JP7165321B2 - exosome production promoter - Google Patents

Description

The present invention relates to an exosome production promoter capable of promoting the production of exosomes in nerve cells.

Alzheimer's disease (AD) is a neurodegenerative disease that accounts for the majority of dementias, and its incidence increases with age. A pathological feature of AD is the appearance of senile plaques, which are extracellular deposits of a protein called amyloid β (Aβ). Aβ is produced by the sequential processing of amyloid precursor protein (APP). Under normal conditions, the balance between Aβ production and degradation/excretion is maintained so that excessive amounts of Aβ do not accumulate in the brain. However, once that balance is disrupted, excessive amounts of Aβ accumulate in the brain, increasing the risk of developing AD.

Drugs that suppress the accumulation of Aβ have been extensively studied so far. For example, Patent Document 1 discloses an inhibitor that suppresses the formation of amyloid fibrils containing a water-soluble peptide-metal complex array. Further, Patent Document 2 discloses an Aβ aggregation-inhibiting composition containing a perilla extract as an active ingredient. Furthermore, Patent Document 3 discloses an inhibitor of Aβ aggregation caused by GNE gene mutation, containing at least one of N-acetylneuraminic acid, N-acetylmannosamine, and sialyllactose.

In addition, recently, it has been reported that exosomes secreted from neurons are involved in the degradation of Aβ (Non-Patent Document 1), and promoting the production of exosomes in neurons is effective in suppressing the accumulation of Aβ. it is conceivable that. Exosomes are small membrane vesicles (approximately 40 to 100 nm in diameter) released from cells, contain mRNA and the like, and are believed to be important for intercellular communication (Non-Patent Document 2).

Previous studies by the present inventors have revealed that extracellularly secreted Aβ binds to exosomes secreted from nerve cells, moves to microglia, and is then degraded. For example, in Non-Patent Document 3, neuron-derived exosomes highly express Aβ-binding glycolipids and have the ability to bind Aβ, and further exosomes in a state where Aβ is bound are taken up by microglia and decomposed. are reporting. Furthermore, Non-Patent Document 4 and Non-Patent Document 5 also report that intracerebral administration of nerve cell-derived exosomes reduces Aβ levels in the brain.

However, drugs that can promote the production of exosomes have not yet been fully investigated.

Yuyama K. , et al. (2012) J. Biol. chem. SimonsM. , et al. (2009) Curr Opin Cell Biol. Monthly "Cells", published by New Science (2016) 48 (1), pp. 44-47 Yuyama K. , et al. (2014) J. Biol. chem. Yuyama K. , et al. (2015) FEBS Lett.

JP 2016-145175 A JP 2016-124865 A JP 2014-224132 A

An object of the present invention is to provide an exosome production promoter that can promote the production of exosomes in nerve cells.

When the present inventors conducted intensive studies to solve the above problems, the sphingoid base has the effect of promoting the production of exosomes in nerve cells and can be used as an exosome production accelerator. The present invention has been completed through further studies based on such findings.

That is, the present invention provides inventions in the following aspects.
Section 1. An exosome production promoter containing a sphingoid base as an active ingredient.
Section 2. Item 1, wherein the sphingoid base is at least one selected from the group consisting of 4-trans, 8-trans-sphingadienine, 4-trans, 8-cis-sphingadienine, and sphingosine. exosome production promoter.
Item 3. Used for preventing or ameliorating diseases caused by accumulation of amyloid β protein, the exosome production promoter according to Item 1 or 2.
Section 4. Used to prevent or improve cognitive decline caused by accumulation of amyloid β protein, exosome production promoter according to Item 1 or 2.
Item 5. Used to prevent or improve memory impairment caused by accumulation of amyloid β protein, exosome production promoter according to item 1 or 2.
Item 6. The exosome production promoter according to any one of Items 1 to 5, which is a food or drink for promoting exosome production.
Item 7. Exosome production promoting agent according to any one of Items 1 to 5, which is a pharmaceutical for promoting exosome production.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide an exosome production promoter that can promote the production of exosomes in nerve cells. In addition, the exosome production promoter of the present invention can be administered or ingested in the form of oral administration, food and drink, so it is non-invasive and less burdensome for patients, and can easily promote exosome production in nerve cells.

It is a graph showing the results of measuring the number of exosome particles contained in the culture supernatant after adding sphingosine to human neuroblastoma SH-SY5Y cells and culturing them for 24 hours. 4-trans, 8-cis-sphingadienine and 4-trans, 8-trans-sphingadienine were added to human neuroblastoma SH-SY5Y cells for 24 hours. It is a graph which shows the result of measuring the number of contained exosome particles.

The present invention is an exosome production promoter containing a sphingoid base as an active ingredient. Hereinafter, the exosome production promoter of the present invention will be described in detail.

[Sphingoid base]
The exosome production promoter of the present invention uses a sphingoid base as an active ingredient. Sphingoid bases are compounds known as base moieties (long-chain aminoalcohols) that constitute sphingolipids.

The type of sphingoid base used in the present invention is not particularly limited, but examples include sphingosine (4-sphingenine), 4-trans, 8-trans-sphingadienine, 4-trans, 8-cis-s fingadienin, 4-cis,8-trans-sphingadienine, 4-hydroxysphinganine (phytosphingosine), 4-hydroxy-8-trans-sphingenine, 4-hydroxy-8-cis-sphingenine, sphinganine, 8-trans-sphingenine, 8-cis-sphingenine, 4-trans-sphingenine, 4-cis, 8-cis-sphingadienine, 4-trans, 14-cis-sphingadienine, 4-trans, 14- and trans-sphingadienine.

These sphingoid bases may be used singly or in combination of two or more.

Among these sphingoid bases, from the viewpoint of more effectively promoting the production of exosomes in nerve cells, preferably sphingosine (4-sphingenine), 4-trans, 8-trans-sphingadienine, 4- trans,8-cis-sphingadienine, more preferably 4-trans,8-trans-sphingadienine and 4-trans,8-cis-sphingadienine.

The sphingoid base may be either plant-derived or animal-derived, but plant-derived is preferred in terms of higher safety.

Specific examples of animals from which the sphingoid base used in the present invention is derived include echinoderms such as sea urchins, starfish, octopuses, and squids, all or part of the tissues of mollusks, and brains of mammals such as horses and bovines. Examples include tissue and skin tissue, milk of mammals such as humans, cows and goats, and fermented products thereof.

Plants from which the sphingoid base used in the present invention is derived include almonds, sea lettuce, green laver, pigweed, acacia, red grape, Japanese red pine, agaricus, almonds, akebi, morning glory, azalea, hydrangea, angelica keiskei, adzuki bean, asparagus, acerola, Asenyak, anise, avocado, amaksa, amacha, amachazul, amaranth, amaryllis, althea, arnica, aloe, angelica, apricot, angkor, angsokkou. rush, iris, rose, yew, fig, ginkgo biloba, ayokan, ylang-ylang, fennel, oolong tea, turmeric, marshmallow, moray eel, udo, Japanese apricot, white beetle, mandarin orange, ageil, shallot, eleuthero, broom, enokitake mushroom, elderflower enso , Orchid, Scutellaria baicalensis, Plantain, Fin thistle, Barley, Okera, Osmanthus, Hypericum, Dead nettle, Orange, Carnation, Cacao, Oyster, Persimmon, Cocktail fruit, Kakon, Kashiwa, Dogtooth violet, Pumpkin, Chamomile, Camu camu, Chamomile, Callus uripei, Raspberry , kiwi, bellflower, cabbage, caraway, cucumber, kiyomi, kumquat, ginkgo nut, guava, wolfberry, kudzu, gardenia, cumin, cranberry, walnut, grapefruit, clementine, clove, kuromaru, black bean, chlorella, ketsumeishi, gennochoko, cowberry, Goose, cosmos, corn, wheat, sesame, komatsuna, rice, coriander, konnyaku, kelp, salmonberry, rhinoceros, pomegranate, sweet potato, taro, sugar cane, sugar beet, saffron, pomelo, hawthorn, Japanese pepper, shiitake, cyclamen, ginger , shiso, shimeji, potato, peony, jasmine, juzdama, chrysanthemum, clove, white oak, daphne, cinnamon, watermelon, sweet pea, sweet spring, horsetail, star anise, star apple, loli, cnidium, japonica, buckwheat, broad bean, radish, Soybean, soybean orange, thyme bamboo shoot, onion, oyster mushroom, tarragon, taro, tankan, tangor, tanjin, tanzero, dandelion, chicory, azalea, horsetail, camellia, centella asiatica, clover, turkweed, tuna, Japanese silverberry, dill, dekopon, pelargonium, Red pepper, hot pepper, angelica, Tokyu kasou, corn, houttuynia cordata, tokon, eucommia, ash, Chinese yam, shepherd's purse, nutmeg, nutmeg, nutmeg, nanten, bitter gourd, wormwood, Chinese chive, carrot, garlic, green onion, yarrow, saw palmetto, nobil, verbena , Palm, Pineapple, Hibiscus, Chickweed, Paprika, Hamamelis, Haruka, Harumi, Harehime, Banpeiyu, Beet, Bell pepper, Amaryllidaceae, Hiba, Water chestnut, Hijiki, Pistachio, Hyssop, Daisy, Corn poppy, Hinoki , Castor, Sunflower, Himenotsuki, Hyuganatsu, Loquat, Phalaenopsis, Fenegreek, Butterbur, Blackberry, Plum, Blueberry, Bilberry, Prune, Buntan, Loofah, Safflower, Beni Madonna, Belladonna, Bergamot, Garden Balsam, Spinach, Physalis, Bodaiju, Button , hops, jojoba, ponkan, maitake, maou, maca, macadamia nuts, marcot, actinidia, marigold, merihime, mango, mitsuba, minneola, mimosa, myoga, myrrh, purple, mace, melissa, melilot, melon, men, Bean sprouts, cornflower, yam, wild lily, sagebrush, eucalyptus, saxifrage, yuzu, lily, yokuinin, yomena, mugwort, lime, rye, lilac, raspberry, groundnut, scallion, apple, gentian, reiko, lychee, lettuce, lemon, astragalus, Examples include lotus root, rosehip, rosemary, bay leaf, spring onion, and wasabi. Among these, preferred are wheat, rice, corn, soybean, konnyaku, maitake and Tamogitake; more preferred is konjac.

The sphingoid base used in the present invention can be obtained from the animals or plants described above by a known production method. Moreover, you may purchase a commercially available sphingoid base.

Sphingoid bases can be produced, for example, by extracting glycosphingolipids from the above-described animals or plants, removing sugars by enzymatic reactions or the like, and then removing fatty acids by chemical reactions or the like. In this case, the glycosphingolipid used as a raw material is not particularly limited as long as the target sphingoid base can be obtained, and any sugar such as glucose or galactose may be bound to ceramide. The enzyme for removing sugar is not particularly limited as long as it can hydrolyze the bond between glycosphingolipid and ceramide, and examples thereof include endoglycosidase (EGCase). Three types of EGCase (EGCaseI, EGCaseII, and EGCaseIII) with different isoelectric points and molecular weights are known, and it is known that the substrate specificity differs depending on the molecular type. The molecular species of EGCase to be used may be appropriately selected according to the structure of the glycosphingolipid used as a substrate.

The produced sphingoid base may be subjected to concentration/purification treatment, if necessary. Examples of the concentration treatment include known concentration treatment using a vacuum concentration device such as an evaporator. Further, examples of the purification treatment include known purification treatments such as ion exchange resin, alkali treatment, solvent fractionation, and silica gel chromatography.

The content of the sphingoid base in the exosome production promoter of the present invention is not limited to an effective amount that can promote the production of exosomes in vivo, and is appropriately adjusted according to the application, dosage form, dosage form, etc. be able to.

[Other additive ingredients]
Exosome production promoter of the present invention, in addition to the sphingoid base described above, in a range that does not impair the effects of the present invention, depending on the dosage form, may contain other additive components. Examples of additive components that can be contained in the exosome production promoter of the present invention include water, oils and fats, waxes, hydrocarbons, fatty acids, higher alcohols, esters, plant extracts, comet polymers, interfaces Active agents, metal soaps, alcohols, polyhydric alcohols, pH adjusters, antioxidants, ultraviolet absorbers, preservatives, fragrances, powders, thickeners, pigments, chelating agents and the like. These additive components may be used singly or in combination of two or more. In addition, the content of these additive components is appropriately set according to the type of additive component to be used and the dosage form of the exosome production promoter of the present invention.

[Dosage form/Formulation/Application]
The dosage form of the exosome production promoter of the present invention is not particularly limited, and may be solid, semi-solid, or liquid, depending on the type, application, administration method, etc. of the exosome production promoter. can be set as appropriate.

The method of administering the agent for promoting exosome production of the present invention is not particularly limited, and may be appropriately selected depending on the type of disease to be applied, and may be systemic administration or local administration. Specific examples include oral, transvascular (intraarterial or intravenous), transdermal, enteral, pulmonary, intranasal administration and the like. Intravascular administration also includes intravascular injection and continuous infusion. Among them, oral administration is preferable from the viewpoint of ease of administration and effective exosome production promoting effect.

The formulation form of the exosome production promoter of the present invention is not particularly limited, and can be appropriately set to a formulation form suitable for the administration method. For example, tablets, capsules, granules, powders, syrups, injections , drops, and suppositories. For example, when the dosage form of the exosome production promoter of the present invention is oral administration, it is not particularly limited as long as it can be administered orally, but specifically includes food and drink and internal medicines.

When the exosome production promoter of the present invention is made into a food or drink formulation, the exosome production promoter of the present invention may be prepared in a desired form as it is or in combination with other food materials or additives. Examples of such food and drink include general food and drink, foods for specified health insurance, dietary supplements, functional foods, foods for the sick, and the like. The form of these foods and drinks is not particularly limited, but specifically, capsules (soft capsules, hard capsules), tablets, downstream agents, powders, jelly agents, supplements such as liposome preparations, nutritional drinks, fruit juice beverages, Beverages such as carbonated beverages and lactic acid beverages; luxury goods such as dumplings, ice creams, chocolates, gummies, and candies; Among these foods and drinks, beverages and supplements are preferred, and beverages and capsules are more preferred.

When the exosome production promoter of the present invention is made into a pharmaceutical formulation for internal use, the exosome production promoter of the present invention may be prepared in a desired form as it is or in combination with other additive components. Specific examples of such drugs for internal use include drinks, capsules (soft capsules, hard capsules), tablets, granules, powders, jellies, syrups, liposome preparations, and the like. Among these medicines for internal use, capsules and drinks are preferable.

When the exosome production promoter of the present invention is in the form of a food or beverage or an internal medicine formulation, the content of the sphingoid base, which is an active ingredient, is not particularly limited as long as it is an effective amount that promotes the production of exosomes. , which may be appropriately set according to the formulation form, for example, 0.1 to 20% by mass, preferably 0.5 to 10% by mass, more preferably 1 to 5% by mass.

The agent for promoting exosome production of the present invention can be applied to diseases whose symptoms are alleviated or improved based on promotion of exosome production in nerve cells. In addition, the exosome production promoter of the present invention can be used for the purpose of preventing or improving diseases caused by accumulation of amyloid β protein.

For example, exosomes secreted from neurons can suppress the accumulation of amyloid β protein, which is one of the causes of Alzheimer's disease. or reduce symptoms. That is, the agent for promoting exosome production of the present invention can suppress the onset of Alzheimer's disease and alleviate symptoms, and can be suitably used as an agent for preventing Alzheimer's disease. Furthermore, the exosome production promoter of the present invention can be used for the purpose of suppressing memory impairment, cognitive decline, etc. caused by accumulation of amyloid β protein.

In addition, other diseases whose symptoms can be improved by promoting the production of exosomes in nerve cells include, for example, Parkinson's disease, frontotemporal degeneration, neurodegenerative diseases such as polyglutamine disease, and the like.

The application amount of the exosome production promoter of the present invention is not particularly limited, and may be appropriately set according to the formulation form, application, administration target, expected effect, and the like. For example, when the exosome production promoter of the present invention is orally ingested, the intake amount is 0.1 to 5 mg, preferably 0.1 to 1 mg per day for adults in terms of the amount of sphingoid base. The exosome production promoter of the present invention may be administered once or divided into several times so that the amount per day falls within the above range.

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited by these examples.

[Experimental example 1]
1. Preparation of sphingoid bases
After treating the konjac extract (provided by Daicel Co., Ltd.) with 10 times the amount of acetone to remove simple lipids, an appropriate amount of chloroform / methanol / 0.4 M KOH aqueous solution (chloroform: methanol: volume ratio of 0.4 M KOH aqueous solution Glycerolipid was saponified in a mixed solvent of 1:1:1), and glucosylceramide was purified by silica gel column chromatography. The purified glucosylceramide was thermally decomposed at 110° C. in an appropriate amount of dioxane/10% barium hydroxide aqueous solution (volume ratio of dioxane:10% barium hydroxide aqueous solution 1:1) to liberate the sphingoid base. The sphingoid bases were finally purified by HPLC equipped with an ODS column to give sphingosine, 4-trans,8-cis-sphingadienine, and 4-trans,8-trans-sphingadienine, respectively.

2. Sphingoid base treatment of human nerve-derived cells and exosome recovery from culture supernatant Human neuroblastoma SH-SY5Y cells (purchased from ATCC) were used for experiments. The cells were seeded in a 6-well plate with a medium (50% Ham's F12/50% E-MEM containing 10% calf serum) at 2.2×10 ⁷ cells/well and cultured at 37° C. for 24 hours. After that, sphingosine (Avanti Polar Lipids), 4-trans,8-cis-sphingadienine, and 4-trans,8-trans-sphingadienine were mixed with 3% bovine serum albumin (BSA). It was suspended in serum-free medium (50% Ham's F12/50% E-MEM) and added to each well so that the final concentration in the well was 5 μM. In addition, dimethyl sulfoxide (DMSO) was added to each well instead of the sphingoid base so that the final concentration in the well was 5 μM, and this was used as a control.

After 24 hours from the addition of the sphingoid base, the culture supernatant (1.5 mL/well) was recovered, and the exosomes therein were recovered. Centrifugation was used to collect exosomes. Specifically, the collected culture supernatant was subjected to stepwise centrifugation at 2,000 g for 10 minutes, 10,000 g for 30 minutes, and 100,000 g for 70 minutes to collect exosomes as a sediment.

3. After suspending exosomes recovered by exosome particle number measurement centrifugation in HEPES / KCl buffer, the particle number was measured with a nanoparticle analyzer qNano (Izon). The measured value was displayed as the number of exosome particles per protein amount (mg) of the derived cells calculated by the BCA method. The number of exosome particles is the average value of each value obtained by performing the measurement twice. In addition, the number of exosome particles was calculated as a ratio (% of control) when the control was 100%. The obtained values were also subjected to a one-way ANOVA method to test for significant difference ( ^*** P<0.001, ^** P<0.01, ^* P<0.05).

4. Results Results are shown in FIGS. Figure 1 shows the number of exosome particles when sphingosine is added, and Figure 2 shows 4-trans, 8-cis-sphingadienine, and 4-trans, 8-trans-sphingadienine added shows the number of exosome particles when

As is clear from FIG. 1, when sphingosine was added, the number of exosome particles in neurons tended to increase relative to the control, and it was confirmed that sphingosine can promote the production of exosomes.

In addition, as is clear from FIG. 2, the addition of 4-trans, 8-cis-sphingadienine, and 4-trans, 8-trans-sphingadienine increased the number of exosome particles in neurons relative to the control. , was confirmed to increase statistically significantly.

From the above results, sphingoid bases have the effect of promoting the production of exosomes in neurons, particularly 4-trans, 8-cis-sphingadienine, and 4-trans, 8-trans-sphingadie It was found that the garlic is excellent in this effect.

Claims (3)

4-trans, 8-trans-sphingadienin and / or 4-trans, 8-cis- exosome production promoter comprising sphingadienin as an active ingredient. Exosome production promoter according to claim 1 , which is food and drink for exosome production promotion. Exosome production promoter according to claim 1 , which is a drug for promoting exosome production.

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