Autophagy inducer containing vaccinium vitis-idaea

Abstract

To provide an autophagy inducer.SOLUTION: An autophagy inducer contains Vaccinium vitis-idaea.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an autophagy inducer comprising bilberry. [Background technology]

[0002] When intracellular proteins and organelles are no longer needed or as needed, they are degraded by autophagy, and the components are reused within the cell. Autophagy activity is low in cells under normal conditions, but becomes active when cells are exposed to stressful environments such as starvation or hypoxia. Autophagy involves a process in which a portion of the cytoplasm is surrounded by an isolation membrane to form a vesicle called an autophagosome, which then fuses with a lysosome to become an autolysosome, which then degrades the enclosed contents (waste products, unnecessary proteins, etc.).

[0003] It is known that decreased autophagy activity is one of the causes of various diseases and conditions, and it has been reported to be involved in, for example, neurodegenerative diseases, infectious diseases, inflammatory diseases, immune diseases, kidney diseases, respiratory diseases, eye diseases, muscle diseases, mitochondrial diseases, lifestyle-related diseases, skin aging, and a variety of other diseases.

[0004] From this perspective, a search has been made for components that induce autophagy, and it has been reported that, for example, neutralized plum flesh extract (Patent Document 1), plant extracts such as hydrangea, ginkgo, Scutellaria, Anacarya, cherry blossom, and orchid (Patent Document 2), Ganoderma lucidum extract (Patent Document 3), and China chinaberry extract (Patent Document 4) have autophagy-inducing effects.

[0005] Thus, although various autophagy inducers have been discovered, their effects are not always sufficient, and there is still a demand for autophagy inducers with superior effects. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2007-143452 A [Patent Document 2] JP 2013-99305 A [Patent Document 3] Special Publication No. 2016-501861 [Patent Document 4] JP 2018-188480 A Summary of the Invention

[0007] The present invention provides an autophagy inducer.

[0008] The present inventors have found that bilberry is useful for promoting the production of LC3II protein, which is known as a marker of autophagy, and the present invention is based on this finding.

[0009] According to the present invention, the following inventions are provided. (1) An autophagy inducer comprising cowberry. (2) The autophagy inducer according to (1), wherein autophagy is carried out in epidermal keratinocytes. (3) A food composition comprising the autophagy inducer according to (1) or (2). (4) An agent for promoting the production of LC3II protein comprising lingonberry. (5) The LC3II protein production promoter according to (4), wherein the promotion of LC3II protein production is carried out in epidermal keratinocytes. (6) A food composition comprising the LC3II protein production promoter according to (4) or (5). (7) Use of cowberry for the manufacture of a drug for the induction of autophagy. (8) The use according to (7) for the manufacture of a drug for inducing autophagy in epidermal keratinocytes. (9) Use of cowberry for the manufacture of a drug intended to promote production of LC3II protein. (10) The use according to (9) for the manufacture of a drug intended to promote production of LC3II protein in epidermal keratinocytes. (11) Use of a composition comprising lingonberry for inducing autophagy. (12) The use according to (11) for inducing autophagy in epidermal keratinocytes. (13) Use of a composition comprising lingonberry to promote production of LC3II protein. (14) The use according to (13) for promoting production of LC3II protein in epidermal keratinocytes. (15) Cowberry to induce autophagy. (16) Cowberry to induce autophagy in epidermal keratinocytes. (17) Cowberry to promote the production of LC3II protein. (18) Cowberry to promote the production of LC3II protein in epidermal keratinocytes. (19) A method for inducing autophagy in a subject, the method comprising administering an effective amount of lingonberry. (20) A method for inducing autophagy in epidermal keratinocytes, comprising administering an effective amount of cowberry. (21) A method for promoting production of LC3II protein in a subject, the method comprising administering an effective amount of lingonberry. (22) A method for promoting production of LC3II protein in epidermal keratinocytes, comprising administering an effective amount of cowberry.

[0010] According to the present invention, an autophagy inducer is provided. Furthermore, according to the present invention, it is also possible to provide an agent for promoting the production of LC3II protein. [Brief description of the drawings]

[0011] [Figure 1]FIG. 1 shows the results of evaluating the amount of melanin in a skin model 3 weeks after the start of culture using a culture medium containing ascorbic acid or bilberry. [Diagram 2] FIG. 2 shows the results of evaluating the amount of LC3II protein in a skin model 3 weeks after the start of culture using a culture medium supplemented with ascorbic acid or bilberry. Description of the Invention

[0012] According to one aspect of the present invention, there is provided an autophagy inducer comprising bilberry. Such an autophagy inducer may be a composition for inducing autophagy.

[0013] According to another aspect of the present invention, there is provided an agent for promoting LC3II protein production, the agent for promoting LC3II protein production comprising bilberry. Such an agent for promoting LC3II protein production may be a composition for promoting the production of LC3II protein.

[0014] Cowberry (Vaccinium vitis-idaea (Chinese name: Yueju) or Vaccinium myrtillus L. (Chinese name: Yueci)), also known as lingonberry, grows abundantly in the Arctic Circle of Northern Europe and Canada and is known to be rich in vitamin C and citric acid. The fruit is eaten raw and is commonly used in processed foods such as juice and jam.

[0015] Parts of cowberry that can be used in the present invention include, for example, fruits (unripe fruits, ripe fruits, dried fruits), leaves, tuberous roots, flowers, seeds, etc., and preferably, fruits are used.

[0016] The cowberry used in the present invention can be either fresh or dried. According to one aspect of the present invention, the "cowberry" is cowberry juice, which may be obtained by squeezing the fruit. According to another aspect of the present invention, the "cowberry" is cowberry extract produced using the whole or part of cowberry.

[0017] The method for producing the cowberry extract of the present invention is not particularly limited, and can be produced according to the method commonly used in the art, for example, by extracting the raw material of the extract. The extraction method is not limited to, but includes, for example, hot water extraction, ultrasonic extraction, filtration, reflux extraction, solvent (e.g., ethanol) extraction, etc. These may be carried out alone, or two or more methods may be used in combination. In addition, the extract may be further extracted one or more times in the same manner to obtain a high-purity extract. In addition, the method for producing the extract without extraction is not limited to, but includes, for example, squeezing and concentrating fruit juice, and can be carried out by any method commonly used in the art.

[0018] The type of solvent used for producing the cowberry extract of the present invention is not particularly limited, and any solvent known in the art may be used as long as it can produce an extract having the desired effect of the present invention. Examples of such solvents include, but are not limited to, water, alcohols having 1 to 4 carbon atoms, ethyl acetate, acetone, chloroform, etc., and two or more of these may be used in combination.

[0019] If lingonberries (including juice, dried powder, extract, etc.) are commercially available, they may be used as long as they have the desired effect of the present invention.

[0020] In the present invention, "autophagy" refers to a system that breaks down a part of the cytoplasm, such as cytoplasmic components containing proteins and intracellular organelles such as mitochondria. Such a system is known to have functions such as breaking down proteins to supply amino acids under starvation conditions and breaking down intracellular waste products. The autophagy system plays an important role in maintaining normal biological functions, and abnormalities in the autophagy system can cause aging, resulting in symptoms caused by the accumulation of intracellular waste products, such as organ and tissue dysfunction. Promotion of autophagy function is expected to lead to the prevention of aging of the skin and the like and the extension of healthy lifespan. Therefore, according to one preferred embodiment of the present invention, the autophagy inducer and LC3II protein production promoter of the present invention are intended for the prevention of aging and the extension of healthy lifespan.

[0021] It is also known that abnormalities in the autophagy system cause various diseases. Examples of such diseases include neurodegenerative diseases, metabolic disorders, immune disorders, infectious diseases, cancer, and other diseases. Examples of neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's chorea, and dementia. Examples of metabolic disorders include type II diabetes, nephropathy, metabolic syndrome, and fatty liver. Examples of immune disorders include Crohn's disease and autoimmune diseases. Examples of infectious diseases include opportunistic infections. Examples of other diseases include arteriosclerosis, muscular atrophy, myopathy, and anemia. It is expected that promoting the function of autophagy will lead to the prevention and improvement of these diseases, and therefore, according to one preferred embodiment of the present invention, the autophagy inducer and the LC3II protein production promoter of the present invention are intended to prevent and improve these diseases.

[0022] Activation of autophagy is induced by the activation of the unc-51 like kinase (ULK) complex, which is induced by the inhibition of the mammalian target of rapamycin (mTOR), a negative regulator, by the intracellular energy sensor AMP-activated protein kinase (AMPK). The ULK complex activates various autophagy-related genes (Atg). For example, Atg7, by its ubiquitin-like enzyme activity, binds phosphatidylethanolamine to the LC3 protein (microtubule-associated protein light chain 3), converting it to the LC3II protein. The LC3II protein induces autophagy by extending and closing the isolation membrane. The LC3II protein in the present invention is synonymous with such an LC3II protein.

[0023] According to a preferred embodiment of the present invention, the promotion of autophagy or LC3II protein production in the present invention is carried out in epidermal keratinocytes. The epidermal keratinocytes in the present invention are not limited to, but may be, for example, cells obtained from a living body, their passaged cells, or established cells, and these cells may be differentiated and used as necessary. The epidermal keratinocytes in the present invention may be cells derived from any animal or plant, preferably animal cells, and preferably human cells. Specific examples of the epidermal keratinocytes in the present invention include normal human neonatal epidermal keratinocytes (NHEK) and normal human epidermal melanocytes (NHEM).

[0024] The epidermal keratinocytes in the present invention may be cells constituting an epidermis model. As the epidermis model in the present invention, for example, a normal skin melanocyte co-culture model can be used.

[0025] The autophagy inducer and LC3II protein production promoter of the present invention may be applied either in vivo or in vitro. When the autophagy inducer and LC3II protein production promoter of the present invention are applied to a target, examples of the target include, but are not limited to, primates including humans and chimpanzees, pet animals such as dogs and cats, livestock animals such as cows, horses, sheep, and goats, and mammals such as rodents such as mice and rats.

[0026] The amount of lingonberry used in the autophagy inducer or LC3II protein production promoter of the present invention can be appropriately determined depending on the application, but preferably the weight of lingonberry juice is 1 mg to 10 g, more preferably 10 mg to 1 g, and more preferably 50 mg to 500 mg per 100 g of the autophagy inducer or LC3II protein production promoter of the present invention. Note that in the present invention, lingonberry juice refers to undiluted and unconcentrated fruit juice obtained by squeezing the fruit (which may include other parts such as seeds and skin).

[0027] The autophagy inducer and LC3II protein production promoter of the present invention may be an oral composition or a topical composition. It is believed that the lingonberry of the present invention is preferably absorbed from the digestive tract by oral ingestion, and the active ingredient acts in the body. Therefore, according to one preferred embodiment of the present invention, the autophagy inducer and LC3II protein production promoter of the present invention is an oral composition.

[0028] When the autophagy inducer and LC3II protein production promoter of the present invention are used as an oral composition, known ingredients (additives) that are incorporated into oral compositions can be further incorporated. Examples of such ingredients include, but are not limited to, emulsifiers, hydrating agents, solvents, emollients, stabilizers, thickeners, preservatives, lubricants, chelating agents, fillers, excipients, powders, fragrances, flavorings, absorbents, dyes, opacifiers, antioxidants, preservatives, vitamins, amino acids, nutrients, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, pectinic acid and its salts, alginic acid and its salts, organic acids, pH regulators, glycerin, alcohol, carbonation agents used in carbonated drinks, etc.

[0029] The oral composition of the present invention may be any of liquid, semi-solid, and solid.

[0030] When the autophagy inducer and LC3II protein production promoter of the present invention are used as a topical composition, known ingredients (additives) that are incorporated into topical compositions can be further incorporated. Examples of such ingredients include, but are not limited to, emulsifiers, hydrating agents, solvents, emollients, stabilizers, thickeners, preservatives, lubricants, chelating agents, fillers, excipients, powders, fragrances, flavorings, absorbents, dyes, opacifiers, antioxidants, vitamins, amino acids, etc.

[0031] The topical compositions of the present invention may be liquid, semi-solid, or solid.

[0032] The autophagy inducer and LC3II protein production promoter of the present invention may be any of a pharmaceutical composition, a cosmetic composition, or a food composition, and is preferably a food composition. EXAMPLES

[0033] The present invention will be described in detail with reference to the following examples, but the present invention is not limited to these examples. Contents are expressed in mass % unless otherwise specified.

[0034] Preparation of drugs to be applied to the skin model The test substance was lingonberry (Chinese lingonberry) juice (purple liquid) (Oryza Oil & Fat Chemical Co., Ltd.) which was diluted to 300μg / mL, 100μg / mL, or 30μg / mL in the culture medium. As a control substance, L-(-)ascorbic acid (Kanto Chemical Co., Ltd.) was diluted to 100μg / mL in the culture medium.

[0035] Cultivation of skin models A melanocyte-containing three-dimensional skin model (MEL-300, Kurabo Co., Ltd.) was purchased and cultured in a 37℃, 5% CO2 incubator according to standard methods. Four wells (n=4) were designed for each condition. EPI-100NMM113 was used as the culture medium, and bFGF, α-MSH, and KGF were added in appropriate amounts as melanin stimulating factors. The culture medium was replaced every other day, and the drug (test substance or control substance) was dissolved to a specified concentration each time the culture medium was replaced. The culture was performed so that each drug was applied from the bottom side of the skin model. The culture was continued for 3 weeks after the start of culture.

[0036] Evaluation of the effect of cowberry on a skin model

[0037] After the skin model was cultured, it was washed with PBS (-) and then divided into two equal parts. One part was used to measure the amount of melanin in the skin model, and the other part was used to measure the amount of LC3II in the skin model.

[0038] The amount of melanin in the skin model was measured according to the following procedure. The collected skin model was extracted by infiltration overnight at 60°C using 0.1N NaOH, and then centrifuged to remove the film of the skin model and the insoluble fraction as a precipitate. The absorbance of the solution fraction was measured at 405 nm, and the amount of melanin was calculated based on the measurement results. The melanin inhibition rate of each group was calculated using the following formula, with the amount of melanin in the control group set at 100%.

number

[0039] The amount of LC3II in the skin model was measured by the following procedure. The collected skin model was crushed and extracted at 4°C using 250μL of T-PER (trademark) Tissue Protein Extraction Reagent buffer (manufactured by Thermo Scientific) to prepare lysate. The crushing and extraction was carried out by adding the skin model, one zirconia ball, and T-PER Buffer to a polypropylene tube, and using a crushing extractor (bead-type cell crusher Micro Smash MS-100R (manufactured by Tommy Seiko Co., Ltd.)) at 4°C, 2000 rpm, and 5 minutes. The concentration of total protein was calculated by a relative comparison with BSA (bovine serum albumin) using a part of the lysate (20μL) by the standard method of BCA protein assay (measured at OD562nm). The amount of LC3II protein contained in 100 μL of lysate was measured based on the OD450nm value using an ELISA kit / Human Microtubule-associated proteins 1A / 1B light chain 3B (MAP1LC3B) ELISA kit CSB-EL013403HU (Cusabi). The measurement results were corrected to values ​​per unit protein amount.

[0040] Statistical analysis was performed using the Dunnett test as a multiple comparison test to determine whether or not there was a significant improvement in the vs. control group. p<0.05 was marked with *, and p<0.01 with **. In addition, 0.05≦p<0.1 was considered to indicate a significant trend, and the p value was indicated.

[0041] The evaluation results of the melanin suppression rate in the skin model are shown in Figure 1. The melanin suppression effect of ascorbic acid and cowberry was confirmed.

[0042] The results of evaluating the amount of LC3II in the skin model are shown in Figure 2. A significant increase in LC3II protein amount was observed in the groups that received 300 μg / mL and 100 μg / mL of bilberry, suggesting the possibility of autophagy activation by bilberry. On the other hand, no significant increase in LC3II protein amount was observed in the group that received 100 μg / mL of ascorbic acid.

Claims

1. An autophagy inducer comprising cowberry.

2. The autophagy inducer according to claim 1 , wherein autophagy is carried out in epidermal keratinocytes.

3. A food composition comprising the autophagy inducer according to claim 1 or 2.

4. An agent for promoting the production of LC3II protein comprising lingonberry.

5. The LC3 II protein production promoter according to claim 4, wherein promotion of LC3 II protein production is carried out in epidermal keratinocytes.

6. A food composition comprising an agent for promoting the production of LC3II protein according to claim 4 or 5.

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