WO2017191838A1

WO2017191838A1 - Safe and stable plasmalogen, formulation thereof, and method for assessing presymptomatic state of dementia

Info

Publication number: WO2017191838A1
Application number: PCT/JP2017/017210
Authority: WO
Inventors: 義剛名達; 永田　仁
Original assignee: 有限会社梅田事務所; 株式会社ピーソリューション
Priority date: 2016-05-02
Filing date: 2017-05-01
Publication date: 2017-11-09

Abstract

The invention provides a safe and stable plasmalogen, a formulation thereof, and a method for assessing presymptomatic dementia. More specifically, the invention comprises: a supplement or a foodstuff as a processed product for the mitigation, improvement, or prevention of dementia having as an active ingredient a plasmalogen with ω-3 highly unsaturated fatty acid (ω-3HUFA) bonds, etc.; a formulation for the treatment of neurodegenerative disease and/or mental illness; and a method for assessing presymptomatic dementia in a subject, where the subject is an adult who has not yet developed dementia (normal subject). The supplement or foodstuff comprises a plasmalogen-containing complex lipid, etc., that has been extracted and separated from the organs and specific sites in the living tissue of a chicken, and has a composition ratio unique to the living tissue in which a DHA-containing ω-3HUFA derivative is transferred to the living tissue of a chicken raised on feed containing the ω-3HUFA derivative.

Description

Safe and stable plasmalogens and their preparations, and methods for determining the unaffected state of dementia

The present invention relates to a safe and stable plasmalogen derived from a safe biological material, a preparation thereof, and a method for determining a non-demented state of dementia in an adult (healthy person) before the onset of dementia. . More specifically, in the present invention, EPA (eicosapentaenoic acid; a kind of ω-3 highly unsaturated fatty acid) is located at the SN-2 position of plasmalogen (hereinafter sometimes referred to as “PLs”) as an essential requirement. Production of safe and stable plasmalogen by minimizing contamination (substantially zero at undetectable level) and increasing the binding ratio of DHA (docosahexaenoic acid; typical ω-3 highly unsaturated fatty acid) And production of safe and stable aqueous preparations (emulsions, jellies, granules, powders) prepared by simply nano-emulsifying (or solubilizing) the PLs using a safe surfactant and supplements or foods The present invention relates to a method for determining the non-morbid state of dementia by ingestion as a new technology and a new product that contributes to long-term suppression, alleviation, and prevention of dementia.

Recently, a family who has a high possibility of having a causative gene for familial Alzheimer's disease has been followed for a long time from the state before onset of Alzheimer's disease (hereinafter referred to as "AD"). In order to find out whether changes can be detected or not, and 2) how long before symptoms appear, the DIAN ^* study (supervised by Professor John Morris) was conducted mainly by the University of Washington in the United States. .
* Dominantly Inherited Alzheimer Network

According to Professor Morris, there are two main research targets: brain image analysis and elucidation of cerebrospinal fluid component dynamics. Specifically, an examination of accumulation in the brain of amyloid β (hereinafter referred to as “A _β ”), which is one of the causes of AD using PET (positron emission tomography), and τ in cerebrospinal fluid It is a test for fluctuations in protein (hereinafter referred to as “τ”) and _Aβ .
Based on the age of the study subject and the age at which their parents developed AD, the study was started by predicting the number of years until the subject's onset. Various substances involved in the development, including A _beta, for example τ is captured how slide into changes between up to the predicted life were compared and the resulting data carrier and non carrier. As a result, 25 around before the onset, in the brain of the carrier it was confirmed clearly that are beginning accumulate A _beta. However, after the onset, the curve started to decrease. In the case of τ, it became clear that τ of carriers began to increase in the cerebrospinal fluid from about 15 years before the onset. This indicates that the nerve cells in the brain are starting to die. Furthermore, the hippocampus, which manages memory, began to gradually become smaller about 15 years before AD began. Ten years before the onset, the decline in memory and other factors gradually began to appear. However, it was only after the onset of AD that forgetfulness, forgetting the names of close people, and forgetting promises with people started to interfere with daily life.

The result of this groundbreaking DIAN study is the strong demand for a “paradigm shift” that requires a radical change in the medical and medical coping strategies related to dementia. That is, when dementia develops in the conventional diagnosis, it has almost reached end-stage disease, indicating that its cure is hopeless. This, in turn, together with the suppression of dementia are coming early prevention becomes essential requirements, again A _beta and accumulation in brain of τ is directly related to disease progression of dementia, including Alzheimer disease It demonstrates that it can be an indicator and that the use of PET that is minimally invasive and can be visualized is essential for these assays.

In recent years, PLs has attracted attention as an active ingredient for the treatment and / or prevention and prevention of neurodegenerative diseases and psychiatric disorders (for example, [Patent Document 1]). PLs are special phospholipids that are resident in vivo, have a vinyl ether bond at the SN-1 position, and therefore have specificity for reducing properties. Although specific, this PLs occupies 18% by mass of the total phospholipid in the human body, and in particular, 20% by mass of the total phospholipid in the brain. It is no exaggeration to say that it is a kind of general-purpose phospholipid because of its high content.

Although this PLs is functionally special in that it exerts an important reducing action in the living body, its existence position is localized in an important film in the living body, and oxidative wear of various films is directly affected. Is defending. Furthermore, PLs has attracted more attention as it has been revealed that it exhibits multifaceted functions particularly in the brain. However, as is apparent from its molecular structure, PLs is easily oxidized by reversible reversal (highly radical scavenging sensitivity) and highly hydrolyzable by hydration under acidic conditions (phospholipid lyso form and aliphatic form). Decomposition into aldehydes). This is considered to be one of the causes that prevent the practical organic synthesis of PLs.

So far, PLs rely exclusively on extraction and purification from living tissues (for example, [Patent Document 2], [Patent Document 3], [Patent Document 4], etc.). In the living body, depending on its importance, stabilization is achieved by various appropriate mechanisms, and the biosynthetic system, that is, replenishment at all times by production from the peroxisome of the endoplasmic reticulum.

After the paradigm shift, there are the following two problems required for PLs.
(1) In vivo, various protective modification actions for maintaining PLs homeostasis work and are automatically stabilized. However, since these protective actions are lacking outside the body, It is essential to make an artificial prescription or formulation. Development of safe and inexpensive stabilization technology for PLs that embodies this.
(2) Development of a technology that normally compensates for and maintains the amount of PLs in the body, especially in the brain, which decreases with age.

[Safe and inexpensive stabilization technology]
In view of the fact that the in-vivo resident structure is the most important component of the cell membrane, it plays an important role in maintaining homeostasis by protecting against various cell membrane attacks that take advantage of its strong antioxidant effect. The normal formation of lipid bilayer membranes (liposomes) as a simulation of cell membranes, in other words, emulsification and dispersion utilizing the self-emulsifying property, especially microemulsification or solubilization type formulation It is judged. Furthermore, in the molecular structure, what kind of molecular species should be introduced at the SN-2 position adjacent to the vinyl ether bond, which is the basis for the expression of strong antioxidant activity, is important. Molecular species capable of exhibiting “effect”, for example, DHA can be exemplified. Another chemical effect of DHA is its antioxidant properties, and the combined physical and chemical action modifies DHA-bound PLs into multiple self-stabilizing types.

[Technology to supplement and maintain the amount of PLs in the brain normally]
The core of maintaining the homeostasis of PLs in vivo is the peroxisome of its biosynthetic system, and this purposeful activation is essential. The synthesis of higher alcohols that form the core of the multi-stage biosynthetic system, as will be described later, is Far1 (fatty
acyl-CoA reductase1) has been identified. As will be described later, it has been reported that DHA phospholipid, especially DHA type PLs, enhances the rate-limiting enzyme expression and thus promotes Pls production in peroxisomes.

The raw material for extracting PLs is mainly poultry, in particular spawning (also called egg collection) adult chickens (for example, [Patent Document 1], [Patent Document 2], [Patent Document 3], [Patent Document 4], etc.) . Furthermore, in recent years, seafood-derived sea squirt viscera ([Patent Document 5] and the like) and aquaculture scallop viscera (eg [Patent Document 6]) have begun to be used as raw materials for extraction. In the case of cultured bivalve molluscs, concerns about the venom accumulated from the environment, especially cadmium (safety concerns) cannot be wiped out. On the other hand, marine PLs are characterized in that DNA and EPA are bound together at the SN-2 position. Since EPA-bound PLs are blocked at the brain blood barrier, they are not taken into the brain, and only the DHA-bound type has attracted attention for its brain functionality. [Method for raising chickens with DHA feed] according to the present invention described later is efficient and safe because only DHA-bound PLs are produced with high selectivity.

The presence form (binding molecule) of DHA in the living body is glycerophospholipid, especially ethanolamine type. DHA is almost bound to the SN-2 position (the only optically active carbon in glycerophospholipid). Among the fatty acids derived from living organisms, DHA has the highest structural specificity. DHA has a long chain length. Therefore, the number of double bonds is as large as 6, and a plurality of bisallyl structures ((—CH═CHCH ₂ —) ₂ CH ₂ —) exist. The methylene carbon is highly oxidatively sensitive and is a major cause of DHA being susceptible to oxidation. However, when this is O / W emulsified (oil-in-water emulsification), the permutation of oxidative stability changes completely as follows (the numbers in parentheses indicate the number of double bonds) (for example, [Non-Patent Document 1]).

* Normal stability permutation;
Linoleic acid (2)> Linolenic acid (3)> Arachidonic acid (4)> EPA (Eicosapentaenoic acid) (5)> DHA (6)
* Stability permutation of O / W emulsion type (oil-in-water type);
DHA (6)> EPA (5)> arachidonic acid (4)> linolenic acid (3)> linoleic acid (2)

It is strongly suggested that DHA is stabilized in a self-emulsified state by binding to glycerophospholipid (amphiphilic and inherently having surface activity) in the living body based on water. Furthermore, in vivo, particularly in the brain, DHA has been reported to be highly selective and binds to the SN-2 position of PLs (eg, [Allower 2]). It is considered that the vinyl ether bond is double-stabilized by the following 1) to 2) by the DHA bonding at the SN-2 position. That is,
1) The bulk conformation of DHA develops a steric hindrance effect and prevents the oxidatively active species from approaching the SN-1 vinyl ether bond.
2) As a result of the molecules being held in the emulsified state by the self-emulsifying function of the DHA-bonded PLs, they are further protected from oxidation and hydration because they exist in the state of being held in the fine capsules of micelles.

Polyunsaturated fatty acids (PUFAs) such as DHA in fish and shellfish are food chains, and zooplankton that has eaten phytoplankton that contains a lot of α-linolenic acid in the ocean is used by EPA and DHA. Inverted and small fish feed on this to start the food chain (eg, Non-Patent Document 3).

It is judged that the most reasonable way to obtain safe and stable PLs is through biological metabolism. For example, as living organisms, adult spawning hens that have already been put to practical use for DHA eggs, and in particular, spawning hens (laying hens that are thinned out due to a decrease in spawning efficiency), more preferably forced molting (short term) Examples of such laying hens include a chicken raising method that regenerates fallen feathers by fasting to improve egg-laying efficiency. The egg-laying chicken is literally a poultry intended for egg-laying, and the same chicken is a different poultry from a broiler for meat-collecting purposes. Due to the distribution of eggs, laying hens are roughly 1/10 of the size of broilers (with a scale of 10 to tens of millions) and are raised in the vicinity of the area where they are consumed. .

Usually, the annual egg-laying amount per bird of this laying hen is 300 or more. Dedicated chicken processing center for semi-governmental and semi-private people located in the vicinity of the poultry farm (about 40 places in the country, millions to 1 thousand) (Processing capacity of 1 million birds) The processed products are used for food, but most of them are distributed as low-cost chicken raw materials for processing in the form of mince etc., except for some. Laying hens have been forced to sell at low prices due to price competition with bulk imported frozen chicken. However, it has been boosted by consumer-oriented safety in recent years, adding value as domestic chicken, and prices are on the rise. However, laying hens are economically unable to compete with vertically integrated poultry farming with tens of millions of meat-only species, and the quality of the larvae is only 50 days old. However, the profitability of the egg-laying poultry industry has remained sluggish.

In the above situation, it should be noted that the average annual number of spawning hens has risen to 90 million and that the price for delivery to the poultry farm is generally low, for example, at 0 yen. That is. Furthermore, the accumulation degree of the generation | occurrence | production is high, and since it is carried as a live chicken to a waste chicken processing center, it can be mentioned that a high freshness processing product equivalent to a broiler is obtained.

In general terms, chickens are artificial breeding products, but belong to birds at the genetic level. In addition, it has a unique “respiratory respiratory organ” ([Non-Patent Document 4]) that is a unique respiratory system, and has a “respiratory respiratory organ” that was almost extinct due to a huge meteorite crash 65 million years ago. It inherits the traits of dinosaur descendants ([Non-Patent Document 5]). Its peculiarity is that the intake and exhaust, which are the characteristics of the air sac type, are performed separately, resulting in a much higher oxygen uptake compared to mammals and a markedly improved metabolic rate. This clearly appears in the amount of feed required to obtain 1 kg of the edible portion, 9.1 kg for a typical domestic pig and 24.5 kg for a beef cattle, which is only 4.5 kg. It is said that only 2.1 kg of cultivated crickets is surpassed in this, although it lacks palatability and versatility (FAO 2013 publication).

Many chicken tissues, parts and organs are unique and different from mammalian livestock. Sand liver, gold crown (immature egg yolk), skin, feathers, intestine, liver, gala (raw bone), chicken crown, etc. (generally treated as industrial waste) and breast meat (like "myocardium", distributed as edible mince), thigh removed Examples include breast with bone (popular name “兜”), egg yolk, bone marrow (contained in raw glass), and the like. If a DHA-containing feed is administered to a laying hen for several weeks, DHA can be transferred to the above tissues, parts, organs and the like. Examples of the DHA source and form added to the test feed include fish oil and finely emulsified (preferably solubilized) and added to the feed.

It is important that the tissue or the like containing the DHA transition type PLs or the like is processed by a unique processing step in which attention is paid to obtaining a target product without deteriorating its specificity. As the processed product, for example, a DHA-bound PLs-containing complex lipid that retains the raw material-specific composition ratios listed above, and a complex lipid composition to which a water-soluble low molecular weight fraction (see * below) is added. In addition, there are exemplified protein / lipid complex compositions in which specific protein fractions (see ** below) are reconstituted, and various aqueous preparations obtained by finely emulsifying (solubilizing) these.

* --- Free amino acids, anserine and carnosine (reducing diminished cognitive function, for example, reducing dipeptides such as [Non-patent Document 19], coenzyme Q10 (involved in electron transfer), carnitine (increased lipid metabolism) It is functional and effective in improving suppression of [hyperlipidemia / hyperglycemia] with high risk factor activity for developing dementia, and can be an adjuvant of plasmalogen).
** --- For example, [myocardial] -like "breast", bone marrow collagen protein, skin collagen protein, etc.

Regarding the various compositions and aqueous preparations described above, dementia including AD, which is regarded as a worldwide general-purpose intractable disease, and is regarded as an urgent global challenge to be held up to the summit ([Non-patent Document 6]) aiming for its cure. It has been reported about the possibility of throwing a stone at the conquest of. Recently, most of chronic neurodegenerative diseases such as AD, Parkinson's disease, amyotrophic lateral sclerosis (hereinafter referred to as “ALS”), multiple sclerosis, or stroke or head It has become clear that acute brain injury such as trauma is accompanied by chronic inflammation of the central nervous system (brain, spinal cord, etc.).

And it is also considered that these diseases may be caused by central inflammation and progress ([Non-Patent Document 7]). For example, it has been reported that central nervous system inflammation may cause neurodegenerative diseases such as AD and cause disease progression (for example, [Non-patent Document 7]). As a result of analyzing the intraperitoneal memory impairment model rats produced by the administration of a substance that causes inflammation LPS (lipopolysaccharide), it was observed accumulation of A _beta peptide in the brain, which is an anti-inflammatory agent Sulindac sulfide It has been reported that the symptoms recovered by (Non-patent Document 8). Furthermore, it has become clear that central nervous system inflammation is found at a high rate even in mental diseases and developmental disorders such as depression and autism, and in the normal aging process.

From the above, by preventing or treating central nervous system inflammation and related neuronal cell death ([Non-Patent Document 9]), inhibition of neurogenesis ([Patent Document 7]), and accumulation in _Aβ brain, Preventing neuropathy caused by damage to nerve cells due to inflammation caused by infection, treatment of neurodegenerative diseases such as AD and Parkinson's disease, or mental diseases such as schizophrenia, depression and autism It is expected that developmental disorders can be treated ([Non-Patent Document 8]). Under such circumstances, a method capable of treating central nervous system inflammation and the like effectively and without side effects is desired more than ever before.

Under such circumstances, the inventor of the present invention uses a unique plasmalogen composition derived from chicken breast by an innovative clinical test method, so that the cognitive function of an adult (on healthy person) before the onset of dementia The inventors have succeeded in demonstrating that the improvement can be improved, and have completed the present invention. The results of the clinical trials will be published in INPROVEMENT IN COGNITIVE FUNCTION BY BY SUPPLEMENT CONTINED PLASMALOGEN FOR HEALTHY JAPANESE-A RAMDOMIZED, DOUBLE-BLIDED, PULSEBO-COND. (Non-patent Document 25). With this innovative achievement, it will be possible to obtain measures to prevent and / or alleviate central nervous system inflammation in the future, and as a result, it will contribute to efficient suppression of central nervous system inflammation. It is thought that.

Japanese Patent No. 5847086 Japanese Patent No. 5062873 Japanese Patent No. 5774816 Japanese Patent No. 5430566 JP 2007-262024 A JP 2006-121957 A Japanese Patent No. 6016363 Japanese Patent No. 5540209 Japanese Patent No. 5360454 Japanese Patent No. 5704493 Japanese Patent No. 5784486 JP 60-51104 A JP-A-57-59629 JP-A-60-64919 Japanese Patent No. 6025568 Japanese Patent No. 40473354 [New Labyrinthula microorganisms with high productivity of docosahexaenoic acid and their use] (Hokkaido University, etc.) Japanese Patent Application No. 2007-148398 [Method for producing DHA-containing phospholipids by microbial fermentation] (Hokkaido University)

The object of the present invention is to provide safe and stable PLs and preparations thereof, uses as foods, cosmetics, pharmaceuticals or feeds containing them, and a method for determining an unaffected state of dementia.

The inventor has surprisingly found that PLs can be safely removed by introducing DHA at the SN-2 position in the PLs molecule, and also by simply emulsifying (solubilizing) PLs and transforming the shape. In addition to being able to stabilize, it has been found that the effects of alleviating and preventing neurodegenerative diseases and psychiatric disorders, as well as improving effects, as well as significantly improving their integrated cost-effectiveness, and further research and development As a result, the present invention has been completed.

That is, the present invention comprises the technical means described in the following section.
(1) From a purified product of total lipids that has been extracted and fractionated from a specific site or organ derived from a living tissue, and the protein fraction and / or water-soluble low-molecular fraction that have been by-produced in the extraction and fractionation step have been purified and removed. A plasmalogen-containing phospholipid (hereinafter referred to as “complex lipid”) having a composition ratio specific to a living tissue,
1) The specific part or organ derived from the biological tissue is a carcass carcass derived from a chicken biological tissue, raw rattle containing bone marrow, chicken yolk, intestine, sand liver, gold crown including ovary and fallopian tube, breast meat, skin Consisting of at least one or more specific parts or organs selected from the group of
2) The plasmalogen-containing phospholipid is generically called an ω-3 highly unsaturated fatty acid (hereinafter referred to as “ω-3HUFA”) derivative (hereinafter referred to as “ω-3HUFA derivative”) containing docosahexaenoic acid (DHA). ) Is extracted and fractionated from a specific part or organ derived from the living tissue to which the ω-3HUFA derivative has been transferred to the biological tissue of a chicken fed with a feed containing The complex lipid, wherein the complex lipid is a plasmalogen-containing phospholipid having a constitutional ratio of ω-3HUFA binding type.
(2) The ω-3HUFA derivative is the following 1) to 9):
1) ω-3HUFA derivative bound to SN-2 of glycerophosphatidyl phospholipid 2) ω-3HUFA derivative bound to SN-2 of 1-alkyl glycerophosphatidyl phospholipid 3) 1-alkenyl glycerophosphatidyl Ω-3HUFA derivative bound to phospholipid SN-2 4) 1-acylglycerophosphatidyl phospholipid SN-2 ω-3HUFA derivative 5) contained in krill shelled and / or dried product 6) ω-3HUFA derivative bound to SN-2 of glycerophosphatidyl phospholipid according to any one of 1) to 4) in the preceding paragraph 6) contained in whole scallop and / or processing residue thereof or meal (dry product) thereof The ω-3 bound to SN-2 of the glycerophosphatidyl phospholipid according to any one of 1) to 4) above UFA derivative 7) SN- of the glycerophosphatidyl phospholipid according to any one of the preceding items 1) to 4), which is contained in whole sea scabbard (sea squirt) and / or processing residue thereof or meal (dry product) thereof Ω-3HUFA derivative bound to 2 8) Organically synthesized ω-3HUFA derivative bound to SN-2 of the glycerophosphatidyl phospholipid according to any one of 1) to 4) above, prepared by fermentation method The composite according to (1), which is any one selected from ω-3HUFA derivatives bound to SN-2 of the glycerophosphatidyl phospholipid according to any one of 1) to 4) above Lipids.
(3) The specific site or organ is chicken egg yolk, and comprises a purified product of total lipids extracted and fractionated from chicken egg yolk containing DHA-PLs biosynthesized in the chicken's living body (1) The complex lipid described.
(4) The above-mentioned (1), comprising a purified product of total lipid extracted and fractionated from a specific part or organ of a living body of a chicken fed with a feed containing an organically synthesized 1-alkylglycerophosphatidylphospholipid The complex lipid described.
(5) The complex lipid described in (1) above has a ω-3HUFA-binding constitutional ratio containing a water-soluble low-molecular fraction produced as a by-product in the extraction fractionation described in (1). Complex lipid composition.
(6) In the presence of saponins, the complex lipid or complex lipid composition having the ω-3HUFA binding type composition ratio described in any one of (1) to (5) above in the aqueous phase is nano-emulsified or An aqueous preparation of a complex lipid composition having a constitutional ratio of complex lipid or ω-3HUFA binding type, characterized by being solubilized.
(7) The complex lipid described in any of (1) to (4) above is enzymatically treated with phospholipase A1 (PLA1) to remove glycerophospholipids including lyso form and extract and fractionate sphingomyelin A method for producing a crude plasmalogen having a purity of 30 to 70% or a high purity plasmalogen having a purity of 30 to 70%, wherein the plasmalogen is removed.
(8) The crude plasma, wherein the crude plasma or high-purity plasmalogen described in (7) above is nano-emulsified or solubilized in the presence of saponins as a substrate. A method for producing an aqueous preparation of rhogen or high-purity plasmalogen.
(9) As an active ingredient, any one or more selected from the complex lipid according to any one of (1) to (8), the complex lipid composition, each plasmalogen, and their aqueous preparations. Food, cosmetics, pharmaceuticals or feed characterized by
(10) The composite lipid according to any one of (1) to (8), the composite lipid composition, each plasmalogen, and at least one selected from each aqueous preparation thereof as an active ingredient. A neurodegenerative disease having a alleviating and preventing action on at least one neurodegenerative disease selected from the group consisting of dementia of neurodegenerative disease, Alzheimer's disease, Parkinson's disease, depression, and schizophrenia Mitigation and prevention supplements.
(11) The composite lipid according to any one of (1) to (8), the composite lipid composition, each plasmalogen, and at least one selected from each aqueous preparation thereof as an active ingredient. A neurodegenerative disease dementia, Alzheimer's disease, Parkinson's disease, depression, and at least one neurodegenerative disease selected from the group consisting of schizophrenia Anti-central nervous system inflammatory preparation for alleviation and prevention or treatment of degenerative diseases.
(12) The composite lipid according to any one of (1) to (8), the composite lipid composition, each plasmalogen, and at least one selected from each of these aqueous preparations is contained as an active ingredient. A neurogenesis agent characterized by the above.
(13) The composite lipid according to any one of (1) to (8), the composite lipid composition, each plasmalogen, and at least one selected from each of these aqueous preparations is contained as an active ingredient. A neuronal apoptosis inhibitor characterized by the above.
(14) The composite lipid according to any one of (1) to (8), the composite lipid composition, each plasmalogen, and at least one selected from each aqueous preparation thereof is contained as an active ingredient. cerebral accumulation inhibitor of a _beta and / or τ, characterized in that.
(15) The composite lipid according to any one of (1) to (8), the composite lipid composition, each plasmalogen, and at least one selected from each aqueous preparation thereof as an active ingredient. , anti CNS inflammation improving action of neurodegenerative diseases and / or psychiatric disorder, neurogenesis, by having at least one action selected from among the suppression as well as a _beta accumulation in brain inhibition of neuronal cell death A processed product as a pharmaceutical product or food for improving the neurodegenerative disease and / or psychiatric disease.
(16) Contains a protein fraction by-produced by the extraction fraction described in any one of (1) to (4) above and a water-soluble low-molecular-weight fraction by-produced by the extraction fraction described in (5) above A protein / lipid composite composition comprising a mixture with a composite lipid composition having a composition ratio specific to living tissue.
(17) The protein / lipid composite composition according to the above (16), wherein the protein / lipid composite composition comprising the mixture of the protein fraction and the composite lipid composition described in the above item is derived from chicken egg yolk.
(18) containing the protein / lipid composite composition according to (16) or (17) as an active ingredient, anti-central nervous system inflammation for improving neurodegenerative diseases and mental disorders, neuronal cell neogenesis,
The processed product as a pharmaceutical or food for ameliorating neurodegenerative disease and / or psychiatric disorder characterized by having at least one action selected from suppression of neuronal cell death and suppression of accumulation in _Aβ brain.
(19) The complex lipid containing the plasmalogen according to any one of (1) to (8) above, a complex lipid composition, a crude plasmalogen or a high-purity plasmalogen, and their respective aqueous preparations An adult before the onset of dementia in a state in which accumulation of physiologically harmless amounts of _Aβ and τ is observed in the brain (hereinafter referred to as “demented non-disease state”) ( Healthy subjects)
(1) using a PET examines the brain accumulation state of A _beta and tau, to determine dementia non pathological condition of the subject, the A _beta and tau on the basis of the SUVR values of the PET image (score) The process of performing the classification of brain accumulation status by rank according to the following:
1) Initial accumulation stage I: 1.0 ± 0,2
2) Intermediate period II: 1.2 ± 0.2
3) Late stage III: 1.4 ± 0.2
(2) Testing the dose response to plasmalogen subjects by said rank at an appropriate frequency over a long period of 3 to 10 months, and setting an appropriate daily dose range or register range as follows:
1) Stage I subjects; “gradual increase” range: 1.0 ± 0.15
2) Stage II subjects; “Zero increase” register range: 1.2 ± 0.15
3) Stage III subjects; “decreasing” register range: 1.4 ± 0.15
(3) Continuing a long-term treatment from the stage before the onset of dementia to the adult (healthy) subject before the onset of dementia, supplements containing the set daily dose of plasmalogen as an active ingredient or processed food products Administering step,
(4) A step of performing a periodic test of the predetermined brain accumulation state of _Aβ and τ by PET diagnosis at an appropriate frequency during the long-term administration,
(5) In parallel with periodic PET diagnosis, performing a hippocampal volume reduction test with MRI diagnosis at an appropriate frequency during the long-term administration period,
(6) Dementia of the subject characterized by determining the non-demented state of the subject by performing the steps (1) to (4) or the steps (1) to (5) A method for determining an unaffected state.

Furthermore, this invention can include the following aspects as invention specific matter which concerns on each said invention.
(1) As an extraction fractionation method, three phases (“insoluble solid matter such as protein minerals” (precipitation), “neutral lipid content” (upper layer) and “hydrous ethanol” which are slowly extracted with excess hydrous ethanol of the pretreated product Separation (separation) into fractions (water-soluble small molecules, polar lipids) ”).
(2) After dealcoholization of the above water-containing ethanol, hexane is added to separate and separate water-soluble low-molecular compounds, and hexane is evaporated to dryness to obtain a polar lipid (complex lipid).
(3) As an enzyme treatment step, the plasma phosphogen is purified by subjecting the phospholipid fraction obtained by evaporating and drying the separated hydrous ethanol phase to PLA1 enzyme treatment.
(4) Each plasmalogen contains ethanolamine plasmalogen and choline plasmalogen.
(5) The mass ratio of ethanolamine plasmalogen and choline plasmalogen contained in each plasmalogen is [1: 5] to [5: 0.01].
(6) The daily oral dose of DHA-containing plasmalogen is 0.01 mg to 10 mg, for example, preferably 0.25 mg (or less than internal medicine).
(7) PET used for accumulation in brain diagnosis of A _beta and τ is, it is a helmet-type PET head only.
(8) The administration time of plasmalogen is from the 30s, more preferably from the 40s.

Here, to explain the present invention, PLs, which are resident phospholipids in the living body, have conventionally been edible for living tissues containing the PLs. Therefore, the PLs extracted from the living tissue according to the present invention are considered to have very high safety with little concern about side effects. However, once PLs is extracted and separated from a living tissue, stability is lost and the vinyl ether bond is exposed to the risk of decomposition. This means that the [strongly reducible] vinyl ether bond is strong [easily oxidizable] (= “easy” oxidatively degradable), and some appropriate defensive measures must be taken to make effective use of it. Application is essential.

The present inventor has found that the three methods are effective as a result of earnest research on a method of rationally overcoming the “antagonism”. One is to rationally emulsify PLs, and the second is to rationally introduce DHA into the PLs molecule, ie, SN-2. Integrating the two, that is, nanoemulsifying DHA-bound PLs, is the strongest rational defense. Here, “reasonable” means that the integrated cost-effectiveness ratio is remarkably high, thereby realizing “safe and practical stabilization”.

Furthermore, the third is to feed chickens with “ether phospholipid” having a molecular structure of [alkylphospholipid] as a feed, using the technique of “several birds with one stone” in both name and reality. This is because 1% by mass of krill-derived alkylphospholipid concentrate, the original research result (Non-patent Document 23), which is said to be the [original effect], was orally administered to rats for 8 days, and alkylphospholipid and PLs in the serum were prominent. While increasing the ratio of DHA and EPA in their SN-2 binding lipid class. That is, the present inventor succeeded in further rationalizing and improving the quality of the present invention by utilizing the [original effect] as follows.

In an appropriate alkylphospholipid-containing material, for example, krill unshelled meat, preferably when the dried product is administered as a feed to an adult chicken, preferably a laying hen, the egg yolk of the laying egg is surprisingly 1-alkenyl-2- It was confirmed that docosahexenoyl-glycerophospholipid and 1-alkyl-2-docosahexenoyl-glycerophospholipid were produced with high selectivity, that is, containing almost no EPA conjugate.
Furthermore, as a result of administering the egg yolk concentrated egg yolk oil to rats, it was found that serum 1-alkenyl-2-docosahexenoyl-2-docosahexenoyl-glycerophospholipid was significantly increased.

The significance and superiority of this method in improving the [integrated cost-effectiveness ratio] of the increase in the actual amount of PLs in vivo is
(1) A selective and highly safe biosynthetic method for DHA-PLs and its precursor [DHA-ether phospholipid],
(2) A so-called so-called so-called so-called so-called so-called “safety-assurance risk” such as ether phospholipids, alkyl glycero phospholipids and their DHA-bound [DHA-ether phospholipids]. Conversion to [biological-derived type] can be put into practical use with a high [cost-effectiveness ratio]
(3) In [conversion to living body-derived type], the chicken is a super artificially reared animal as described above, and the rate of turnover is unacceptable.
[Highly rational but low safety [artificial synthesis]] x [Safety but low productivity [biosynthesis]] is a safe and inexpensive product.

By adopting the above configuration, the present invention has the following special effects.
(1) The present invention uses an adult (healthy person) before the onset of dementia in a state in which accumulation of physiologically harmless amounts of _Aβ and τ is recognized in the brain (demented state). This is useful as a method for determining the non-demented state of a subject.
(2) The present invention is useful for providing a processed product as a supplement or food for alleviating and preventing dementia and the like.
(3) Supplements for mitigation and prevention of neurodegenerative diseases (dementia, AD, Parkinson's disease, depression, and schizophrenia) containing the safe and stabilized PLs of the present invention as active ingredients ([non- Patent Document 16]), anti-central nervous system inflammation preparation ([Non-Patent Document 8] and [Patent Document 1]), neuronal cell neoplasia preparation ([Patent Document 7]), neuronal apoptosis inhibitor preparation ([Non-Patent Document 8]). 9]) and _Aβ and τ accumulation-inhibiting preparations in the brain ([Non-patent Document 8]) to prevent, alleviate and improve symptoms of cognitive dysfunction and dysfunction in general and supplements or preparations for treatment Can be provided.
(4) As one of the causes for causing inflammation of the central nervous system, it is considered that activated glial cells present in the central nervous system release inflammatory cytokines. The anti-central nervous system inflammation of the present invention The preparation has an inhibitory effect on the increase and activation of glial cells that accompany the development of inflammation in the central nervous system, and it is possible to alleviate, prevent, improve and treat central nervous system inflammation. it can.

(5) Diseases considered to be caused by inflammation of the central nervous system, for example, chronic neurodegeneration such as dementia (particularly AD), Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, etc. The anti-central nervous system inflammatory preparation of the present invention can also be suitably used for the treatment of diseases and mental disorders such as schizophrenia, depression, and autism.
(6) PLs is a component that is abundantly contained in living tissue, and the safety has been demonstrated since the living tissue containing PLs has been used for food, and has been extracted from living tissue. of the present invention including the PLs, supplements for mitigation and prevention of cerebral dysfunction, anti-CNS inflammatory preparations, neurogenesis formulation, cerebral accumulation inhibiting formulation apoptosis suppression formulations and a _beta and τ in neurons, side effects The safety is extremely high.

3 shows questions for selection of subjects regarding cognitive function. An MMSE sheet is shown. A collection of questions for cognitive function diagnosis. The test result of MMSE is shown. The result of the UK test is shown.

Hereinafter, the present invention will be described in more detail.
The present invention relates to safe and stable PLs and preparations thereof, as well as supplements for alleviating and preventing cognitive impairment, anti-central nervous system inflammation preparations, neuronal neoplasia preparations, neuronal apoptosis inhibitor preparations, and brains of _Aβ and τ. The present invention relates to an internal accumulation-suppressing preparation and a method for determining an undemented state of dementia that contributes to prevention of onset of dementia through oral intake thereof.

Here, PLs will be further described. PLs is a kind of ether phospholipid, and refers to glycerophospholipid having a long-chain alkenyl group via a vinyl ether bond in SN-1. In addition, PLs is a general term for special phospholipids having a strong reducing ability while being a general-purpose type accounting for 18% by mass of human phospholipids. This peculiarity is responsible for imparting remarkable oxidative and hydrolytic properties to PLs. For this reason, when PLs is put to practical use, a typical “twisting” phenomenon is confronted and its rational breakthrough is required. The present inventor has succeeded in achieving the stabilization of the “antiprotest” phenomenon rationally, that is, while making the most of the original functionality of PLs, by an inexpensive and safe means, The present invention has been established.

The PLs used in the present invention are preferably those extracted and fractionated from chicken biological tissues. A biological tissue is a tissue containing PLs in a living organism. Here, to explain in general terms, examples of the organism include animals and microorganisms. As the microorganism, an anaerobic bacterium is preferable, and for example, an enterobacteria acidaminococaceae bacterium is particularly preferable. In the case of bacteria, “living tissue” is bacteria itself. As animals, birds, mammals, fish, krill, shellfish and the like are suitable. For mammals, livestock is preferable from the viewpoint of both supply stability and safety, and examples thereof include cattle, pigs, horses, sheep, and goats.

The main tissues of mammals for raw materials include skin, brain, intestine, heart, genital organs, muscles, vertebrae, milk, etc. PLs can be extracted from these tissues (organs, sites). Examples of birds include chicken, domestic duck, frog, duck, eagle, eagle bird and turkey. From the viewpoint of procurement, cost, and abundant eating experience, laying hens including chickens, especially chickens and breeding hens are particularly suitable. The tissue to be used is not particularly limited, but for example, breast, chicken skin, internal organs (especially, intestine, ovarian fallopian tube, sand liver, liver), egg, gala (minc preparation byproduct), feathers, etc. are used. Is preferred.

In the present invention, PLs extracted and separated from chicken tissue are used as PLs extracted and separated from living tissue. Conventionally edible chickens are suitable because their safety has been confirmed and stable supply is easy. The method for extracting and fractionating PLs from living tissue is not particularly limited as long as PLs can be extracted (and purified if necessary), but from the viewpoint of simplicity and cost, extraction and purification as described below. Is preferred. In addition, the extraction and purification method is preferable in that the diacyl glycerophospholipid can be decomposed and removed, and thus the purity of PLs can be further increased.

Specific examples of the process for extracting and purifying PLs include the following processes 1) to 5).
(1) A step of extracting and fractionating from a biological tissue into three phases of a total lipid fraction (including a low molecular weight water-soluble fraction), a protein fraction and a neutral lipid fraction.
Specifically, the following steps are exemplified.
1) Step of mincing and slowly freezing the live tissue 2) Forced thawing of frozen mince and subsequent dehydration of “overheated water” (Aquagas ^RTM ; [Patent Literature 8], [Patent Literature 9], [Patent Literature 10], [Patent Document 11]) High-speed cooking sterilization and vacuum high-speed cooling (cooling and dehydration in an oxygen-free atmosphere) 3) After the dehydration treatment, 3 times (V / W) amount of deaerated (deoxygenated) ethanol is added. Step 4) Repeat extraction for 12 hours in a sealed anoxic atmosphere 4) Repeat the above steps 5) Combine the ethanol, distill off the ethanol in an oxygen-free atmosphere, and centrifuge to remove the aqueous layer (water-soluble) To obtain a total lipid fraction (water-soluble low molecular weight fraction is lyophilized separately)

(2) A step of extracting and fractionating complex lipids having a tissue-specific composition ratio from the total lipid fraction.
(3) A step of fractionating a water-soluble low molecular weight fraction from the above steps and adding this to the complex lipid fraction to obtain a complex lipid composition.
(4) A step of separating the protein fraction from the above steps and adding the complex lipid composition described in the previous section to obtain a protein / lipid complex composition.
(5) The enzyme is added to the complex lipid fraction to hydrolyze SN-1-linked fatty acid, and the mixed diacylglycerophospholipid is converted into a lyso form and extracted with a hydrophilic solvent together with by-product fatty acid to purify PLs. Process.

Extraction is preferably performed with an organic solvent (for example, ethanol, acetone, hexane, etc., and at least two kinds of mixed solvents selected from the group consisting of these and other food-compatible materials) or a water-containing organic solvent. Moreover, the chicken tissue to be subjected to extraction is preferably exemplified by a method of treating in the steps (1) and (2) above. That is, in order to minimize the amount of ethanol used in the subsequent three-phase fractionation with ethanol, it is required to perform deoiling and dehydration as much as possible from a living tissue in an oxygen-free atmosphere at low temperature and in a short time.

As for the extraction treatment conditions, the important thing is to perform a short stirring process at a low temperature in a sealed anoxic atmosphere in order to minimize the oxidative degradation and hydrolysis of the contained PLs. As a suitable example, ethanol is added to the peeled minced meat of the laying hen that has been treated in the above-mentioned pretreatments (1) and (2), and it is allowed to stand still or slowly at 30 ° C. or more and 50 ° C. or less for 180 minutes or more. The method of performing stirring is illustrated. Add 2 to 4 L of ethanol that has been degassed (deoxygenated) in advance to 1 kg of the breast meat, and leave it under sealing or slowly agitate.

Among the three phases, the water-containing ethanol phase is obtained by evaporating and concentrating ethanol to separate the aqueous layer, and then adding degassed hexane in advance to extract the phospholipid fraction. Combine the separated water layer and hexane-insoluble layer, add deaerated water, leave at 4 ° C, centrifuge at low temperature to remove insoluble parts, freeze-dry, 18 g is obtained. On the other hand, the hexane solution is evaporated to dryness by a conventional method to obtain 7 g of a complex lipid fraction. The low molecular weight water-soluble fraction is added to the phospholipid fraction to obtain 25 g of a complex lipid composition.

The phospholipid fraction can be subjected to an enzymatic hydrolysis treatment step to hydrolyze the diacyl phospholipid, so that PLs can be preferably concentrated. Examples of such hydrolysis treatment include enzyme treatment with phospholipase A1 (hereinafter referred to as “PLA1”) ([Patent Document 4]). If treated with PLA1, the mixed diacyl glycerophospholipid is decomposed into free fatty acid and lysophospholipid, and if these are extracted and distributed with acetone and hexane, the plasmalogen can be purified. Removal of free fatty acids and lysophospholipids can be performed by partitioning with, for example, acetone and hexane.

The PLA1 is not particularly limited in its origin as long as the above-described effects can be obtained, and examples thereof include PLA1 derived from Aspergus oryzae. The PLA1 can be purchased from, for example, Mitsubishi Chemical Foods Corporation. The amount used can be appropriately set according to the amount of complex lipid obtained. Preferably, 0.2 to 200 units / mg of complex lipid can be used, and more preferably, 2 to 200 units / mg of complex lipid can be used. In addition, 1 unit is an amount that changes 1 μmol of substrate (complex lipid) per minute, and means 1 μmol / min.

The buffer to be used can also be appropriately selected according to PLA1. For example, 0.1 M citrate-hydrochloric acid buffer (pH 4.5) can be used by dissolving 1 g of complex lipid in 1 to 30 ml, preferably 5 to 15 ml per 1 g of complex lipid, and adding a predetermined amount of PLA1. . The reaction is carried out in a nitrogen gas atmosphere using a degassed buffer for as short a time as possible, preferably for 1 hour, and at a temperature as low as possible, preferably at 50 ° C. with stirring as an enzyme reaction.

Avoid enzyme deactivation by warming. After the reaction is complete, immediately cool to room temperature, add 2 to 3 times the amount of hexane that has been degassed beforehand, and centrifuge to recover the upper hexane layer. To remove the enzyme buffer and the enzyme protein. The hexane solution is distributed by appropriately adding acetone and water, and further, the solution is distributed by water or an aqueous solution to remove lysophospholipid and purify the plasmalogen. That is, neutral lipids other than phospholipids are removed with acetone, and plasmalogen and lysophospholipid are separated by aqueous solution partition. The plasmalogen derived from a biological tissue thus obtained is preferably a supplement for alleviating and preventing cognitive dysfunction according to the present invention, an anti-central nervous system inflammatory preparation, a neuronal neoplastic preparation, and neuronal apoptosis. It can be used as an active ingredient of an inhibitory preparation and / or an Aβ brain accumulation inhibitory preparation.

The complex lipids derived from biological tissues (including complex lipid compositions) and the phospholipids of PLs and their composition ratios have unique characteristics depending on the biological tissues. Below, the composition ratio in the case of a laying hen is demonstrated.

1. Skin (1) PLs; [Ethanolamine type]: [Choline type] = [1-10]: 1
(2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: [1.5 to 15]
(3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: [0.5 to 5]
(4) [Total glycerophospholipid]: [Total sphingomyelin] = [1.5-10]: 1

2.兜 carcass (peeling)
(1) PLs; [ethanolamine type]: [choline type] = [0.5 to 5]: 1
(2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: [2-15]
(3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: [0.5 to 5]
(4) [Total glycerophospholipid]: [Total sphingomyelin] = [3-20]: 1

3. Egg yolk (1) PLs; [ethanolamine type]: [choline type] = 1: [0.1 to 1.5]
(2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: [2 to 20]
(3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: [10-50]
(4) [Total glycerophospholipid]: [Total sphingomyelin] = [40-350]: 1

4). Gizzard (1) PLs; [Ethanolamine type]: [Choline type] = [2-15]: 1
(2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: [1 to 10]
(3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: [0.5-6]
(4) [Total glycerophospholipid]: [Total sphingomyelin] = [1-10]: 1

5). Intestine (1) PLs; [ethanolamine type]: [choline type] = [2-15]: 1
(2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: [2 to 20]
(3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: [1-12]
(4) [Total glycerophospholipid]: [Total sphingomyelin] = [1-10]: 1

6). Gala (1) PLs; [ethanolamine type]: [choline type] = [1 to 10]: 1
(2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: [2 to 20]
(3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: [1-10]
(4) [Total glycerophospholipid]: [Total sphingomyelin] = [3-25]: 1

7). Gold crown (including ovary)
(1) PLs; [ethanolamine type]: [choline type] = 1: [0.0001 to 0.1]
(2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: [1.5 to 15]
(3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: [15 to 130]
(4) [Total glycerophospholipid]: [Total sphingomyelin] = [20-200]: 1

8). Bone marrow (1) PLs; [ethanolamine type]: [choline type] = 1: [0.0001-0.1]
(2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: [1 to 10]
(3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: [0.5-6]
(4) [Total glycerophospholipid]: [Total sphingomyelin] = [30-3]: 1
9. Breast (1) PLs; [ethanolamine type]: [choline type] = 1: [0.5 to 5]
(2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: [2 to 10]
(3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: [0.5 to 5]
(4) [Total glycerophospholipid]: [Total sphingomyelin] = [5-50]: 1

The following 1) to 4) have high specificity.
1) Breast ([PL-PE] <[PL-PC]) with [myocardium] 2) Gold crown ([PL-PE] >>>> [PL-PC]) with [PL-PC] almost zero 3 ) Raw Gala (High incidence, low price, zero or less, composition close to [breast], and also contains bone marrow (internal peripheral tissue of the brain), potential added value (Brain function improvement function) is expected, and the integrated price-to-performance ratio is considered to be remarkably high.
4) The carcass carcass ([breast] + [bone marrow]) is considered to be equivalent to [breast] with a high integrated [price to performance ratio].

The complex lipids and PLs extracted from the living tissue used in the present invention have the following ethanol content PLs and choline PLs content as 1) to 2) in terms of dry mass.
1) The upper limit of the complex lipid is 50% by mass, preferably 10% by mass or more, more preferably 20% by mass or more, still more preferably 30% by mass or more, and 40% by mass or more. Is still preferred.
2) In PLs, 50% by mass or more is preferable, 60% by mass or more is more preferable, 70% by mass or more is more preferable, 80% by mass or more is further more preferable, and 90% by mass or more. Even more preferred is that of 92% by weight or more.

The mass ratio and content of ethanolamine PLs and choline PLs can be determined by analyzing complex lipids or plasmalogens extracted from biological tissues by high performance liquid chromatography (HPLC). Specifically, in HPLC, a chromatogram is obtained by evaporative light scattering detection (ELSD; Evaporating Light Scattering Detector), and a mass ratio is obtained by determining respective peak area ratios indicating ethanolamine PLs and choline PLs in the chromatogram. Can be calculated. Further, the content can be obtained by calculating what percentage of the peak area of the entire chromatogram the peak areas showing ethanolamine PLs and choline PLs correspond to.

In the present invention, it is important to modify the living body of the chicken used for the purpose. That is, in the present invention, by feeding a chicken with a ω-3HUFA derivative-containing feed, ω-3HUFA is transferred with high selectivity to lipids, particularly PLs, of tissues, organs and parts thereof. Transesterification of SN-2 conjugated fatty acid and transition ω-3HUFA (Non-patent Document 26) produces ω-3HUFA-conjugated PLs-containing complex lipids and PLs. Examples of ω-3HUFA-binding PLs suggest a relationship with cognitive dysfunction, which is exemplified below.

1. Correlation between the amount of DHA in the brain and the amount of PLs DHA in the brain exists as a glycerophospholipid, especially as a PLs-binding type (Non-patent Document 26), and is a biosynthesis system of peroxisomal PLs that are PLs-producing endoplasmic reticulum. Restriction enzyme fatty
It is known to be involved in increased expression of acyl-CoA reductase1 (Far1) (Non-patent Document 27) (Non-patent Document 10).
2. Correlation between the decrease in brain DHA and PLs concentrations and the onset of neurodegenerative diseases and psychiatric disorders (Non-Patent Document 10).

1) When [ ³ H] DHA is added into the brain, DHA binds to the SNs of PLs existing mostly between the membranes in the brain.
2) The bound DHA is greatly reduced in cell lines that block the PLs production system.
3) When sir1-hexadecylyglycol is added to the culture system, the amount of PLs-bound DHA and the amount of PLs produced are restored.

4) The decrease in brain DHA content and the decrease in brain PLs production are related to the onset of neurodegenerative diseases and psychiatric disorders 1) to 7) below.
(1) Peroxisome ataxia (2) AD
(3) Depression (4) ADHD (Attention Deficit Hyperactivity Disorder)
(5) Stroke (6) Hyperactivity (7) Schizophrenia

5) It has been suggested that the intake of DHA-enhanced foods is related to the possibility of improving information transmission related to behavioral abnormalities and decreased learning ability, as well as deterioration of mental status, which are characteristic of 1) to 7) above. Yes.
Furthermore, DHA is susceptible to oxidative decomposition due to its structure, and it is common to oxidize and give off malodor, but it is not surprisingly known that it can be simply solved by emulsifying (nano-emulsifying) it. Hereinafter, this will be described by way of example.

(1) When a highly unsaturated fatty acid (HUFA) and its ester (including glyceride) are dispersed (emulsified) in an aqueous solution, the higher the degree of unsaturation, the more the one that is left in the air is oxidized. It becomes difficult. In an underwater dispersion system, the oxidative decomposability of DHA and EPA is extremely low ([Non-Patent Document 1]). The reason is that the three-dimensional structure such as emulsified DHA becomes a barrier against the approach of the oxidized active species, and the so-called “steric hindrance” effect blocks the collision of the active species with the oxidized target site (bisallyl-bonded carbon atom). It is thought that this prevents the oxidation reaction. At that time, the presence of water molecules is important, and it is considered that the water molecules are indirectly protected by covering (covering) the vicinity of the oxidation target portion.

(2) In the emulsified O / W emulsion, the smaller the micelle particle size, the better the oxidation stability of PUFA such as DHA, and nanoemulsification (solubilization) is more preferable than ordinary microemulsification. It should be noted that the emulsification may be a heavy load, for example, avoiding the use of a high-pressure homogenizer or the like, and simple and slow stirring is preferable. High load emulsification impairs the oxidation stability of PUFA.
(3) In the cell culture system, linoleic acid (LA), arachidonic acid (AA) and DHA were incorporated and oxidized, and in LA and AA addition systems, the amount of peroxide produced increased. There was no increase.

(4) When LA-PC (phosphatidylcholine), AA-PC and DHA-PC were added to the cultured cell system and oxidized, the amount of peroxide generated specifically in DHA was similar to the above. Results were less than control. As described above, the characteristic oxidative stability of DHA in cultured cells is completely different from that in air or solvent systems. DHA is less susceptible to oxidation in vivo than previously considered. It is not stable.
(5) When fish oil is administered to rats, no change is observed in the lipid peroxidation level in the living body unless the fish oil dose is extremely large.
(6) This suggests that as long as an appropriate amount of fish oil is ingested, there is almost no increase in lipid peroxidation in the living body and any adverse effects associated therewith.

(7) From the above, by directly binding DHA to SN-2 of PLs, it is expected that the following 1) to 2) of the direct stabilizing effect in the PLs molecule is expressed.
1) In vitro, the steric hindrance of the adjacent bond DHA to the vinyl ether bond of PLs effectively blocks the attack of the oxidatively active substance, and the DHA-binding molecule, ie, PLs itself, is also an emulsified form unique to the bonded DHA. It is thought that oxidative stability can be obtained through a specific molecular structure.
2) In vivo (established cell culture system), especially in the brain (intracellular), DHA binds to phospholipids and, in particular, ethanolamine-type PLs, and PLs biosynthetic endoplasmic reticulum "peroxisome" It is considered that localization in the membrane promotes the production of PLs by enhancing the expression of key biosynthetic enzymes (restriction enzymes).

In addition to the above, the correlation between PLs and brain dysfunction will be exemplified below.
1. Correlation with AD 1) The PLs content in the brain of AD patients is significantly reduced (Non-patent Documents 11 and 12).
2) Serum PLs content of AD patients is significantly reduced (Non-patent Document 13).
3) The erythrocyte PLs content of AD patients is significantly reduced (Non-patent Document 14).
4) In the central nervous system inflammation model mouse (LPS intraperitoneal injection causes inflammation-induced cognitive impairment), PLs in the brain decrease (Non-patent Document 8).

2. In vivo kinetics of PLs 1) Plasma plasmalogens in orally administered rats increase (Non-patent Document 15).
2) The PLs content in the brain of LPS-induced central nervous system inflammation model mice increases (Non-patent Document 8).
3) Blood ethanolamine PLs in AD patients with oral administration of PLs increases (Non-patent Document 16).

3. Prevention, alleviation and therapeutic effects of cognitive dysfunction 1) Promote neurogenesis in aging model rats (SAMP8) (Patent Document 7).
2) A _beta suppresses spatial perception learning dysfunctions bilateral injection rats (Non-Patent Document 16).
3) Relieve symptoms of LPS-induced central nervous system inflammation mice (Non-patent Document 8).
(1) Suppression of microglia activation.
(2) Increase in brain cytokine, TNFα-mRNA and suppression of brain IL-2β expression.
(3) A _beta accumulation suppression.

4) Inhibition of central nervous system inflammation and _Aβ accumulation in mice with central nervous system inflammation induced by LPS (intraperitoneal injection) (Non-patent Document 8).
(1) Inhibition of glial cell activation.
(2) Suppression of _Aβ accumulation in the prefrontal cortex and hippocampus.
It suppresses the decrease in PLs due to inflammation induced by intraperitoneal injection of LPS (Non-patent Document 8).

5) In vitro suppression of neuronal cell death (Non-patent Document 9).
6) Other relations (1) Suppression of hyperglycemia and hyperlipidemia, which are risk factors for developing cognitive impairment (Non-patent Document 18).
(2) Suppression of cognitive decline due to carnosine diet (Non-patent Document 19).
In the above-described PLs efficacy, the above-mentioned DHA-bound PLs also has a markedly increased anti-cognitive dysfunction efficacy due to the combination of direct stabilization within the molecule and the synergistic effect on improvement / correction of cognitive dysfunction. It is considered a thing.

An example of a method in which chickens are fed with a ω-3HUFA derivative-containing feed and ω-3HUFA is efficiently transferred to the living tissue is shown below.
1. Selection of biological species Chickens, particularly laying hens are preferred, male and female laying hens are more preferred, and laying laid hens including “forced molting” laying hens are particularly preferred. The basis for this is illustrated below.
1) Birds are the only animals that have inherited the “dinosaur-specific respiratory system“ air sac type ””, and have the following remarkable specificity compared to animals.
(1) Oxygen intake is doubled (since intake and exhaust are separate systems).
(2) Due to the above, the energy-related metabolic rate is doubled.
(3) Based on the above, the feed required to produce 1 kg of edible portion is 4.5 kg for broilers, while 9.1 kg for domestic pigs and 25.0 kg for beef cattle are significantly more efficient. It is said that only 2.1 kg of crickets is surpassed, although it lacks versatility (FAO 2013 publication).

2) Procurability (integrated price to performance ratio) is remarkably superior due to the following factors.
(1) Poultry farming is a global industry with high quality, uniform quality, low price and high concentration;
* Broiler is a vertically integrated industry * Abandoned laying hens (about 700 days old) (about 70% thinned annually; about 750 days old) is a dedicated slaughter and demolition site The center (with 30 locations all over the country, with an average processing capacity of around 3 million a year and over 10 million) is characterized by high freshness, low cost, domestic production and safety. * National policy for egg price stabilization No-tachiko (Japan-specific)
(2) Cumulative total annual number of wings in the world: 20 billion; growing in China (3) Globally compatible with several varieties; broilers and laying hens (chicken eggs)
(4) Currently growing (5) Good freshness, slaughtered even in abandoned chickens in an active state (6) Livestock grows specifically fast; Broiler 50 days, laying hens produce nearly 1 egg daily throughout the year Lay

(7) “Forced molting” is an artificial means of regenerating laying hens whose egg-laying efficiency has dropped, and fasting old hens about 700 days old for about 10 days. A poultry farming style in which the animals are bred again after being in condition. Although it is a single piece of paper with animal cruelty, it is acquiesced as an emergency measure when egg prices are sluggish or sales volume is sluggish. It is interesting as a specific individual for biofunctional regeneration, for example, as a chicken for transferring ω-3HUFA derivative containing DHA. One of the peculiarities of poultry is that this type of measure can be realized on a large scale.
(8) The egg yolk derived from the laying hen has a PLs composition ratio of PL-PC >> PL-PE (substantially zero) specifically in the gold crown of its precursor. There is a very mysterious phenomenon that PLs are not included.
However, as a result of the transfer breeding of the ω-3HUFA derivative containing DHA, the following * was surprisingly found in the egg yolk of the laying hen.
* PLs were detected * Most of them were DHA-PLs * However, the composition ratio was PL-PE >> PL-PC, as opposed to the gold crown of the precursor. Thus, it is considered that DHA produced PLs in the yolk as a result of enhancing the expression of the rate-limiting enzyme Far1 in the biosynthetic pathway of the PLs neoplasmic reticulum “peroxisome” in vivo. Furthermore, it is interpreted that the PLs specifically supplemented DHA in egg yolk to generate DHA-PLs.

2. Selection of suitable tissue for extraction of PLs The following is exemplified.
1) Egg yolk of laying hen (frozen)
2) Raw glass (freeze mince)
3) Salmon (Frozen mince)
4) Gizzard (freeze mince)
5) Intestine (freezing mince)
6) Skin (freezing mince)
7) Gold crown (frozen)
8) Breast (freeze mince)

3. Selection of ω-3HUFA derivatives to be tested Meat derived from seafood, sardines, mackerel, sanma, tuna, bonito, scallops, squirts, krill, etc., and combinations of two or more of these, leftover seafood-derived meal by-products Examples include meals derived from reproductive tissues of seafood, by-product oil on the left, derived from fermentation medium of microorganisms, and the like.
4). ω-3HUFA-containing feed and feeding conditions The following are exemplified.
1) Chicken species Julia species and / or Boris Brown species 2) Feed Corn 62%, ω-3HUFA-containing feed 15% register, vegetable oil 15%, basic animal feed 6% and other grains 2% register 3) Feeding conditions ( 1) Feeding period is 1 to 5 weeks, preferably 1 to 3 weeks (2) Chickens are 40 weeks old (3) Feeding place is Kyushu poultry farm (4) The number of breeding birds is about 50 and 4 ) Slaughter and dismantlement primary processing storage Kyushu waste chicken processing center

Details of nanoemulsification using a complex lipid derived from a living tissue and its composition and PLs as a substrate are shown below.
1. Basic Formula A suitable formulation is illustrated below.
By nanoemulsifying the complex lipid in the presence of saponins, a complex lipid or PLs-containing aqueous preparation characterized in that the complex lipid or PLs is solubilized in the aqueous phase is obtained. ([Patent Document 12])
In that case, one kind selected from saponin, preferably Quillaja saponin as an essential component, fatty acid monoglyceride, fatty acid monoglyceride having 6 to 12 carbon atoms, polyglycerol fatty acid ester, polyhydroxy compound, syrup, etc. The above auxiliary components are used as appropriate.

2. Nano-emulsification step Add the above complex lipid into a degassed aqueous Quillaja saponin solution while stirring with a magnetic stirrer under a nitrogen gas atmosphere and stir until it is no longer cloudy to obtain a transparent solubilized solution. It is done.
3. Properties of nanoemulsified solution The prepared solution is diluted 10,100,1000 times, and the transparency is visually confirmed.
4). Stability test ([Patent Document 12])
For example, the prepared solution is diluted to 500 times with a transparent aqueous solution, adjusted to pH 4 using citric acid, visually checked for change, and heated at 95 ° C. for 30 minutes, and then returned to room temperature. Visually check for changes. Furthermore, after evaporating the heated liquid to dryness, a hexane extract is obtained by a conventional method, and an unheated solubilized liquid is used as a control, and a peak area comparison analysis is performed on an HPLC / ELSD chart to test residual plasmalogen. .

5). Average particle size measurement test ([Patent Document 12])
The average particle diameter of the oil phase particles in the prepared solubilized solution is measured with a commercially available submicron analyzer.
6). Preparation test of powder preparation and residual properties of ingredients ([Patent Document 14])
After the starch hydrolyzate of the excipient is dissolved in the solubilized solution, spray drying is performed using a desktop mini spray dryer. The transparency of the solution is visually determined by, for example, 100-fold dilution with water, and the solubilization is confirmed.
In addition, as described above, with the unheated solubilized solution as a control, a peak area comparative analysis is performed on an HPLC / ELSD chart to confirm the remaining amount of PLs.

7). Preparation test of solubilized jelly ([Patent Document 13])
1) Sodium caseinate is dissolved by heating in glycerin, and, for example, a 1% complex lipid δ tocopherol solution is added with stirring to obtain a transparent jelly-like lysate. After evaporating to dryness, extraction with hexane is performed, and the residual PLs is confirmed by HPLC / ELSD as in the previous section from a comparative analysis of the peak area ratio. Further, for example, a 100-fold water dilution of the jelly is prepared, and the average particle size distribution is measured with a commercially available submicron analyzer.
2) For example, 2 g of dried egg white is dissolved in 8 g of water, and upper white sugar is appropriately dissolved therein. If an appropriate amount of, for example, a 0.5% δ tocopherol solution of PLs is added to this solution under stirring in a nitrogen gas atmosphere, a transparent jelly-like solubilized product can be obtained.

Complex lipids according to the present invention, compositions thereof and PLs and aqueous preparations thereof, protein / lipid composite compositions, complex lipids containing ω-3HUFA-binding PLs, compositions thereof, PLs, and aqueous preparations thereof, Furthermore, protein-lipid complexes composition (hereinafter sometimes referred to as "various PLs such".) the central nervous system inflammation, neurogenesis ataxia, brain apoptosis diseases and a _beta and τ in neurons accumulation It can be used in foods, cosmetics, pharmaceuticals or feeds as an active ingredient for alleviating, preventing, improving and treating symptom (hereinafter referred to as “cognitive dysfunction”).

When the PLs preparation according to the present invention is used as a supplement and / or a general food (hereinafter sometimes referred to as “various foods”), the various foods may be various PLs preparations themselves, A material, a carrier, an additive acceptable in food hygiene, and other components and materials that can be used as food may be appropriately blended. Examples of such various food forms include, but are not limited to, liquid, powder, flake, granule, and paste foods.

When the PLs preparation according to the present invention is used as a food or drink, the preparation is appropriately mixed with PLs and ingredients / materials that can be used as food hygiene-acceptable substrates, carriers, additives, and other foods. (Ie, a food composition containing various PLs). For example, processed foods, beverages, health foods (nutrient functional foods, foods for specified health use, etc.), foods for sick people (hospital foods, sick foods, nursing foods, etc.) and the like including various PLs preparations can be exemplified.

The types of supplements and foods are not particularly limited, and include, for example, processed foods such as hamburger, meatballs, wieners, bird rags, chicken skin chips, etc., which are mixed with various PLs preparations, processed meat foods, etc. Examples include health foods (functional nutritional foods, foods for specified health use), supplements, foods for the sick, and the like. In addition, various PLs preparations, for example, powdered, beverages (juice etc.), confectionery (eg gum, chocolate, candy, biscuits, cookies, rice crackers, rice crackers, pudding, jelly-like confectionery, Examples thereof include apricot tofu and the like, breads, soups (including powdered soups), and various foods and beverages such as processed foods.

In addition, when preparing foods and drinks related to the present invention as health foods (nutrient functional foods, foods for specified health use, etc.) and supplements, for example, granules, capsules, tablets (chewable agents) so as to facilitate continuous intake Etc.), beverages (drinks, etc.), etc. are preferably prepared. Among them, capsules, tablets, tablets, jellies and the like are more preferable from the viewpoint of easy intake. The form of granules, capsules, tablets, jelly and the like can be appropriately prepared according to a conventional method using a pharmaceutically and / or food hygienically acceptable carrier.

The blending amount of PLs in the food or drink according to the present invention is preferably 0.00005 to 100% by mass, more preferably 0.0005 to 75% by mass, and still more preferably 0.005 to 50% by mass. The food / beverage products according to the present invention can be used for the prevention / mitigation / improvement of cognitive impairment. Moreover, it is preferable that the measurement of the intake amount, the intake target, the amount of contained PLs, and the like are the same as, for example, the pharmaceutical product according to the present invention described later.

The hospital food is a meal provided when hospitalized, the sick food is a meal for the sick, and the care food is a meal for the care recipient. The food / beverage products according to the present invention can be used as hospital foods, sick foods or nursing foods for patients who are hospitalized, treated at home due to the above-exemplified diseases, or who are receiving nursing care. In addition, a person who is highly likely to suffer from the above-illustrated diseases such as the elderly can be ingested prophylactically.

When the anti-cognitive dysfunction agent of the present invention is used in the pharmaceutical field, the preparation may be composed solely of PLs, or may be formulated with other components (ie, a pharmaceutical containing PLs). For example, in the pharmaceutical product according to the present invention, various PLs preparations which are active ingredients are optionally added to pharmaceutically acceptable bases, carriers, additives (for example, excipients, binders, disintegrants, Lubricants, solvents, sweeteners, colorants, flavoring agents, surfactants, humectants, preservatives, pH adjusters, thickeners, and the like can be blended. In addition, it is prepared by a conventional method such as tablets, coated tablets, powders, granules, fine granules, capsules, pills, solutions, suspensions, emulsions, jellies, chewables, soft tablets, etc. be able to. In particular, it may be prepared as a solution, suspension, emulsion, etc. and used as an injection or infusion, or as an oral preparation.

The amount of PLs in the pharmaceutical product according to the present invention is not particularly limited as long as the anticognitive dysfunction is exerted, and can be appropriately set according to the preferred daily intake of PLs. The blending amount is preferably 0.0005 to 100% by mass, more preferably 0.005 to 90% by mass, and still more preferably 0.05 to 80% by mass. The subject to which the pharmaceutical product according to the present invention is administered is preferably a subject suffering from cognitive dysfunction. Examples of such diseases include neurodegenerative diseases and mental diseases. Specific examples of neurodegenerative diseases include AD, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis.

Specific examples of mental illness include depression, mania, manic depression, schizophrenia, autism, and contact disorder. Furthermore, the pharmaceutical product according to the present invention may be administered to a subject who is likely to suffer from such a disease in the future. For example, it can be administered prophylactically to subjects who are genetically highly likely to suffer from the above exemplified diseases, elderly people (especially 60 years or older) and the like. The subject to which the pharmaceutical product according to the present invention is administered is not limited to humans but may be other mammals for feed. Examples of such mammals include animals raised as livestock and pets, and examples include dogs, cats, cows, horses, pigs, sheep, goats, monkeys, rabbits, mice, rats, and hamsters. .

The administration time of the anticognitive dysfunction agent according to the present invention is required to be administered preferably 5 years ago, more preferably 10 years ago, and even more preferably 15 years before the onset of the cognitive dysfunction. It has been clarified that end-stage disease has been reached at the time of onset diagnosis ([Non-Patent Document 20]). A preferred dosage form is oral administration in view of its long administration period. The dosage of the anticognitive dysfunction agent according to the present invention is appropriately selected according to the age of the patient, the degree of symptoms of the patient, other conditions, and the like. Usually, the amount of PLs in the agent is preferably within a range of 0.001 to 1000 mg, more preferably 0.01 to 100 mg per day for an adult. The administration can be performed once or a plurality of times (preferably 2 to 3 times) a day.

The present invention also includes a method for treating cognitive dysfunction comprising a step of administering (in particular, oral administration or transvascular administration) an effective amount of the anticognitive dysfunction agent of the present invention to a subject suffering from cognitive dysfunction. provide. Furthermore, the present invention administers an effective amount of the anticognitive dysfunction agent of the present invention to a subject suffering from or likely to suffer from a neurodegenerative disease or psychiatric disorder (particularly oral administration or transvascular administration). And a method for preventing or treating these diseases. Specifically, this method is carried out by administering the aforementioned anticognitive dysfunction agent of the present invention. In addition, each condition, such as a control | contrast and intake, in the said method is as above-mentioned.

The following 1) to 7) can be exemplified as unique clinical test methods for the predetermined efficacy (prevention and treatment of neurodegenerative diseases and psychiatric disorders) of PLs and their preparations according to the present invention.
1) The subject is an adult (healthy person) before the onset of dementia who is in a state where accumulation of physiologically innocuous amounts of _Aβ and τ is recognized in the brain. It is characterized by being a subject.
In research in recent years in the United States (Non-Patent Document 20]), brain accumulation of A _beta and τ protein than traditional onset diagnosis period, at least 10 years, usually accumulated from 15-20 years ago is started in As a result, a kind of “paradigm shift” has occurred. Early prevention is the decisive factor for suppression of the onset, and this is why it is important to show the efficacy in adults (determined healthy people) to non-disease subjects before the onset of dementia.
2) It is characterized by using safe and stable PLs and / or preparations thereof.

Based on the above, safe, stable and inexpensive plasmalogen is an essential requirement for ingredients for prevention, alleviation, improvement and treatment of neurodegenerative diseases and psychiatric disorders, as compared with conventional ingredients It is said.
3) More preferably, safe and stable ω-3HUFA-binding PLs and / or preparations thereof are used.
4) The safe and stable ω-3HUFA-binding PLs are safely extracted from chickens safely fed with feed containing a safe ω-3HUFA source.
5) It is characterized by safe and simple oral administration.

6) The administration period is at most 2 years, preferably 18 months, more preferably 12 months, and preferably 6 months or less, compared to 1 year or more of the usual clinical trial period for the disease subject. It is as short as more than a month, and can reduce the burden on the subject.
7) An innovative method for the prevention and treatment of neurodegenerative and psychiatric disorders, characterized in that the main essential measurement efficacy includes three or more items selected from the following groups marked with *. .
* Uchida / Kraepelin test * MMSE
* Resting functional MRI (rs-fMRI) image analysis (Non-patent Document 17)
* Measurement of PLs content in erythrocytes (Non-patent Document 24)
* PSOL "Cognitive function self-diagnosis test"
* RBANS ([Non-Patent Document 21])
* Cognitax ([Non-Patent Document 21])
* Wexler memory test ([Non-Patent Document 22])

Note that the rs-fMRI image is useful for analysis and evaluation of the default mode network (DMN) of the brain. In other words, in the past, it was thought that the brain was also resting when conscious activity was not performed, but in recent years, a surprising fact has been revealed by functional brain imaging research, and important activities have been carried out even at rest. It is said that the energy consumed for the activity in the basal state of the brain reaches 20 times the brain energy used for the conscious reaction ([Non-Patent Document 17]). At the heart of this brain activity is a network composed of a plurality of brain regions called DMN, which acts to synchronize with various neural activities in the brain. It should be noted that the brain area where significant atrophy is observed in AD patients almost overlaps with the main brain area that constitutes DMN, which is a clue for understanding new neurological diseases by fMRI image analysis at rest. Is expected to be.

Recently, it has been clarified that the small-world nature and modularity of the resting brain functional network decrease with age, and these functional changes generally precede anatomical changes such as brain atrophy. Therefore, it has been announced that it may be a useful marker in the study of aging and dementia (Non-patent Document 17).

Clinical trials that meet the following requirements for judging the cognitive function improvement effect of the latest PLs based on the above-mentioned basic concept by the present inventors;
* Entrusted to JACTTA (Tokyo), a specialized organization,
* Random, double-blind, placebo-controlled,
* Innovative PLs composition using cognitive function-improving food * Carefully selected healthy subjects (135 applicants registered for institutional registrants ⇒ Cognitive function 3-level interview selection 81
⇒ 75 people selected for interview by the person in charge of the examination ⇒ 71 who excluded ineligible persons found during the examination)
* Extremely low daily doses, 0.25 mg and 0.5 mg,
* Within a very short 12 weeks,
However, in only 6 weeks, a significant and immediate improvement effect of cognitive function was confirmed (Non-patent Document 25), which is evaluated as a surprising result.

The conventional implementation method of [Phase II clinical trials] related to dementia has been difficult to rationalize the test results because the subject is the brain. As for the direct image analysis method, there has been no scientific recognition because the apparatus is too expensive, the rationality of image analysis is lacking, or there is a problem in reproducibility.
But to directly test the dynamics of the _A β and τ protein brain, it is necessary to perform a "lumbar puncture", is high, there is no versatility extremely invasive.
According to a recent report, PET (Positoron
Emission Tomography (Positron Emission Tomography) has been improved and miniaturized, and a dedicated head PET has been developed at the National Institute of Radiological Sciences, Quantum Science and Technology Research and Development Organization. It is said that the sensitivity in the vicinity of the hippocampus has also been successfully improved (Non-patent Document 28).
Further, in the orthotopic have became to be observed by [tau protein] also succeeded in developing a test agent [pBB3] for viewing by PET, [tau protein] with A _beta also PET.
The present inventor has performed the [DHA-PLs] vs. [PLs] efficacy evaluation [clinical study] in the cognitive improvement of concern using the [amyloid PET-PIB], and obtained extremely interesting results. .

Hereinafter, the present invention will be specifically described based on verification examples, preparation examples, and examples, but the present invention is not limited to the following examples.

Preparation Example 1 [Production of PLs-containing phospholipid for biological tissue extraction]
[Production of biological tissue-extracted PLs-containing phospholipids]
1. Preparation of complex lipids from the skins of the laying hens The complex lipids were prepared from the freshly peeled pupae of the laying laying hens, which were the chicken tissues (the carcass from which the "thigh" had been removed).
Skin peeled rice was procured from the waste chicken processing center to prepare 1 kg of minced meat of about 8 mm. The mince was stored frozen slowly. In use, it was dehydrated and deoiled by pressing after forced thawing with warm running water. This was cooked with [superheated water] (Aquagas; registered trademark) in an “oxygen-free atmosphere” (oxygen 0.5% by volume or less) for 5 minutes, and then subjected to rapid dehydration by vacuum cooling. The obtained dehydrated / deoiled / cooked / sterilized breast meat was subjected to a three-phase ethanol separation step after low-temperature grinding.

[Three-phase separation process]
Put the cooked and sterilized crushed skin peeled rice obtained in the above into a container and deaerate it. Add 800 ml of degassed ethanol to this container and keep it gently stirred at 35 ° C for 10 hours. Thereafter, the mixture was allowed to stand under ice cooling to separate the upper layer chicken oil and the solid precipitated protein, etc. to obtain a water-containing ethanol fraction (sealed refrigerated storage). 800 ml of degassed ethanol was added to the protein, etc., and the same extraction operation was repeated. The ethanol phase was separated by centrifugation, ice-cooled in a sealed state, and concentrated under reduced pressure. The solid content of the water-soluble low-molecular fraction was filtered off and evaporated to dryness under reduced pressure to obtain 7 g of PLs-containing phospholipid (complex lipid).

1) Fractionation of total lipid The total lipid extracted, distributed and separated by the Folch method was 26.5% by mass.
2) Phospholipid profile assay The phospholipid profile assay was performed by the conventional method of HPLC / ELSD.
3) List notation of [mg / 100g skin peel] of the above results TL (total lipid): 26.5% by mass, TPL (total phospholipid): 0.6% by mass
PL-PE (ethanolamine type PLs): 104, PE (phosphatidylethanolamine): 43, PL-PC (choline type PLs): 49, PC (phosphatidylcholine): 327, SM (sphingomyelin): 74

4) Composition ratio of skin peel-specific phospholipids (1) PLs; [ethanolamine type]: [choline type] = 2.1: 1
(2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: 7.6
(3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: 2.4
(4) [Total glycerophospholipid]: [Total sphingomyelin] = 7: 1

2. Preparation of complex lipids from raw chicken waste.
1) Separation of lipids Total lipids extracted and distributed by the Folch method were 14% by mass.
2) Phospholipid profile assay The phospholipid profile assay was performed by the conventional method of HPLC / ELSD.
3) List notation of [mg / 100g raw glass] of the above results TL (total lipid): 14% by mass, TPL (total phospholipid): 0.73%
PL-PE (ethanolamine type PLs): 149, PE (phosphatidylethanolamine): 119, PL-PC (choline type PLs): 42, PC (phosphatidylcholine): 283, SM (sphingomyelin): 60

4) Composition ratio of raw arabe specific phospholipid (1) PLs; [ethanolamine type]: [choline type] = 3.5: 1
(2) Acylglycerophospholipid; [ethanolamine type]: [choline type] = 1: 2.4
(3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: 2.1
(4) [Total glycerophospholipid]: [Total sphingomyelin] = 10: 1

3. Preparation of complex lipids from the intestines of slaughtered chickens.
1) Separation of lipids Total lipids extracted and distributed by the Folch method were 32% by mass.
2) Phospholipid profile assay The phospholipid profile assay was performed by the conventional method of HPLC / ELSD.
3) List notation of [mg / 100g raw glass] of the above results TL (total lipid): 32% by mass, TPL (total phospholipid): 1% by mass
PL-PE (ethanolamine type PLs): 169, PE (phosphatidylethanolamine): 88, PL-PC (choline type PLs): 14, PC (phosphatidylcholine): 379, SM (sphingomyelin): 86

4) Composition ratio of intestinal specific total lipid (1) PLs; [ethanolamine type]: [choline type] = 12: 1
(2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: 4.3
(3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: 2.6
(4) [Total glycerophospholipid]: [Total sphingomyelin] = 7, 6: 1

4). Preparation of complex lipids from gizzards of waste chickens was processed according to the same method.
1) Separation of lipids Total lipids extracted and distributed by the Folch method were 7.9% by mass.
2) Phospholipid profile assay The phospholipid profile assay was performed by the conventional method of HPLC / ELSD.
3) List notation in [mg / 100 g gizzard] of the above results TL (total lipid): 7.9% by mass, TPL (total phospholipid): 0.88% by mass
PL-PE (ethanolamine type PLs): 111, PE (phosphatidylethanolamine): 91, PL-PC (choline type PLs): 23, PC (phosphatidylcholine): 255, SM (sphingomyelin): 118

4) Specific composition ratio of sand liver (1) PLs; [ethanolamine type]: [choline type] = 4.8: 1
(2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: 2.8
(3) Ether glycerophospholipid]: [diacylglycerophospholipid] = 1: 2.6
(4) [Total glycerophospholipid]: [Total sphingomyelin] = 4.1: 1

5). Preparation of complex lipids from gold crowns 1) Treated according to the gold crown of spawning hens.
(1) Separation of lipids Total lipids extracted and distributed by the Folch method were 31% by mass.
(2) Phospholipid profile assay The phospholipid profile assay was performed by a conventional method of HPLC / ELSD.
(3) List notation in [mg / 100 g gold crown] of the above results TL (total lipid): 31% by mass, TPL (total phospholipid): 9.5% by mass
PL-PE (ethanolamine type PLs): 200, PE (phosphatidylethanolamine): 1,574, PL-PC (choline type PLs): 0, PC (phosphatidylcholine): 7,542, SM ( Sphingomyelin): 145

(4) Composition ratio of gold crown-specific phospholipid 1) PLs; [ethanolamine type]: [choline type] = 200: 0
2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: 4.8
3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: 45
4) [Total glycerophospholipid]: [Total sphingomyelin] = 64: 1

2) Golden crown of seed chicken female (1) Separation of lipid Total lipid extracted and distributed by the Folch method was 39.3% by mass.
(2) Phospholipid profile assay The phospholipid profile assay was performed by a conventional method of HPLC / ELSD.
(3) List notation in [mg / 100 g gold crown] of the above results TL (total lipid): 39.3 mass%, TPL (total phospholipid): 12.2 mass%
PL-PE (ethanolamine-type PLs): 551, PE (phosphatidylethanolamine): 2, 236, PL-PC (choline-type PLs): 0, PC (phosphatidylcholine): 10,592, SM ( Sphingomyelin): 339

(4) Composition ratio of gold crown-specific phospholipid 1) PLs; [ethanolamine type]: [choline type] = 551: 0
2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: 4.7
3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: 23.3
4) [Total glycerophospholipid]: [Total sphingomyelin] = 40: 1

6). Preparation of complex lipids from the skins of slaughtered chickens.
1) Fractionation of lipid The total lipid extracted and distributed by the Folch method was 46% by mass.
2) Phospholipid profile assay The phospholipid profile assay was performed by the conventional method of HPLC / ELSD.
3) List of the above results in [mg / 100g skin] TL (total lipid): 46% by mass, TPL (total phospholipid): 0.72% by mass
PL-PE (ethanolamine type PLs): 58, PE (phosphatidylethanolamine): 21, PL-PC (choline type PLs): 17, PC (phosphatidylcholine): 95, SM (sphingomyelin): 55

4) Composition ratio of skin-specific phospholipid (1) PLs; [ethanolamine type]: [choline type] = 3.4: 1
(2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: 4.5
(3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: 1.5
(4) [Total glycerophospholipid]: [Total sphingomyelin] = 3.5: 1

7). Preparation of complex lipid from waste chicken breast Freshly peeled chicken breast of egg-laying chicken which is a chicken tissue was procured from a waste chicken processing center to prepare 1 kg of minced meat of about 8 mm. The mince was stored frozen slowly. In use, it was dehydrated and deoiled by pressing after forced thawing with warm running water. This was cooked with [superheated water] (Aqua gas; RTM) in an “anoxic atmosphere” (oxygen 0.5% by volume or less) for 5 minutes, and then subjected to rapid dehydration by a vacuum cooling method. The obtained dehydrated / deoiled / cooked / sterilized breast meat was subjected to a three-phase ethanol separation step after low-temperature grinding.
[Three-phase separation process]
The cooked and sterilized crushed breast meat obtained above is put in a container and degassed, and 800 ml of degassed ethanol is added to the container and sealed, and the mixture is slowly stirred at 35 ° C. for 10 hours. Thereafter, the mixture was allowed to stand under ice cooling to separate the upper layer chicken oil and the solid precipitated protein, thereby obtaining a water-containing ethanol fraction (sealed refrigerated storage). 800 ml of degassed ethanol was added to the protein, and the same extraction operation was repeated. The ethanol phase was separated by centrifugation, ice-cooled under sealing, and concentrated under reduced pressure. The solid content of the water-soluble low-molecular fraction was filtered off and evaporated to dryness under reduced pressure to obtain 7 g of PLs-containing phospholipid (complex lipid).

(1) Fractionation of total lipid The total lipid extracted, distributed and separated by the Folch method was 1.8% by mass.
(2) Phospholipid profile assay Measurement was carried out by HPLC / ELSD method of Nana et al.
(3) List of the above results in [mg / 100 g breast] TL (total lipid): 1.8% by mass, TPL (total phospholipid): 0.7% by mass
PL-PE (ethanolamine type PLs): 61, PE (phosphatidylethanolamine): 44, PL-PC (choline type PLs): 124, PC (phosphatidylcholine): 276, SM (sphingomyelin): 32
(4) Composition ratio of breast-specific phospholipid 1) PLs; [ethanolamine type]: [choline type] = 1: 2
2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: 6.3
3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: 1.7
4) [Total glycerophospholipid]: [Total sphingomyelin] = 16: 1

Preparation Example 2
[Preparation of purified PLs from complex lipids]
1. Preparation of Purified PLs from Waste Chicken Skin Lipid Complex Lipid 20 g of the above PLs-containing phospholipid (complex lipid) was added to 400 ml of phospholipase A1 (Mitsubishi Chemical Foods) solution (10 mg / ml 0.1 M citric acid-hydrochloric acid buffer). The mixture was dispersed and stirred at 50 ° C. for 2 hours under nitrogen gas filling. Under ice cooling, 2 volumes of hexane was added, and the mixture was stirred and distributed twice. The upper layer was collected and concentrated to dryness. Furthermore, the operation of adding 60 ml of acetone to the dried product, stirring and centrifuging, and collecting the precipitate was repeated twice.

Furthermore, 60 ml of hexane / acetone (7: 3) was added to the precipitate, followed by stirring and centrifugation, and the liquid layer was recovered. The liquid layer was concentrated to dryness, 240 ml of hexane / acetone (1: 1) was added and transferred to a separatory funnel, and 36 ml of water was added, stirred and distributed. The lower layer was discarded, and 96 ml of acetone / water (5: 3) was added to the upper layer and stirred and distributed. The upper layer was collected and quickly dried under reduced pressure to obtain purified PLs derived from chicken skin peeler.
[Assessment of purity of purified PLs from waste chicken skin peeler]
Purity test was performed according to the technique of Nana et al. ([Patent Document 1]). The purity was 93.6% by mass.

2. Preparation of purified PLs from waste chicken gull complex lipid 20 g of the above plasmalogen-containing phospholipid was dispersed in 400 ml of phospholipase A1 (Mitsubishi Chemical Foods) solution (10 mg / ml 0.1 M citric acid-hydrochloric acid buffer), and nitrogen was added. It stirred at 50 degreeC under gas filling for 2 hours. Under ice cooling, 2 volumes of hexane was added, and the mixture was stirred and distributed twice. The upper layer was collected and concentrated to dryness. Furthermore, the operation of adding 60 ml of acetone to the dried product, stirring and centrifuging, and collecting the precipitate was repeated twice. Furthermore, 60 ml of hexane / acetone (7: 3) was added to the precipitate, followed by stirring and centrifugation, and the liquid layer was recovered.

The liquid layer was concentrated to dryness, 240 ml of hexane / acetone (1: 1) was added and transferred to a separatory funnel, and 36 ml of water was added, stirred and distributed. The lower layer was discarded, and 96 ml of acetone / water (5: 3) was added to the upper layer and stirred and distributed. The upper layer was collected and quickly dried under reduced pressure to obtain purified PLs derived from raw chicken waste.
[Assessment of Purity of Purified PLs from Waste Raw Chicken]
Purity test was performed according to the method of Nana et al. The purity was 94.3% by mass.

3. Preparation of purified PLs from waste chicken intestinal complex lipid 20 g of the above-mentioned PLs-containing phospholipid was dispersed in 400 ml of phospholipase A1 (Mitsubishi Chemical Foods) solution (10 mg / ml 0.1 M citric acid-hydrochloric acid buffer) and filled with nitrogen gas The mixture was stirred at 50 ° C. for 2 hours. Under ice cooling, 2 volumes of hexane was added, and the mixture was stirred and distributed twice. The upper layer was collected and concentrated to dryness. Furthermore, the operation of adding 60 ml of acetone to the dried product, stirring and centrifuging, and collecting the precipitate was repeated twice. Furthermore, 60 ml of hexane / acetone (7: 3) was added to the precipitate, followed by stirring and centrifugation, and the liquid layer was recovered.

The liquid layer was concentrated to dryness, 240 ml of hexane / acetone (1: 1) was added and transferred to a separatory funnel, and 36 ml of water was added, stirred and distributed. The lower layer was discarded, and 96 ml of acetone / water (5: 3) was added to the upper layer and stirred and distributed. The upper layer was collected and quickly dried under reduced pressure to obtain purified PLs derived from waste chicken intestine.
[Purification of purified plasmalogen derived from the intestines of waste chicken]
Purity test was performed according to the method of Nana et al. The purity was 91% by mass.

4). Preparation of purified plasmalogen from complex lipids of waste chicken sand liver Disperse 20 g of the above-mentioned phospholipid containing PLs in 400 ml of phospholipase A1 (Mitsubishi Chemical Foods) solution (10 mg / ml 0.1 M citric acid-hydrochloric acid buffer), and nitrogen It stirred at 50 degreeC under gas filling for 2 hours. Under ice cooling, 2 volumes of hexane was added, and the mixture was stirred and distributed twice. The upper layer was collected and concentrated to dryness. Furthermore, the operation of adding 60 ml of acetone to the dried product, stirring and centrifuging, and collecting the precipitate was repeated twice. Furthermore, 60 ml of hexane / acetone (7: 3) was added to the precipitate, followed by stirring and centrifugation, and the liquid layer was recovered.

The liquid layer was concentrated to dryness, 240 ml of hexane / acetone (1: 1) was added and transferred to a separatory funnel, and 36 ml of water was added, stirred and distributed. The lower layer was discarded, and 96 ml of acetone / water (5: 3) was added to the upper layer and stirred and distributed. The upper layer was collected and quickly dried under reduced pressure to obtain 3 g of purified plasmalogen derived from waste chicken sand liver.
[Purification of purified PLs derived from chicken gizzards]
Purity test was performed according to the method of Nana et al. The purity was 95.4% by mass.

5). Preparation of purified PLs from chicken gold crown complex lipids 1) Gold hen derived from waste chickens 20 g of the above-mentioned PLs-containing phospholipids are dispersed in 400 ml of phospholipase A1 (Mitsubishi Chemical Foods) solution (10 mg / ml 0.1 M citric acid-hydrochloric acid buffer). The mixture was stirred at 50 ° C. for 2 hours under nitrogen gas filling. Under ice cooling, 2 volumes of hexane was added, and the mixture was stirred and distributed twice. The upper layer was collected and concentrated to dryness. Furthermore, the operation of adding 60 ml of acetone to the dried product, stirring and centrifuging, and collecting the precipitate was repeated twice. Furthermore, 60 ml of hexane / acetone (7: 3) was added to the precipitate, followed by stirring and centrifugation, and the liquid layer was recovered.

The liquid layer was concentrated to dryness, 240 ml of hexane / acetone (1: 1) was added and transferred to a separatory funnel, and 36 ml of water was added, stirred and distributed. The lower layer was discarded, and 96 ml of acetone / water (5: 3) was added to the upper layer and stirred and distributed. The upper layer was collected and quickly dried under reduced pressure to obtain purified PLs derived from a waste chicken crown.
[Purification of Purified Plasmalogen from Waste Chicken Gold Crown]
Purity test was performed according to the method of Nana et al. The purity was 95.6% by mass.

2) Seedling female gold crown Based on the above, purified PLs derived from seeded chicken female crown was prepared.
[Purity test of purified PLs derived from abandoned chicken crown]
Purity test was performed according to the method of Nana et al. The purity was 96.7% by mass.

6). Preparation of purified PLs from waste chicken skin complex lipids Disperse 20 g of the above PLs-containing phospholipids in 400 ml of phospholipase A1 (Mitsubishi Chemical Foods) solution (10 mg / ml 0.1 M citric acid-hydrochloric acid buffer) and fill with nitrogen gas The mixture was stirred at 50 ° C. for 2 hours. Under ice cooling, 2 volumes of hexane was added, and the mixture was stirred and distributed twice. The upper layer was collected and concentrated to dryness. Furthermore, the operation of adding 60 ml of acetone to the dried product, stirring and centrifuging, and collecting the precipitate was repeated twice. Furthermore, 60 ml of hexane / acetone (7: 3) was added to the precipitate, followed by stirring and centrifugation, and the liquid layer was recovered.
The liquid layer was concentrated to dryness, 240 ml of hexane / acetone (1: 1) was added and transferred to a separatory funnel, and 36 ml of water was added, stirred and distributed. The lower layer was discarded, and 96 ml of acetone / water (5: 3) was added to the upper layer and stirred and distributed. The upper layer was collected and quickly dried under reduced pressure to obtain purified PLs derived from waste chicken skin.
[Assessment of Purity of Purified PLs from Waste Chicken Skin]
Purity test was performed according to the method of Nana et al. The purity was 96.7% by mass.

7). Preparation of purified PLs from waste chicken breast complex lipid Disperse 20 g of the above PLs-containing phospholipid (complex lipid) in 400 ml of phospholipase A1 (Mitsubishi Chemical Foods) solution (10 mg / ml 0.1 M citric acid-hydrochloric acid buffer). The mixture was stirred at 50 ° C. for 2 hours under nitrogen gas filling. Under ice cooling, 2 volumes of hexane was added, and the mixture was stirred and distributed twice. The upper layer was collected and concentrated to dryness. Furthermore, the operation of adding 60 ml of acetone to the dried product, stirring and centrifuging, and collecting the precipitate was repeated twice.
Furthermore, 60 ml of hexane / acetone (7: 3) was added to the precipitate, followed by stirring and centrifugation, and the liquid layer was recovered. The liquid layer was concentrated to dryness, 240 ml of hexane / acetone (1: 1) was added and transferred to a separatory funnel, and 36 ml of water was added, stirred and distributed. The lower layer was discarded, and 96 ml of acetone / water (5: 3) was added to the upper layer and stirred and distributed. The upper layer was collected and quickly dried under reduced pressure to obtain purified PLs derived from chicken fillet.
[Purification of purified plasmalogen derived from waste chicken fillet]
Purity test was performed according to the technique of Nana et al. ([Patent Document 1]). The purity was 94% by mass.

Preparation Example 3
Production of laying hens fed with ω-3HUFA derivative-containing feed and preparation of phospholipids (composite lipids) having site-specific composition ratios and containing ω-3HUFA-binding PLs
-Feeding conditions-
a. Laying hen species and number of flocks; 30 Julia b. Same as above; 700 days old c. Average weight as above; 1.8kg
d. ω-3HUFA derivative-containing feed; DHA-TG 25% (EPA-TG 6%)-containing bonito oil 5% commercial egg-laying chicken feed (corn 62%, vegetable oil 20%, animal basic feed 6%, others 12%)
e. Administration period as above; 4 weeks f. Rearing environment; cage rearing g. Slaughter day; 735 days h. Results after breeding;
[Profile analysis of phospholipids of complex lipids in ω-3HUFA transitioning tissue / site and assay of its purified PLs-bound DHA ratio]

1. Skin peeling 1) Separation of each lipid; Total lipid: 26.5% by mass Total phospholipid: 0.6% by mass
2) Phospholipid profile (1) Relyed on HPLC / ELSD method.
(2) The profile is described below as mg / 100 g (兜).
PL-PE: 114, PE: 43, PL-PC: 54, PC: 276, SM: 74

3) PHA binding DHA assay (1) Preparation of PLs The purity of PLs obtained by following the technique of Nana et al. Was 94.6% by mass (breakdown, PL-PC: 30. 4%, PL-PE: 64.2%).
(2) Fatty acid composition analysis of PLs The PLs were methyl esterified by a conventional method, and fatty acid composition analysis was performed. The composition ratio of DHA in the SN-2-binding fatty acid of PLs was 64%.

2. Raw Gala 1) Separation of each lipid; Total lipid: 14% by mass Total phospholipid: 0.73% by mass
2) Phospholipid profile (1) Relyed on HPLC / ELSD method.
(2) The profile is described below as mg / 100 g (raw glass).
PL-PE: 164, PE: 119, PL-PC: 46, PC: 283, SM: 60
3) Assay of bound DHA of raw rat PLs (1) Preparation of plasmalogen The purity of PLs obtained by following the method of Nara et al. Was 94.4% by mass (breakdown, PL-PC: 20. 8%, PL-PE: 73.6%).
(2) Fatty acid composition analysis of PLs The PLs were methyl esterified by a conventional method, and fatty acid composition analysis was performed. The composition ratio of DHA in the SN-2-binding fatty acid of PLs was 69%.

3. Intestine 1) Separation of each lipid; TL: 32% TPL: 1%
2) Phospholipid profile (1) Relyed on HPLC / ELSD method.
(2) The profile is described below as mg / 100 g (intestine).
PL-PE: 186, PE: 88, PL-PC: 16, PC: 379, SM: 86
3) Assay of bound DHA of intestinal PLs (1) Preparation of PLs The purity of PLs obtained by following the method of Nara et al. Was 92.4% by mass (breakdown, PL-PC: 7.3% , PL-PE: 85.1%).
(2) Fatty acid composition analysis of PLs The PLs were methyl esterified by a conventional method, and fatty acid composition analysis was performed. The composition ratio of DHA in the SN-2-binding fatty acid of PLs was 70%.

4). Sand liver 1) Separation of each lipid; Total lipid: 7.9% by mass Total phospholipid: 0.9% by mass
2) Phospholipid profile (1) Relyed on HPLC / ELSD method.
(2) The profile was described below as mg / 100 g (gizzard).
PL-PE: 122, PE: 91, PL-PC: 25, PC: 255, SM: 118
3) Assay of bound DHA of PLs in sand liver (1) Preparation of plasmalogen The purity of PLs obtained by following the method of Nana et al. Was 95% by mass (breakdown, PL-PC: 16.2% , PL-PE: 78.8%).
(2) Fatty acid composition analysis of PLs The PLs were methyl esterified by a conventional method, and fatty acid composition analysis was performed. The composition ratio of DHA in the SN-2-binding fatty acid of PLs was 69%.

5). Gold crown [Growing chicken]
1) Separation of each lipid; Total lipid: 31% by mass Total phospholipid: 9.5% by mass
2) Phospholipid profile (1) Relyed on HPLC / ELSD method.
(2) The profile is described below as mg / 100 g (gold crown).
PL-PE: 220, PE: 1574, PL-PC: 0, PC: 7,542, SM: 145
3) Assay for binding DHA of PLs in gold crown (1) Preparation of PLs The purity of PLs obtained by following the method of Nana et al. Was 96% by mass (breakdown, PL-PC: 0%, PL-PE : 96%).
(2) Fatty acid composition analysis of PLs The PLs were methyl esterified by a conventional method, and fatty acid composition analysis was performed. The composition ratio of DHA in the SN-2-binding fatty acid of PLs was 69%.

[Seedling female]
1) Separation of each lipid; Total lipid: 39% by mass Total phospholipid: 12.2% by mass
2) Phospholipid profile (1) Relyed on HPLC / ELSD method.
(2) The profile is described below as mg / 100 g (gold crown).
PL-PE: 551, PE: 2, 238, PL-PC: 0, PC: 10, 592, SM: 339
3) Binding DHA assay of gold crown PLs (1) Preparation of PLs The purity of PLs obtained by following the method of Nana et al. Was 97% by mass (breakdown, PL-PC: 0%, PL-PE : 97%).
(2) Fatty acid composition analysis of PLs The PLs were methyl esterified by a conventional method, and fatty acid composition analysis was performed. The composition ratio of DHA in the SN-2-binding fatty acid of PLs was 70%.

6). Skin (skin)
1) Separation of each lipid; Total lipid: 31% by mass Total phospholipid: 9.5% by mass
2) Phospholipid profile (1) Relyed on HPLC / ELSD method.
(2) The profile is described below as mg / 100 g (skin).
PL-PE: 58, PE: 21, PL-PC: 17, PC: 95, SM: 55
3) Assay for binding DHA of skin PLs (1) Preparation of PLs The purity of PLs obtained by following the method of Nara et al. Was 96% by mass (breakdown, PL-PC: 21.8%, PL -PE: 74.2%).
(2) Fatty acid composition analysis of PLs The PLs were methyl esterified by a conventional method, and fatty acid composition analysis was performed. The composition ratio of DHA in the SN-2-binding fatty acid of PLs was 67%.

7). Breast (1) Fractionation of each lipid; TL: 1.9%, TPL: 0.8%
(2) Phospholipid profile 1) Dependent on HPLC / ELSD method.
2) The profile was described below as mg / 100 g (breast).
PL-PE: 67, PE: 44, PL-PC: 136, PC: 276, SM: 32
(3) Assay for Bind DHA of PLs of Breast 1) Preparation of Purified PLs The purity of PLs prepared by following the method of Nara et al. Was 95.4% by mass (breakdown, PL-PC: 36. 8%, PL-PE: 58.6%).
2) Fatty acid composition analysis of PLs The above PLs was methyl esterified by a conventional method, and fatty acid composition analysis was performed. The composition ratio of DHA in the SN-2-binding fatty acid of PLs was 65%.

8). Egg yolk of chicken egg 1) Separation of each lipid; Total lipid: 32.1% by mass Total phospholipid: 9.8% by mass
2) Phospholipid profile (1) Relyed on HPLC / ELSD method.
(2) The profile is described below as mg / 100 g (gold crown).
PL-PE: 100, PE: 1,624, PL-PC: 5, PC: 7, 942, SM: 133
3) Assay for binding DHA of PLs in gold crown (1) Preparation of PLs The purity of PLs obtained by following the method of Nara et al. Was 97% by mass (breakdown, PL-PC: 5%, PL-PE : 92%).
(2) Fatty acid composition analysis of PLs The PLs were methyl esterified by a conventional method, and fatty acid composition analysis was performed. The composition ratio of DHA in the SN-2-binding fatty acid of PLs was 75%.

Preparation Example 4
[Extraction and separation of egg yolk-derived PLs-containing phospholipids (complex lipids)]
100 g of raw egg yolk is mixed with 20 g of ethanol and allowed to stand at room temperature for 10 minutes, and then 120 g of 20% aqueous ethanol is added and stirred. When this is centrifuged at 2000 rpm for 5 minutes, it is separated from the upper layer into three phases: a yellow transparent egg yolk oil phase, a yellow-white complex lipid emulsion phase, and an almost white egg yolk protein precipitate. The egg yolk oil phase and the emulsified phase are separated, the precipitated phase is extracted with 50 g of 20% aqueous ethanol, centrifuged in the same way, and the supernatant is combined with the emulsified phase and concentrated under reduced pressure to give 11.5 g of yellow egg yolk complex lipid ( Acetone insoluble part 80%) was obtained.

1) Separation of lipids Total lipids extracted and distributed by the Folch method were 33.5% by mass.
2) Phospholipid profile assay The phospholipid profile was measured by the HPLC / ELSD method of Nana et al.
3) List of the above results in [mg / 100g egg yolk] TL (total lipid): 33.5%
TPL (total phospholipid): 9.5%
PLs ([PL-PE] + [PL-PC] (ethanolamine type PLs + choline type PLs)): 0
PE (phosphatidylethanolamine): 1,574
PC (phosphatidylcholine): 7,542
SM (Sphingomyelin): 145

4) Composition ratio of phospholipids specific to egg yolk complex lipid (1) PLs; [ethanolamine type]: [choline type] =-
(2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: 4.8
(3) [ether glycerophospholipid]: [diacylglycerophospholipid] = −
(4) [Total glycerophospholipid]: [Total sphingomyelin] = 62.9: 1

[Extraction and separation of PLs-containing phospholipids (composite lipids) derived from female gold crowns]
After mixing 20 g of ethanol with 100 g of the gold crown and allowing to stand at room temperature for 10 minutes, 120 g of 20% aqueous ethanol is further added and stirred. When this is centrifuged at 2000 rpm for 5 minutes, it is separated from the upper layer into three phases of yellow transparent gold crown oil phase, yellowish white complex lipid emulsion phase, and almost white egg yolk protein precipitate. The gold crown oil phase and the emulsified phase are separated, the precipitated phase is extracted with 50 g of 20% aqueous ethanol, centrifuged in the same manner, and the supernatant is combined with the emulsified phase and concentrated under reduced pressure to give 12.2 g of yellow gold crown complex lipid ( Acetone insoluble part 80%) was obtained.

1) Separation of lipid The total lipid extracted and distributed by the Folch method was 39.3% by mass.
2) Phospholipid profile assay The phospholipid profile was measured by the HPLC / ELSD method of Nana et al.
3) List of the above results in [mg / 100 g egg yolk] TL (total lipid): 39.3%
TPL (total phospholipid): 12.2%
PLs ([PL-PE] + [PL-PC] (ethanolamine type PLs + choline type PLs)): 551
PE (phosphatidylethanolamine): 2, 233
PC (phosphatidylcholine): 10,592
SM (Sphingomyelin): 339

4) Composition ratio of phospholipids specific to complex lipid of gold crown (1) PLs; [ethanolamine type]: [choline type] = 1: [˜0.1]
(2) Diacylglycerophospholipid; [ethanolamine type]: [choline type] = 1: 4.7
(3) [ether glycerophospholipid]: [diacylglycerophospholipid] = 1: 23
(4) [Total glycerophospholipid]: [Total sphingomyelin] = 40: 1

Preparation Example 5
[Composition of two products made from waste chicken fillet]
1. Complex lipid composition; mg / g
PLs 6-60, non-plasmalogen phospholipids 9.6-84.3, anserine 0.4-3.6, carnosine 0.07-0.6, free amino acids 0.24-2.4, vitamin E0. 12-1.2, carnitine 0.3-3, coenzyme Q10
0.12 to 1.2
2. Protein / lipid complex composition Protein content 87.1, complex lipid content 1.4, low molecular water soluble fraction 3.2

Verification example 1
1. Example of measurement (assay) of effects of chicken-derived PLs on cognitive function and related events The PLs for measurement test were prepared as follows.
[Chicken species and their parts]
Skinned breast minced minced hens were procured from an abandoned chicken processing center.
[Preparation of total lipids]
The mince was externally freeze-dried and slowly extracted with ethanol in the usual manner to prepare breast-derived total lipids.
[Preparation of complex lipid]
The composite lipid was prepared by appropriately adding water to the total lipid ethanol solution and degumming (de-neutral lipid).
[Preparation of glycerophospholipid]
The complex lipid was treated with hexane / acetone (7: 3) as usual to prepare de-sphingomyelin and purified glycerophospholipid.
[Preparation of high purity PLs]
As usual, diacylglycerophospholipid was converted to a lyso form with PLA1 enzyme to prepare PLs having a purity of about 90% by mass.
2. Example of verification of digestibility and absorption of orally administered waste chicken fillet PLs

(1) The effectiveness of oral administration was confirmed by the increase in blood PLs in rats (Non-patent Document 15).
(2) By increasing the PLs concentration in the brain of LPS-induced central nervous system inflammation model mice, the absorption of orally administered PLs from the brain blood barrier was confirmed (Non-patent Document 8).
(3) The effectiveness of oral administration for humans, especially AD patients, was verified by increasing the blood PLs concentration in AD patients (Non-patent Document 16).
3. Example of suppression (prevention) and alleviation of treatment of cognitive dysfunction of breast chicken breast meat PLs and verification of therapeutic effects (1) Cognition by waste chicken breast PLs due to renewal of neurons in aging model rats (SAMP8) The possibility of alleviating dysfunction and its therapeutic effect was verified (Patent Document 7).
(2) A suppressive effect expression of the spatial cognitive learning dysfunctions by waste chicken breast plasmalogen against either side injection model rat _β is to demonstrate the potential therapeutic effects of cognitive dysfunction waste chicken breast PLs (Non-patent document 16).

4). Example of verification of the alleviation of inflammatory symptoms in LPS-induced central nervous system inflammation model mice (Non-patent Document 8)
1) Suppression of microglia activation by waste chicken fillet PLs demonstrates the possibility of alleviating and treating cognitive impairment through central nervous system inflammation.
2) Increase of TNFα m-RNA in brain cytokine and suppression of IL-2β expression in brain by waste chicken fillet PLs alleviates and treats cognitive impairment through central nervous system inflammation by waste chicken fillet PLs This proves the possibility.
3) accumulation inhibition in A _beta brain by waste chicken breast PLs is to demonstrate the possibility of alleviating or treating cognitive dysfunction through its central nervous system inflammation.
5). Inhibition of central nervous system inflammation and _Aβ accumulation in a mouse model of central nervous system inflammation induced by intraperitoneal LPS ([Non-patent Document 8])
1) The extinction of central nervous system inflammation through suppression of central nervous system glial cell activation by waste chicken fillet PLs demonstrates the possibility of preventing and alleviating cognitive dysfunction of waste chicken fillet PLs. .
2) inhibitory effect of A _beta accumulation in the waste chicken breast PLs according prefrontal and hippocampus, which demonstrates the possibility of inhibiting the effect of cognitive disorders through the inhibitory effect of A _beta accumulation of waste chicken breast PLs It is.
3) Waste chicken fillet PLs demonstrates the possibility of an inhibitory effect on cognitive dysfunction through suppression of decrease in PLs caused by inflammation induced by intraperitoneal injection of LPS.
4) Waste chicken fillet PLs demonstrates the possibility of exerting an effect of directly treating cognitive impairment associated with nerve cell death through suppression of nerve cell death in a nerve cell culture system. (Non-patent document 9)
5) Others (1) The composite lipid fraction derived from waste chicken fillet has the potential to exert an action to delay the onset of cognitive dysfunction through the suppression of hyperglycemia and hyperlipidemia, which are risk factors for the development of cognitive dysfunction It is proved. ([Non-Patent Document 18])
(2) The waste chicken fillet-derived complex lipid composition demonstrates the possibility of preventing and / or alleviating cognitive dysfunction through the suppression of cognitive decline caused by the carnosine diet. (Non-patent document 19)

Verification example 2
1. Verification example of whether or not the suppression effect of cognitive dysfunction of chicken-derived DHA-binding PLs corresponding to the suppression effect of cognitive dysfunction of waste chicken fillet PLs is increased. PLs are the main phospholipids that store DHA ([Non-Patent Document 26]).
2) Verification of biochemical / physiological specificity of DHA-PLs (1) In the case of a single molecule (free), the aqueous system (emulsion) is more stable ([Non-Patent Document 1]).
(2) In a complex system (lipid bilayer), since it is a main membrane constituent, it is far more stable than a monomolecular system.
(3) DHA-PLs sterically hinder the approach to vinyl ether bond sites such as radicals and active acidic water by DHA, which is a bulk fatty acid bonded adjacent to an unstable vinyl ether bond. As a result, it is considered to be stabilized ([Non-Patent Document 1]).
(4) The fatty acid residue bonded to the SN-2 position of glycerophospholipid is the least susceptible to hydrolysis, the digestion absorption system passes well without being decomposed and is absorbed, and PLs are also taken into the blood Therefore, it is determined that DHA-PLs efficiently migrate into the blood.

(5) Regarding the ability of the brain blood barrier to enter the brain, DHA cannot be transferred into the brain with a glyceride structure, phospholipid structure is considered to be essential, and PLs have binding selectivity with DHA. Largely (described above), it is no exaggeration to say that the DHA-PLs structure is the most likely molecular species form that passes through the blood-brain barrier.
(6) After moving into the brain, DHA is the PLs biosynthetic rate-limiting enzyme Far1 (fatty)
By promoting the expression of acyl-CoA reductase 1), PLs production is enhanced ([Non-Patent Document 10]).
(7) Increase in the concentration of PLs in the brain promotes suppression of cognitive dysfunction expression, mitigation and treatment mainly through suppression of central nervous system inflammation (Non-Patent Document 7, Non-Patent Document 8, Non-Patent Document) 10 and Patent Document 1).
As mentioned above, DHA-PLs can be said to be the most suitable molecular species for improvement, maintenance and improvement of cognitive function due to the combination of direct stabilization within the molecule and the synergistic effect on improvement / correction of cognitive dysfunction. .

Verification example 3
(1) as a breakthrough of DIAN ^* research project that prevention is necessary 1) Morris professor of Washington University was responsible from adulthood, it is that the brain accumulation of _A β and τ protein is happening from 40 years of age As a result, it has been clarified that there is almost no treatment for the patient with dementia because the “terminal state” has been reached at the time of dementia diagnosis.
* Dominantly Inherited Alzheimer Network (heritable Alzheimer's disease network): Going to AD by tracking family members who are likely to have a causative gene of familial AD (over 100 people) from the pre-AD state in the long term The goal is to find out if changes can be detected and how long before the symptoms appear, the change will appear in the brain. Starting in 2005, the above breakthrough results were obtained in 2012 seven years later. (Non-Patent Document 30 Morris, JC. Et, al. Clinical and biomarkers changes in dominant Alzheimer's disease. N Engl J Med. Non-Patent Document 20] [Naose Alzheimer's disease!]
NHK Special Coverage Group (Housewife and Seikatsusha 2014))
2) First, with top priority to suppress the accumulation of a pioneering symptoms of AD onset A _beta, said interlock to happen tau in neurons of protein aggregation [A _beta accumulation] ([tau tangling]; Inhibition of neuronal death) is also required.
3) The onset of the above symptoms activates microglial cells into the “combat mode” and results in the death of neurons due to the attack of the released strong oxidizing factors and cytokines. It is required to suppress “microglia activation”.

(2) contributes spawning correction of the brain abnormalities adult chickens breast PLs (hereinafter, referred to as "breast PLs".) Various effects 1) breast PLs of suppressing the accumulation in the brain A _beta] (Patent Document No. 1).
2) Breast PLs suppress the activation of brain microglia (Non-patent Document 8).
3) Breast PLs suppresses neuronal apoptosis (Non-patent Document 9).
4) Breast PLs suppresses neuronal wear by suppressing TNFα-mRNA up-regulation of brain cytokine and IL-2β expression (Non-patent Document 8).
5) Breast PLs promotes neurogenesis (Patent Document 7).
The various effects of breast PLs are as follows: [A _β accumulation] suppression and intraneuronal aggregation [τ protein] -induced cell death augmentation by neurogenesis, long-term PPLs intake from the 40s Can be an effective prevention method.

(3) Attached to “Yan ^* ” of breast meat PLs; * Reliable, safe, stable and inexpensive 1) Reliable
i) The human experience of breast meat is long and the consumption is also significant.
ii) Breast meat that requires long-term activity during flight is considered to be equivalent to mammalian myocardium.
Therefore, since it is an energy consuming organ, there are many by-product highly oxidizing substances, and it is inevitable to construct an antioxidant system that appropriately protects them. The core is this strongly reducing PLs, which is a cell membrane component, and is therefore considered to have a high content.
iii) Based on the above, PLs are important components of the cell membrane structure in vivo, and are “generic” phospholipids that account for 18% of all human phospholipids.
iv) PLs are usually produced in the intracellular endoplasmic reticulum [peroxisome], but their biosynthetic ability decreases with age, so their supplementation is required.
2) Safety
i) In the production of breast PLs, all chemical treatments are eliminated, and a safe and mild extraction treatment and similar phospholipid groups contained in the purification are only decomposed with naturally occurring enzymes.
ii) Raw breast meat is edible meat that is divided from freshly laid egg-laying carcasses, and is peeled and manufactured. Heavy metal has been recorded for certification.
iii) Laying eggs are domestic products.
3) Stable
i) Stabilize and antioxidant by dissolving in edible grade tocopherol
ii) Enteric-coated soft capsules for external sales or
iii) This is gently emulsified with only natural edible ingredients,
iv) Nanoemulsion
(I) soft encapsulation,
(Ii) Add predetermined amount to existing foods,
(Iii) Add excipients and spray dry to form granules and powders.
(Iv) Tableting this, or (v) Dry blending with a solid food or the like.
4) Inexpensive
i) Since the daily dose is 0.5 mg, it can be provided at a reasonable cost.
ii) With the above stabilization, the storage stability is good, and it is not necessary to add water to the basic unit
iii) Raw laying hens (domestic) are the cheapest in poultry
iv) Breast is the cheapest chicken meat, usually distributed as bulk minced for processing
v) Breast meat has the highest ratio among chicken meat parts and has excellent procurement

Verification example 4
[Productivity of DHA phospholipid of Rabinchura microorganisms]
1) The amount of DHA contained in 12B (total DHA) / 100g of Rabinchura microorganisms isolated from microalgae collected from mangrove brackish waters in Okinawa is an extremely high 21g (total DHA) / 100g (see fish data below) Associate professor Okuyama and others of Hokkaido University revealed that the DHA content actually exceeds 50% (Patent Literature 16, Permissible Literature 17).
(1) Fish-containing DHA (g / edible part 100 g) (Non-patent Document 29)
1) Bluefin tuna Toro; 3.2
2) Mackerel (Atlantic); 2.3
3) White salmon salmon roe; 2.0
4) Yellowtail; 1.6
5) Saury; 1.6

(2) Phospholipids are dramatically improved and DHA content is further increased by the new technology “Glucose-starved fermentation method”. The Rabinchura microorganism 12B strain accumulates a large amount of fat when fermented in the presence of glucose. Then, as a result of glucose starvation, the accumulated fat is converted to phospholipid (13% ⇒67%), resulting in an increase in DHA content.
2) Targets for practical use of DHA phospholipid (1) Current status (bench level) DHA phospholipid; 11% of dry weight of culture solution (1 g / L)
(2) Target DHA phospholipid; 20% of the dry weight of the culture solution (5-10 g / L)
3) The fermentation broth and / or its dried product is very suitable as a ω-3HUFA derivative (1) Activation of peroxisomes in the in vivo plasmalogen production system (2) Easily transferred from the brain blood barrier to the brain (3 ) In-situ DHA structure in the brain Next, the present invention will be specifically described based on examples.

Example 1
[Preparation of purified plasmalogen derived from egg yolk of laying hens]
Based on the method of Nana et al. ([Patent Document 4]), plasmalogen (PLs) having a purity of 95.6% by mass was prepared.
[Preparation of purified PLs derived from egg yolk of breeder female]
In accordance with the method of Nana et al. ([Patent Document 4]), PL of 96.7% by mass purity was prepared.

Example 2
[Nano-emulsification of purified egg yolk-derived PLs and its stability test]
A nano-emulsion was obtained by adding 5 g of a 10 mass% δ tocopherol solution of 95.6 mass% PLs to 95 g of an aqueous solution containing 15 g of Quillaja saponin while stirring with a magnetic stirrer and continuing stirring until no turbidity occurred. . It was 58 nm as a result of measuring the average particle diameter of the oil phase particle | grains in the said nano emulsion liquid with a submicron analyzer. A solution obtained by diluting the nano-emulsion solution 500 times with water is transparent and has no change even when adjusted to pH 4 with citric acid. There was no change in transparency. The nanoemulsion diluted solution was evaporated to dryness at a low temperature, and the purity of PLs was tested by the method of the above patent (HPLC / ELSD). As a result, it was confirmed to be 94% by mass.

Example 3
[Shape conversion and stability of nano-emulsified purified egg yolk derived PLs]
1) Spray drying of 0.5 mass% nanoemulsified liquid of 92 mass% PLs prepared in Example 1 30 g of starch hydrolyzate (for example, Amikol 6H manufactured by Nissho Kagaku Co., Ltd.) was added to 100 g of the nanoemulsified liquid. After dissolving, spray drying using a desktop mini spray dryer (for example, manufactured by Yamato Scientific Co., Ltd.) at an inlet hot air temperature of 110 ° C. and an outlet temperature of 60 ° C. to contain 92% by mass of PL-PE 1% by mass A powdery formulation was obtained. The 100-fold diluted solution of this powdery preparation was slightly bluish but transparent. As a result of measuring the particle size of the oil phase particles in this water dilution, the average particle size was 61 nm. Furthermore, the aqueous dilution did not change at all to pH 4 using citric acid, and did not change such as turbidity when heated to 95 ° C. for 30 minutes and then returned to room temperature. This nanoemulsion diluted solution was evaporated to dryness at a low temperature, and the purity of PLs was tested by the method of the above patent (HPLC / ELSD). As a result, it was confirmed to be 90% by mass.

2) Preparation and properties of egg yolk-derived PL-PE-containing jelly Dry egg white was dissolved in 8 g of water, and 14 g of super white sugar was added thereto and dissolved. While stirring, 120 g of a 10% by mass δ tocopherol solution of 95% PLs was added and homogenized to obtain a translucent jelly-like solubilized product. When this was put into water, a substantially transparent aqueous solution was obtained.

Example 4
[Extraction and purification of DHA-bound PLs of PLs from DHA-transferred yolk obtained from eggs laid by laying hens prepared in Preparation Example 3]
In the same manner as in Preparation Example 3, an egg yolk complex lipid to which DHA was transferred was prepared.

Example 5
[Preparation of Purified PLs Derived from Egg Yolk Transferred DHA]
PLs containing 96% pure DHA-bound PLs were prepared based on the HPLC / ELSD method ([Patent Document 4]) of Nana et al.

Example 6
[Measurement of DHA binding ratio of PLs in previous paragraph]
According to a conventional method, the PLs was methyl esterified, and the fatty acid composition was measured. As a result, it was found that the DHA binding ratio (mol%) in the SN-2-bound fatty acid of PLs was 75%.

Example 7
[Nanoemulsification of 96% pure DHA-bound (75%) PLs and its stability test]
In the same manner as in Example 2, a nanoemulsion was obtained. It was 37 nm as a result of measuring the average particle diameter of the oil phase particle in the said nano emulsion liquid with a submicron analyzer.
A solution obtained by diluting the nanoemulsion solution 500 times with water is transparent, and there is no change even when it is adjusted to pH 4 with citric acid. There was no change in transparency. The nanoemulsion diluted solution was evaporated to dryness at a low temperature, and the purity of PLs was tested by the method of the above patent (HPLC / ELSD). As a result, it was confirmed to be 95% by mass.

Example 8
[Shape conversion of nanoemulsion and its stability]
Following Example 3, it implemented as follows.
1) Spray drying of a 0.5% by mass nanoemulsion solution of 96% by mass PLs prepared in Example 1 To 100 g of the nanoemulsion solution, 30 g of starch hydrolyzate (for example, Amycol 6H manufactured by Nissho Kagaku Co., Ltd.) After dissolving, spray drying using a desktop mini spray dryer (for example, manufactured by Yamato Scientific Co., Ltd.) at an inlet hot air temperature of 110 ° C. and an outlet temperature of 60 ° C., and a powder containing 1% by mass of 96% by mass PLs A shaped formulation was obtained. The 100-fold diluted solution of this powdery preparation was slightly bluish but transparent. As a result of measuring the particle size of the oil phase particles in the diluted water, the average particle size was 40 nm. Furthermore, the aqueous dilution did not change at all to pH 4 using citric acid, and did not change such as turbidity when heated to 95 ° C. for 30 minutes and then returned to room temperature. This nanoemulsion diluted solution was evaporated to dryness at a low temperature, and the purity of PLs was tested by the method of the above patent (HPLC / ELSD). As a result, it was confirmed to be 94% by mass.

2) Preparation and properties of DHA-bound PLs-containing jelly Dry egg white was dissolved in 8 g of water, and 14 g of sucrose was added thereto and dissolved. When 120 g of a 10% by mass δ tocopherol solution of 96% PLs was added to the solution while stirring, a translucent jelly-like solubilized product was obtained. This 100-fold diluted solution of water was almost transparent, and there was no change even at pH 4 using citric acid, and there was no change in turbidity even after heating at 95 ° C. for 30 minutes and then returning to room temperature. This nanoemulsion diluted solution was evaporated to dryness at a low temperature, and the purity of PLs was tested by the method of the above patent (HPLC / ELSD). As a result, it was confirmed to be 95% by mass.

Example 9
[Stability comparison test of 92% by mass PLs and 96% by mass PLs with a DHA bond ratio of 75 mol%]
1) Test at 50 ° C. with 10% by weight δ tocopherol solution sealed under light shielding Compared to half life of content, 92% by weight PLs halved in one week on average, whereas 96% by weight PLs of DHA bond was 4% There was no wear even during the week.
2) 10% δ Tocopherol solution 0.1% by weight nanoemulsion solution sealed under light shielding at 60 ° C standing test Compared with the half-life of content, 92% by weight PLs were halved in an average of 5 days, whereas DHA binding 96% by mass of PLs showed almost no wear after 4 weeks.

Example 10
[Examination of egg yolk modification effect of unshelled krill meal]
~ Test conditions ~
・ Bird bred species; Boris Brown 42-week-old 56 birds, 28 birds / district / test group; control group / test group / feeding;

Test plot 85% by mass of commercial feed + 15% by mass of unshelled krill meal
・ Culture period: 1 week
-Processing of produced egg: After egg breaking, the yolk was separated and freeze-dried. The lipid fraction was separated by extraction with ethanol.
-Lipid assay for lipid fractions; gas chromatography and HPLC / ELSD
Test items: EPA, DHA, PLs
~ Test results ~
Table 1 shows the results of the study.

-Additive effect of unshelled krill meal; contrast with control-
(1) EPA; 0
(2) DHA: 3.3 times up
(3) PLs: A significant improvement effect of egg yolk by unshelled krill meal was observed by 2.5 times or more.

Example 11
[Clinical study on the improvement effect of cognitive function of PLs derived from chicken breast waste]
[Production of PLs-containing phospholipids extracted from waste chicken skins]
1. Preparation of complex lipid from waste chicken skinned meat In accordance with Preparation Example 1-7, an edible [complex lipid] derived from breast meat was prepared.
[Preparation of purified PLs from complex lipids]
2. Preparation of purified PLs from edible [composite lipid] of scraped chicken skin fillet Purified PLs was prepared according to Preparation Example 2-7.
[Preparation of edible [PLs specimen] for clinical trials]
[Compound lipid] and [purified PLs] prepared above were appropriately mixed to prepare a edible [PLs sample] for testing.
[Clinical trial]
Improvement of cognitive function by administration of supplements containing edible PLs specimens selected from healthy Japanese subjects-Randomized, double-blind, placebo-controlled trial (for details, see Non-patent document 21) Conduct clinical trials.
This study is based on the Japan Clinical Trial Association (JACTA), which is a specialized organization in the field of clinical trials.
(Tokyo) The implementation period was 12 weeks from April 5 to June 29, 2016, and was conducted in compliance with ethical principles based on the Declaration of Helsinki. Consents for this study were obtained from all healthy subjects and 135 healthy women aged 40-79 years.

I. Preparation of test sample 1) Preparation of sample food [PLs specimen] As described above.
2) Preparation of stock solutions for soft capsules containing 0.25 mg of PLs (hereinafter referred to as “plasmalogen EX”) and soft capsules containing 0.50 mg of PLs (hereinafter referred to as “plasmalogen ES”) depending on the addition of edible [PLs specimen]. Based on the formulation shown in Table 1, plasmalogen EX and plasmalogen ES soft capsule stock solutions were prepared.
3) Preparation of placebo soft capsule stock solution Based on the formulation shown in Table 1, edible [PLs sample] was extracted from the plasmalogen EX stock solution, starch was added to adjust the weight, and a placebo soft capsule stock solution was prepared.
4) Soft encapsulation of each undiluted solution Two types of test capsules and placebo soft capsules are produced by consignment manufacturing of oval spherical soft capsules coated with caramel-colored standard gelatin for foods that are indistinguishable from their appearance. did. Table 2 shows the specifications of the test capsules.

II. Selection of test subjects A total of 135 people, CROee of the monitor bank
Inc. (Tokyo) selected from voluntary registrants between March and April 2016 by document screening.
1) Selection criteria;
(1) General healthy Japanese men and women aged 40 to 79 years (2) Cognitive function interviews in Figure 1 reveal that there is no experience of taking appropriate drugs in the past even though it is not completely normal 2) Exclusion criteria; excluding 54 people (1) Those who are being treated for cognitive dysfunction (2) Those who are taking Chinese medicines (3) Pregnant women and fear of becoming pregnant during this study A woman (4) Person who was judged by the person in charge of this study to be ineligible as a subject of this study

As a result of the above, 54 subjects were excluded from 135 people, resulting in 81 subjects.
As a result, they were randomly assigned to three groups, group A (n = 26), group B (n = 28), and group C (n = 27). At this time, consideration was given so that the gender and age were not offset.
* Group A is the placebo group * Group B is the test-1 (0.25 mg) group * Group C is the test-2 (0.5 mg) group Soft capsules) were taken for 12 weeks while living a normal life in preparation for overdrinking and eating, and a diary describing the physical and forgetfulness status was submitted to the supervisor of this study.

III. Test Outline 1) Test Schedule Table 3 shows the test schedule.

2) MMSE
See FIG. The highest score is 30.
3) Uchida-Kreppelin test (hereinafter referred to as “UK test”)
A simple one-digit addition is continuously performed for a certain period of time, and the calculation ability test that determines "ability when a person calculates" and "characteristics when the calculation ability is demonstrated" by a curve expressed by the amount of calculation. It is. Usually, 2 sets of 1 minute × 15 times are performed.
4) PSOL cognitive function self-diagnosis test Refer to Fig. 3 in the form of answering a highly comprehensive question on cognitive function originally devised. 2 is the reference point (baseline) in a four-step evaluation from 0 to 4. It is good evaluation when it exceeds 2.
5) Safety evaluation of test sample (3 types of soft capsules) The evaluation was made in a daily report from the subjects.
6) Data analysis method In this test, it was carried out using all the analysis results without arranging the number of specimens.
All statistically processed values are expressed in mean ± SD.
The difference between the measured values at 0, 6 and 12 weeks was statistically processed using the paired t test. The comparative evaluation of the measured values of the MMSE, UK test, and PSOL cognitive function self-diagnostic test within the same group was performed using the pair t test.
Using the Student t test, intergroup comparisons were made between the measured values at 0, 6 and 12 weeks and the deviation from baseline (Δ0-6 weeks and Δ0-12 weeks). Background comparison of subjects within the group was performed using unidirectional square deviation analysis.
This was not adjusted for fluctuations associated with the implementation period. Mislisted subjects were completely excluded from statistical processing. Statistical processing was performed using Statcell 4 and Excel statistics. The test statistical processing result between two samples was judged to be significant at <5%.

IV. Test results 1) Statistical information of subjects The ingestion test started by dividing 81 people into 3 wards, but 6 people dropped out. The reason is that poor physical condition 2, sudden work convenience 3, housework convenience 1, and 75 people completed the test after all. The breakdown was test-1; 27, test-2; 23, placebo; 25, but there was no significant difference between age, sex and UK test (Table 3).

Table 4 shows the contents of statistical processing on subjects.

2) MMSE
The results are shown in FIG. A significant difference was observed in the group comparison between Test-1 and Test-2 at 12 weeks. In addition, at the 6th week, a significant difference was recognized in the group comparison in the placebo group. However, there was no significant difference between the groups, test-1 vs. placebo and test-2 vs. placebo.

3) UK test The results are shown in FIG. There was no significant difference among the three groups. However, in the comparison between groups, a trend of significant difference was observed between Test-1 at 6 weeks and placebo.

4) Cognitive function self-diagnosis The results are shown in Table 5.

In Table 5, a significant difference was observed between the following items in test 1 and placebo, and in test 2 and placebo in the change contrast analysis between groups:
# 1 (Test-1, Test-2), # 5 (Test-1) at 6th week,
# 7 (Test-1, Test-2), # 8 (Test-1), # 12 (Test-1), # 13 (Test-1), # 17 (Test-1, Test-2), # 18
(Test-1), # 25 (Test-1), # 26 (Test-2), and # 27 (Test-1, Test-2)
On the other hand, in the 12th week, # 1 (test-1), # 2 (test-1, test-2),
# 4 (Test-1, Test-2), # 5 (Test-1, Test-2), # 8 (Test-1, Test-2), # 12 (Test-1), # 15 (Test-2 ),
# 16 (Test-2), # 17 (Test-2), # 20 (Test-2), # 21 (Test-2), # 23 (Test-2), # 24 (Test-1, Test-2 ),
There was a significant difference between # 26 (test-2) and # 27 (test-1, test-2).
In test-1 vs. placebo, the number of significantly different items at week 6 decreased to 6 at week 12,
In Test-2 vs. placebo, the number of significant difference items at 6 weeks increased significantly to 14 at 12 weeks.

5) The cognitive function self-diagnosis results and the analysis results are shown in Tables 6-9.

6) Safety evaluation As far as we can see in the daily report, there were no questions that suggested any doubt in the safety of the test samples, and there was no problem with safety.

V. Evaluation of test results 1) General remarks It was confirmed that it was effective in improving cognitive functions related to language and status of healthy subjects who took plasmalogen-containing supplements for 12 weeks. Furthermore, the soft capsules under test did not cause any safety problems during the 12-week test period.
Although no significant difference was observed in the results of the UK test, a significant difference tendency was observed between the high-dose intake group and the placebo group at 6 weeks.
Results suggested a dose dependence between the high and low dose groups and a significant trend between them and the placebo group.
The dosage is very low as a food, especially as a supplement, and it is considered to be less than an internal medicine. It is considered to be less than 0.5 mg per tablet, and it is healthy for 3 months. It is a surprising discovery that results were obtained in a short time and at 6 weeks suggesting an immediate improvement in computing power.

2) Detailed discussion (1) MMSE
Within the 0.5 mg group and the 0.25 mg group, a significant difference within the group was observed at 12 weeks. However, a significant difference within the group was found at 6 weeks in the placebo group.
(2) UK test Immediately, there was a significant difference between the high-dose group and the placebo group at 6 weeks, and there was a dose-dependent trend between the high-dose group and the low-dose group. It is worth noting.
(3) PSOL cognitive function self-diagnostic test From the 12th week and the 6th week, significant differences were observed within the high and low dose groups, between the groups, and between the placebo groups. There were significant differences between the high and low dose groups, but the results were not dose-dependent.
3) Summary evaluation * Random, double-blind, placebo-controlled trial,
* Using an innovative edible plasmalogen composition,
* For carefully selected healthy subjects (135 institutional registrants' document screening 135 ⇒ Cognitive function 3 level interview selection 81 ⇒ Examiner's interview selection 75 ⇒ Excludes ineligible persons found during the study 71)
* Extremely low daily doses, 0.25 mg and 0.5 mg, and
* Within an extremely short period of 12 weeks,
In only 6 weeks, a significant and immediate improvement effect of cognitive function was confirmed, which is evaluated as a surprising result.

Example 12
Single-blind open trial of [ω-3HUFA _"A β and accumulation in brain transition elucidation of τ in comparison with -PLs derived from the gold crown of derivatives transition species chicken female and the breast meat derived from PLs and placebo-ku set of laying waste chicken] Clinical trial]
1) Specimen (1) DHA-PLs; 96.7% purity, DHA binding rate is 75 mol%
According to Preparation Example 2 5.2) in [DHA migrating chicken female gold crown] (2) PLs; 92% by mass [derived from waste chicken fillet]
2) Subjects and n number 15 men and women (3 DHA-PLs x [0.25 mg and 0.5 mg], 3 PLs x [0.25 mg and 0.5 mg] and 3 placebo)) Selected at 60 ± 5 years old. The composition of men and women is 5 men and 10 women.
3) Condition (1) Daily dose: 0.25 mg (morning) and 0.5 mg (0.25 mg × twice morning and evening)
(2) Dosage form: Soft capsule (3) Period: 40 weeks (4) Examination interval: 0, 20 weeks, 40 weeks 4) Evaluation method A It depends on PET image analysis of _β protein. Incidentally, the test agents of the A _beta PET using PIB (Non-Patent Document 31).
The amount of accumulation is an SUVR (Standardized Uptake Value Ratio) of PET images corresponding to the degree of progression of cognitive dysfunction. Tables 10 to 11 show the SUVR scores of the PET images.
5) Results <0.25 mg section>
(1) Outline of findings of PET image at the start [A _β image]
Since the test subject was not afflicted with dementia, no blue color was observed, and a development of “yellow + green” including “red” slightly with “blue” in the background was observed.
(4) Summary Evaluation 1) [DHA-PLs] and [PLs] are both accumulation inhibitory effect on A _beta in the subjects of dementia non-pathological condition ingestion of low daily dose has been suggested. Regarding the reduction of accumulated _Aβ , all of these suggested a reduction effect at 40 weeks.
2) In contrast to [PLs], [DHA-PLs] showed the same tendency in the above.

<0.5mg ward>
(1) Observation outline of PET image at the start [A _β image]
Since the test subject was MCI, a single blue color was not recognized, and the development of “yellow + green” including “red” slightly with “blue” in the background was observed.
(2) Evaluation 1) [PLs] and [DHA-PLs] is, A _beta accumulation-inhibiting and accumulation reducing effect of dementia non-sick ingestion of low daily dose has been suggested.
2) In comparison with [PLs], [DHA-PLs] was suggested to have a higher efficacy in the above.
(3) Overall evaluation Based on the above, [PLs] and [DHA-PLs], especially [DHA-PLs], are A in a person with no dementia even at an oral very low daily dose of 0.25 mg. It was suggested that _β has accumulation suppression and accumulation reduction effects.

Example 13
[Elucidation of changes in hippocampal volume reduction in comparison with -PLs derived from gold crowns of ω-3HUFA derivative transfer females, breast-derived PLs derived from laying hens, and placebo settings] Single-blind open trial clinical trial ]
1) Specimen (1) DHA-PLs; 96.7% purity, DHA binding rate is 75 mol%
According to Preparation Example 2 5.2) in [DHA migrating chicken female gold crown] (2) PLs; 92% by mass [derived from waste chicken fillet]
2) Men and women of 15 subjects and 5 wards (3 DHA-PLs x [0.25 mg, 0.5 mg], 3 PLs x [0.25 mg, 0.5 mg] and 3 placebo)) Selected at 60 ± 5 years old. The composition of men and women is 5 men and 10 women.
3) Condition (1) Daily dose: 0.25 mg (morning) and 0.5 mg (0.25 mg × twice morning and evening)
(2) Dosage form: Soft capsule (3) Period: 40 weeks (4) Examination interval: 0, 20 weeks, 40 weeks 4) Evaluation method The volume reduction state of “hippocampus” in the brain MRI image of the subject was examined.
5) Results The results are shown in Tables 12-13.
(1) 0.25 mg section 1) It was suggested that both [PLs] and [DHA-PLs] have low inhibitory effects on hippocampal volume reduction in subjects who are not demented by oral intake at a low daily dose.

(2) Evaluation result of 0.5 mg section 1) Both [PLs] and [DHA-PLs] are hippocampal volume reductions in brain MRI of subjects with no dementia by oral ingestion at low daily dose for 40 weeks This suggests a tendency to suppress
(3) Overall evaluation Both [PLs] and [DHA-PLs] suggest a tendency to suppress hippocampal volume reduction in brain MRI of subjects with no dementia by oral intake at low daily doses for 40 weeks. It was.

As described above in detail, the present invention relates to a safe and stable plasmalogen and its preparation and use. According to the present invention, 1) a safe and stable plasmalogen derived from a safe biological material, more specifically, Provides a safe and stable plasmalogen-containing complex lipid formed by binding DHA (docosahexaenoic acid) to SN-2 of plasmalogen as an essential requirement. 2) Uses a safe surfactant for the plasmalogen complex lipid. Provides safe and stable aqueous preparations (emulsions, jellies, granules, powders) that are easily nano-emulsified (or solubilized) using 3) Specific to living tissue in the aqueous phase in the presence of saponins Complex lipid and ω-3 HUFA-conjugated complex lipid having a structural composition ratio, and complex lipid and ω-3 HUFA-binding wherein the complex lipid composition is nanoemulsified or solubilized Type complex lipids and aqueous preparations of these complex lipid compositions, 4) provide their use, 5) neurodegenerative diseases containing ω-3HUFA-bound crude or high-purity plasmalogen, etc. as active ingredients, and Provided with various effects such as providing various processed products, supplements, preventive or therapeutic agents for the prevention or treatment of mental illness, 6) providing a method for determining the non-demented state of dementia, etc. The present invention has industrial applicability.

Claims

A living body comprising a purified product of a total lipid that is extracted and fractionated from a specific site or organ derived from a living tissue and from which a protein fraction and / or a water-soluble low-molecular fraction that are by-produced in the extraction and fractionation step are purified and removed. A plasmalogen-containing phospholipid (hereinafter referred to as “complex lipid”) having a tissue-specific composition ratio,
1) The specific part or organ derived from the biological tissue is a carcass carcass derived from a chicken biological tissue, raw rattle containing bone marrow, chicken yolk, intestine, sand liver, gold crown including ovary and fallopian tube, breast meat, skin Consisting of at least one or more specific parts or organs selected from the group of
2) The plasmalogen-containing phospholipid is generically called an ω-3 highly unsaturated fatty acid (hereinafter referred to as “ω-3HUFA”) derivative (hereinafter referred to as “ω-3HUFA derivative”) containing docosahexaenoic acid (DHA). ) Is extracted and fractionated from a specific part or organ derived from the living tissue to which the ω-3HUFA derivative has been transferred to the biological tissue of a chicken fed with a feed containing The complex lipid, wherein the complex lipid is a plasmalogen-containing phospholipid having a constitutional ratio of ω-3HUFA binding type.
ω-3HUFA derivatives are the following 1) to 9);
1) ω-3HUFA derivative bound to SN-2 of glycerophosphatidyl phospholipid 2) ω-3HUFA derivative bound to SN-2 of 1-alkyl glycerophosphatidyl phospholipid 3) 1-alkenyl glycerophosphatidyl Ω-3HUFA derivative bound to phospholipid SN-2 4) 1-acylglycerophosphatidyl phospholipid SN-2 ω-3HUFA derivative 5) contained in krill shelled and / or dried product 6) ω-3HUFA derivative bound to SN-2 of glycerophosphatidyl phospholipid according to any one of 1) to 4) in the preceding paragraph 6) contained in whole scallop and / or processing residue thereof or meal (dry product) thereof The ω-3 bound to SN-2 of the glycerophosphatidyl phospholipid according to any one of 1) to 4) above UFA derivative 7) SN- of the glycerophosphatidyl phospholipid according to any one of the preceding items 1) to 4), which is contained in whole sea scabbard (sea squirt) and / or processing residue thereof or meal (dry product) thereof Ω-3HUFA derivative bound to 2 8) Organically synthesized ω-3HUFA derivative bound to SN-2 of the glycerophosphatidyl phospholipid according to any one of 1) to 4) above, prepared by fermentation method The complex lipid according to claim 1, which is any one selected from ω-3HUFA derivatives bound to SN-2 of the glycerophosphatidyl phospholipid according to any one of 1) to 4) above .
2. The complex lipid according to claim 1, wherein the specific site or organ is chicken egg yolk and consists of a purified product of total lipid extracted and fractionated from chicken egg yolk containing DHA-PLs biosynthesized in the chicken's living body. .
The complex lipid according to claim 1, comprising a purified product of a total lipid extracted and fractionated from a specific part or organ of a living body of a chicken fed with a feed containing an organically synthesized 1-alkylglycerophosphatidylphospholipid. .
A complex lipid composition characterized in that the complex lipid according to claim 1 contains a water-soluble low-molecular-weight fraction by-produced by the extraction fractionation according to claim 1 and has a constitutional ratio of ω-3HUFA binding type.
The complex lipid or complex lipid composition having the ω-3HUFA-binding composition ratio according to any one of claims 1 to 5 is nanoemulsified or solubilized in the aqueous phase in the presence of saponins. An aqueous preparation of a complex lipid composition having a constitutional ratio of complex lipid or ω-3HUFA binding type, characterized in that
The complex lipid according to any one of claims 1 to 4 is treated with phospholipase A1 (PLA1) to remove glycerophospholipids including lyso form and extract and remove sphingomyelin. A method for producing a high-purity plasmalogen having a purity of 30 to 70% or more.
The crude plasmalogen or high purity, characterized in that, in the presence of saponins, nanoemulsification or solubilization is carried out in the aqueous phase using the crude plasmalogen or high purity plasmalogen according to claim 7 as a substrate, respectively. A method for producing an aqueous preparation of plasmalogen.
9. A food comprising as an active ingredient any one or more selected from the complex lipid according to any one of claims 1 to 8, the complex lipid composition, each plasmalogen, and their aqueous preparations, Cosmetics, pharmaceuticals or feed.
Cognitive of neurodegenerative diseases, comprising as an active ingredient at least one selected from the complex lipid according to any one of claims 1 to 8, the complex lipid composition, each plasmalogen, and their respective aqueous preparations A supplement for alleviating and preventing neurodegenerative diseases characterized by having an alleviating and preventing action on at least one neurodegenerative disease selected from the group of Alzheimer's disease, Alzheimer's disease, Parkinson's disease, depression, and schizophrenia .
Cognitive of neurodegenerative diseases, comprising as an active ingredient at least one selected from the complex lipid according to any one of claims 1 to 8, the complex lipid composition, each plasmalogen, and their respective aqueous preparations Alleviation and prevention of said neurodegenerative disease characterized by having at least one neurodegenerative disease alleviation and prevention or treatment action selected from the group of Alzheimer's disease, Parkinson's disease, depression and schizophrenia Or an anti-central nervous system inflammation preparation for treatment.
A nerve comprising at least one selected from the complex lipid according to any one of claims 1 to 8, the complex lipid composition, each plasmalogen, and each of their aqueous preparations as an active ingredient Cytogenic agent.
A nerve comprising at least one selected from the complex lipid according to any one of claims 1 to 8, the complex lipid composition, each plasmalogen, and their aqueous preparations as an active ingredient Cell apoptosis inhibitor.
A compound comprising at least one selected from the complex lipid according to any one of claims 1 to 8, the complex lipid composition, each plasmalogen, and their respective aqueous preparations as an active ingredient A β and / or τ brain accumulation inhibitor.
A complex lipid according to any one of claims 1 to 8, a complex lipid composition, each plasmalogen, and at least one selected from their respective aqueous preparations as an active ingredient, comprising a neurodegenerative disease and / or or anti CNS inflammation improving action of psychiatric disorders, neurogenesis, said neurodegenerative, characterized in that it comprises at least one action selected from among the suppression as well as a beta accumulation in brain inhibition of neuronal cell death Processed products as drugs or foods for improving diseases and / or mental disorders.
A tissue-specific composition comprising the protein fraction by-produced by the extraction fraction according to any one of claims 1 to 4 and the water-soluble low-molecular fraction by-produced by the extraction fraction according to claim 5. A protein / lipid composite composition comprising a mixture with a composite lipid composition having a ratio.
17. The protein / lipid composite composition according to claim 16, wherein the protein / lipid composite composition comprising the mixture of the protein fraction and the composite lipid composition described in the preceding paragraph is derived from chicken egg yolk.
18. A protein / lipid composite composition according to claim 16 or 17, comprising as an active ingredient, an anti-central nervous system inflammation for improving neurodegenerative diseases and psychiatric disorders;
The processed product as a pharmaceutical or food for ameliorating neurodegenerative disease and / or psychiatric disorder characterized by having at least one action selected from suppression of neuronal cell death and suppression of accumulation in Aβ brain.
A complex lipid containing the plasmalogen according to any one of claims 1 to 8, a complex lipid composition, a crude plasmalogen or a high-purity plasmalogen, and one selected from these aqueous preparations An adult (healthy person) before the onset of dementia who is in a state where accumulation of physiologically harmless amounts of Aβ and τ is recognized in the brain (hereinafter referred to as “non-demented condition”) ,
(1) using a PET examines the brain accumulation state of A beta and tau, to determine dementia non pathological condition of the subject, the A beta and tau on the basis of the SUVR values of the PET image (score) The process of performing the classification of brain accumulation status by rank according to the following:
1) Initial accumulation stage I: 1.0 ± 0,2
2) Intermediate period II: 1.2 ± 0.2
3) Late stage III: 1.4 ± 0.2
(2) Testing the dose response to plasmalogen subjects for each rank at an appropriate frequency over a long period of 3 to 10 months, and setting an appropriate daily dose range or register range as follows:
1) Stage I subjects; “gradual increase” range: 1.0 ± 0.15
2) Stage II subjects; “Zero increase” register range: 1.2 ± 0.15
3) Stage III subjects; “decreasing” register range: 1.4 ± 0.15
(3) Continuing for a long period from the stage before the onset of dementia to the adult (healthy person) subjects before the onset of dementia, supplements containing the plasmalogen of the set daily dose as an active ingredient or processed products as food Administering step,
(4) performing a periodic test of the predetermined brain accumulation state of Aβ and τ by PET diagnosis at an appropriate frequency during the long-term administration;
(5) In parallel with periodic PET diagnosis, performing a hippocampal volume reduction test with MRI diagnosis at an appropriate frequency during the long-term administration period,
(6) Dementia of the subject characterized by determining the non-demented state of the subject by performing the steps (1) to (4) or the steps (1) to (5) A method for determining an unaffected state.