WO2022075096A1 - Raldh2 expression enhancing agent - Google Patents

Raldh2 expression enhancing agent Download PDF

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WO2022075096A1
WO2022075096A1 PCT/JP2021/035162 JP2021035162W WO2022075096A1 WO 2022075096 A1 WO2022075096 A1 WO 2022075096A1 JP 2021035162 W JP2021035162 W JP 2021035162W WO 2022075096 A1 WO2022075096 A1 WO 2022075096A1
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Prior art keywords
raldh2
cells
present
raldh2 expression
expression enhancer
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PCT/JP2021/035162
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French (fr)
Japanese (ja)
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優美 白井
喜範 片倉
辰也 金
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株式会社 資生堂
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Priority to CN202180055005.2A priority Critical patent/CN116096389A/en
Priority to JP2022555371A priority patent/JPWO2022075096A1/ja
Publication of WO2022075096A1 publication Critical patent/WO2022075096A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/63—Arthropods
    • A61K35/64—Insects, e.g. bees, wasps or fleas
    • A61K35/644—Beeswax; Propolis; Royal jelly; Honey
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
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    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K36/18—Magnoliophyta (angiosperms)
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    • A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
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    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention provides a RALDH2 expression enhancer.
  • Treg Regulatory T cells
  • Treg is known to differentiate in the thymus and intestine, and retinoic acid has been reported as a factor contributing to Treg differentiation.
  • Tretinoin acid is a metabolite of vitamin A, and it has been reported that a specific lactic acid bacterium is effective as a retinoin acid-producing agent (Patent Document 5).
  • Patent Document 5 a specific lactic acid bacterium is effective as a retinoin acid-producing agent
  • RALDH2 an enzyme that an enzyme called RALDH2 is involved in the metabolism of retinoic acid, and that increasing the expression of RALDH2 in dendritic cells via intestinal cells promotes differentiation into Tregs.
  • Non-Patent Document 2 Although specific lactic acid bacteria have been reported (Non-Patent Document 2), the search for substances that promote the expression of RALDH2 has not been carried out much.
  • Japanese Unexamined Patent Publication No. 2019-80497 Japanese Unexamined Patent Publication No. 2017-127846 Japanese Unexamined Patent Publication No. 2018-188436 Japanese Unexamined Patent Publication No. 2018-188408 Japanese Patent No. 6543614
  • An object of the present invention is to provide a RALDH2 expression enhancer.
  • the present inventors have quercetin, kaempferol, honeybee, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, barley young leaf, goji, baobab fruit, broccoli. , Moringa, broccoli, goji, Houttuynia cordata, maca, olive leaf, one or more components selected from the group, have been found to function as RALDH2 expression enhancers. Furthermore, the present inventors have also found that these substances act on monocyte cells via intestinal cells.
  • RALDH2 expression can be promoted by administration of the RALDH2 expression enhancer of the present invention. According to the present invention, it is possible to provide a composition or an internal preparation containing a RALDH2 expression enhancer.
  • FIG. 1 is a schematic diagram of Experiment 3.
  • FIG. 2 shows the expression level of RALDH2 in THP-1 cells when each sample was added in Experiment 3 as a relative value with 1 when a negative control (DMSO only) was added.
  • Non-Patent Document 3 Various cytokines are produced from dendritic cells (DC) by antigens that have invaded from the intestinal tract. Undifferentiated T cells present in the intestinal membrane are differentiated into activated (T1, T2, T17) or inhibitory T cells (Treg) by stimulation with these cytokines. It has been reported that Treg suppresses self-tolerance and excessive immune response by suppressing the immune response (Non-Patent Document 3).
  • RALDH2 increasing the expression of RALDH2 in dendritic cells via intestinal cells suppresses the production of inflammatory T cells, promotes differentiation into Tregs, and maintains a good immune balance. It has been suggested that it promotes and inhibits the migration of T cells to the skin (Non-Patent Documents 1, 4, and 5).
  • RALDH2 retinaldehyde dehydrogenase 2, retinaldehyde dehydrogenase
  • Retinoic acid CAS number: 302-79-4
  • retinoic acid is an important substance for controlling cell proliferation and differentiation at the developmental stage, and it also contributes to the improvement of defense power in the mucous membrane, and in recent years, it has been clarified that it contributes to the differentiation of Treg.
  • the RALDH2 expression enhancer of the present invention promotes Treg differentiation by promoting RALDH2 expression of monocyte cells via the intestinal tract, especially by oral administration, improving immune balance, suppressing allergies, suppressing inflammatory T cell activity, and skin. It is expected that the condition will improve.
  • the improvement of the skin condition refers to the improvement of the skin condition by enhancing the expression of RALDH2 in monocyte cells such as suppression of inflammation and allergic reaction in the skin.
  • the improvement of the skin condition may be mediated by promotion of Treg differentiation by enhancing RALDH2 expression in monocyte cells, suppression of inflammatory T cell production, inhibition of T cell migration to the skin, and the like.
  • the enhancement of RALDH2 expression can mean that the expression level of RALDH2 is increased when the RALDH2 expression enhancer is given, as compared with the state (control) where nothing is given, for example. For example, it may mean an increase with a statistically significant difference (eg Student's t-test) with a significance level of 5%.
  • the enhancement of RALDH2 expression in the present invention means that the expression level of RALDH2 when a RALDH2 expression enhancer is given is, for example, 5% or more, 10% or more, as compared with the state (control) in which nothing is given.
  • RALDH2 is not limited to any known technique, for example, a method of introducing a GFP gene downstream of the RALDH2 promoter of monocyte cells and determining a change in EGFP fluorescence derived from RALDH2p-EGFP as described in Examples. Can be obtained by.
  • the RALDH2 expression enhancer of the present invention enhances the expression of RALDH2 in monocyte cells, especially via intestinal cells.
  • "Enhancement of RALDH2 expression in monocytes via intestinal cells” means that the active ingredient of the present invention is administered to intestinal cells and absorbed by the intestinal cells so that the same ingredient as the active ingredient is directly produced as monocytes. Reaching the cells, or reaching the monocyte cells in a state where the active ingredient is absorbed by the intestinal cells and decomposed or modified, or the intestinal cells take up the active ingredient to release another different component, and the different components It refers to the ultimate enhancement of RALDH2 expression in monocyte cells via direct or indirect pathways such as acting on monocyte cells.
  • enhancing RALDH2 expression of monocyte cells via intestinal cells there is an example of enhancing RALDH2 expression of monocyte cells by oral administration.
  • Increasing RALDH2 expression in monocyte cells by oral administration means that the active ingredient of the present invention is absorbed through the intestine by ingesting the RALDH2 expression enhancer of the present invention orally or enterally, and is absorbed from the intestine. It means that the taken up substance acts directly or indirectly, and finally the expression of RALDH2 in monocyte cells is enhanced.
  • Non-Patent Documents 6-8 carnosine taken up from the intestine does not directly act on the brain, but activates CREB in intestinal cells, resulting in enhanced BDNF production. Indirect effects such as BDNF activating nerve cells and improving brain function have been reported. Furthermore, it has been suggested that such cerebral-intestinal correlation activation is mediated by exosomes. In other words, it is unclear what kind of action the component has on the target cells even if a certain component is given to the intestine, so give the test substance to the intestinal cells and then confirm what kind of action it will have on the target cells. There is a need.
  • Such actions via intestinal cells can be measured by various methods including in vivo, in vitro, ex vivo and the like.
  • the expression level of RALDH2 in monocyte cells such as THP-1 when the test substance is administered to a culture medium containing intestinal cells such as Caco-2 as in the examples in the present specification and the culture medium is given. It can be determined by an in vitro method such as requesting.
  • the measuring method is not limited to the above method, and any other method may be adopted.
  • the Transwell method in which a test substance is added to one side of a layer of intestinal cells and the test substance is passed through this layer to measure the expression level of RALDH2 in monocyte cells existing on the opposite side of the layer, or in animals such as humans.
  • An in vivo method such as measuring the activity of RALDH2 in monocytes after oral ingestion may be adopted.
  • the present invention relates to kelsetin, kaempferol, honeybee, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, young barley leaf, dokudami, baobab fruit, broccoli, moringa, obaco, goji.
  • a RALDH2 expression enhancer containing one or more components selected from the group consisting of broccoli, maca, and olive leaf as an active ingredient.
  • the present invention relates to kelsetin, kaempferol, honeybee, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, young barley leaf, goji, baobab fruit, broccoli, moringa, obaco,
  • a composition comprising one or more ingredients selected from the group consisting of goji, ashitaba, maca, and olive leaves as active ingredients.
  • the RALDH2 expression enhancer and composition of the present invention may be in the form of an oral preparation or an enteral preparation, or may be for beautiful skin.
  • Quercetin (CAS number: 849061-97-8) used in the present invention is a kind of flavonoid and is a component contained in vegetables such as onions. It is known to have antioxidant, anti-inflammatory, blood flow improving, and lipolytic effects. It may be quercetin itself or it may be in the form of glycosides such as rutin and quercitrin.
  • Kaempferol (CAS number: 520-18-3) used in the present invention is a kind of flavonoid and is a component contained in tea and vegetables. Known for its antioxidant, anti-inflammatory, anti-cancer and anti-diabetic effects. Kaempferol itself may be in the form of glycosides such as kaempferitrin and astragalin.
  • Coix lacryma-jobi Linne var. Mayuen Stapf (Gramineae) used in the present invention is a grain of the genus Coix of the Gramineae family. It is preferable to use the seed portion excluding the seed coat of Coix seeds.
  • the bark is said to be effective in improving skin warts and whitening.
  • the bee pollen used in the present invention is a pollen grain (pollen load) collected by honeybees. There are reports that it contains nutrients such as vitamins and minerals. It may be any pollen such as cistus, oak, rose, echium, and chrysanthemum pollen. The place where bee pollen is collected is not limited to Europe such as Spain and Asia such as China.
  • Narco lily used in the present invention is a perennial herb belonging to the genus Solomon's seal in the family Liliaceae. It is preferable to use it as a crude drug with dried rhizomes. It is said to be effective for nourishing and tonic.
  • the hop (Humulus lupulus) used in the present invention is a vine perennial plant of the family Cannabaceae. It is preferable to use cones. It is said to have a healthy stomach and a sedative effect.
  • the chenpi used in the present invention is a dried mandarin orange peel.
  • Citrus (genus Citrus) is an evergreen shrub of the genus Citrus of the family Rutaceae, Sapindaceae. It is said to be effective in improving blood flow, indigestion, loss of appetite, etc.
  • the cabbage (Brassica oleracea var. Capitata) used in the present invention is a vegetable belonging to the Brassicaceae genus Brassicaceae. It is preferable to use cabbage leaves. Cabbage is rich in dietary fiber, vitamin U, and vitamin C, and is said to have the effect of keeping the gastrointestinal health.
  • the celery used in the present invention is an annual plant of the Umbelliferae family (Apium graveolens var. Dulce). It is preferable to use celery foliage. It is said that the foliage of celery has the effects of preventing anorexia, stabilizing the mind, and suppressing the rise in blood pressure.
  • Okra Abelmoschus esculentus used in the present invention is a plant belonging to the genus Abelmoschus manihot. It is preferable to use fruits. It has been reported that fruits contain mucin and pectin and have an effect of adjusting gastrointestinal tone.
  • the parsley (Petroselinum crispum) used in the present invention is a biennial plant of the Umbelliferae family. It is preferable to use foliage.
  • the foliage is rich in nutrients such as vitamins and minerals, and is said to be effective against stomach upset, recovery from fatigue, and anemia.
  • Asparagus spp. Used in the present invention is a general term for various species belonging to the genus Asparagus. It is preferable to use stems.
  • the stem is rich in nutrients such as oligosaccharides, vitamins and minerals, and is known to have diuretic, stomachic and intestinal regulating effects.
  • Angelica keiskei used in the present invention is a plant belonging to the genus Ashitaba in the Umbelliferae family. It is preferable to use foliage. It is rich in vitamins, minerals and dietary fiber, and is known to have constipation-preventing, diuretic, hypertensive-preventing and tonic effects.
  • the clove (Syzygium aromaticum) used in the present invention is a flower bud of the tree Clove of the Myrtaceae family. Clove is eaten as a spice and is said to have pain and stomachic effects.
  • the young barley leaves used in the present invention are young leaves before heading of barley (Hordeum vulgare), which is a plant belonging to the genus Hordeum of the Gramineae family. It contains various vitamins, minerals, folic acid, SOD enzyme, etc., has a high nutritional effect, and is used as a material for green juice and the like.
  • Houttuynia cordata Thunberg used in the present invention is a perennial plant belonging to the genus Houttuynia in the family Houttuynia. It is preferable to use the above-ground part of Houttuynia cordata. It is used as an internal medicine and tea, and is said to be effective for gastrointestinal diseases, detoxification, food poisoning, diarrhea, constipation, diuresis, etc.
  • the baobab fruit Used in the present invention is a fruit of a tree belonging to the genus Baobab of the Malvaceae family.
  • Baobab fruit contains vitamins, minerals, dietary fiber, etc., and is known as superfruit.
  • Broccoli (Brassica oleracea var. Italica) used in the present invention is a green-yellow vegetable belonging to the genus Brassicaceae. It is preferable to use an above-ground part or the like. It contains sulforaphane and has been reported to have effects such as cancer prevention and suppression of Helicobacter pylori.
  • Moringa (Moringa oleifera LAM. (M. pterygosperma)) used in the present invention is a plant belonging to the genus Moringa of the Moringa family, and it is preferable to use Moringa leaves. It is used for food and medicine, and is said to be effective for nutritional supplementation. It contains various vitamins and minerals and is known as a superfood.
  • the plantain (Plantago asiatica) used in the present invention is a perennial plant belonging to the genus Plantago of the family Plantain family, and it is preferable to use the whole plantago. It is rich in dietary fiber and is said to have effects such as stomach upset, swelling improvement, anti-inflammatory, diuretic, antidiarrhea, and cough.
  • the wolfberry fruit used in the present invention is a fruit of wolfberry (Lycium chinense), which is a deciduous shrub of the Solanaceae family, and contains zeaxanthin, betaine, polyphenol, and the like. Lycium chinense has been reported to have a nourishing tonic effect, a blood flow improving effect, a whitening effect, and a DNA damage repairing effect.
  • Candelabra aloe used in the present invention is a kind of succulent plant of the genus Aloe. It is preferable to use the leaves of Kidachi aloe. It has been reported that external use is effective against burns and cuts, and internal use is effective in improving gastrointestinal pain and constipation.
  • Maca (Lepidium meyenii) is a perennial plant of the Brassicaceae family. It is preferable to use roots. It contains amino acids, minerals, dietary fiber, vitamins, etc., and is known to have effects such as nutritional tonicity, endurance improvement, and anti-fatigue.
  • Olive leaves are the leaves of olive (Olea europaea), an evergreen tree of the family Oleaceae. Olive leaves contain polyphenols such as oleuropein and are known to have antibacterial and viral effects.
  • the compound such as quercetin and kaempferol may be synthesized, a commercially available product may be used, or a compound contained in a crude drug or the like may be used.
  • Hatomugi bee pollen, narco lily, hops, chenpi, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, barley young leaves, Houttuynia cordata, baobab fruit, broccoli, moringa, obaco, goji nuts, kidachi aloe, maca, olive leaves
  • Crude drugs are known substances, which can be easily dried, purified, extracted, etc. by known methods, and commercially available products are easily available.
  • It can be used raw or dried, but it can also be used as an extract, a dried product, a dried powder, a powdered raw material, a juice squeezed liquid, or the like from the viewpoint of usability, formulation, and the like.
  • a dried product e.g., a dried powder
  • a powdered raw material e.g., a powdered raw material
  • a juice squeezed liquid e.g., a juice squeezed liquid, or the like
  • the extraction method of the extract can be performed, for example, by solvent extraction.
  • solvent extraction the whole plant or various parts (leaves, flowers, roots, etc.) of the plant are dried as needed, shredded or crushed as needed, and then an aqueous extractant, water, for example, cold water.
  • Hot water hot water having a boiling point or a lower temperature, or a hydrous organic solvent, an organic solvent such as ethanol, methanol, ether, 1,3-butylene glycol, etc. It is extracted by selecting it at room temperature or by heating it.
  • the extraction method is not limited to solvent extraction, and may be a conventional method known in the art, and the extraction method of the extract and the form of the extract used in the present invention are not limited to the effect of the present invention.
  • the form of the extract may be not only the extract itself but also appropriately diluted or concentrated by a conventional method, and is a powdery or lumpy solid obtained by drying the extract. May be good.
  • a water-containing lower alcohol such as water-containing ethanol
  • the water content in that case is, for example, 0 to 10v / v%, 10 to 40v / v%, 20 to 30v / v%, 30. It may be ⁇ 50v / v%, 50-80v / v%, 80-99.5v / v%, or the like.
  • a dry powder As a method for obtaining a dry powder, the whole plant or various parts (leaves, flowers, roots, etc.) of the plant are shredded or crushed and then dried, or the plant is dried and then shredded or crushed to obtain a dry powder. There is a way to get it. Further, a method of shredding or crushing the plant, subjecting it to fermentation or enzyme treatment, drying it, and further crushing it to a predetermined particle size, if necessary, can be appropriately adopted.
  • the RALDH2 expression enhancer of the present invention is preferably taken orally or enterally, it does not exclude other routes of administration such as transdermal administration.
  • the RALDH2 expression enhancer of the present invention is administered by various routes of administration, it is preferable to apply the active ingredient of the present invention in an amount such that the RALDH2 expression enhancer effect is sufficiently exhibited.
  • the blending amount of the components of the present invention can be appropriately determined according to their types, purposes, forms, usage methods and the like.
  • the dry weight of the plant or its solvent extract is about 0.001 to 100% by weight based on the total amount of the oral preparation or the enteral preparation. It is preferably prepared so as to be, more preferably about 0.01 to 10% by weight, still more preferably about 0.1 to 1.0% by weight.
  • the concentration in the intestine may be adjusted to be about 1 to 10000 ⁇ g / ml, more preferably about 1 to 5000 ⁇ g / ml, and even more preferably about 5 to 2000 ⁇ g / ml.
  • RALDH2 expression enhancer of the present invention When used as an oral preparation or an enteric preparation, kercetin, kaempferol, adlay, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, etc. per adult.
  • the intake of asparagus, ashitaba, adlay, young barley leaves, Houttuynia cordata, baobab fruit, broccoli, moringa, obaco, goji, kidachi aloe, maca, olive leaf plant or its solvent extract is, for example, 1 mg per day.
  • the frequency of ingestion is not limited, but it may be taken once, but according to one example, for example, once every two weeks, once a week, once every three days, once every two days, one day. It can be taken once, twice a day, three times a day, four times a day, and the like. In addition, it may be ingested each time, continuously, or intermittently, for example, at intervals of several months.
  • the RALDH2 expression enhancer of the present invention can be contained in a composition for oral or enteral ingestion, for example, a food composition.
  • a composition for oral or enteral ingestion for example, a food composition.
  • powdery, liquid, solid such as tablets, granules, granules, pastes, gels and the like can be arbitrarily selected.
  • Additives can be arbitrarily selected and used in combination with the RALDH2 expression enhancer and composition of the present invention, if necessary. Excipients and the like can be included as the additive.
  • the excipient may be any starch as long as it is usually used in the desired dosage form, for example, starches such as wheat starch, rice starch, corn starch, potato starch, dextrin, cyclodextrin, and crystalline celluloses.
  • Lactose Lactose, starch, sugar, reduced malt sugar, water candy, fructo-oligosaccharide, emulsified oligosaccharide and other sugars, sorbitol, erythritol, xylitol, lactitol, mannitol and other sugar alcohols.
  • excipients can be used alone or in combination of two or more.
  • colorants preservatives, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, pH adjusters, etc. Can be selected and used as appropriate.
  • the present invention relates to quercetin, kaempferol, honeybee, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, young barley leaf, houttuynia cordata, baobab fruit, broccoli, moringa, obaco, goji.
  • a method of promoting RALDH2 expression in monocytic cells by administering one or more components selected from the group consisting of Houttuynia cordata, maca, and olive leaf, for example, by oral or enteric route.
  • the present invention also includes quercetin, kaempferol, honeybee, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, young barley leaves, houttuynia cordata, baobab fruit, broccoli, moringa, and obaco.
  • quercetin kaempferol, honeybee, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, young barley leaves, houttuynia cordata, baobab fruit, broccoli, moringa, and obaco.
  • To improve immune balance through enhanced RALDH2 expression in monocytic cells for example by administration of one or more components selected from the group consisting of goji, ashitaba, maca and olive leaves, for example by oral or enteric route.
  • the method of the present invention is a method
  • the present invention relates to quercetin, kaempferol, honeybee, bee pollen, narco lily, hop, husk, cabbage, celery, okra, parsley, asparagus, in the manufacture of pharmaceuticals such as oral or enteric agents for improving immune balance. Also provided is the use of one or more ingredients selected from the group consisting of Ashitaba, Chouji, young barley leaves, Houttuynia cordata, Baobab fruit, broccoli, moringa, obaco, goji, kidachi aloe, maca and olive leaves.
  • the present invention comprises quercetin, kaempferol, ashitaba, bee pollen, broccoli, hops, broccoli, for use in methods of improving immune balance by promoting RALDH2 expression in monocytic cells, for example by oral or enteral administration.
  • quercetin kaempferol, ashitaba, bee pollen, broccoli, hops, broccoli
  • peanuts kaempferol
  • ashitaba bee pollen
  • broccoli hops
  • broccoli for use in methods of improving immune balance by promoting RALDH2 expression in monocytic cells, for example by oral or enteral administration.
  • Experiment 1 Preparation of samples As candidate samples for RALDH2 expression enhancer, quercetin, kaempferol, honeybee, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, barley young leaves, Houttuynia cordata, Baobab fruit, broccoli, moringa, cabbage, goji, ashitaba, maca, and olive leaves were prepared as shown in the table below.
  • a total of 147 types of candidate samples were prepared, including naturally derived components such as extracts of animals and plants and synthetic components.
  • the sample was adjusted to 100 ⁇ g / ml using DMSO.
  • DMSO containing nothing was used as a negative control.
  • Experiment 2 Construction of a screening system for components that promote RALDH2 expression
  • Experiment 2-1 Culture of human acute monocytic leukemia-derived cell line THP-1 cells Human acute monocyte leukemia-derived cell line THP-1 cells Using. THP-1 cells were subjected to 10% Fetal bovine serum (FBS; Life Technologies, CA, USA) -added RPMI 1640 medium (Nissui, Tokyo, Japan), Petri dish (FALCON, Tokyo, Japan) at 37 ° C, 5%. Subcultured in the presence of CO 2 .
  • FBS Fetal bovine serum
  • RPMI 1640 medium Nasui, Tokyo, Japan
  • Petri dish FALCON, Tokyo, Japan
  • RPMI 1640 medium is prepared by dissolving 5.1 g of RPMI 1640 powder in 500 mL of Milli-Q water, streptomycin sulfate 0.1 g titer (Meiji. Tokyo, Japan), penicillin G potassium 100,000 U (Meiji), 10 % ⁇ 3 (Wako, Osaka, Japan) 9 mL was added, and a 0.22 ⁇ m filter sterilized (Toyo Roshi kaisha, Tokyo, Japan) was used as RPMI 1640 medium and stored at 4 ° C.
  • Experiment 2-2 Culture of human colon cancer-derived cell line Caco-2 cells Human colon cancer-derived Caco-2 cells were used as a human intestinal epithelial model. Caco-2 cells were cultured in a cell culture dish (Greiner) using Dulbecco's Modified Eagle Medium (DMEM) (Nissui, Tokyo, Japan) containing deactivated 10% Fetal Bovine Serum (FBS) (Life Technologies, CA, USA). Bio-one, Tokyo, Japan) was subcultured under the conditions of 37 ° C and 5% CO 2 .
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS Fetal Bovine Serum
  • DMEM medium 10.0 g of DMEM powder is dissolved in 1 L of Milli-Q water, 100 U / mL penicillin (Meiji), 0.1 mg / mL streptomycin (Meiji), 1M HEPES 2.38 g (DOJINDO, Kumamoto, Japan). ), 10% NaHCO 3 2.0 g (Wako, Osaka, Japan) was added, and 0.22 ⁇ m filter sterilized (Tokyo Roshi Kaisha, Tokyo, Japan) was used.
  • Experiment 2-3 Preparation of plasmid (RALDH2-EGFP) and introduction into THP-1 cells
  • the plasmid RALDH2-EGFP was constructed and introduced into THP-1 cells prepared in Experiment 2-1.
  • the human RALDH2 promoter (Forward: ATTAATAACTGACTTACCAGCCTCGT (SEQ ID NO: 1), Reverse: GCTAGCGGCGATCTCGCTGGAAGTCA (SEQ ID NO: 2)
  • the amplified fragment was cloned into pEGFP-C3 (TaKaRa, Japan) and its CMV promoter was removed by digestion with restriction enzymes AseI and NheI.
  • the obtained plasmid was designated as RALDH2p-EGFP.
  • This plasmid was stably transfected into THP-1 cells prepared in Experiment 2-1 to obtain THP-1 (RALDH2p-EGFP), which was used for evaluation of human RALDH2 promoter activity. Then, the obtained RALDH2p-EGFP was induced to differentiate by PMA (48hPMA100ng / ml).
  • Experiment 3 Screening of test substance Caco-2 cells cultured in Experiment 2-2 were seeded on a 24-well plate at 2.0 ⁇ 10 5 cells / mL, and 24 hours later, each sample prepared in Experiment 1 as shown in FIG. was added to the final concentration of 10 ⁇ g / mL. 100 ⁇ L of the supernatant of Caco-2 cells was added to THP-1 (RALDH2p-EGFP) prepared in Experiment 2-3. Twenty-four hours after the addition of the supernatant, the EGFP fluorescence intensity of THP-1 cells was evaluated with IN Cell Analyzer 2200 (Cytiva) to evaluate the promoter activity of RALDH2.
  • THP-1 RALDH2p-EGFP

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Abstract

Provided is a novel RALDH2 expression enhancing agent.　The present invention provides a RALDH2 expression enhancing agent containing, as an active ingredient, one or a plurality of components selected from the group consisting of quercetin, kaempferol, adlay, bee pollen, Polygonatum falcatum, hop, Citrus unshiu peel, cabbage, celery, okra, parsley, asparagus, Angelica keiskei, clove, barley grass, Houttuynia cordata, baobab fruit, broccoli, moringa, Plantago asiatica, Lycium chinense berry, Aloe arborescens, maca, and olive leaf.

Description

ＲＡＬＤＨ２発現増強剤RALDH2 expression enhancer
　本発明はRALDH2発現増強剤を提供する。 The present invention provides a RALDH2 expression enhancer.
　アレルギー疾患に罹患している患者数は年々増加している。アレルギーは体の免疫バランス（Th1・Th2）の崩壊が主因となり引き起こされる疾患であり、人体に於いては免疫に関わる細胞や抗体の約6割が腸に集まっている。体内の免疫バランス（Th1・Th2）を整える制御性T細胞（以下、Tregと称する）が近年発見され、Tregの誘導により過剰な免疫応答が制御されることが分かってきた。例えば、マウスにおいてTregを誘導したところアレルギー性皮膚炎や炎症性腸疾患の症状が緩和したことが報告されている。Tregの分化を誘導する物質として特定の乳酸菌、リコピン、トレハロース等が有効でることが報告されている（特許文献１～４）。 The number of patients suffering from allergic diseases is increasing year by year. Allergy is a disease caused mainly by the disruption of the body's immune balance (Th1 and Th2). In the human body, about 60% of cells and antibodies involved in immunity are concentrated in the intestine. Regulatory T cells (hereinafter referred to as Treg) that regulate the immune balance (Th1 and Th2) in the body have been discovered in recent years, and it has become clear that the induction of Treg controls an excessive immune response. For example, it has been reported that induction of Treg in mice alleviated the symptoms of allergic dermatitis and inflammatory bowel disease. It has been reported that specific lactic acid bacteria, lycopene, trehalose and the like are effective as substances that induce the differentiation of Treg (Patent Documents 1 to 4).
　Tregは胸腺や腸で分化する事が知られており、Treg分化に寄与する因子としてレチノイン酸が報告されている。レチノイン酸はビタミンAの代謝物質であり、特定の乳酸菌がレチノイン酸産生剤として有効であることが報告されている（特許文献５）。更に、レチノイン酸の代謝にはRALDH2という酵素が関与し、腸管細胞を介し樹状細胞のRALDH2の発現を上昇させるとTregへの分化が促されることが示唆されている（非特許文献１）。しかしながら、特定の乳酸菌が報告されている(非特許文献２)ものの、RALDH2の発現を促進する物質の探索はあまり行われていない。 Treg is known to differentiate in the thymus and intestine, and retinoic acid has been reported as a factor contributing to Treg differentiation. Tretinoin acid is a metabolite of vitamin A, and it has been reported that a specific lactic acid bacterium is effective as a retinoin acid-producing agent (Patent Document 5). Furthermore, it has been suggested that an enzyme called RALDH2 is involved in the metabolism of retinoic acid, and that increasing the expression of RALDH2 in dendritic cells via intestinal cells promotes differentiation into Tregs (Non-Patent Document 1). However, although specific lactic acid bacteria have been reported (Non-Patent Document 2), the search for substances that promote the expression of RALDH2 has not been carried out much.
特開2019-80497号公報Japanese Unexamined Patent Publication No. 2019-80497 特開2017-127846号公報Japanese Unexamined Patent Publication No. 2017-127846 特開2018-188436号公報Japanese Unexamined Patent Publication No. 2018-188436 特開2018-188408号公報Japanese Unexamined Patent Publication No. 2018-188408 特許第6543614号公報Japanese Patent No. 6543614
　本発明の課題は、RALDH2発現増強剤の提供にある。 An object of the present invention is to provide a RALDH2 expression enhancer.
　本発明者らは、鋭意研究の結果、ケルセチン、ケンフェロール、ハトムギ、ビーポーレン、ナルコユリ、ホップ、陳皮、キャベツ、セロリ、オクラ、パセリ、アスパラガス、アシタバ、チョウジ、大麦若葉、ドクダミ、バオバブフルーツ、ブロッコリー、モリンガ、オオバコ、クコの実、キダチアロエ、マカ、オリーブ葉からなる群より選択される1つまたは複数の成分が、RALDH2発現増強剤として機能することを見出した。さらに、本発明者らは、これらの物質は腸管細胞を介して単球細胞に作用することも見出した。以上の発見により、以下の発明を完成するに至った：
（１）ケルセチン、ケンフェロール、ハトムギ、ビーポーレン、ナルコユリ、ホップ、陳皮、キャベツ、セロリ、オクラ、パセリ、アスパラガス、アシタバ、チョウジ、大麦若葉、ドクダミ、バオバブフルーツ、ブロッコリー、モリンガ、オオバコ、クコの実、キダチアロエ、マカ、オリーブ葉からなる群より選択される１つまたは複数の成分を有効成分として含有するRALDH2発現増強剤。
（２）前記RALDH2発現増強剤は、腸管細胞を介して単球細胞のRALDH2発現を増強する、（１）に記載のRALDH2発現増強剤。
（３）前記RALDH2発現増強剤は、内服により単球細胞のRALDH2発現を増強する、（１）又は（２）に記載のRALDH2発現増強剤。
（４）（１）～（３）のいずれか１項に記載のRALDH2発現増強剤を含有する組成物。 As a result of diligent research, the present inventors have quercetin, kaempferol, honeybee, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, barley young leaf, goji, baobab fruit, broccoli. , Moringa, broccoli, goji, Houttuynia cordata, maca, olive leaf, one or more components selected from the group, have been found to function as RALDH2 expression enhancers. Furthermore, the present inventors have also found that these substances act on monocyte cells via intestinal cells. The above discoveries have led to the completion of the following inventions:
(1) Kercetin, kaempferol, honeybee, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, young barley leaf, dokudami, baobab fruit, broccoli, moringa, obaco, goji , Kidachi aloe, okra, RALDH2 expression enhancer containing one or more components selected from the group consisting of olive leaves as an active ingredient.
(2) The RALDH2 expression enhancer according to (1), wherein the RALDH2 expression enhancer enhances the RALDH2 expression of monocyte cells via intestinal cells.
(3) The RALDH2 expression enhancer according to (1) or (2), wherein the RALDH2 expression enhancer enhances the RALDH2 expression of monocyte cells by oral administration.
(4) A composition containing the RALDH2 expression enhancer according to any one of (1) to (3).
　本発明のRALDH2発現増強剤の投与により、RALDH2発現を促進することができる。本発明によれば、RALDH2発現増強剤を含有する組成物や内服剤を提供することができる。 RALDH2 expression can be promoted by administration of the RALDH2 expression enhancer of the present invention. According to the present invention, it is possible to provide a composition or an internal preparation containing a RALDH2 expression enhancer.
図１は、実験３の概略図である。FIG. 1 is a schematic diagram of Experiment 3. 図２は、実験３において各試料を添加した場合のTHP-1細胞におけるRALDH2発現量を、陰性対照（DMSOのみ）を添加した場合を1とした相対値として示す。FIG. 2 shows the expression level of RALDH2 in THP-1 cells when each sample was added in Experiment 3 as a relative value with 1 when a negative control (DMSO only) was added.
　腸管より侵入した抗原によって種々のサイトカインが樹状細胞(DC)から産生さる。これらサイトカインの刺激により腸管膜に存在する未分化のT細胞が活性化型（T1,T2,T17）もしくは抑制型T細胞(Treg)へと分化する。Tregは免疫応答を抑制することで自己寛容や過剰な免疫応答を抑制することが報告されている（非特許文献３）。ここで、腸管細胞を介し樹状細胞のRALDH2の発現を上昇させると炎症性T細胞の産生を抑制し、Tregへの分化が促され免疫バランスが良好に保たれる、粘膜における抗体IgA産生の促進、皮膚へのT細胞の遊走を阻害する、ことが示唆されている（非特許文献１、４、５）。 Various cytokines are produced from dendritic cells (DC) by antigens that have invaded from the intestinal tract. Undifferentiated T cells present in the intestinal membrane are differentiated into activated (T1, T2, T17) or inhibitory T cells (Treg) by stimulation with these cytokines. It has been reported that Treg suppresses self-tolerance and excessive immune response by suppressing the immune response (Non-Patent Document 3). Here, increasing the expression of RALDH2 in dendritic cells via intestinal cells suppresses the production of inflammatory T cells, promotes differentiation into Tregs, and maintains a good immune balance. It has been suggested that it promotes and inhibits the migration of T cells to the skin (Non-Patent Documents 1, 4, and 5).
　RALDH2（retinaldehyde dehydrogenase 2、レチンアルデヒド脱水素酵素）は、レチナールをレチノイン酸に変換する酵素である。レチノイン酸（CAS番号：302-79-4）は、ビタミンAの代謝産物であり、体内に取り込まれたビタミンAはレチナールに変換され、レチナールがさらにレチノイン酸に変換され体内で様々な作用を及ぼすことが知られている。レチノイン酸は、発生段階における細胞の増殖や分化の制御に重要な物質であることが知られており、粘膜における防御力の向上にも寄与し、近年ではTregの分化に寄与することも明らかになっている（非特許文献１、５）。 RALDH2 (retinaldehyde dehydrogenase 2, retinaldehyde dehydrogenase) is an enzyme that converts retinal into retinoic acid. Retinoic acid (CAS number: 302-79-4) is a metabolite of vitamin A, and vitamin A taken into the body is converted to retinal, which is further converted to retinoic acid and exerts various actions in the body. It is known. It is known that retinoic acid is an important substance for controlling cell proliferation and differentiation at the developmental stage, and it also contributes to the improvement of defense power in the mucous membrane, and in recent years, it has been clarified that it contributes to the differentiation of Treg. (Non-Patent Documents 1 and 5).
　本発明のRALDH2発現増強剤は、とりわけ内服により腸管を介し単球細胞のRALDH2発現を促進することによりTregの分化が促進され免疫バランスの改善、アレルギーの抑制、炎症性T細胞の活性抑制、肌状態の改善などが期待される。本発明において、肌状態の改善とは、皮膚における炎症やアレルギー反応の抑制といった単球細胞におけるRALDH2発現の増強による皮膚状態の改善を指す。皮膚状態の改善は、単球細胞におけるRALDH2発現の増強によるTregの分化促進、炎症性T細胞の産生抑制、皮膚へのT細胞の遊走阻害等を介するものであってもよい。 The RALDH2 expression enhancer of the present invention promotes Treg differentiation by promoting RALDH2 expression of monocyte cells via the intestinal tract, especially by oral administration, improving immune balance, suppressing allergies, suppressing inflammatory T cell activity, and skin. It is expected that the condition will improve. In the present invention, the improvement of the skin condition refers to the improvement of the skin condition by enhancing the expression of RALDH2 in monocyte cells such as suppression of inflammation and allergic reaction in the skin. The improvement of the skin condition may be mediated by promotion of Treg differentiation by enhancing RALDH2 expression in monocyte cells, suppression of inflammatory T cell production, inhibition of T cell migration to the skin, and the like.
　RALDH2発現の増強とは、例えば何も付与していない状態（コントロール）に比べて、RALDH2発現増強剤を付与した場合のRALDH2の発現量が増加していることを意味し得る。例えば、有意水準を5％とした統計学的有意差（例えばスチューデントのｔ検定）をもって増加していることを意味することもある。あるいは、本発明におけるRALDH2発現の増強とは、例えば何も付与していない状態（コントロール）に比べて、RALDH2発現増強剤を付与した場合のRALDH2の発現量が、例えば5％以上、10％以上、20％以上、30％以上、40％以上、50％以上、60％以、70％以上、80％以上、90％以上、100％以上、200％以上、300％以上、400％以上、又は500％以上亢進していることを意味し得る。RALDH2の発現量は、任意の公知技術、例えば、限定されないものの、実施例に記載のように単球細胞のRALDH2プロモーター下流にGFP遺伝子を導入しRALDH2p-EGFPに由来するEGFP蛍光の変化を求める方法により求めることができる。 The enhancement of RALDH2 expression can mean that the expression level of RALDH2 is increased when the RALDH2 expression enhancer is given, as compared with the state (control) where nothing is given, for example. For example, it may mean an increase with a statistically significant difference (eg Student's t-test) with a significance level of 5%. Alternatively, the enhancement of RALDH2 expression in the present invention means that the expression level of RALDH2 when a RALDH2 expression enhancer is given is, for example, 5% or more, 10% or more, as compared with the state (control) in which nothing is given. , 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 200% or more, 300% or more, 400% or more, or It can mean that it is increased by 500% or more. The expression level of RALDH2 is not limited to any known technique, for example, a method of introducing a GFP gene downstream of the RALDH2 promoter of monocyte cells and determining a change in EGFP fluorescence derived from RALDH2p-EGFP as described in Examples. Can be obtained by.
　本発明のRALDH2発現増強剤は、とりわけ腸管細胞を介して単球細胞のRALDH2の発現を増強する。「腸管細胞を介して単球細胞のRALDH2発現を増強する」とは、本発明の有効成分を腸管細胞に投与し、腸管細胞に吸収されることにより、有効成分と同一の成分が直接単球細胞に到達する、あるいは有効成分が腸管細胞に吸収され分解又は修飾された状態で単球細胞に到達する、あるいは腸管細胞が有効成分を取り込むことにより別の異なる成分を放出し、その異なる成分が単球細胞に作用するといった直接的又は間接的な経路を介して最終的に単球細胞におけるRALDH2発現が増強されることを指す。「腸管細胞を介して単球細胞のRALDH2発現を増強する」ことの一例として、内服により単球細胞のRALDH2発現を増強すること等が挙げられる。「内服により単球細胞のRALDH2発現を増強する」とは、本発明のRALDH2発現増強剤を経口又は経腸的に摂取することにより、本発明の有効成分が腸管を介して吸収され、腸から取り込まれた物質が直接的又は間接的に作用し最終的に単球細胞におけるRALDH2発現が増強されることを指す。 The RALDH2 expression enhancer of the present invention enhances the expression of RALDH2 in monocyte cells, especially via intestinal cells. "Enhancement of RALDH2 expression in monocytes via intestinal cells" means that the active ingredient of the present invention is administered to intestinal cells and absorbed by the intestinal cells so that the same ingredient as the active ingredient is directly produced as monocytes. Reaching the cells, or reaching the monocyte cells in a state where the active ingredient is absorbed by the intestinal cells and decomposed or modified, or the intestinal cells take up the active ingredient to release another different component, and the different components It refers to the ultimate enhancement of RALDH2 expression in monocyte cells via direct or indirect pathways such as acting on monocyte cells. As an example of "enhancing RALDH2 expression of monocyte cells via intestinal cells", there is an example of enhancing RALDH2 expression of monocyte cells by oral administration. "Increasing RALDH2 expression in monocyte cells by oral administration" means that the active ingredient of the present invention is absorbed through the intestine by ingesting the RALDH2 expression enhancer of the present invention orally or enterally, and is absorbed from the intestine. It means that the taken up substance acts directly or indirectly, and finally the expression of RALDH2 in monocyte cells is enhanced.
　例えば、ある成分が内服により腸から取り込まれてもその物質が直接標的細胞へ到達されるわけではなく腸管細胞から分泌された別の物質が標的細胞に到達し間接的に作用することがある。例えば、非特許文献６～８に記載のように、腸から取り込まれたカルノシンが直接的に脳に作用を及ぼすのではなく、腸管細胞においてCREBを活性化し、その結果、BDNF産生が増強され、BDNFが神経細胞を活性化し脳の機能改善をもたらすというような間接的な作用が報告されている。さらには、このような脳腸相関活性化はエクソソームが介在するという示唆もされている。つまり、腸にある成分を与えてもその成分が標的とする細胞でどのような作用を及ぼすのかは不明であるため、被験物質を腸管細胞に与え、その後標的細胞においていかなる作用を奏するのか確認する必要がある。 For example, even if a certain component is taken up from the intestine by oral administration, the substance does not reach the target cell directly, but another substance secreted from the intestinal cell may reach the target cell and act indirectly. For example, as described in Non-Patent Documents 6-8, carnosine taken up from the intestine does not directly act on the brain, but activates CREB in intestinal cells, resulting in enhanced BDNF production. Indirect effects such as BDNF activating nerve cells and improving brain function have been reported. Furthermore, it has been suggested that such cerebral-intestinal correlation activation is mediated by exosomes. In other words, it is unclear what kind of action the component has on the target cells even if a certain component is given to the intestine, so give the test substance to the intestinal cells and then confirm what kind of action it will have on the target cells. There is a need.
　このような腸管細胞を介した作用は、in vivo、in vitro、ex vivo等を含む各種方法で測定できる。例えば、本明細書における実施例のように被験物質をCaco-2等の腸管細胞を含む培養液に投与し、その培養液を与えた場合のTHP-1等の単球細胞におけるRALDH2発現量を求めるといったin vitroの方法により決定することができる。しかしながら測定方法は上記方法に限定されず、他の任意の方法を採用してもよい。例えば、腸管細胞の層の一方の側に試験物質を添加し、この層を通過して、層の逆側に存在する単球細胞におけるRALDH2発現量を測定するTranswell法や、ヒト等の動物に経口的に摂取させた後に、単球におけるRALDH2の活性を測定するといったin vivoの方法を採用してもよい。 Such actions via intestinal cells can be measured by various methods including in vivo, in vitro, ex vivo and the like. For example, the expression level of RALDH2 in monocyte cells such as THP-1 when the test substance is administered to a culture medium containing intestinal cells such as Caco-2 as in the examples in the present specification and the culture medium is given. It can be determined by an in vitro method such as requesting. However, the measuring method is not limited to the above method, and any other method may be adopted. For example, the Transwell method, in which a test substance is added to one side of a layer of intestinal cells and the test substance is passed through this layer to measure the expression level of RALDH2 in monocyte cells existing on the opposite side of the layer, or in animals such as humans. An in vivo method such as measuring the activity of RALDH2 in monocytes after oral ingestion may be adopted.
　本発明は、ケルセチン、ケンフェロール、ハトムギ、ビーポーレン、ナルコユリ、ホップ、陳皮、キャベツ、セロリ、オクラ、パセリ、アスパラガス、アシタバ、チョウジ、大麦若葉、ドクダミ、バオバブフルーツ、ブロッコリー、モリンガ、オオバコ、クコの実、キダチアロエ、マカ、オリーブ葉からなる群より選択される1つまたは複数の成分を有効成分として含有するRALDH2発現増強剤を提供する。さらに、本発明は、ケルセチン、ケンフェロール、ハトムギ、ビーポーレン、ナルコユリ、ホップ、陳皮、キャベツ、セロリ、オクラ、パセリ、アスパラガス、アシタバ、チョウジ、大麦若葉、ドクダミ、バオバブフルーツ、ブロッコリー、モリンガ、オオバコ、クコの実、キダチアロエ、マカ、オリーブ葉からなる群より選択される１つまたは複数の成分を有効成分として含有される、組成物も提供する。本発明のRALDH2発現増強剤及び組成物は、経口剤又は経腸剤の形態であってもよく、美肌のためのものであってもよい。 The present invention relates to kelsetin, kaempferol, honeybee, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, young barley leaf, dokudami, baobab fruit, broccoli, moringa, obaco, goji. Provided is a RALDH2 expression enhancer containing one or more components selected from the group consisting of broccoli, maca, and olive leaf as an active ingredient. Further, the present invention relates to kelsetin, kaempferol, honeybee, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, young barley leaf, goji, baobab fruit, broccoli, moringa, obaco, Also provided is a composition comprising one or more ingredients selected from the group consisting of goji, ashitaba, maca, and olive leaves as active ingredients. The RALDH2 expression enhancer and composition of the present invention may be in the form of an oral preparation or an enteral preparation, or may be for beautiful skin.
　本発明で用いられるケルセチン（CAS番号:849061-97-8）は、フラボノイドの一種で、タマネギなどの野菜に含まれる成分である。抗酸化、抗炎症、血流改善、脂肪分解などの作用が知られている。ケルセチンそのものであってもルチン、クエルシトリンなどの配糖体の形態であってもよい。 Quercetin (CAS number: 849061-97-8) used in the present invention is a kind of flavonoid and is a component contained in vegetables such as onions. It is known to have antioxidant, anti-inflammatory, blood flow improving, and lipolytic effects. It may be quercetin itself or it may be in the form of glycosides such as rutin and quercitrin.
　本発明で用いられるケンフェロール（CAS番号:520-18-3）は、フラボノイドの一種で茶や野菜などに含まれる成分である。抗酸化、抗炎症、抗がん、抗糖尿病などの作用が知られている。ケンフェロールそのものであってもケンペリトリン、アストラガリンなどの配糖体の形態であってもよい。 Kaempferol (CAS number: 520-18-3) used in the present invention is a kind of flavonoid and is a component contained in tea and vegetables. Known for its antioxidant, anti-inflammatory, anti-cancer and anti-diabetic effects. Kaempferol itself may be in the form of glycosides such as kaempferitrin and astragalin.
　本発明で用いられるハトムギ（Coix lacryma-jobi Linne var．mayuen Stapf (Gramineae)）は、イネ科ジュズダマ属の穀物である。ハトムギの種皮を除いた種子部分を用いることが好ましい。樹皮には皮膚のイボ改善や美白等に効果があるといわれている。 Coix lacryma-jobi Linne var. Mayuen Stapf (Gramineae) used in the present invention is a grain of the genus Coix of the Gramineae family. It is preferable to use the seed portion excluding the seed coat of Coix seeds. The bark is said to be effective in improving skin warts and whitening.
　本発明で用いられるビーポーレンはミツバチが集めた花粉の粒（花粉荷）である。ビタミンやミネラルなどの栄養素が含まれるという報告がある。シスタス、オーク、バラ、エキウム、キクの花粉など任意の花粉であってよい。ビーポーレンが採取される場所もスペインといったヨーロッパ、中国といったアジア等限定されない。 The bee pollen used in the present invention is a pollen grain (pollen load) collected by honeybees. There are reports that it contains nutrients such as vitamins and minerals. It may be any pollen such as cistus, oak, rose, echium, and chrysanthemum pollen. The place where bee pollen is collected is not limited to Europe such as Spain and Asia such as China.
　本発明で用いられるナルコユリ（Polygonatum falcatum）はユリ科アマドコロ属に属する多年草草本である。根茎を乾燥させた生薬として使用するのが好ましい。滋養強壮などに効果があると言われている。 Narco lily (Polygonatum falcatum) used in the present invention is a perennial herb belonging to the genus Solomon's seal in the family Liliaceae. It is preferable to use it as a crude drug with dried rhizomes. It is said to be effective for nourishing and tonic.
　本発明で用いられるホップ（Humulus lupulus）はアサ科のつる性の多年草植物である。球果を使用するのが好ましい。健胃、鎮静効果があると言われている。 The hop (Humulus lupulus) used in the present invention is a vine perennial plant of the family Cannabaceae. It is preferable to use cones. It is said to have a healthy stomach and a sedative effect.
　本発明で用いられる陳皮はミカンの果皮を乾燥させたものである。ミカン（Citrus属）は、ムクロジ目ミカン科ミカン属の常緑低木である。血流改善、消化不良、食欲不振等に効果があると言われている。 The chenpi used in the present invention is a dried mandarin orange peel. Citrus (genus Citrus) is an evergreen shrub of the genus Citrus of the family Rutaceae, Sapindaceae. It is said to be effective in improving blood flow, indigestion, loss of appetite, etc.
　本発明で用いられるキャベツ（Brassica oleracea var. capitata）はアブラナ科アブラナ属の野菜である。キャベツの葉を使用するのが好ましい。キャベツは、食物繊維やビタミンＵ、ビタミンＣを豊富に含み、胃腸を健康に保つ効果があると言われている。 The cabbage (Brassica oleracea var. Capitata) used in the present invention is a vegetable belonging to the Brassicaceae genus Brassicaceae. It is preferable to use cabbage leaves. Cabbage is rich in dietary fiber, vitamin U, and vitamin C, and is said to have the effect of keeping the gastrointestinal health.
　本発明で用いられるセロリは、セリ科（Apium graveolens var. dulce）の一年草である。セロリの茎葉を使用するのが好ましい。セロリの茎葉には、食欲不振防止、精神安定、血圧上昇抑制効果があると言われている。 The celery used in the present invention is an annual plant of the Umbelliferae family (Apium graveolens var. Dulce). It is preferable to use celery foliage. It is said that the foliage of celery has the effects of preventing anorexia, stabilizing the mind, and suppressing the rise in blood pressure.
　本発明で用いられるオクラ（Abelmoschus esculentus）はアオイ科トロロアオイ属に属する植物である。果実を用いるのが好ましい。果実にはムチンやペクチンなどが含まれており胃腸の調子を整える作用があることが報告されている。 Okra (Abelmoschus esculentus) used in the present invention is a plant belonging to the genus Abelmoschus manihot. It is preferable to use fruits. It has been reported that fruits contain mucin and pectin and have an effect of adjusting gastrointestinal tone.
　本発明で用いられるパセリ（Petroselinum crispum）はセリ科の二年草である。茎葉を用いるのが好ましい。茎葉にはビタミン、ミネラルなどの栄養素が豊富に含まれており健胃、疲労回復、貧血などに対する効果があると言われている。 The parsley (Petroselinum crispum) used in the present invention is a biennial plant of the Umbelliferae family. It is preferable to use foliage. The foliage is rich in nutrients such as vitamins and minerals, and is said to be effective against stomach upset, recovery from fatigue, and anemia.
　本発明で用いられるアスパラガス（Asparagus spp.）はキジカクシ属に属する各種の総称である。茎を用いるのが好ましい。茎にはオリゴ糖、ビタミン、ミネラルなどの栄養素が豊富に含まれており利尿、健胃、整腸作用があることが知られている。 Asparagus spp. Used in the present invention is a general term for various species belonging to the genus Asparagus. It is preferable to use stems. The stem is rich in nutrients such as oligosaccharides, vitamins and minerals, and is known to have diuretic, stomachic and intestinal regulating effects.
　本発明で用いられるアシタバ（Angelica keiskei）はセリ科シシウド属の植物である。茎葉を用いるのが好ましい。ビタミン、ミネラル、食物繊維が豊富で便秘防止や利尿・高血圧予防・強壮作用があることが知られている。 Angelica keiskei used in the present invention is a plant belonging to the genus Ashitaba in the Umbelliferae family. It is preferable to use foliage. It is rich in vitamins, minerals and dietary fiber, and is known to have constipation-preventing, diuretic, hypertensive-preventing and tonic effects.
　本発明で用いられるチョウジ（Syzygium aromaticum）はフトモモ科の樹木チョウジノキの花蕾である。チョウジは、香辛料として食用され、疼痛、健胃作用などがあると言われている。 The clove (Syzygium aromaticum) used in the present invention is a flower bud of the tree Clove of the Myrtaceae family. Clove is eaten as a spice and is said to have pain and stomachic effects.
本発明で用いられる大麦若葉はイネ科オオムギ属の植物である大麦（Hordeum vulgare）の出穂前の若い葉である。各種ビタミン、ミネラル類、葉酸、SOD酵素などが含まれ、栄養効果が高く、青汁等の材料として使用されている。 The young barley leaves used in the present invention are young leaves before heading of barley (Hordeum vulgare), which is a plant belonging to the genus Hordeum of the Gramineae family. It contains various vitamins, minerals, folic acid, SOD enzyme, etc., has a high nutritional effect, and is used as a material for green juice and the like.
　本発明で用いられるドクダミ（Houttuynia cordata Thunberg）はドクダミ科ドクダミ属の多年草である。ドクダミの地上部を用いることが好ましい。内服薬や茶として利用され、胃腸病、解毒、食あたり、下痢、便秘、利尿などに有効があるといわれている。 Houttuynia cordata Thunberg used in the present invention is a perennial plant belonging to the genus Houttuynia in the family Houttuynia. It is preferable to use the above-ground part of Houttuynia cordata. It is used as an internal medicine and tea, and is said to be effective for gastrointestinal diseases, detoxification, food poisoning, diarrhea, constipation, diuresis, etc.
　本発明で用いられるバオバブフルーツ（Adansonia spp.)はアオイ科バオバブ属の樹木の果実である。バオバブフルーツには、ビタミン、ミネラル、食物繊維等が含まれ、スーパーフルーツとして知られている。 The baobab fruit (Adansonia spp.) Used in the present invention is a fruit of a tree belonging to the genus Baobab of the Malvaceae family. Baobab fruit contains vitamins, minerals, dietary fiber, etc., and is known as superfruit.
　本発明で用いられるブロッコリー（Brassica oleracea var. italica）はアブラナ科アブラナ属の緑黄色野菜である。地上部等を使用することが好ましい。スルフォラファンを含み、がん予防、ピロリ菌抑制などの効果等が報告されている。 Broccoli (Brassica oleracea var. Italica) used in the present invention is a green-yellow vegetable belonging to the genus Brassicaceae. It is preferable to use an above-ground part or the like. It contains sulforaphane and has been reported to have effects such as cancer prevention and suppression of Helicobacter pylori.
　本発明で用いられるモリンガ（Moringa oleifera LAM.（M. pterygosperma））はワサビノキ科ワサビノキ属の植物であり、モリンガの葉を用いることが好ましい。食用や薬用に使用され、栄養補給などに効果があると言われている。各種ビタミン・ミネラル等が含まれ、スーパーフードとして知られている。 Moringa (Moringa oleifera LAM. (M. pterygosperma)) used in the present invention is a plant belonging to the genus Moringa of the Moringa family, and it is preferable to use Moringa leaves. It is used for food and medicine, and is said to be effective for nutritional supplementation. It contains various vitamins and minerals and is known as a superfood.
　本発明で用いられるオオバコ（Plantago asiatica）はオオバコ科オオバコ属の多年草であり、全草を用いることが好ましい。食物繊維を豊富に含んでおり、健胃、むくみ改善、消炎、利尿、下痢止め、咳止めなどの効果等があるといわれている。 The plantain (Plantago asiatica) used in the present invention is a perennial plant belonging to the genus Plantago of the family Plantain family, and it is preferable to use the whole plantago. It is rich in dietary fiber and is said to have effects such as stomach upset, swelling improvement, anti-inflammatory, diuretic, antidiarrhea, and cough.
本発明で用いられるクコの実はナス科の落葉低木であるクコ（Lycium chinense）の果実であり、ゼアキサンチン、ベタイン、ポリフェノールなどが含まれている。クコの実には滋養強壮作用、血流改善作用、美白効果、DNA損傷修復作用等が報告されている。 The wolfberry fruit used in the present invention is a fruit of wolfberry (Lycium chinense), which is a deciduous shrub of the Solanaceae family, and contains zeaxanthin, betaine, polyphenol, and the like. Lycium chinense has been reported to have a nourishing tonic effect, a blood flow improving effect, a whitening effect, and a DNA damage repairing effect.
本発明で用いられるキダチアロエ（Aloe arborescens）はアロエ属の多肉植物の一種である。キダチアロエの葉を使用することが好ましい。外用では火傷、切り傷に対する効果に、内用では胃腸痛、便秘改善効果等が報告されている。 Candelabra aloe (Aloe arborescens) used in the present invention is a kind of succulent plant of the genus Aloe. It is preferable to use the leaves of Kidachi aloe. It has been reported that external use is effective against burns and cuts, and internal use is effective in improving gastrointestinal pain and constipation.
　マカ（Lepidium meyenii）はアブラナ科の多年生植物である。根を使用することが好ましい。アミノ酸をはじめ、ミネラル、食物繊維、ビタミンなどを含み、滋養強壮、持久力向上、抗疲労などの効果が知られている。 Maca (Lepidium meyenii) is a perennial plant of the Brassicaceae family. It is preferable to use roots. It contains amino acids, minerals, dietary fiber, vitamins, etc., and is known to have effects such as nutritional tonicity, endurance improvement, and anti-fatigue.
オリーブ葉はモクセイ科の常緑高木であるオリーブ（Olea europaea）の葉である。オリーブの葉はオレウロペイン等のポリフェノールを含み、抗菌・ウイルス作用が知られている。 Olive leaves are the leaves of olive (Olea europaea), an evergreen tree of the family Oleaceae. Olive leaves contain polyphenols such as oleuropein and are known to have antibacterial and viral effects.
　ケルセチン、ケンフェロール等の化合物は、合成してもよく、市販品を用いてもよく、生薬などに含まれる態様で用いてもよい。ハトムギ、ビーポーレン、ナルコユリ、ホップ、陳皮、キャベツ、セロリ、オクラ、パセリ、アスパラガス、アシタバ、チョウジ、大麦若葉、ドクダミ、バオバブフルーツ、ブロッコリー、モリンガ、オオバコ、クコの実、キダチアロエ、マカ、オリーブ葉の生薬は公知の物質であり、公知の方法により容易に乾燥、精製、抽出等ができ、また市販品を容易に入手可能である。生のままでも乾燥したものでも使用することができるが、使用性、製剤化等の観点から抽出物、乾燥物、乾燥粉末、原料の粉末物、搾汁液等として用いることもできる。原料により、いすれの形態を用いるかは適宜選択することができ、必要に応じて殺菌等の処理を施してもよい。 The compound such as quercetin and kaempferol may be synthesized, a commercially available product may be used, or a compound contained in a crude drug or the like may be used. Hatomugi, bee pollen, narco lily, hops, chenpi, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, barley young leaves, Houttuynia cordata, baobab fruit, broccoli, moringa, obaco, goji nuts, kidachi aloe, maca, olive leaves Crude drugs are known substances, which can be easily dried, purified, extracted, etc. by known methods, and commercially available products are easily available. It can be used raw or dried, but it can also be used as an extract, a dried product, a dried powder, a powdered raw material, a juice squeezed liquid, or the like from the viewpoint of usability, formulation, and the like. Depending on the raw material, it is possible to appropriately select whether to use the form of the squirrel, and if necessary, sterilization or the like may be performed.
　抽出物として用いる場合、その抽出物の抽出方法は例えば溶媒抽出により行うことができる。溶媒抽出の場合には、植物の全草あるいは各種部位（葉、花、根等）を必要に応じて乾燥させ、更に必要に応じて細断又は粉砕した後、水性抽出剤、水、例えば冷水、温水、又は沸点若しくはそれより低温の熱水、あるいは含水有機溶媒、有機溶媒、例えばエタノール、メタノール、エーテル、１，３－ブチレングリコール等を原料の性質や組成物の用途等により好ましい溶媒を適宜選択して常温で又は加熱して用いることにより抽出される。しかしながら、抽出方法は溶媒抽出に限定されず、当業界で知られている常用の手法によってもよく、本発明で用いる抽出物の抽出方法や抽出物の形態は、本発明の効果を損なわない限り任意である。上記抽出物の形態は、抽出液自体だけでなく、常用の手法により適宜希釈又は濃縮したものであってもよく、更に、抽出液を乾燥することによって得られる粉状あるいは塊状の固体であってもよい。 When used as an extract, the extraction method of the extract can be performed, for example, by solvent extraction. In the case of solvent extraction, the whole plant or various parts (leaves, flowers, roots, etc.) of the plant are dried as needed, shredded or crushed as needed, and then an aqueous extractant, water, for example, cold water. , Hot water, hot water having a boiling point or a lower temperature, or a hydrous organic solvent, an organic solvent such as ethanol, methanol, ether, 1,3-butylene glycol, etc. It is extracted by selecting it at room temperature or by heating it. However, the extraction method is not limited to solvent extraction, and may be a conventional method known in the art, and the extraction method of the extract and the form of the extract used in the present invention are not limited to the effect of the present invention. Optional. The form of the extract may be not only the extract itself but also appropriately diluted or concentrated by a conventional method, and is a powdery or lumpy solid obtained by drying the extract. May be good.
　含水有機溶媒の例として、含水エタノール等の含水低級アルコールを用いてもよく、その場合の含水率は、例えば0～10v/v％、10～40v/v％、20～30v/v％、30～50v/v％、50～80v/v％、80～99.5v/v％等であってもよい。 As an example of the water-containing organic solvent, a water-containing lower alcohol such as water-containing ethanol may be used, and the water content in that case is, for example, 0 to 10v / v%, 10 to 40v / v%, 20 to 30v / v%, 30. It may be ~ 50v / v%, 50-80v / v%, 80-99.5v / v%, or the like.
　乾燥粉末を得る方法としては、植物の全草あるいは各種部位（葉、花、根等）を細断又は粉砕し、その後に乾燥する方法や植物を乾燥した後に細断又は粉砕して乾燥粉末を得る方法がある。また、植物を細断又は粉砕し、発酵や酵素処理を施した後、乾燥し、更に必要に応じて所定の粒径にすべく粉砕する方法等を適宜採ることができる。 As a method for obtaining a dry powder, the whole plant or various parts (leaves, flowers, roots, etc.) of the plant are shredded or crushed and then dried, or the plant is dried and then shredded or crushed to obtain a dry powder. There is a way to get it. Further, a method of shredding or crushing the plant, subjecting it to fermentation or enzyme treatment, drying it, and further crushing it to a predetermined particle size, if necessary, can be appropriately adopted.
　本発明のRALDH2発現増強剤は、経口又は経腸摂取が好ましいものの、経皮投与といった他の投与経路を排除するものではない。本発明のRALDH2発現増強剤を各種投与経路で投与する場合、本発明の有効成分をRALDH2発現増強効果が十分発揮されるような量で適用することが好ましい。本発明の成分の配合量は、それらの種類、目的、形態、利用方法などに応じて、適宜決めることができる。 Although the RALDH2 expression enhancer of the present invention is preferably taken orally or enterally, it does not exclude other routes of administration such as transdermal administration. When the RALDH2 expression enhancer of the present invention is administered by various routes of administration, it is preferable to apply the active ingredient of the present invention in an amount such that the RALDH2 expression enhancer effect is sufficiently exhibited. The blending amount of the components of the present invention can be appropriately determined according to their types, purposes, forms, usage methods and the like.
　本発明のRALDH2発現増強剤を経口剤又は経腸剤として利用する場合には、経口剤又は経腸剤全量に対して、植物体又はその溶媒抽出物の乾燥重量が、0.001～100重量％程度になるように調製することが好ましく、0.01～10重量％程度がより好ましく、0.1～1.0重量％程度だと更に好ましい。あるいは、腸内での濃度が1～10000μg/ml程度、より好ましくは1～5000μg/ml程度、更に好ましくは5～2000μg/ml程度になるように調整してもよい。 When the RALDH2 expression enhancer of the present invention is used as an oral preparation or an enteral preparation, the dry weight of the plant or its solvent extract is about 0.001 to 100% by weight based on the total amount of the oral preparation or the enteral preparation. It is preferably prepared so as to be, more preferably about 0.01 to 10% by weight, still more preferably about 0.1 to 1.0% by weight. Alternatively, the concentration in the intestine may be adjusted to be about 1 to 10000 μg / ml, more preferably about 1 to 5000 μg / ml, and even more preferably about 5 to 2000 μg / ml.
　また、本発明のRALDH2発現増強剤を経口剤又は経腸剤として利用する場合には、成人１人当たりのケルセチン、ケンフェロール、ハトムギ、ビーポーレン、ナルコユリ、ホップ、陳皮、キャベツ、セロリ、オクラ、パセリ、アスパラガス、アシタバ、チョウジ、大麦若葉、ドクダミ、バオバブフルーツ、ブロッコリー、モリンガ、オオバコ、クコの実、キダチアロエ、マカ、オリーブ葉の植物体又はその溶媒抽出物の摂取量は、例えば１日あたり、1mg～50000mｇ（乾燥重量換算）程度、より好ましくは10mg～5000mｇ、更に好ましくは100mg～500mｇ（乾燥重量換算）程度になるように調製するのが好ましい。また、摂取頻度は、限定されないものの、１回の摂取でもよいが、一例によれば、例えば２週間に１回、１週間に１回、３日に１回、２日に１回、１日１回、１日２回、１日３回、１日４回等の頻度で摂取することができる。また、都度摂取するものであっても、継続的に摂取するものであっても、例えば数か月の間隔を空け断続的に摂取するものであってもよい。 When the RALDH2 expression enhancer of the present invention is used as an oral preparation or an enteric preparation, kercetin, kaempferol, adlay, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, etc. per adult. The intake of asparagus, ashitaba, adlay, young barley leaves, Houttuynia cordata, baobab fruit, broccoli, moringa, obaco, goji, kidachi aloe, maca, olive leaf plant or its solvent extract is, for example, 1 mg per day. It is preferably prepared to be about 50,000 mg (in terms of dry weight), more preferably about 10 mg to 5000 mg, and even more preferably about 100 mg to 500 mg (in terms of dry weight). The frequency of ingestion is not limited, but it may be taken once, but according to one example, for example, once every two weeks, once a week, once every three days, once every two days, one day. It can be taken once, twice a day, three times a day, four times a day, and the like. In addition, it may be ingested each time, continuously, or intermittently, for example, at intervals of several months.
　また、本発明のRALDH2発現増強剤は、経口又は経腸摂取用の組成物、例えば食品組成物に含有できる。本発明のRALDH2発現増強剤及び組成物の形態としては、例えば、粉末状、液体状、錠剤等の固形、顆粒、粒状、ペースト状、ゲル状など任意に選択することができる。 Further, the RALDH2 expression enhancer of the present invention can be contained in a composition for oral or enteral ingestion, for example, a food composition. As the form of the RALDH2 expression enhancer and the composition of the present invention, for example, powdery, liquid, solid such as tablets, granules, granules, pastes, gels and the like can be arbitrarily selected.
　本発明のRALDH2発現増強剤及び組成物には、必要に応じて添加剤を任意に選択し併用することができる。添加剤としては賦形剤等を含ませることができる。賦形剤としては、所望の剤型としたときに通常用いられるものであれば何でも良く、例えば、コムギデンプン、コメデンプン、トウモロコシデンプン、バレイショデンプン、デキストリン、シクロデキストリンなどのでんぷん類、結晶セルロース類、乳糖、ブドウ糖、砂糖、還元麦芽糖、水飴、フラクトオリゴ糖、乳化オリゴ糖などの糖類、ソルビトール、エリスリトール、キシリトール、ラクチトール、マンニトールなどの糖アルコール類が挙げられる。これら賦形剤は、単独で又は二種以上組み合わせて使用できる。 Additives can be arbitrarily selected and used in combination with the RALDH2 expression enhancer and composition of the present invention, if necessary. Excipients and the like can be included as the additive. The excipient may be any starch as long as it is usually used in the desired dosage form, for example, starches such as wheat starch, rice starch, corn starch, potato starch, dextrin, cyclodextrin, and crystalline celluloses. , Lactose, starch, sugar, reduced malt sugar, water candy, fructo-oligosaccharide, emulsified oligosaccharide and other sugars, sorbitol, erythritol, xylitol, lactitol, mannitol and other sugar alcohols. These excipients can be used alone or in combination of two or more.
　その他の着色剤、保存剤、増粘剤、結合剤、崩壊剤、分散剤、安定化剤、ゲル化剤、酸化防止剤、界面活性剤、保存剤、ｐＨ調整剤等については、公知のものを適宜選択して使用できる。 Other known colorants, preservatives, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, pH adjusters, etc. Can be selected and used as appropriate.
　本発明は、ケルセチン、ケンフェロール、ハトムギ、ビーポーレン、ナルコユリ、ホップ、陳皮、キャベツ、セロリ、オクラ、パセリ、アスパラガス、アシタバ、チョウジ、大麦若葉、ドクダミ、バオバブフルーツ、ブロッコリー、モリンガ、オオバコ、クコの実、キダチアロエ、マカ、オリーブ葉からなる群より選択される1つまたは複数の成分を例えば経口又は経腸経路で投与することにより単球細胞のRALDH2発現を促進する方法も提供する。また、本発明は、ケルセチン、ケンフェロール、ハトムギ、ビーポーレン、ナルコユリ、ホップ、陳皮、キャベツ、セロリ、オクラ、パセリ、アスパラガス、アシタバ、チョウジ、大麦若葉、ドクダミ、バオバブフルーツ、ブロッコリー、モリンガ、オオバコ、クコの実、キダチアロエ、マカ、オリーブ葉からなる群より選択される1つまたは複数の成分を例えば経口又は経腸経路で投与することにより単球細胞のRALDH2発現増強を介する免疫バランスの改善のための方法も提供する。本発明の方法は、美容を目的とする方法であり、医師や医療従事者による治療ではないことがある。 The present invention relates to quercetin, kaempferol, honeybee, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, young barley leaf, houttuynia cordata, baobab fruit, broccoli, moringa, obaco, goji. In fact, there is also provided a method of promoting RALDH2 expression in monocytic cells by administering one or more components selected from the group consisting of Houttuynia cordata, maca, and olive leaf, for example, by oral or enteric route. The present invention also includes quercetin, kaempferol, honeybee, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, young barley leaves, houttuynia cordata, baobab fruit, broccoli, moringa, and obaco. To improve immune balance through enhanced RALDH2 expression in monocytic cells, for example by administration of one or more components selected from the group consisting of goji, ashitaba, maca and olive leaves, for example by oral or enteric route. Also provides the method of. The method of the present invention is a method for the purpose of cosmetology and may not be treated by a doctor or a medical professional.
　さらに、本発明は、免疫バランスの改善のための経口剤又は経腸剤といった医薬の製造におけるケルセチン、ケンフェロール、ハトムギ、ビーポーレン、ナルコユリ、ホップ、陳皮、キャベツ、セロリ、オクラ、パセリ、アスパラガス、アシタバ、チョウジ、大麦若葉、ドクダミ、バオバブフルーツ、ブロッコリー、モリンガ、オオバコ、クコの実、キダチアロエ、マカ、オリーブ葉からなる群より選択される1つまたは複数の成分の使用も提供する。本発明は、例えば経口又は経腸投与により、単球細胞のRALDH2発現を促進することにより、免疫バランスを改善する方法に使用するためのケルセチン、ケンフェロール、ハトムギ、ビーポーレン、ナルコユリ、ホップ、陳皮、キャベツ、セロリ、オクラ、パセリ、アスパラガス、アシタバ、チョウジ、大麦若葉、ドクダミ、バオバブフルーツ、ブロッコリー、モリンガ、オオバコ、クコの実、キダチアロエ、マカ、オリーブ葉からなる群より選択される1つまたは複数の成分も提供する。 Furthermore, the present invention relates to quercetin, kaempferol, honeybee, bee pollen, narco lily, hop, husk, cabbage, celery, okra, parsley, asparagus, in the manufacture of pharmaceuticals such as oral or enteric agents for improving immune balance. Also provided is the use of one or more ingredients selected from the group consisting of Ashitaba, Chouji, young barley leaves, Houttuynia cordata, Baobab fruit, broccoli, moringa, obaco, goji, kidachi aloe, maca and olive leaves. The present invention comprises quercetin, kaempferol, ashitaba, bee pollen, broccoli, hops, broccoli, for use in methods of improving immune balance by promoting RALDH2 expression in monocytic cells, for example by oral or enteral administration. One or more selected from the group consisting of cabbage, celery, okra, parsley, asparagus, ashitaba, chow, barley young leaves, houttuynia cordata, baobab fruit, broccoli, moringa, obaco, goji, kidachi aloe, maca, olive leaf. Also provides the ingredients of.
　次に実施例によって本発明をさらに詳細に説明する。なお、本発明はこれにより限定されるものではない。 Next, the present invention will be described in more detail by way of examples. The present invention is not limited thereto.
　実験１：試料の調製
　RALDH2発現増強剤の候補試料として、ケルセチン、ケンフェロール、ハトムギ、ビーポーレン、ナルコユリ、ホップ、陳皮、キャベツ、セロリ、オクラ、パセリ、アスパラガス、アシタバ、チョウジ、大麦若葉、ドクダミ、バオバブフルーツ、ブロッコリー、モリンガ、オオバコ、クコの実、キダチアロエ、マカ、オリーブ葉を、以下の表のように調製した。 Experiment 1: Preparation of samples As candidate samples for RALDH2 expression enhancer, quercetin, kaempferol, honeybee, bee pollen, narco lily, hop, bark, cabbage, celery, okra, parsley, asparagus, ashitaba, chow, barley young leaves, Houttuynia cordata, Baobab fruit, broccoli, moringa, cabbage, goji, ashitaba, maca, and olive leaves were prepared as shown in the table below.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　その他動植物の抽出物といった天然由来成分や合成成分を含め、合計147種類の候補試料を調製した。試料はDMSOを用いて100μg/mlとなるように調整した。陰性対照として何も含まないDMSOを用いた。 A total of 147 types of candidate samples were prepared, including naturally derived components such as extracts of animals and plants and synthetic components. The sample was adjusted to 100 μg / ml using DMSO. DMSO containing nothing was used as a negative control.
　実験２：RALDH2発現を促進する成分のスクリーニングシステムの構築
　実験２-1：ヒト急性単球性白血病由来株化細胞THP-1細胞の培養
　ヒト急性単球性白血病由来株化細胞THP-1細胞を用いた。THP-1細胞は、10% Fetal bovine serum（FBS; Life Technologies, CA, USA） 添加RPMI 1640 培地（Nissui, Tokyo, Japan）、ペトリディッシュ（FALCON, Tokyo, Japan）を用いて37℃、5％ CO2存在下で継代培養した。RPMI 1640培地は、500 mLのMilli-Q 水に対して、RPMI 1640粉末5.1gを溶解し、硫酸ストレプトマイシン0.1 g 力価（Meiji. Tokyo, Japan）、ペニシリンGカリウム10 万U（Meiji）、10% NaHCO3（Wako, Osaka, Japan）9mL を添加し、0.22μmフィルター滅菌（Toyo Roshi kaisha, Tokyo, Japan）したものをRPMI 1640培地とし、4℃で保存した。 Experiment 2: Construction of a screening system for components that promote RALDH2 expression Experiment 2-1: Culture of human acute monocytic leukemia-derived cell line THP-1 cells Human acute monocyte leukemia-derived cell line THP-1 cells Using. THP-1 cells were subjected to 10% Fetal bovine serum (FBS; Life Technologies, CA, USA) -added RPMI 1640 medium (Nissui, Tokyo, Japan), Petri dish (FALCON, Tokyo, Japan) at 37 ° C, 5%. Subcultured in the presence of CO 2 . RPMI 1640 medium is prepared by dissolving 5.1 g of RPMI 1640 powder in 500 mL of Milli-Q water, streptomycin sulfate 0.1 g titer (Meiji. Tokyo, Japan), penicillin G potassium 100,000 U (Meiji), 10 % י 3 (Wako, Osaka, Japan) 9 mL was added, and a 0.22 μm filter sterilized (Toyo Roshi kaisha, Tokyo, Japan) was used as RPMI 1640 medium and stored at 4 ° C.
　実験２-２：ヒト結腸ガン由来細胞株Caco-2細胞の培養
　ヒト腸管上皮モデルとしてヒト結腸ガン由来Caco-2細胞を用いた。Caco-2細胞は、非働化した10% Fetal Bovine Serum (FBS)(Life Technologies, CA, USA)を含むDulbecco’s Modified Eagle Medium (DMEM)(Nissui, Tokyo, Japan)を用いて、細胞培養ディッシュ（Greiner bio-one, Tokyo, Japan）にて、37℃、5%CO2条件下で継代培養した。DMEM培地としては、1LのMilli-Q水に対して、DMEM粉末10.0gを溶解し、100U/mL ペニシリン(Meiji)、0.1mg/mL ストレプトマイシン(Meiji)、1M HEPES 2.38g(DOJINDO, Kumamoto, Japan)、10% NaHCO3 2.0g(Wako, Osaka, Japan)を添加し、0.22μmフィルター滅菌 (Tokyo Roshi Kaisha, Tokyo, Japan) したものを用いた。 Experiment 2-2: Culture of human colon cancer-derived cell line Caco-2 cells Human colon cancer-derived Caco-2 cells were used as a human intestinal epithelial model. Caco-2 cells were cultured in a cell culture dish (Greiner) using Dulbecco's Modified Eagle Medium (DMEM) (Nissui, Tokyo, Japan) containing deactivated 10% Fetal Bovine Serum (FBS) (Life Technologies, CA, USA). Bio-one, Tokyo, Japan) was subcultured under the conditions of 37 ° C and 5% CO 2 . As the DMEM medium, 10.0 g of DMEM powder is dissolved in 1 L of Milli-Q water, 100 U / mL penicillin (Meiji), 0.1 mg / mL streptomycin (Meiji), 1M HEPES 2.38 g (DOJINDO, Kumamoto, Japan). ), 10% NaHCO 3 2.0 g (Wako, Osaka, Japan) was added, and 0.22 μm filter sterilized (Tokyo Roshi Kaisha, Tokyo, Japan) was used.
　実験２-３：プラスミド(RALDH2-EGFP)の調製およびTHP-1細胞への導入
　プラスミドRALDH2-EGFPを構築し、実験２－１で調製したTHP-1細胞に導入した。具体的には、マウスゲノムDNAをテンプレートとして使用したPCRにより、ヒトRALDH2プロモーター（Forward: ATTAATAACTGACTTACCAGCCTCGT（配列番号１）, Reverse: GCTAGCGGCGATCTCGCTGGAAGTCA（配列番号２））を増幅した。増幅断片をpEGFP-C3（TaKaRa、日本）にクローニングし、そのCMVプロモーターを制限酵素AseIおよびNheI消化により除去した。得られたプラスミドをRALDH2p-EGFPとした。このプラスミドを実験２－１で調製したTHP-1細胞に安定的にトランスフェクトし、THP-1（RALDH2p-EGFP）とし、ヒトRALDH2プロモーター活性の評価に使用した。その後、得られたRALDH2p-EGFPをPMAにより分化誘導（48hPMA100ng/ml）した。 Experiment 2-3: Preparation of plasmid (RALDH2-EGFP) and introduction into THP-1 cells The plasmid RALDH2-EGFP was constructed and introduced into THP-1 cells prepared in Experiment 2-1. Specifically, the human RALDH2 promoter (Forward: ATTAATAACTGACTTACCAGCCTCGT (SEQ ID NO: 1), Reverse: GCTAGCGGCGATCTCGCTGGAAGTCA (SEQ ID NO: 2)) was amplified by PCR using mouse genomic DNA as a template. The amplified fragment was cloned into pEGFP-C3 (TaKaRa, Japan) and its CMV promoter was removed by digestion with restriction enzymes AseI and NheI. The obtained plasmid was designated as RALDH2p-EGFP. This plasmid was stably transfected into THP-1 cells prepared in Experiment 2-1 to obtain THP-1 (RALDH2p-EGFP), which was used for evaluation of human RALDH2 promoter activity. Then, the obtained RALDH2p-EGFP was induced to differentiate by PMA (48hPMA100ng / ml).
　実験３：被験物質のスクリーニング
　実験２－２で培養したCaco-2細胞を24 wellプレートに2.0×105cells/mLで播種し、24時間後に図１に示すように実験１で調製した各試料を1μL添加し最終濃度10μg/mLとなるようにした。実験２－３で作成したTHP-1（RALDH2p-EGFP）にCaco-2細胞の上清を100μL添加した。上清添加から24時間経過後、THP-1細胞のEGFP蛍光強度をインセルアナライザー2200（IN Cell Analyzer 2200、Cytiva社）にて評価し、RALDH2のプロモーター活性を評価した。 Experiment 3: Screening of test substance Caco-2 cells cultured in Experiment 2-2 were seeded on a 24-well plate at 2.0 × 10 5 cells / mL, and 24 hours later, each sample prepared in Experiment 1 as shown in FIG. Was added to the final concentration of 10 μg / mL. 100 μL of the supernatant of Caco-2 cells was added to THP-1 (RALDH2p-EGFP) prepared in Experiment 2-3. Twenty-four hours after the addition of the supernatant, the EGFP fluorescence intensity of THP-1 cells was evaluated with IN Cell Analyzer 2200 (Cytiva) to evaluate the promoter activity of RALDH2.
　結果：
　結果を図２に示す。図２に示すように、本発明の成分を投与すると、陰性対照（DMSO）と比べてTHP-1細胞内のRALDH2の発現量が有意に増加していた。したがって、本発明の成分は単球細胞におけるRALDH2発現増強効果が優れていることがわかる。 result:
The results are shown in FIG. As shown in FIG. 2, when the component of the present invention was administered, the expression level of RALDH2 in THP-1 cells was significantly increased as compared with the negative control (DMSO). Therefore, it can be seen that the component of the present invention has an excellent effect of enhancing RALDH2 expression in monocyte cells.

Claims (4)

  1. 　ケルセチン、ケンフェロール、ハトムギ、ビーポーレン、ナルコユリ、ホップ、陳皮、キャベツ、セロリ、オクラ、パセリ、アスパラガス、アシタバ、チョウジ、大麦若葉、ドクダミ、バオバブフルーツ、ブロッコリー、モリンガ、オオバコ、クコの実、キダチアロエ、マカ、オリーブ葉からなる群より選択される１つまたは複数の成分を有効成分として含有するRALDH2発現増強剤。 Kersetin, Kaempferol, Hatomugi, Bee Paulen, Narco lily, Hop, Chen bark, Cabbage, Celoli, Okra, Parsley, Asparagus, Ashitaba, Chouji, Barley young leaves, Dokudami, Baobab fruit, Broccoli, Moringa, Okra, Goji, Kidachi aloe, A RALDH2 expression enhancer containing one or more components selected from the group consisting of maca and olive leaves as an active ingredient.
  2. 　前記RALDH2発現増強剤は、腸管細胞を介して単球細胞のRALDH2発現を増強する、請求項１に記載のRALDH2発現増強剤。 The RALDH2 expression enhancer according to claim 1, wherein the RALDH2 expression enhancer enhances the RALDH2 expression of monocyte cells via intestinal cells.
  3. 　前記RALDH2発現増強剤は、内服により単球細胞のRALDH2発現を増強する、請求項１又は２に記載のRALDH2発現増強剤。 The RALDH2 expression enhancer according to claim 1 or 2, wherein the RALDH2 expression enhancer enhances the RALDH2 expression of monocyte cells by oral administration.
  4. 　請求項１～３のいずれか１項に記載のRALDH2発現増強剤を含有する組成物。 A composition containing the RALDH2 expression enhancer according to any one of claims 1 to 3.
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