Autophagy activator and protein aggregate degradation accelerator

Abstract

The objective of this disclosure is to provide an autophagy activator that can activate autophagy in vivo. [Solution] An autophagy activator containing Seihaito extract as an active ingredient.

Description

Technical Field

[0001] One embodiment of the present disclosure relates to an autophagy activator that can activate autophagy in vivo. Another embodiment of the present disclosure relates to an accelerator for promoting the degradation of protein aggregates in vivo.

Background Art

[0002] Autophagy is an intracellular degradation mechanism in which a part of the cytoplasm is surrounded by an isolation membrane to form a membrane structure called an autophagosome, and this autophagosome fuses with a lysosome to become an autolysosome, thereby degrading waste products, unnecessary proteins, and the like. In recent years, autophagy has played an important role in physiological or pathological functions in the living body, and many diseases and symptoms caused by a decrease in autophagy activity have been reported. Such diseases and symptoms include, for example, neurodegenerative diseases, infectious diseases, inflammatory diseases, immune diseases, kidney diseases, respiratory diseases, eye diseases, muscle diseases, mitochondrial diseases, lifestyle diseases, skin aging, and various other diseases or symptoms.

[0003] Therefore, conventionally, various pharmaceuticals and foods containing active ingredients that activate autophagy have been developed for the prevention or treatment of diseases and symptoms caused by a decrease in autophagy activity. For example, Patent Document 1 reports that memantine, clemastine, and their salts can be used as autophagy inducers. Patent Document 2 reports that a specific pyrroloquinoline quinone can be used as an autophagy inducer. Patent Document 3 reports that cyclic glucooligosaccharides can be used as autophagy inducers. Patent Document 4 discloses that a hydrophobic organic solvent extract of ume flesh extract or a neutralized product of ume flesh extract can be used as a food or drink for inducing autophagy. However, in order to respond to diversification of formulation and further improvement of autophagy activation effect, development of new active ingredients for activating autophagy is desired.

[0004] Furthermore, it is known that when proteins that have not been able to adopt a normal higher-order structure due to unfolding or misfolding form aggregates and accumulate both inside and outside cells, they can cause neurodegenerative diseases and conformational diseases such as amyloidosis. It has also been reported that protein aggregates induced by cigarette smoke are a cause of chronic obstructive pulmonary disease and emphysema (Non-Patent Literature 1). Therefore, if the protein aggregate degradation function of autophagy can be enhanced, it is expected that the effect of suppressing conformational diseases and maintaining the health of the body will be increased. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2019-94304 [Patent Document 2] Japanese Patent Publication No. 2018-83790 [Patent Document 3] International Publication No. 2018 / 173653 [Patent Document 4] Japanese Patent Publication No. 2007-143452 [Non-patent literature]

[0006] [Non-Patent Document 1] Ian Tran et al., Role of Cigarette Smoke-Induced Aggresome Formation in Chronic Obstructive Pulmonary Disease-Emphysema Pathogenesis, Am. J. Respir Cell Mol. Biol., Vol.53, Iss.2, pp.159-173, 2015 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] One embodiment of this disclosure aims to provide an autophagy activator that can activate autophagy in vivo. Another embodiment of this disclosure aims to provide a protein aggregate degradation accelerator that can promote the degradation of protein aggregates in vivo. [Means for solving the problem]

[0008] The inventors of the present invention conducted diligent research to solve the aforementioned problems and discovered that Seihaito extract has excellent autophagy-activating properties and can be used as an active ingredient in autophagy activators. Furthermore, the inventors discovered that Seihaito extract also has excellent properties for promoting the degradation of protein aggregates and can be used as an active ingredient in protein aggregate degradation accelerators. The present invention was completed by further research based on these findings.

[0009] One embodiment of the present disclosure provides an invention relating to an invention for autophagy activation, in the form described below. Item 1-1. An autophagy activator containing Seihaito extract as an active ingredient. Item 1-2. Autophagy activators as described in Item 1-1, used in combination with pharmaceuticals. Sections 1-3. Pharmaceuticals for activating autophagy, including the autophagy activators described in Section 1-1. Sections 1-4. Use of Qingfei Tang extract for the manufacture of autophagy activators. Sections 1-5. Qingfei Tang extract, used in treatments to activate autophagy. Sections 1-6. A method for activating autophagy, comprising administering an effective amount of Qingfei Tang extract to a person who requires activation of autophagy. Sections 1-7. Non-therapeutic use of Qingfei Tang extract to activate autophagy.

[0010] Another embodiment of the present disclosure relates to an invention for promoting the degradation of protein aggregates, and provides an invention in the following aspects. Item 2-1. A protein aggregate decomposition accelerator containing Seihaito extract as an active ingredient. Item 2-2. A protein aggregate degradation accelerator described in Item 2-1, used in combination with pharmaceuticals. Section 2-3. Protein aggregate degradation accelerators as described in Section 2-1 or 2-2, used for the prevention or improvement of conformational diseases. Item 2-4. A pharmaceutical product for promoting the degradation of protein aggregates, comprising the protein aggregate degradation accelerator described in Item 2-1. Item 2-5. A pharmaceutical product for promoting the degradation of protein aggregates as described in Item 2-4, used for the prevention or improvement of conformational diseases. Section 2-6. Use of Qingfei Tang extract for the manufacture of a protein aggregate decomposition accelerator. Section 2-7. Qingfei Tang extract, used in treatments to promote the breakdown of protein aggregates. Section 2-8. A method for promoting the degradation of protein aggregates, comprising administering an amount of Qingfei Tang extract effective in promoting the degradation of protein aggregates to a person who requires promotion of protein aggregate degradation. Section 2-9. Non-therapeutic use of Qingfei Tang extract to promote the breakdown of protein aggregates. [Effects of the Invention]

[0011] The autophagy activator disclosed herein can exhibit excellent autophagy activating effects, making it possible to provide products that can prevent or improve diseases and symptoms caused in part by decreased autophagy activity.

[0012] Furthermore, since the protein aggregate degradation accelerator of this disclosure can effectively exert a protein aggregate degradation-promoting effect, it becomes possible to provide products that can prevent or improve diseases and symptoms caused in part by the accumulation of protein aggregates. [Modes for carrying out the invention]

[0013] In this disclosure, the notation "X~Y" for numerical ranges means the range between X and Y.

[0014] 1. Autophagy activators The autophagy activator of the present disclosure is characterized by having Seihakuto extract as an active ingredient. In the present disclosure, the "autophagy activator" refers to a component used for the purpose of activating autophagy. Hereinafter, the autophagy activator of the present disclosure will be described in detail.

[0015] [Active ingredient] Seihakuto is a mixed crude drug containing Scutellaria baicalensis Georgi, Platycodon grandiflorum, Glycyrrhiza glabra, Coptis japonica, Panax ginseng, Prunella vulgaris, Poria cocos, Zingiber officinale, Mentha haplocalyx, Bupleurum falcatum, Paeonia lactiflora, Cinnamomum cassia, Mori cortex, Asarum sieboldii, and Glycyrrhiza uralensis. The standards and used parts of these crude drugs are defined in the Japanese Pharmacopoeia and the specifications for crude drugs outside the Japanese Pharmacopoeia. The preparation of Seihakuto that can be used in the present disclosure can be carried out in accordance with the "Manual of Kampo Prescriptions for General Use" (supervised by the Pharmaceutical Affairs Bureau of the Ministry of Health, Labor and Welfare, edited by the Kampo Special Committee of the Japan Pharmaceutical Manufacturers Association, published by Yakugyo Jiho Co., Ltd.) or the "Revised Manual of Kampo Prescriptions for General Use" (supervised by the Japan Public Document Association, edited by the Japan Kampo Crude Drug Preparation Association, published by Jihou Co., Ltd.).

[0016] Regarding the mixing ratio of each crude drug contained in Seihakuto, there is no particular limitation, but usually, by weight ratio, Scutellaria baicalensis Georgi 2 - 2.5, Platycodon grandiflorum 2 - 2.5, Glycyrrhiza glabra 2 - 2.5, Coptis japonica 2 - 2.5, Panax ginseng 2 - 2.5, Prunella vulgaris 2 - 2.5, Poria cocos 2 - 2.5, Zingiber officinale 2 - 2.5, Mentha haplocalyx 2 - 2.5, Bupleurum falcatum 2 - 2.5, Paeonia lactiflora 2 - 2.5, Cinnamomum cassia 3, Mori cortex 3, Asarum sieboldii 0.5 - 1, Zingiber officinale 1, and Glycyrrhiza uralensis 1 can be mentioned.

[0017] The Seihakuto extract used in the present disclosure is an extract obtained by subjecting Seihakuto to an extraction process.

[0018] The extraction solvent used in the extraction process of Qingfei Tang is not particularly limited, but examples include water, ethanol, and mixtures thereof. The extraction conditions for Qingfei Tang are not particularly limited, but for example, one method involves adding 5 to 25 times, preferably 10 to 20 times, the amount of extraction solvent relative to the total weight (dry weight) of the crude drugs, and then heating and decocting it at 70 to 100°C for 30 minutes to 2 hours.

[0019] After the extraction process of Qingfei Tang, the solid matter is removed by solid-liquid separation to obtain an extract of Qingfei Tang. The extract obtained from the extraction process may be used as is as a non-concentrated extract, but it may also be subjected to a concentration process to be used as a soft extract, or further subjected to a drying process to be used as an extract powder. The extract powder may also be formed into granules as needed.

[0020] [How to use autophagy activators] The autophagy activators of this disclosure may be administered in vivo via any of the following routes: oral, transdermal, transpulmonary, transmucosal, enteral, intravenous, or nasal, but oral administration is preferred.

[0021] The autophagy activator of this disclosure may be used as is, or it may be used in the form of a product in which the active ingredient is formulated together with other ingredients. The dosage form of the product containing the autophagy activator of this disclosure may be solid, semi-solid, liquid, etc., and may be set as appropriate depending on the type and use of the product. The product containing the autophagy activator of this disclosure is not particularly limited, but examples include pharmaceuticals.

[0022] When the autophagy activator of this disclosure is used in a pharmaceutical product (including quasi-drugs) (i.e., when provided as a pharmaceutical product for autophagy activation), the active ingredient may be prepared in the desired form either as is or in combination with other additives. Examples of pharmaceutical products include oral pharmaceuticals such as tablets, pills, powders, granules, capsules (including hard and soft capsules), lozenges, chewable tablets, extracts (including soft extracts and dried extracts), jellies, syrups, alcoholic preparations, elixirs, and liposomal preparations; external pharmaceuticals such as ointments, creams, lotions, gels, sprays, patches, emulsions, suspensions, poultices, liniments, aerosols, ointments, packs, inhalants, and suppositories; and injectable preparations. Among these, oral pharmaceuticals are a preferred example.

[0023] When the autophagy activator of this disclosure is used in a pharmaceutical product, the amount of the active ingredient in the pharmaceutical product can be appropriately set according to the type of pharmaceutical product, the daily dose of the active ingredient, etc. For example, the amount of Qingfei Tang extract contained in an oral pharmaceutical product is 20 to 100% by weight, preferably 30 to 90% by weight, and more preferably 40 to 75% by weight on a dry weight basis.

[0024] [Applications, dosage, etc.] The autophagy activators disclosed herein are used to enhance the activity of autophagy in vivo.

[0025] Since improving autophagy activity in vivo can prevent or improve diseases and symptoms caused in part by decreased autophagy activity, the autophagy activator of this disclosure can be used as a preventive or ameliorative agent for such diseases or symptoms.

[0026] Diseases and symptoms that may result from a decrease in autophagy activity include, 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 various other diseases or symptoms.

[0027] Specifically, the diseases and symptoms in question include respiratory diseases such as cystic fibrosis, asthma, bronchiectasis, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis; muscular atrophy, myopathy, Alzheimer's disease, Parkinson's disease (including neuronal ceroid lipofuscinosis, multiple system atrophy (MSA), etc.), Huntington's disease, Leigh syndrome, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Lewy body dementia (DLB), Creutzfeldt-Jakob disease, and idiopathic acute pulmonary fibrosis. Neurodegenerative diseases such as rapid eye movement sleep behavior disorder; infections caused by pathogens killed by autophagy (e.g., Group A Streptococcus, Mycobacterium tuberculosis, Staphylococcus aureus, Shigella, etc.); inflammatory diseases such as Crohn's disease, pancreatitis, and sinusitis; immune diseases such as Graves' disease, autoimmune thyroiditis, Hashimoto's disease, type 1 diabetes, myasthenia gravis, Goodpasture's disease, systemic lupus erythematosus, systemic sclerosis, psoriasis, atopic dermatitis, and Sjögren's syndrome; glomerulosclerosis and renal failure Renal diseases such as; eye diseases such as dry eye and age-related macular degeneration; muscle diseases such as muscle weakness, muscle atrophy, cachexia, sarcopenia, muscular dystrophy, and myopenia; diabetic hearing loss syndrome (DAD), Leber's hereditary optic nerve atrophy, neuropathy, ataxia, retinitis pigmentosa syndrome (NARP), mitochondrial neurogastrointestinal encephalopathy syndrome (MNGIE), myoclonus epilepsy with red ragged fibers (MERRF), mitochondrial encephalomyopathy / lactic acidosis Examples of conditions that may be considered include: mitochondrial diseases such as stroke-like seizure syndrome (MELAS); lifestyle-related diseases such as type 2 diabetes, arteriosclerosis, hepatic fibrosis, cirrhosis, hepatitis, alcoholic liver disease, fatty liver, non-alcoholic steatohepatitis, metabolic syndrome, hyperlipidemia, and obesity; skin aging such as melanin accumulation, age spots, and wrinkles; and other diseases and symptoms such as brain swelling, lysosomal storage diseases, Down syndrome, heart failure, anemia, fatigue, sleep deprivation, cold sensitivity, constipation, muscle weakness, and delayed wound healing.

[0028] For example, when the autophagy activator of this disclosure is administered orally, the daily dose of the active ingredient for adults should be an amount effective in activating autophagy, such as approximately 2000 to 6500 mg per day in terms of dry weight of the Qingfei Tang extract. The daily dose may be administered orally once a day or divided into multiple doses per day.

[0029] 2. Protein aggregate degradation accelerator The protein aggregate degradation accelerator of this disclosure is characterized by containing Seihaito extract as an active ingredient. In this disclosure, "protein aggregate degradation accelerator" refers to an ingredient used to promote the degradation of protein aggregates that occur in living organisms. The protein aggregate degradation accelerator of this disclosure (sometimes simply referred to as "degradation accelerator") will be described in detail below.

[0030] [Active ingredients] The active ingredient of the decomposition accelerator in this disclosure is Qingfei Tang extract. The Qingfei Tang extract used in the decomposition accelerator in this disclosure is as described in the "1. Autophagy Activator" section above.

[0031] [How to use decomposition accelerators] The degradation accelerators of this disclosure may be applied in vivo via any route, such as orally, transdermally, transpulmonaryly, transmucosally, enterally, intravenously, or transnasally, but oral application is a preferred example.

[0032] The degradation accelerator of this disclosure may be used in the form of the active ingredient as is, or it may be used in the form of a product in which the active ingredient is formulated together with other ingredients. The dosage form of the product containing the degradation accelerator of this disclosure may be solid, semi-solid, liquid, etc., and may be set as appropriate depending on the type and use of the product. The product containing the degradation accelerator of this disclosure is not particularly limited, but examples include pharmaceuticals.

[0033] When the degradation accelerator of this disclosure is used in a pharmaceutical product (including quasi-drugs) (i.e., when provided as a pharmaceutical product for autophagy activation), the active ingredient may be prepared in the desired form either as is or in combination with other additives. Specific examples of pharmaceutical products and the amount of Seihaito extract contained in them are the same as in the case of "1. Autophagy Activators" described above.

[0034] [Applications, dosage, etc.] The degradation accelerators of this disclosure are used to accelerate the degradation of protein aggregates in vivo. The types of protein aggregates whose degradation is accelerated by the degradation accelerators of this disclosure are not particularly limited, but examples include ubiquitin-positive protein aggregates (protein aggregates to which ubiquitin is bound), and more specifically, protein aggregates containing K63-linked polyubiquitin.

[0035] By promoting the degradation of protein aggregates in vivo, the degradation accelerator of this disclosure can be used as a preventive or ameliorative agent for diseases or symptoms caused in vivo by the accumulation of protein aggregates. Examples of diseases caused in vivo by the accumulation of protein aggregates include conformational diseases. Conformational diseases are a general term for diseases caused by the formation and accumulation of aggregates of proteins that have adopted incorrect three-dimensional structures, and are also called folding disorders or protein misfolding diseases. Specifically, conformational diseases include respiratory diseases such as chronic obstructive pulmonary disease and emphysema; neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, diffuse Lewy body disease, prion diseases, polyglutamine diseases, and amyotrophic lateral sclerosis; and osteogenesis imperfecta and systemic amyloidosis.

[0036] Furthermore, it is known that the accumulation of protein aggregates in the body is associated with type 2 diabetes, non-alcoholic fatty liver disease, chronic kidney disease, glaucoma, age-related macular degeneration, and arteriosclerosis. Therefore, the degradation accelerators of this disclosure can also be used as preventive or ameliorative agents for these diseases and symptoms.

[0037] For example, when the degradation accelerator of this disclosure is administered orally, the daily dose of the active ingredient for adults should be an amount effective in promoting the degradation of protein aggregates, such as approximately 2000 to 6500 mg of Qingfei Tang extract per day in terms of dry weight. The daily dose may be administered orally once a day or divided into multiple doses per day. [Examples]

[0038] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0039] 1. Manufacturing of Seihaito extract Based on dry weight, the following ingredients were mixed in the following proportions: 2 parts by weight of Scutellaria baicalensis, 2 parts by weight of Platycodon grandiflorus, 2 parts by weight of Morus alba bark, 2 parts by weight of Apricot kernel, 2 parts by weight of Gardenia fruit, 2 parts by weight of Angelica acutiloba, 2 parts by weight of Fritillaria thunbergii, 2 parts by weight of Citrus unshiu peel, 2 parts by weight of Jujube, 2 parts by weight of Bambusa nigrum, 3 parts by weight of Poria cocos, 3 parts by weight of Angelica sinensis, 3 parts by weight of Ophiopogon japonicus, 0.5 parts by weight of Schisandra chinensis, 1 part by weight of Zingiber officinale, and 1 part by weight of Glycyrrhiza uralensis. These were then extracted using five times the amount of water as the total amount (dry weight of the raw materials) at approximately 95°C for 1 hour, and the extract was obtained by centrifugation. The obtained extract was filtered (150 mesh) to obtain Qingfei Tang extract. The obtained Qingfei Tang extract was concentrated under reduced pressure at 60°C or below according to a conventional method, and dried using a spray-drying method to prepare Qingfei Tang extract powder. The obtained Qingfei Tang extract powder was used in the following tests.

[0040] 2. Evaluation of autophagy activation effect 2-1. Test Method In this study, the autophagy activation effect was evaluated using an autophagy flux assay. Specifically, first, Calu-3 cells (human bronchial epithelial cells) were suspended in 10% fetal bovine serum (FBS)-EMEM medium (FBS-EMEM medium), and a solution was prepared in a 3 × 10⁻¹⁶ solution. 5 Seeds were seeded into 6-well plates to achieve a cell / well ratio and cultured at 37°C and 5% CO2 for 3 days.

[0041] Next, the culture medium was removed, and 2 mL of FBS-EMEM medium containing 200 μg / mL of cigarette smoke extract (CSE) was added to each well. The cells were then incubated at 37°C in 5% CO2 for 3 days (CSE-treated group). The same incubation process was also performed in FBS-EMEM medium without cigarette smoke extract (untreated group).

[0042] Cells in the wells of the CSE-treated group were washed once with PBS(-), and 2 mL of FBS-EMEM medium containing 0.01 w / v% Qingfei Tang extract powder was added to each well and incubated at 37°C in 5% CO2 for 24 hours (CSE-treated group with Qingfei Tang extract). The same incubation procedure was also performed for the CSE-treated group and the untreated group using FBS-EMEM medium without Qingfei Tang extract powder (control CSE-treated group, control untreated group).

[0043] Next, chloroquine diphosphate (a lysosomal inhibitor) was added to each well to a final concentration of 10 μM, and the cells were incubated at 37°C and 5% CO2 for a further 4 hours (chloroquine-added group). In addition, cells were incubated at 37°C and 5% CO2 for a further 4 hours under conditions where chloroquine diphosphate was not added to each well (chloroquine-free group).

[0044] Subsequently, the supernatant from each well was removed, washed once with PBS(-), and the cells were collected suspended in PBS(-) and centrifuged (1000×g, 5 min, 25°C) to obtain a cell pellet. The obtained cell pellet was then treated with RIPA buffer (Fujifilm Wako Pure Chemical Industries) containing a 1% protease inhibitor cocktail, sonicated, and centrifuged to obtain a cell lysate (supernatant).

[0045] The obtained cell lysates were adjusted to a protein concentration of 15 μg / mL by adding 4× sample buffer (BIO-RAD), and then subjected to Western blotting using anti-LC3B antibody and anti-β-actin antibody to determine the amounts of LCB-II and β-actin. The amounts of LCB-II and β-actin were determined by quantifying the intensity of the corresponding bands using ImageJ. The amount of LCB-II was standardized using β-actin as a loading control, and the autophagy flux value was determined according to the following formula, after which the autophagy activity value was calculated.

[0046]

number

[0047] 2-2. Test Results The results are shown in Table 1. These results confirm that Seihaito extract has a high autophagy activity level and excellent autophagy-activating properties.

[0048] [Table 1]

[0049] 3. Evaluation of the effect of promoting the degradation of protein aggregates 3-1. Test Method When evaluating the effect of promoting the degradation of protein aggregates, a method is known that uses K63-linked polyubiquitin as an indicator to measure the degree of degradation (S. Tan, E. Wong, Kinetics of Protein Aggregates Disposal by Aggrephagy, Methods in Enzymology, (2017) 588, ISSN 0076-6879, http: / / dx.doi.org / 10.1016 / bs.mie.2016.09.084; Nicha Puangmalai et al., Lysine 63-linked ubiquitination of tau oligomers contributes to the pathogenesis of Alzheimer's disease, J. Biol. Chem. (2022) 298(4) 101766, https: / / doi.org / 10.1016 / j.jbc.2022.101766). Furthermore, chemical substances contained in tobacco are known to promote the formation of protein aggregates. Therefore, in this study, the effect of Seihaito extract on promoting the degradation of protein aggregates was evaluated by first promoting the formation of protein aggregates with tobacco smoke extract, and then adding Seihaito extract to evaluate the amount of K63-bound polyubiquitin.

[0050] First, Calu-3 cells (human bronchial epithelial cells) were suspended in EMEM medium containing 10% fetal bovine serum (FBS) (FBS-EMEM medium), and the resulting solution was prepared in 3 × 10⁻⁶ units. 5 Seeds were seeded in a 12-well plate with sterile coverslips placed in each well to achieve a cell / well ratio, and then cultured at 37°C and 5% CO2 for 3 days.

[0051] Next, the culture medium was removed, and 1.0 mL of FBS-EMEM medium containing 200 μg / mL of cigarette smoke extract (CSE) was added to each well. The cells were then incubated at 37°C in 5% CO2 for 3 days to promote the production of K63-bound polyubiquitin in the cells (CSE-treated group). The same incubation process was also performed in FBS-EMEM medium without cigarette smoke extract (non-CSE-treated group).

[0052] Cells in the wells of the CSE-treated group were washed once with PBS(-), and 1.0 mL of FBS-EMEM medium containing 0.01 w / v% Qingfei Tang extract powder was added to each well and cultured at 37°C and 5% CO2 for 5 days (Qingfei Tang extract-treated CSE group). In addition, the CSE-treated group and the CSE-untreated group were cultured similarly using FBS-EMEM medium without Qingfei Tang extract powder (control CSE-treated group, control CSE-untreated group).

[0053] After culturing, the coverslips were removed from the wells, and the cells attached to the coverslips were fixed with 4% paraformaldehyde. Next, the cells were permeabilized with PBS(-) containing 0.1% Triton X-100, followed by blocking with PBS(-) containing 3% bovine serum albumin (BSA). The blocked cells were treated with mouse anti-K63 conjugated polyubiquitin antibody as the primary antibody, and Alexa Fluor as the secondary antibody. TM Immunostaining was performed using 568-labeled anti-mouse IgG antibody. Immunostained cells were observed under a fluorescence microscope, and fluorescence images were obtained. Using analysis software, the average fluorescence area per cell was calculated from the obtained fluorescence images. The average fluorescence area was calculated from the fluorescence area measurements of more than 700 cells.

[0054] 3-2. Test Results The results are shown in Table 2. The CSE-treated group, treated with Qingfei Tang extract, showed significantly lower levels of K63-bound polyubiquitin compared to the control CSE-treated group. This result clearly indicates that Qingfei Tang extract has an effect of promoting the degradation of protein aggregates.

[0055] [Table 2]

Claims

1. An autophagy activator containing Seihaito extract as its active ingredient.

2. An autophagy activator according to claim 1, which is used by being incorporated into a pharmaceutical product.

3. A protein aggregate decomposition accelerator containing Seihaito extract as its active ingredient.

4. A protein aggregate degradation accelerator according to claim 3, which is used by being incorporated into a pharmaceutical product.

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