetone solvent, or a hexane
`solvent,
`the
`phosphatidyl
`choline
`and,
`using
`adsorption column chromatography with a silica gel
`as
`a
`filler,
`phosphatidyl
`ethanol
`amine
`are
`fractionated and then isolated using a
`faction
`collector‘.
`
`First step: Blocks of raw krill quick-frozen
`at sea have a water content of 90% or more, so drying
`is a problem. With the present invention, a vacuum
`
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`Japanese Unexamined Patent Application Publication H2-2l535l (3)
`
`freezing and drying machine is used as a pre-process
`to
`extraction
`using
`adsorption
`column
`chromatography to achieve dewatered, dried krill. It
`is desirable for dewatering and drying to reduce the
`water content
`to 6% or
`less. This minimizes
`
`admixture of water-soluble proteins in the ethanol
`extract,
`allowing a greater purity of separated
`components.
`lipids are extracted by
`Second step: Total
`homogenizing the dried krill obtained in the first step
`with ethanol.
`
`Third step: As much of the ethanol as
`possible is removed from the total lipids next, and an
`elution is obtained with an acetone solvent or a
`
`hexane solvent, fractionated into a soluble section
`and an insoluble section. For example, in the case of
`an acetone solvent, most of the phospholipids are in
`the insoluble section, so if the solvent is washed off,
`crude phospholipids are obtained.
`Fourth step: An elution is formed of the
`crude phospholipids with an ethanol
`solvent, an
`acetone solvent, or a hexane solvent, which is then
`fractionated
`into
`phosphatidyl
`choline
`and
`phosphatidyl ethanol amine using adsorption column
`chromatography, from which the phospholipids are
`isolated using a faction collector with a high purity of
`90% or more and around 95%.
`
`a method for
`invention is
`The present
`extracting krill phospholipids such as phosphatidyl
`choline and phosphatidyl ethanol amine at high
`purities using the method above.
`the high-
`that
`Next, focusing on the fact
`purity phosphatidyl choline or phosphatidyl ethanol
`amine isolated from the krill using the above method
`is a biofunction activating substance that enhances
`brain functions, the present invention is a functional
`food product having a brain function enhancing
`effect, wherein at least one or more of phosphatidyl
`choline or phosphatidyl ethanol amine isolated from
`krill or a derivative of these is mixed into a base food
`
`ingredient as an active ingredient.
`also a brain
`The present
`invention is
`function enhancing agent, wherein at
`least one or
`more ofphosphatidyl choline or phosphatidyl ethanol
`amine isolated from krill or a derivative of these is
`
`contained as an active ingredient. In this case the
`brain function enhancing agent can be made into a
`pharmaceutical product in tablet, capsule, granular, or
`liquid form.
`[Operation]
`With Alzheimer's-type dementia, it is known
`that production of acetyl choline, a neurotransmitter,
`drops significantly as a neurochemical change in the
`brain. To prevent and treat this ailment, an attempt is
`made to restore biological function by supplementing
`metabolism of the reduced choline.
`
`in the case of humans, when
`In particular,
`choline or lecithin, a naturally-produced compound
`which dissociates into choline, is administered orally,
`it
`is known that
`the resulting biofunction is an
`increase in the blood choline sufficient to promote
`synthesis and release of acetyl choline in the brain
`and an increase in the amount of choline in the
`
`cerebrospinal fluid.
`the present
`an object of
`Accordingly,
`invention is to expect prevention and treatment of
`Alzheimer's-type dementia and other neurological
`disorders by supplying acetyl choline to the brain by
`extracting
`phosphatidyl
`choline, which
`is
`a
`phospholipid, from krill
`in as safe and efficient a
`manner as possible and ingesting this as a food or
`pharmaceutical product.
`[Examples]
`The present invention is described in detail
`below, with reference to examples.
`<Example l.>
`20 kg of raw krill that was quick-frozen at
`sea was dried until a water content of around 4% was
`
`achieved using a vacuum drying machine, obtaining
`2.2 kg of dried krill. Table 1 gives the results of an
`iatroscan analysis of the lipid composition of the
`dried krill which was the raw material.
`
`Next, 2 kg of the dried krill thus obtained
`was homogenized using 40 kg of ethanol and the total
`lipids were extracted. Extraction was repeated,
`this
`time with 20 kg ofethanol.
`The total
`lipids, which were the extract,
`were condensed and as much of the ethanol as
`
`lipids were
`total
`removed. The
`possible was
`dissolved in acetone and fractionated into a soluble
`
`section and an insoluble section. The majority of the
`phospholipids were fractionated into the insoluble
`section. Accordingly, the substances fractionated into
`the insoluble section were repeatedly washed with
`acetone, obtaining 408 g of crude phospholipids.
`
`Table l. Lipid Composition of Dried Krill
`
`Lipid Composition
`Phosphatidyl choline
`phosphatidyl ethanol amine
`Triglycerides
`Free fatty acids
`Other
`
`Wt. %
`3 l .1
`7.5
`43 .2
`6.5
`5.7
`
`Next, 400 g of the crud phospholipids was dissolved
`in 2000 ml of ethanol and 20 ml per batch was
`automatically injected into an extraction column
`(column length x width: 50 cm x 50 mm, cross-
`sectional area:
`19.6 cm3) mounted on a fully-
`automated extraction-type high-performance liquid
`chromatography [sic] filled with spherical silica gel
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`Japanese Unexamined Patent Application Publication H2-215351 (4)
`
`(adsorption agent) having a particle diameter of 10
`pm. The elution was introduced with 100% ethanol at
`a flowrate of 30 ml/min. The column constant
`
`temperature tank was at 40°C. Peak detection was
`monitored using a UV absorption detector (205 pm).
`The chromatograph shown in FIG.
`1 was obtained.
`The fraction section of the first peak is A and the
`fraction section of the second large peak is B. These
`were extracted using a fraction collector. The purity
`of the phosphatidyl choline in the fraction section B
`was 98% or higher according to the iatroscan
`analysis. The cycle time per batch was 30 min. The
`base solution was automatically filled every 30 min,
`repeated over 100 cycles for a total time of around 50
`hr. As a result, approximately 239 g of high-purity
`phosphatidyl choline was obtained from 2 kg of dried
`krill.
`
`high-purity
`of
`g
`45
`Approximately
`phosphatidyl ethanol amine with a purity of 95% or
`higher was
`similarly obtained from the fraction
`section A.
`
`<Example 2.>
`Wexler memory and intelligent quotient
`tests were performed. A patient with memory loss,
`having a memory quotient of 123, was orally
`administered 10 g of the high-purity phosphatidyl
`choline (98% purity) obtained from the krill
`in the
`first example by mixture into food at every meal,
`three times a day for six weeks.
`Blood samples were taken from the patient
`before the test
`treatment and after six weeks of
`
`phosphatidyl
`high-purity
`the
`administration of
`choline. The blood plasma was separated and frozen
`and analyzed using an ordinary radiation oxygen
`method to determine the choline content. As a result,
`the blood plasma choline content in the blood taken
`before the test treatment was 13.4::2.5 nanomol/ml.
`
`After four weeks of high-purity phosphatidyl choline
`
`administration, the blood plasma choline content had
`increased to 31.3::2.5 nanomol/ml (P<0.01).
`Moreover,
`the patient's memory quotient
`had improved to l42 after six weeks of high-purity
`phosphatidyl choline administration.
`
`[Effects]
`
`The
`
`invention which is
`
`submitted for
`
`is an extraction
`protection according to claim 1
`method wherein krill, which is an untapped marine
`resource,
`is used as a raw material
`to refine and
`isolate useful phosphatidyl choline and phosphatidyl
`ethanol amine therein to a high purity of 90% or
`higher. This extraction method not only achieves a
`high purity in the refined and isolated components
`thereof, but also uses no solvents having toxicity in
`the refinement process. Therefore,
`this method is
`characterized in being able to be used safely for food
`and pharmaceutical products.
`inventions which are
`Furthermore,
`the
`submitted for protection according to claims 2 and 3
`are of a functional
`food product having a brain
`function enhancing effect and a brain function
`enhancing agent, because the phosphatidyl choline
`and phosphatidyl ethanol amine, which are the krill
`phospholipids
`thus
`obtained,
`have
`biological
`functions for enhancing brain functions which can be
`expected to be used in preventing and treating
`Alzheimer's disease.
`4. BRIEF DESCRIPTION or THE DRAWINGS
`
`is a fraction table of components
`1
`FIG.
`obtained from a chromatogram according to the
`present invention.
`
`Patent Applicant
`Agent
`attorney
`[SEAL: illegible]
`
`Taiyo Fishery Co., Ltd.
`Hiroo
`Otsu,
`patent
`
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`Japanese Unexamined Patent Application Publication H2-215351 (5)
`
`FIG. 1
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`0 mm
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`Japanese Unexamined Patent Application Publication H2-215351 (6)
`
`Con‘t from first page
`
`(51) Int. Cl.5
`A6lK 31/685
`37/22
`
`Identification codes
`AAM
`
`JPO file numbers
`7431-4C
`8615-4C
`
`(72) Inventor
`
`Michio Nonaka
`
`“/0 Taiyo Fishery C0,, Ltd.
`3-2-9 Tsukijirna, Chuo-ku, Tokyo-to
`
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