TW202114705A - Composition for promoting myelin formation capable of acting synergistically to significantly increase p-21.5kDaMBP which holds the key to myelin formation - Google Patents

Abstract

This invention provides a composition for promoting myelin formation. The composition for promoting myelin formation of this invention comprises a first active ingredient which is at least one compound selected from a group consisting of glycerophosphocholine (G) and pharmaceutically acceptable salts thereof; a second active ingredient which is at least one compound selected from a group consisting of hesperidin (H), narirutin (N), and pharmaceutically acceptable salts thereof; and a third active ingredient which is at least one compound selected from a group consisting of cinnamyl ester, cinnamyl alcohol, cinnamaldehyde (C), cinnamic acid (C), and pharmaceutically acceptable salts thereof. These active ingredients act synergistically to significantly increase p-21.5kDaMBP which holds the key to myelin formation.

Description

Composition to promote the formation of myelin

The present invention relates to a composition for promoting the formation of myelin, which can be used to treat and/or prevent patients with myelinogenesis disorders such as Alzheimer's disease.

In today’s ageing society, the number of patients diagnosed with dementia by medical institutions is rapidly increasing. According to the 2015 Alzheimer’s report, there were already 46.8 million dementias worldwide in 2015. It is estimated that it will increase to 131.5 million by 2050. This value doubles every 20 years, which brings the following warning signs: dementia is a disease that will cause a world-scale crisis. Therefore, the establishment of effective treatment and prevention methods for dementia has become a top priority.

Alzheimer’s disease is the largest cause of dementia. The main symptom is persistent dementia. As a pathological feature, most age spots and neurofibrillary changes can be confirmed in the brain. Atrophy of the brain due to the shedding of neurons can be confirmed. Therefore, it has been considered that the degeneration and shedding of nerve cells in the gray matter sites are the cause of Alzheimer's disease. In other words, it is believed that although the abnormality of the metabolism of amyloid precursor protein causes agglutination and accumulation of β-amyloid protein in the brain (hereinafter referred to as "Aβ" for β-amyloid protein) and the formation of age spots, The agglutination and accumulation of Aβ will cause the formation of neurofibril changes, the disappearance of neurons, and the decrease of cognitive function. Such a theory is called the "amyloid-like tandem hypothesis." So far, treatment and/or prevention methods for Alzheimer’s disease based on this hypothesis for the purpose of reducing the expression of Aβ have been extensively studied, but it is still difficult to accurately predict Alzheimer’s disease based on this hypothesis Or the development of productive treatments. Therefore, the hypothesis has now been criticized. In addition, the etiology of Alzheimer's disease is being studied to replace the amyloid-like tandem hypothesis, and various etiologies such as demyelination have been proposed (see Non-Patent Document 1 (J. Neurochem. (2017) 143, 432-444)).

The theory that demyelination is the cause of Alzheimer's disease is called the "myelin hypothesis." This hypothesis assumes that there are white matter abnormalities in the brains of Alzheimer’s patients. This is based on the fact that myelin is particularly significantly reduced, and it is not only in the corpus callosum at the initial myelin formation site and the corpus callosum knee at the late myelin formation site. Anomalies of white matter can be found even in the lower part of the hippocampus CAI.

In addition, based on the myelin hypothesis, for the purpose of restoring demyelination, a method of treatment and/or prevention of Alzheimer’s disease has been developed, which uses a kind of ginseng yangrong soup, which nourishes ginseng Rong Tang is a mixture of 12 kinds of herbs. The herbs are ginseng, angelica, peony, rehmannia, atractylodes, tuckahoe, cinnamon, astragalus, tangerine peel, polygala, schisandra and licorice. Non-Patent Document 2 (J neurosci Res 85:954-966, 2007) reported the following research results: phosphorylated myelin basic protein, especially phosphorylated myelin basic protein with a molecular mass of 21.5kDa (Hereinafter, myelin basic protein is denoted as "MBP", phosphorylated MBP is denoted as "p-MBP", MBP with a molecular weight of XkDa is denoted as "XkDaMBP", and phosphorylated XkDaMBP is denoted as "p- XkDaMBP”) was significantly reduced during the period of demyelination induced by the application of cuprizone, which is an aging and demyelination-inducing drug; and, in cuprizone-treated mice and aged mice The recovery of demyelination can be achieved by administering ginseng Yangrong soup. In addition, Non-Patent Document 3 (Evid Based Complement Alternative Med.2011.[doi:10.1093/ecam/neq 001]) reported the following research results: Among the 12 kinds of herbs that make up ginseng Yangrong soup, the source Tangerine peel from Wenzhou mandarin is the main component to restore demyelination caused by aging; the restoration of demyelination is achieved by remyelination, rather than inhibiting demyelination; and it is further reported that if in The main components of the above-mentioned tangerine peel, namely, hesperidin and/or naringin (narirutin) are cultured in the presence of oligodendritic cell precursor cells that will develop into myelin, and the same results will be obtained when cultured in the presence of tangerine peel. That is, p-21.5kDaMBP will be produced hyperactively, and the proliferation and differentiation of oligodendritic cell precursor cells will proceed rapidly.

Furthermore, the inventors of the present invention have found that in the brains of demyelinated elderly mice, especially p-21.5kDa, will be significantly reduced through research using Alzheimer’s disease model mice, but if they are used in combination Hesperidin and/or naringin shown in Non-Patent Document 3, and L-α-glycerylphosphorylcholine (L-α-glycerylphosphorylcholine) or glycerylphosphorylcholine called α-GPC ( Hereinafter, glycerophosphocholine (glycerophosphocholine) is expressed as "α-GPC"). Through the multiplicative effect of these components, the expression level of p-21.5kDa will be significantly increased, and by this significant increase, Myelination is significantly increased and demyelination is restored. Therefore, Patent Document 1 (WO2018/190146A1) proposes a composition for the treatment and/or prevention of Alzheimer’s disease, which includes: as the first The active ingredient is at least one compound in the group consisting of glycerophosphocholine and its pharmacologically acceptable salts; and, as the second active ingredient, it is composed of hesperidin and naringo At least one compound in the group consisting of cortin and these pharmacologically acceptable salts. α-GPC is a natural compound found in the brain and milk. In the choline metabolism system, it is present in the cell membrane of the cells responsible for the formation of myelin, that is, the ENPP6 (choline specificity) of the oligodendritic cells. Phosphodiesterase) is metabolized to choline, and the choline produced is used by oligodendritic cells for lipid synthesis and myelination (see Non-Patent Document 4 (Scientific reports|6:20995|Doi:10.1038/ srep20995)). However, based on Non-Patent Documents 3 and 4, it is impossible to predict the synergistic effect produced by the combined use of hesperidin and/or naringin and α-GPC. [Previous Patent Document] (Patent Document)

Patent Document 1: WO2018/190146A1. Patent Document 2: Japanese Patent Application Laid-Open No. 2006-206584. Patent Document 3: Japanese Patent Application Laid-Open No. 2010-106001. Patent Document 4: Japanese Patent Application Laid-Open No. 2016-079125. Patent Document 5: Japanese Patent Application Laid-Open No. 2016-088851. (Non-patent literature)

Non-Patent Document 1: J. Neurochem. (2017) 143, 432-444. Non-Patent Document 2: J neurosci Res 85:954-966, 2007. Non-Patent Document 3: Evid Based Complement Alternative Med. 2011. [doi:10.1093/ecam/neq001]. Non-Patent Document 4: Scientific reports|6:20995|Doi:10.1038/srep20995. Non-Patent Document 5: J. Neurosci., July 2, 2003･23(13):5963-5973. Non-Patent Document 6: J. Neurosci., June 1, 2003･23(11): 4549-4559. Non-Patent Document 7: J. Neurosci., June 17, 2009･29(24):7743-7752. Non-Patent Document 8: Acta Neuropathologica Communications (2018) 6:144. Non-Patent Document 9: J Biosci Vol. 43, No. 5, December 2018, pp. 1055-1068 | DOI: 10.1007/s12038-018-9811-0.

[The problem to be solved by the invention] As described above, in Patent Document 1, it is expected that Alzheimer's disease can be effectively treated and/or prevented by the synergistic effect of the proposed first active ingredient and the second active ingredient. However, there is a continuing need for rapid treatment and/or prevention effects.

Therefore, the present inventors focused on oligodendritic cells, which are cells responsible for forming myelin sheath, and stellate cells connected to the oligodendritic cells. Stellate cells release nutrient factors to improve the myelin-forming ability of oligodendritic cells, and have the function of removing debris generated by demyelination when re-myelinating, so it is a kind of remyelination In addition to qualitatively indispensable cells, stellate cells can also play various roles such as forming blood-brain barriers, maintaining the constancy of the extracellular environment, and ingesting neurotransmitters. Figure 1 is a diagram conceptually showing stellate cells, which are located between oligodendritic cells and oligodendritic cells and blood vessels. The oligodendritic cells myelinate around the axons of neurons Quality wraps up. It can be seen from Figure 1 that stellate cells, oligodendritic cells, and stellate cells and oligodendritic cells are connected by gap junction channels (denoted as "GJC" in Figure 1). The gap junction channel can promote direct cytoplasmic contact between adjacent cells, and play a role in promoting the movement of low molecular weight molecules related to cell message transmission and metabolism, and the gap junction channel is connected by a hemichannel The hemichannel is formed by 6 oligomer protein subunits (oligomer protein subunits), each provided by adjacent cells and called connexin (represented as "Cx" in the first figure) Constituted. The gap junction channel between stellate cells is composed of connexin 43/connexin 43 and connexin 30/connexin 30. The gap junction channel between oligodendritic cells is composed of connexin 47/connexin 47 and connexin 32. / Connexin 32, the gap junction channel between astrocytes and oligodendritic cells is formed by connexin 43/connexin 47 and connexin 30/connexin 32.

Moreover, the relationship between these connexins and demyelination has been reported so far. Non-Patent Document 5 (J. Neurosci., July 2, 2003･23(13):5963-5973) reported that although there is a lack of either connexin 47 or connexin 32 that can be expressed in oligodendritic cells In one case, the mice can still survive, but the double gene knockout mice lacking these two connexins will die within 6 weeks after birth due to severe abnormalities in the central myelin sheath, thus recording the relationship between gap junctions and Normal formation of myelin is extremely important. The severe abnormalities of the above-mentioned central myelin are characterized by thin or non-existent myelin, hollowing, enlargement of the diameter around axons, death of oligodendritic cells, and loss of axons. sudden. Non-Patent Document 6 (J. Neurosci., June 1, 2003･23(11): 4549-4559) reported the following results: the white matter of the central nervous system was analyzed by electron microscopy in mice with connexin 47 deficiency, It was found that the nerve fibers in the optic nerve site had significant hollowing, the optic nerve site was where nerve fibers, especially axons and oligodendritic cells first contacted and began to form myelin; and, connexin 32/connexin 47 The double-gene knockout mice produced trembling symptoms and, on average, died within 51 days after birth.

Non-Patent Document 7 (J. Neurosci., June 17, 2009･29(24):7743-7752) reported that the double gene knockout mice of connexin 43 and connexin 30 that can be expressed in stellate cells found In addition, extensive abnormalities appeared in the white matter area containing the hollowed oligodendritic cells and the edema in the myelin sheath; the number of CC1-positive mature oligodendritic cells decreased; and the MBP decreased. In addition, Non-Patent Document 7 also It is stated that the loss of stellate cell gap junctions may be related to the white matter disease symptoms with functional defects. On the other hand, Non-Patent Document 8 (Acta Neuropathologica Communications (2018) 6:144) in addition to showing the increase in the expression of connexin 43 and the use of the clinical dementia rating scale (CDR, Clinical Dementia Rating) and simple intelligence The scores of Mini-Mental Status Examination (MMSE) are related to the severity of cognitive impairment, and a working hypothesis has also been proposed. During the onset of occasional Alzheimer’s disease, the brain Increased neural activity will be the cause of nerve cell depletion and ultimately death. This increase in neural activity is due to the chronic and long-term upward regulation of connexin 43.

Furthermore, Non-Patent Document 9 (J Biosci Vol. 43, No. 5, December 2018, pp. 1055-1068 | DO I: 10.1007/s12038-018-9811-0) is a literature review, but It shows that the connexin 43 of stellate cells and the connexin 47 of oligodendritic cells that are connected to their partner cells are very important in maintaining the myelin of the central nervous system and the functions of oligodendritic cells.

Therefore, in order to rapidly treat Alzheimer’s disease through remyelination (restoration of demyelination), it is necessary to activate the signal transmission between stellate cells and oligodendritic cells by the following methods, and to make contact with oligodendritic cells. The expression of p-21.5kDAMBP, which is related to the maturation, differentiation and survival of dendritic cells and the key to the formation of myelin sheath, increases. The expression of connexin 43 and connexin 30, which reduces the amount of connexin 43 that can be used to form a healthy myelin sheath, is aimed at oligodendritic cells, and ensures the amount of connexin that can be used to form a healthy myelin sheath. The performance of 47 and connexin 32, especially the performance of connexin 47, further maintains the sound connection produced by connexin 43 of stellate cells and connexin 47 of oligodendritic cells.

In addition, defects in myelin formation are not only found in Alzheimer’s disease, in addition to dementia and mild cognitive impairment other than Alzheimer’s disease, multiple sclerosis and subacute sclerosis are known. Pan-encephalitis and other demyelinating diseases in which the temporarily formed myelin sheath is destroyed for unknown reasons. In addition, in neurodegenerative diseases such as epilepsy, Down’s syndrome, and amyotrophic lateral sclerosis, and mental disorders Mental illnesses such as symptom have also been pointed out to be related to defects in myelin formation. As long as a method for promptly promoting myelin formation can be found, it is expected that it can be widely and effectively applied to the treatment and/or prevention of the aforementioned diseases in which myelin formation defects occur, and is not limited to the treatment and prevention of Alzheimer’s disease. / Or prevention.

Therefore, the object of the present invention is to provide a composition for promoting the formation of myelin, which can meet the above-mentioned requirements. [Technical means to solve the problem]

The present inventors found that based on the composition containing the first active ingredient and the second active ingredient disclosed in Patent Document 1, and further used the third active ingredient in combination with the composition, the above requirements can be met, and the present invention has been completed. The first active ingredient is at least one compound selected from the group consisting of glycerophosphocholine and its pharmacologically acceptable salts, and the second active ingredient is selected from hesperidin, naringin and the At least one compound in the group consisting of pharmacologically acceptable salts, the third active ingredient is selected from the group consisting of cinnamic esters, cinnamyl alcohol, cinnamic aldehyde, cinnamic acid and these pharmacologically acceptable salts At least one compound in the group consisting of. It is known that cinnamyl ester, cinnamyl alcohol and cinnamaldehyde can be metabolized into cinnamic acid in the human body.

Thus, the present invention relates to a composition for promoting the formation of myelin, which comprises: The first active ingredient is at least one compound selected from the group consisting of glycerophosphocholine and pharmacologically acceptable salts thereof; The second active ingredient is at least one compound selected from the group consisting of hesperidin, naringin and these pharmacologically acceptable salts; and, The third active ingredient is at least one compound selected from the group consisting of cinnamyl ester, cinnamyl alcohol, cinnamaldehyde, cinnamic acid and these pharmacologically acceptable salts.

In the present invention, the effect of the third active ingredient is mainly used to reduce the expression level of connexin 43 that is up-regulated due to the onset of Alzheimer’s disease, mainly by the combined use of the first active ingredient and the second active ingredient , To increase the expression of connexin 32, and by the synergistic effect of the first active ingredient, the second active ingredient, and the third active ingredient, significantly increase the expression of connexin 47, and significantly increase the control of myelin The expression of p-21.5kDaMBP is the key to plasm formation.

Since the third active ingredient is derived from cinnamon, it is also contained in the ginseng Yangrong soup shown in Non-Patent Document 2 and Non-Patent Document 3. However, in these documents, there is no special description about the compound listed as the third active ingredient or the action of the cinnamon containing the third active ingredient. In addition, there are previous documents on the treatment and/or prevention of Alzheimer's disease, which use compounds listed as the third active ingredient or cinnamon containing the third active ingredient. For example, Patent Document 2 (Japanese Patent Laid-Open No. 2006-206584) describes the Aβ aggregation inhibitory effect and the decomposing effect of agglutinated Aβ produced by cinnamon bark and/or its extract. Patent Document 3 (Japanese Patent Publication No. 2010-106001) describes a method for preventing or ameliorating Alzheimer’s disease, which is a peroxisome proliferation-responsive receptor activator prepared by using cinnamic acid and its derivatives and The activator was carried out. Patent Document 4 (Japanese Patent Application Laid-Open No. 2016-079125) describes a β-secretase inhibitory action of cinnamic aldehyde, and Patent Document 5 (Japanese Patent Application Laid-Open No. 2016-088851) describes A composition for inhibiting phosphorylation of Tau protein and a treatment method for Alzheimer's disease using the composition, the composition containing cinnamon. However, in these documents, there is no record related to the expression of connexin or p-21.5kDaMBP. In addition, these documents do not describe the combined use of the first active ingredient, the second active ingredient, and the third active ingredient.

As mentioned above, the composition of the present invention can significantly increase the expression level of p-21.5kDaMBP, which is the key to the formation of myelin. Therefore, the composition of the present invention can be widely and effectively applied to the above-mentioned defects in myelin formation. The treatment and/or prevention of the disease is not limited to the treatment and/or prevention of Alzheimer’s disease.

The composition for promoting the formation of myelin of the present invention preferably contains 10-200 mg of the first active ingredient, 30-350 mg of the second active ingredient, and 0.6-350 mg of the first active ingredient as the daily dosage per adult. 15mg of the third active ingredient.

The composition for promoting the formation of myelin of the present invention preferably contains both hesperidin and naringin as the second effective ingredient. In addition, in the composition for promoting myelin formation of the present invention, it is preferable that at least a part of the second active ingredient is tangerine peel derived from Wenzhou tangerine, an extract of the orange peel, Jabara citrus, Jabala citrus extract and any of these mixtures are included. The tangerine peel derived from the Wenzhou tangerine contains a large amount of hesperidin and naringin, and it is possible to easily include hesperidin and naringin in the composition. Jabala mandarin is a citrus that contains a large amount of naringin but hardly contains hesperidin, and can easily contain naringin in the composition. Furthermore, in the composition for promoting myelin formation of the present invention, it is preferable that at least a part of the third active ingredient is contained in the form of cinnamon bark or an extract of cinnamon bark. Cinnamon bark or a cinnamon bark extract particularly contains a large amount of cinnamic aldehyde, and it is possible to easily include cinnamic aldehyde in the composition. Of course, the compounds themselves listed as the second effective ingredient and the third effective ingredient can also be used to obtain the composition for promoting myelin formation of the present invention.

When the composition for promoting myelin formation of the present invention is formulated, it may be in the form of a formulation containing all of the first active ingredient, the second active ingredient, and the third active ingredient, or it may be a kit In a form, the set is composed of a plurality of medicines, and the medicines include any one or two of the first active ingredient, the second active ingredient, and the third active ingredient. The formulation agent and each agent constituting the set may contain ingredients other than the first active ingredient, the second active ingredient, and the third active ingredient within a range that adversely affects the effects of the present invention. In the case of the kit form, there is no restriction on the order of administration, and any medicine can be administered first.

The composition for promoting the formation of myelin of the present invention can be administered in any form of oral or parenteral administration, and can also be administered in the form of pharmaceuticals, quasi-drugs, health foods (including nutritional supplements), etc. Form to vote. The composition for promoting the formation of myelin of the present invention can be continuously administered without worrying about harmful side effects, and from the viewpoint of simplicity, it is preferable to prepare a healthy food for oral administration. [Effects of the invention]

According to the composition for promoting the formation of myelin sheath of the present invention, the synergistic effect of the first effective ingredient, the second effective ingredient, and the third effective ingredient can ensure the amount that can be used to form a healthy myelin sheath. The performance of connexin 43, connexin 30, connexin 47, and connexin 32 ensure the sound connection formed by connexin 43 of stellate cells and connexin 47 of oligodendritic cells, and significantly increase and The maturation, differentiation and survival of oligodendritic cells are related to the expression of p-21.5kDaMBP, which is the key to myelin formation. Thereby, myelin formation can proceed quickly.

The composition for promoting the formation of myelin of the present invention includes: The first active ingredient is at least one compound selected from the group consisting of glycerophosphocholine and pharmacologically acceptable salts thereof; The second active ingredient is at least one compound selected from the group consisting of hesperidin, naringin and these pharmacologically acceptable salts; and, The third active ingredient is at least one compound selected from the group consisting of cinnamyl ester, cinnamyl alcohol, cinnamaldehyde, cinnamic acid and these pharmacologically acceptable salts. In the present invention, the effect of the third active ingredient is mainly used to reduce the expression level of connexin 43 that is up-regulated due to the onset of Alzheimer’s disease, mainly by the combined use of the first active ingredient and the second active ingredient, Increase the expression level of connexin 32, and through the synergistic effect of the first active ingredient, the second active ingredient, and the third active ingredient, significantly increase the expression level of connexin 47, and significantly increase the amount of expression with oligodendritic cells Maturation, differentiation and survival are related to the expression of p-21.5kDaMBP, which is the key to myelin formation. In addition, since the composition of the present invention can significantly increase the expression level of p-21.5kDaMBP, which is the key to myelin formation, the composition of the present invention can be widely and effectively applied to diseases in which myelin formation defects occur. The treatment and/or prevention is not limited to the treatment and/or prevention of Alzheimer's disease.

Α-GPC as the first active ingredient can be synthesized by, for example, hydrolyzing phospholipid choline obtained by extracting and refining soybean oil with lipase, and it can also be purchased as a commercially available product. α-GPC can be used in the form of pharmacologically acceptable salts, such as hydrochloride, phosphate, citrate, acetate, and carbonate, and can also be used in the form of solvates. As the second active ingredient, hesperidin and naringin can also be purchased commercially, and pharmacologically acceptable salt forms such as hydrochloride, phosphate, citrate, acetate, and carbonate can be used. A solvate form can also be used, and in order to further improve the water solubility, a transglycosidated form can be used. Transglycosidation can be hydrolyzed into hesperidin or naringin in the body. Furthermore, for the second active ingredient, it is also possible to use a large amount of hesperidin and naringin derived from tangerine tangerine peel from Wenzhou, or an extract obtained by subjecting the peel to hot water extraction or other extraction treatments. Since dry extracts dried with hot water extracts are commercially available, it is easy to use dry extracts. Furthermore, for the second active ingredient, it is also possible to use Jabala citrus containing a large amount of naringin but almost no hesperidin, or an extract obtained by subjecting the Jabala citrus to an extraction process such as hot water extraction. Dried products or extracts of citrus species other than Wenzhou mandarin orange and Jabala mandarin can also be used as the second effective ingredient. As the third active ingredient, cinnamyl ester, cinnamyl alcohol, cinnamaldehyde, and cinnamic acid can also be purchased commercially, and pharmacologically acceptable salt forms such as hydrochloride, phosphate, citrate, and acetate can be used. , Carbonate, solvate form can also be used. In addition, as the third active ingredient, an extract obtained by extraction treatment such as cinnamon bark or hot water extraction can be used.

The composition for promoting the formation of myelin of the present invention is a combination of the first active ingredient, the second active ingredient, and the third active ingredient in a desired amount, and these can be in either oral or parenteral form. The administration can also be administered in the form of pharmaceuticals, quasi-drugs, health foods (including nutritional supplements), etc. The composition for promoting the formation of myelin of the present invention can be continuously administered without worrying about harmful side effects, and from the viewpoint of simplicity, it is preferable to prepare a nutritional supplement for oral administration.

When it is formulated into a formulation, it may be in the form of a formulation containing all of the first active ingredient, the second active ingredient, and the third active ingredient, or it may be in the form of a kit consisting of a plurality of drugs. The medicine contains any one or two of the first active ingredient, the second active ingredient, and the third active ingredient. In the form of a set, the order of administration is not restricted. Any medicine can be administered first, and each medicine can be administered continuously or at intervals.

In the case of formulations, or in the case of each drug constituting the set, it can be formulated into the following forms according to the purpose of use: capsules, chewables, lozenges, powders, granules, syrups, etc. for oral administration Preparations for pre-use; or, preparations for parenteral administration such as injections, drips, enema. In the case of a kit form, the dosage form of each agent may be the same or different.

In the process of manufacturing these preparations, generally speaking, the first effective ingredient, the second effective ingredient, and the third effective ingredient can be mixed with pharmacologically acceptable excipients selected according to the purpose of use, and the dosage form is For specific shapes, the excipients are: solid excipients such as dextrin and lactose; liquid excipients such as water, saline, and glycerin. These preparations may also contain other ingredients within the range that does not adversely affect the effects of the present invention, such as vitamins such as vitamin C and vitamin E; minerals such as iron and zinc; Functional ingredients such as dicosahexaenoic acid (DHA); additives other than the above ingredients, which are commonly used as binders, tackifiers, lubricants, flavors, sweeteners, buffers, preservatives, fungicides, etc. Additives.

Furthermore, the composition for promoting the formation of myelin of the present invention can also be formulated in various foods and beverages such as mineral water and refreshing water; dairy products such as cheese and yogurt; jelly, biscuits, candies, etc. Dim sum etc.

The dosage of the composition for promoting the formation of myelin of the present invention can be based on the symptoms, age, weight, etc. of the patient suffering from the myelin formation deficiency disease, or the dosage form, number of times of administration, and other drugs. It is appropriately determined by the combination and the like. As the daily dosage per adult, it is generally preferable to combine 10 to 200 mg of the first active ingredient, 30 to 350 mg of the second active ingredient, and 0.6 to 15 mg of the third active ingredient. Active ingredients. When both hesperidin and naringin are used as the second active ingredients, the daily dosage per adult is preferably in the range of 20-300 mg hesperidin and 4-50 mg naringin内组合。 Within the combination. Among the cinnamyl esters, cinnamyl alcohol, cinnamaldehyde, and cinnamic acid as the third active ingredient, because cinnamyl esters, cinnamyl alcohol, and cinnamaldehyde are metabolized into cinnamic acid in the body, even when cinnamyl ester, cinnamyl alcohol, and cinnamaldehyde are used When two or more kinds of cinnamic acid are used as the third active ingredient, the daily dosage per adult should be 0.6 to 1.5 mg in total. In the present invention, even with such a small amount of administration, a sufficient improvement effect can be expected, and harmful side effects can be avoided.

The composition of the present invention significantly increases the expression level of p-21.5kDaMBP, which is related to the maturation, differentiation, and survival of oligodendritic cells, and grasps the key to myelin formation, so it can be rapidly remyelinated. Treatment and/or prevention of Alzheimer’s disease. In addition, because it significantly increases the expression level of p-21.5kDaMBP, which is the key to myelin formation, the composition of the present invention can be widely and effectively used to treat and/or prevent myelin formation. Defective diseases. As examples of such diseases, dementia or mild cognitive impairment other than Alzheimer's disease include: multiple sclerosis, neuromyelitis optica, and acute diffuse encephalomyelitis and other inflammatory demyelinating diseases; Progressive multifocal leukoencephalopathy, HTLV-1 (Human T-lymphotropic virus 1, human T-lymphotropic virus) associated myelitis, subacute sclerosing panencephalitis and other viral demyelinating diseases; osmotic demyelination syndrome Toxic demyelinating diseases such as subacute compound spinal cord degeneration and toxic leukoencephalopathy; demyelinating diseases in which the temporarily formed myelin will be destroyed for unknown reasons, which are arteriosclerosis and chronic hypertension Such vascular diseases, etc.; epilepsy and neurodegenerative diseases such as Down’s syndrome and amyotrophic lateral sclerosis; even schizophrenia, bipolar disorder, severe depression, autism, and lack of attention Mental diseases such as hyperactivity, obsessive-compulsive disorder, post-traumatic stress syndrome and drug dependence. [Example]

The following examples are used to illustrate the present invention, but the present invention is not limited to the following examples.

(1) Research using Alzheimer's disease model mice (A) Animals and breeding environment The Alzheimer's disease model mice used in the following experiments were transgenic with Swedish mutant APP (APP) _K670N/M671L ) gene transgenic mouse Tg2576, which was purchased from The Jackson Laboratory in the United States. In a breeding facility with a room temperature of 25±1°C and a relative humidity of 55±1%, the purchased ones are reared under a 12-hour/12-hour light-dark lighting cycle (lights on at 7:00 and lights off at 19:00) Tg2576 mice. The animal experiment is carried out in the animal experiment facility of Keio University in accordance with the Keio University Animal Experiment Code. The animal experiment code is based on the NIH Code which stipulates the correct use and management of laboratory animals.

(B) Administration of α-GPC/hesperidin and naringin/cinnamaldehyde and cinnamic acid 15-month-old Tg2576 mice (average body weight of 30 g) were divided into 4 groups, and artificial bait was administered to each group and raised for 1 month. In addition, the amount of artificial bait shown below is the amount given to each mouse daily, and it is confirmed that each mouse eats it within one day. Control group: artificial bait of 5.26g feed GHN administration group: an artificial bait prepared by mixing 20 mg of a composition with 5.26 g of feed. The composition is 0.833 mg of α-GPC (G), 1.458 mg of hesperidin (H) in total, and Naringin (N) is mixed with conventional additives such as reduced maltose syrup and starch. C administration group: 5 mg (the total of cinnamic aldehyde and cinnamic acid is 0.06625 mg) of cinnamon powder (containing 1.3% by mass of cinnamic aldehyde and 0.025% by mass of cinnamic acid) is mixed with 5.26 g of feed. GHN+C administration group: 20 mg of the composition and 5 mg (the total of cinnamic aldehyde and cinnamic acid is 0.06625 mg) of cinnamon powder (containing 1.3% by mass of cinnamic aldehyde and 0.025% by mass of cinnamic acid) mixed with 5.26 g of feed An artificial bait made of 0.833 mg of α-GPC (G), 1.458 mg of hesperidin (H) and naringin (N) in total, and customary additives such as reduced maltose syrup and starch Blended.

In this dosing experiment, the amount of α-GPC, the total amount of hesperidin and naringin, and the total amount of cinnamic aldehyde and cinnamic acid in this dosing experiment are used to convert the coefficient "10" ( Refer to "Regul.Toxicol.Pharmacol.24,108-120") to convert into the dosage of a 50kg human adult, which is the following dosage: 138.8mgα-GPC/day, 243mg hesperidin and naringin in total /Day and the total amount of 11.0mg cinnamic aldehyde and cinnamic acid per day.

(C) Solubilization of the brain with cell lysis buffer After feeding for 1 month, the control group mice, the GHN administration group mice, the C administration group mice, and GHN+C administration were administered under inhalation anesthesia using isoflurane (manufactured by Wako Pure Chemical Industries, Ltd.) Group mice were euthanized, and the whole brain was removed immediately after decapitation. Measure 1/2 weight of the extracted brain, add 10 mL of ice-cold phosphate buffered saline (PBS) per 1 g of brain weight, and further add 10 mL of specific liquid, then use a homogenizer to solubilize the brain. The liquid is a 100-fold dilution of 100x protease inhibitor cocktail (Protease inhibitor cocktail, manufactured by Merck Co., Ltd.) and 10% by volume of RIPA buffer (20 mM ginseng (hydroxymethyl) aminomethane (Tris, Nacalai Tesque)). Co., Ltd.), 150 mM NaCl (manufactured by Sigma Orange Japan Co., Ltd.), 1% poly(oxyethylene) octyl phenyl ether (trade name Tx-100, manufactured by Sigma Orange Japan Co., Ltd.) , 1% sodium deoxycholate (manufactured by Sigma Orange Japan Co., Ltd.), and 0.1% sodium lauryl sulfate (SDS, manufactured by Sigma Orange Japan Co., Ltd.)). The obtained homogenized brain suspension was recovered in a 1.5 mL Eppendorf tube, and centrifuged at 4° C. and 20000×G for 20 minutes. After centrifugation, the supernatant was separated as the soluble part and the precipitate was separated as the insoluble part, and then stored at -80°C.

(D) Electric swimming (SDS-PAGE) Laemmli sample buffer (manufactured by Merck Millipore, containing 62.5mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, 0.01% bromophenol blue), will be obtained from the above step (C) The soluble part was diluted 4 times and placed in a constant temperature bath at 100°C for 10 minutes to obtain a sample for SDS-PAGE.

SDS-PAGE is performed using gradient gel (Gradient gel, manufactured by TFCO Co., Ltd.). Install the gel plate in the electromobility device, and pour the migration buffer (25mM Tris (manufactured by Nacalai Tesque Co., Ltd.), 192mM glycine (manufactured by Sigma Orich Japan Co., Ltd.), 0.1% After SDS (manufactured by Sigma Orange Japan Co., Ltd., pH 8.3), 25 μL of the sample for SDS-PAGE was injected into the sample well, and electrophoresis was performed at room temperature and 5 mA for 30 minutes, and the Electrophoresis was performed for 90 minutes under the condition of 25mA.

(E) Immunoblotting method Put a polyvinylidene fluoride (PVDF) membrane (trade name: immobilon-p; pore size 0.45μm; manufactured by Merck Millipore) into the transfer solution (31mM Tris, Nacalai Tesque Co., Ltd.) Manufacture), 0.24M glycine (manufactured by Sigma Orich Japan Co., Ltd.), 20% methanol (Wako Pure Chemical Industries Co., Ltd.)) for 15 minutes to make the recovered PVDF membrane and ) The obtained colloidal contact after electrophoresis is applied, and a current is passed ^{under the conditions of 20 mA/cm 2} at room temperature to transfer the protein to the PVDF membrane. Use a blocking buffer to block the transferred PVDF membrane for 1 hour at room temperature. The blocking solution is to dissolve 5% skimmed milk powder (Defco Co., Ltd.) in a 10-fold dilution 10x Tris buffered saline (TBS, 0.5M Tris (pH8.1, manufactured by Nacalai Tesque Co., Ltd.), 1.5M NaCl (manufactured by Sigma Orich Japan Co., Ltd.), 1N hydrochloric acid (Wako Pure Chemical Industries Co., Ltd. The company)).

As the primary antibody, an internal control mouse monoclonal antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (manufactured by Proteintech Co., Ltd., 1/1000 dilution), and an anti-p-MBP monoclonal antibody were used Antibody PC12 (manufactured by Merck Millipore, diluted 1/500), Purified Mouse Anti-Connexin-43 (purified mouse anti-connexin 43 antibody, manufactured by BD Transduction Laboratories), Connexin 30 Polyclonal Antibody (more than 30 strains of connexin) Antibodies, manufactured by Thermo Fisher Scientific), Connexin 47 Monoclonal Antibody (manufactured by Invitrogen), and Connexin 32 Polyclonal Antibody (manufactured by Invitrogen), resist the resistance at 4°C The treated PVDF undergoes an antibody reaction overnight. For the dilution of each antibody, the above blocking solution was used, and a blocking solution made by dissolving 1% skimmed milk powder in 10 times diluted 10xTBS was used, and the PVDF membrane after the primary antibody reaction was performed three times for 10 minutes each Of washing. Then, using Alkaline Phosphatase-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (manufactured by Jackson ImmunoResearch Laboratories) that has been diluted 800 to 1000 times with blocking solution, the secondary antibody reaction was performed at room temperature for 2 hours, and Using a blocking solution made by dissolving 1% skimmed milk powder in 10 times diluted 10xTBS, the PVDF membrane after the secondary antibody reaction is washed three times for 10 minutes, and then the alkaline phosphatase reaction is carried out. Antigen detection. The alkaline phosphatase reaction is carried out by the following method: 5-bromo-4-chloro-3-indole phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) as a substrate for alkaline phosphatase is used as a color developing agent. Nitro blue tetrazolium (manufactured by Wako Pure Chemical Industries, Ltd.) and buffer (0.1M Tris (manufactured by Nacalai Tesque Co., Ltd.), 0.1M NaCl (manufactured by Sigma Orich Japan Co., Ltd.), 0.05 M MgCl ₂ (manufactured by Sigma Orange Japan Co., Ltd.)), the reaction is performed under light shielding for 30 minutes to 1 hour. In addition, the results of antigen detection are evaluated as the average value ± standard deviation of 3 independent experiments.

(F) Experimental results Figure 2 is a graph showing the abundance of connexin 30 normalized by GAPDH using an internal reference in the form of an abundance ratio in which the abundance in the brain of a control group mouse is set to 1. It can be seen from Fig. 2 that although the expression level of connexin 30 will be increased by the administration of GHN and C, respectively, the increase in the expression level is not significant. In addition, the increase in connexin 30 caused by the administration of GHN+C is roughly equivalent to the sum of the increase caused by the administration of GHN and the increase caused by the administration of C, so it is considered Has a synergistic effect.

Figure 3 is a graph showing the abundance of connexin 43 normalized by GAPDH using an internal reference in the form of an abundance ratio in which the abundance in the brain of a control group mouse is set to 1. It can be seen from Figure 3 that the expression level of connexin 43 is reduced to 0.86 times the expression level of the control group due to the administration of GHN. In contrast, the reduction in the expression level of connexin 43 caused by the administration of C was extremely large, even 0.41 times the expression level of the control group. Since it has been reported that the onset of Alzheimer's disease will cause the up-regulation of connexin 43 (see Non-Patent Document 8), the reduction in the expression level of connexin 43 is better for the treatment and/or prevention of Alzheimer's disease of. Although the expression level of connexin 43 produced by the administration of GHN+C is 0.62 times, although it is not as good as the case of administering C alone, by using C in addition to GHN, the expression level produced by the administration of GHN can still be compared. Obtain the result of reduced performance.

Figure 4 is a graph showing the abundance of connexin 32 normalized by GAPDH using an internal reference in the form of an abundance ratio in which the abundance in the brain of a control group mouse is set to 1. It can be seen from Figure 4 that although the expression level of connexin 32 is significantly increased by the administration of GHN, the increase in the expression level caused by the administration of C is very small. In addition, the increase in expression of connexin 32 caused by the administration of GHN+C is roughly equivalent to the sum of the increase caused by the administration of GHN and the increase caused by the administration of C, so it is considered to have a phase Multiplication effect. Connexin 32 can be expressed in oligodendritic cells (refer to Figure 1). The increase in connexin 32 produced by the administration of GHN is consistent with the result of the enhancement of maturation and differentiation of oligodendritic cells produced by the composition of Patent Document 1.

Figure 5 is a graph showing the abundance of connexin 47 normalized by GAPDH using an internal reference in the form of an abundance ratio in which the abundance in the brain of a control group mouse is set to 1. It can be seen from Fig. 5 that the expression level of connexin 47 will increase to 1.18 times the expression level of the control group due to the administration of GHN, and will increase to 1.10 times the expression level of the control group due to the administration of C. Connexin 47 can be expressed in oligodendritic cells (refer to Figure 1). The increase in connexin 47 produced by the administration of GHN is consistent with the result of the enhancement of maturation and differentiation of oligodendritic cells produced by the composition of Patent Document 1. And unexpectedly, in addition to GHN, the use of C can also increase the expression of connexin 47 by 1.52 times that of the control group. This increase is significantly greater than the sum of the increase due to the GHN administration and the increase due to the C administration, and is therefore considered to be a multiplicative effect. It has been reported that the connexin 43 of stellate cells and the connexin 47 of oligodendritic cells that are connected to its partner cells are very important in maintaining the myelin of the central nervous system and the functions of oligodendritic cells (see Non-Patent Document 9), therefore, the synergistic effect is excellent for treating and/or preventing Alzheimer's disease through remyelination.

Figure 6 shows a photo of PVDF membrane after p-MBP was detected by alkaline phosphatase reaction. Tg2576 mice have four MBP isoforms with molecular masses of 21.5kDa, 18.5kDa, 17kDa and 14kDa. It can be seen from Figure 6 that the expression levels of the isomers p-18.5kDaMBP, p-17kDaMBP and p-14kDaMBP in the GHN administration group, C administration group and GHN+C administration group, compared with the control group There is no difference. In contrast, only the expression level of p-21.5kDaMBP will vary greatly depending on the type of administration. In addition, it has been reported that when demyelination occurs, especially p-21.5kDaMBP decreases, and the expression of p-21.5kDaMBP increases to remyelination (see Patent Document 1). Therefore, it is necessary to pay attention to the increase in the expression level of p-21.5kDaMBP, which allows remyelination to proceed.

Figure 7 is a graph showing the abundance of p-21.5kDaMBP normalized by GAPDH using an internal reference in the form of an abundance ratio that sets the abundance in the brain of a control group mouse to 1. It can be seen from Figure 7 that although the expression level of p-21.5kDaMBP is still only 1.07 times that of the control group due to the administration of C, the expression level produced by the administration of GHN is significantly increased, even reaching the control group's expression level. 1.28 times the performance. In addition, by using C in addition to GHN, the expression level of p-21.5kDaMBP even increased 1.61 times that of the control group. This increase is significantly greater than the sum of the increase due to the administration of GHN and the increase due to the administration of C. Therefore, it is considered to be a synergistic effect of GHN and C. This synergistic effect is excellent for treating and/or preventing Alzheimer's disease through remyelination.

From the above results, it can be seen that by combining α-GPC as the first active ingredient, hesperidin and naringin as the second active ingredient, and cinnamic aldehyde and cinnamic acid as the third active ingredient for treatment And/or the composition for preventing Alzheimer’s disease, mainly through the action of cinnamic aldehyde and cinnamic acid, reduces the expression of connexin 43 that is up-regulated due to the onset of Alzheimer’s disease, mainly through the combined use α-GPC and hesperidin and naringin increase the expression of connexin 32, and the synergistic effects of α-GPC, hesperidin and naringin, cinnamic aldehyde and cinnamic acid significantly increase connexin 32 47, and significantly increase the expression of p-21.5kDaMBP, which is related to the maturation, differentiation and survival of oligodendritic cells and masters the formation of myelin sheath. Therefore, the composition of the present invention is extremely effective for treating and/or preventing Alzheimer's disease.

(2) Application for people with Down syndrome According to the gene expression study of the pathogenesis of Down's syndrome using model mice of Down's syndrome, it is reported that there are abnormalities of oligodendritic cells and myelin sheath (2016 Mar 16;89(6):1208-1222. doi: 10.1016/j.neuro n.2016.01.042). On the other hand, because the composition of the present invention can significantly increase the expression level of p-21.5kDaMBP, which is the key to myelin formation, it can be expected that the composition of the present invention is effective for improving Down’s syndrome. . Therefore, for 7 adults with degenerative symptoms but no organic syndrome with Down’s syndrome, after taking the following composition daily for 6 months, the evaluation of social degenerative symptoms and cognitive function was carried out. Contains: 40 mg of α-GPC, 70 mg of hesperidin and naringin in total, and cinnamon extract powder containing cinnamic acid and cinnamic aldehyde in a total of 0.6 mg.

The social degeneration symptoms are evaluated using the following social degeneration symptom scale developed by Kuroki and others. As long as 1 point is added for each item that meets a separate item, it can be judged as follows: 6 points or more means obvious degradation, 4 to 5 points high degradation degree, and 3 points or less means no degradation. On the other hand, the evaluation of cognitive function is carried out using the points of the Cognitive Function Evaluation Scale (CS-DS) developed by the British team Carla Startin and others to evaluate Down’s syndrome patients. The higher the score of the evaluation, the better the cognitive function can be evaluated. Figures 8 and 9 show the evaluation results of 7 patients with Down syndrome (indicated by A to G). [Table 1] Social Degeneration Symptom Scale 1 Movement becomes slow 2 Become lack of expression 3 Reduced conversations and speeches 4 Lack of response in interpersonal relationships 5 Less interest in things 6 What can be done in the past becomes impossible 7 Problems with urination and defecation 8 Staying at home and not going out 9 Sleep disturbance 10 Tell the same thing repeatedly or ask questions repeatedly 11 Emotional ups and downs become intense

In the evaluation of social degenerative symptoms, with the exception of 1 case showing deterioration (increased points), improvement results were confirmed in 6 cases. In addition, in the evaluation of cognitive function, results of improvement in 3 cases, no change in 3 cases, and deterioration in 1 case were confirmed. From these results, it is expected that the degenerative symptoms of Down's syndrome can be improved by taking the composition of the present invention. [Industrial availability]

With the present invention, it is possible to treat or prevent diseases in which myelin formation defects occur.

no

Figure 1 is a diagram conceptually showing oligodendritic cells surrounding the axons of neurons and enveloping myelin, and stellate cells between the oligodendritic cells and blood vessels. Figure 2 shows the disease model mice for Alzheimer's disease, administered with a composition containing α-GPC, hesperidin and naringin (GHN), cinnamon containing cinnamic aldehyde and cinnamic acid (C), In the case of both (GHN+C), the result of investigating the amount of connexin 30 in the mouse brain. Figure 3 shows the disease model mice for Alzheimer's disease, administered with a composition containing α-GPC, hesperidin and naringin (GHN), cinnamon containing cinnamic aldehyde and cinnamic acid (C), And both (GHN+C), a graph showing the results of investigating the amount of connexin 43 in the mouse brain. Figure 4 shows the disease model mice for Alzheimer’s disease, administered with a composition containing α-GPC, hesperidin and naringin (GHN), cinnamon containing cinnamic aldehyde and cinnamic acid (C), And both (GHN+C), a graph showing the results of investigating the amount of connexin 32 in the mouse brain. Figure 5 shows the disease model mice for Alzheimer’s disease, administered with a composition containing α-GPC, hesperidin and naringin (GHN), cinnamon containing cinnamic aldehyde and cinnamic acid (C), In the case of both (GHN+C), the result of investigating the amount of connexin 47 in the mouse brain. Figure 6 shows the disease model mice for Alzheimer’s disease, administered with a composition containing α-GPC, hesperidin and naringin (GHN), cinnamon containing cinnamic aldehyde and cinnamic acid (C), In the case of both (GHN+C), the result of detecting the expression of p-MBP in the mouse brain by the immunoblotting method. Figure 7 shows the disease model mice for Alzheimer's disease, administered with a composition containing α-GPC, hesperidin and naringin (GHN), cinnamon (C) containing cinnamic aldehyde and cinnamic acid, In the case of both (GHN+C), the result of investigating the amount of p-21.5kDaMBP in the mouse brain. Figure 8 is a graph showing the results of evaluating changes in social degeneration symptoms when the composition of the present invention is administered to a patient with Down syndrome. Figure 9 is a graph showing the results of evaluating changes in cognitive function when the composition of the present invention is administered to a patient with Down syndrome.

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Claims (9)

A composition for promoting the formation of myelin sheath, which comprises: The first active ingredient is at least one compound selected from the group consisting of glycerophosphocholine and pharmacologically acceptable salts thereof; The second active ingredient is at least one compound selected from the group consisting of hesperidin, naringin and these pharmacologically acceptable salts; and, The third active ingredient is at least one compound selected from the group consisting of cinnamyl ester, cinnamyl alcohol, cinnamaldehyde, cinnamic acid and these pharmacologically acceptable salts. The composition for promoting the formation of myelin as described in claim 1, which is a composition for treating and/or preventing Alzheimer's disease. The composition for promoting the formation of myelin according to claim 1 or 2, wherein the daily dosage per adult includes: 10 to 200 mg of the first active ingredient and 30 to 350 mg of the second Active ingredient and 0.6-15mg of the third active ingredient. The composition for promoting myelin formation according to any one of claims 1 to 3, wherein the second active ingredient contains hesperidin and naringin. The composition for promoting the formation of myelin according to any one of claims 1 to 4, wherein at least a part of the second active ingredient is tangerine peel derived from Wenzhou tangerine, an extract of the orange peel, and It is contained in any form of extracts of Bala mandarin, Jabala mandarin, and mixtures thereof. The composition for promoting myelin formation according to any one of claims 1 to 5, wherein at least a part of the third active ingredient is contained in the form of cassia bark or an extract of cinnamon bark. The composition for promoting the formation of myelin according to any one of claims 1 to 6, wherein the composition is formulated to include all of the first active ingredient, the second active ingredient, and the third active ingredient Agent for formulation. The composition for promoting myelin formation according to any one of claims 1 to 6, wherein the composition is formulated as a kit, the kit is composed of a plurality of drugs, and the drug Contains any one or two of the first effective ingredient, the second effective ingredient, and the third effective ingredient. The composition for promoting the formation of myelin according to any one of claims 1 to 8, wherein the composition is administered as a health food.

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