Endoplasmic reticulum stress suppressing composition and liver function improving composition

Abstract

To provide an endoplasmic reticulum stress suppressing composition and a liver function improving composition.SOLUTION: A composition for suppressing endoplasmic reticulum stress and / or a composition for improving hepatic functions, comprising one or more plants selected from the group consisting of Plantago asiatica, Hamilton's spindle tree, panicled hydrangea, Japanese plum yew, Japanese mallotus, and Papaver rhoeas, and / or an extract thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for suppressing endoplasmic reticulum stress and a composition for improving liver function. [Background technology]

[0002] In recent years, endoplasmic reticulum stress, which causes the accumulation of defective protein synthesis products within cells, has been attracting attention as one of the causes of aging and diseases (cancer, diabetes, Alzheimer's disease, etc.). In particular, the number of patients with NASH / NAFLD (non-alcoholic fatty liver disease / hepatitis) has been increasing in recent years, and endoplasmic reticulum stress is believed to be the cause (Non-Patent Document 1). Compounds such as 4-phenylbutyrate (4-PBA), tauroursodeoxycholic acid (TUDCA), 7-methylsulphonyl-2-phenylimidazo[2,1-b]benzothiazole (IBT21), and oleic acid are known to suppress endoplasmic reticulum stress (Non-Patent Documents 2 to 4). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] C. Lebeaupin et al.2018.J Hepatol.Volume 69, Issue 4, p927-947. [Non-patent document 2] U. Ozcan et al.2006.Science.313(5790):1137-40. [Non-patent document 3] K. Kitakaze et al.2019.eLife,8:e43302. [Non-patent document 4] X. Zeng et al.2020.Nutr Metab(Lond).17:11. Summary of the Invention [Problem to be solved by the invention]

[0004] However, the above techniques are still insufficient, and further development of means to suppress endoplasmic reticulum stress is desired. For example, 4-PBA and TUDCA are pharmaceuticals and cannot be easily ingested. Furthermore, the safety of IBT21 has not been confirmed in humans. Oleic acid is a type of fatty acid, and excessive intake can lead to increased calorie intake, raising concerns about lifestyle-related diseases. An object of the present invention is to provide a composition for suppressing endoplasmic reticulum stress and a composition for improving liver function that can solve the above problems. [Means for solving the problem]

[0005] The present inventors have found that the above-mentioned problems can be solved by a composition containing a specific plant and / or an extract thereof. The present invention includes the following aspects [1] to [5]. [1] A composition for suppressing endoplasmic reticulum stress and / or a composition for improving liver function, comprising one or more plant bodies and / or extracts thereof selected from the group consisting of Plantago asiatica, Mayumi, Hydrangea paniculata, Cordyceps gracilis, Mallotus japonicus, and Mitsuba. [2] The composition according to [1], which is a tablet, capsule, granular dosage form or syrup. [3] The composition according to [1] or [2], which is a food product. [4] The composition according to [1] or [2], which is a pharmaceutical product. [5] The composition according to [1] or [2], which is a cosmetic. [Effects of the Invention]

[0006] The present invention provides a novel composition for suppressing endoplasmic reticulum stress and a novel composition for improving liver function. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 shows the results of the UPRE reporter assay. [Figure 2] FIG. 2 shows the results of the Western blot study. [Figure 3] FIG. 3 shows the results of the Western blot study. [Figure 4] FIG. 4 shows the results of the Western blot study. DETAILED DESCRIPTION OF THE INVENTION

[0008] [Endoplasmic reticulum stress inhibitor and / or liver function improver] The endoplasmic reticulum stress inhibitor and / or liver function improver of the present invention comprises one or more plants and / or extracts thereof selected from the group consisting of Plantago asiatica, Mayumi, Hydrangea paniculata, Cordyceps gracilis, Mallotus japonicus, and Mitsuba.

[0009] Endoplasmic reticulum stress refers to the phenomenon in which incompletely folded proteins (misfolded proteins) accumulate in the endoplasmic reticulum of cells due to various factors. ER stress is a risk factor or cause of diseases in organs such as the liver, pancreas, and nervous system. Pathologies caused by ER stress include liver dysfunction such as NASH / NAFLD (non-alcoholic fatty liver disease / hepatitis), diabetes and diabetes-associated diseases such as type 1 diabetes, type 2 diabetes, Wolcott-Rallison syndrome, and Wolfram syndrome, neurodegenerative diseases such as Alzheimer's disease, Creutzfeldt-Jakob disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease, metabolic diseases such as obesity and dyslipidemia, cardiovascular diseases such as atherosclerosis and myocardial infarction, various cancers, hypertension, renal disorders, and diseases associated with skeletal dysplasia such as Wolcott-Rallison syndrome and Coffin-Lowry syndrome.

[0010] The occurrence of ER stress can be detected, for example, by using known markers. Specifically, ER stress can be detected by detecting increased expression of CHOP protein, BiP protein (also known as GRP78 protein), truncated XBP-1 protein, ATF4 protein, etc., using an immunochemical technique using antibodies.

[0011] The one or more plant bodies and / or extracts thereof selected from the group consisting of Plantago asiatica, Mayumi, Hydrangea paniculata, Cordyceps gracilis, Mallotus japonicus, and Mitsuba, which constitute the endoplasmic reticulum stress inhibitor and / or liver function improver of the present invention, have excellent endoplasmic reticulum stress inhibitory effects. Furthermore, due to its ability to suppress endoplasmic reticulum stress, it also has the effect of improving or treating liver dysfunction caused by endoplasmic reticulum stress.

[0012] <Active ingredient> The endoplasmic reticulum stress inhibitor and / or liver function improver of the present invention contains as an active ingredient a plant body and / or an extract thereof of one or more plants selected from the group consisting of Plantago asiatica, Mayumi, Hydrangea paniculata, Cordyceps gracilis, Mallotus japonicus, and Mitsuba. As the active ingredient, the above-mentioned plant body and / or extract thereof can be used alone or in combination of two or more kinds. (Plantain) Plantain, whose scientific name is Plantago asiatica L., is a perennial plant of the Plantaginaceae family. In the natural environment, it grows mainly in East Asia. (Mayumi) The scientific name of Mayumi is Euonymus sieboldianus Blume, and it is a woody plant in the Celastraceae family. In the natural environment, it grows mainly in East Asia. (Deutzia paniculata) The scientific name for panicle hydrangea is Hydrangea paniculata Siebold, and it is a deciduous shrub in the Hydrangeaceae family. In the natural environment, it grows mainly in East Asia. (Mitsuba) Mitsuba, whose scientific name is Cryptotaenia japonica Hassk., is a perennial plant of the Apiaceae family. It grows wild mainly in East Asia. It is also cultivated for food. (Mallotus japonicus) Mallotus japonicus (Lf) Muell. Arg. is a deciduous tall tree of the Euphorbiaceae family. It grows naturally mainly in East Asia. (grassgrass) The scientific name of the Cephalotaxus harringtonia is Cephalotaxus harringtonia (Knight ex Forbes) K. Koch var. nana (Nakai) Rehder, and it is an evergreen shrub in the Cephalotaxaceae family. In the natural environment, it grows mainly in East Asia.

[0013] (Plant and / or its extract) The active ingredient of the present invention uses the plant body and / or an extract thereof of the above-mentioned plant. The plant body may be any part of the plant body, including the whole plant body, or any or all of the above-ground parts of the plant body such as flowers, leaves, stems, fruits, pericarp, seeds (also called seeds), seed coats, branches, bark, etc., and any or all of the underground parts of the plant such as roots, etc. The plant body parts may be used singly or in combination of two or more types. From the viewpoint of the effects of the present invention, it is preferable to use the above-ground parts of the plant as the plantain. From the viewpoint of the effects of the present invention, it is preferable to use the above-ground parts of Mayumi, and more preferably the above-ground parts (excluding fruits and seeds). Fruits, branches, and / or leaves can also be used. These Mayumi parts can be used alone or in combination of two or more types. From the viewpoint of the effects of the present invention, leaves of Hydrangea paniculata are preferably used. From the viewpoint of the effects of the present invention, branches are preferably used as the grass. From the viewpoint of the effects of the present invention, it is preferable to use the above-ground parts of the plant (excluding the bark) as Mallotus japonicus, but seeds can also be used. From the viewpoint of the effects of the present invention, it is preferable to use the plant body of the mitsuba, and it is more preferable to use the above-ground parts. The preferred moieties described above can be used alone or in combination with other moieties. The plant body can be used fresh as collected or dried. If necessary, it can be cut and crushed before use.

[0014] The plant extract can be obtained by extracting the whole or part of the plant as a raw material using water, an organic solvent, or a combination thereof, by a known method. Examples of organic solvents include alcohols that are liquid at room temperature, such as lower alcohols (e.g., methanol, ethanol, n-propanol, isopropanol, n-butanol), and polyhydric alcohols (e.g., 1,3-butylene glycol, propylene glycol, glycerin), esters (e.g., butyl acetate, ethyl acetate), and ketones (e.g., acetone, ethyl methyl ketone), and extraction can be carried out using one or more of these. Among these, from the viewpoints of operability and environmental friendliness, alcohols that are liquid at room temperature, particularly lower alcohols having 1 to 4 carbon atoms, are preferred. Specific extraction methods include, for example, adding the target plant to room temperature or heated water, organic solvent, or a combination thereof under normal or elevated pressure, and extracting while immersing or stirring, or extracting while refluxing in water, organic solvent, or a combination thereof. The extraction temperature is preferably between 5°C and the boiling point of the solvent used, and the extraction time varies depending on the type of solvent used and the extraction conditions, but is preferably about 30 minutes to 72 hours. When extracting by reflux, it is preferable to use a solvent with a low boiling point to prevent denaturation or thermal decomposition of the extracted components.

[0015] The extract thus obtained may be used as it is as a plant extract, or may be dried and powdered by methods such as concentration, freeze-drying, or spray-drying, as needed, and then used as a plant extract. Specific drying methods may be any method that does not cause denaturation or thermal decomposition of the target plant and its components, such as filtration, centrifugation, centrifugal filtration, spray drying, spray cooling, drum drying, vacuum drying, freeze drying, etc. These methods may be used alone or in combination. The endoplasmic reticulum stress inhibitor and / or liver function improver can be ingested and / or administered alone, and can also be used as an active ingredient in an endoplasmic reticulum stress-inhibiting composition and a liver function-improving composition, which will be described later.

[0016] [Endoplasmic reticulum stress suppression composition] [Liver function improving composition] The endoplasmic reticulum stress-suppressing composition and / or liver function-improving composition of the present invention contains the above-mentioned endoplasmic reticulum stress-suppressing agent and / or liver function-improving agent as an active ingredient. Specifically, in one embodiment, the composition is provided as an endoplasmic reticulum stress-suppressing composition containing the endoplasmic reticulum stress-inhibitor and / or liver function-improving agent as an active ingredient. In another embodiment, the composition is provided as a liver function-improving composition containing the endoplasmic reticulum stress-inhibitor and / or liver function-improving agent as an active ingredient. The endoplasmic reticulum stress-suppressing composition and liver function-improving composition are, for example, but not limited to, foods, pharmaceuticals, cosmetics, or feeds.

[0017] The endoplasmic reticulum stress-suppressing composition and / or liver function-improving composition of the present invention has an excellent effect of suppressing endoplasmic reticulum stress by containing the above-mentioned endoplasmic reticulum stress inhibitor and / or liver function-improving agent as an active ingredient. Furthermore, due to its ability to suppress endoplasmic reticulum stress, it also has the effect of improving or treating liver dysfunction caused by endoplasmic reticulum stress. In particular, it is preferable to use it in subjects experiencing endoplasmic reticulum stress and / or subjects requiring improvement in liver function.

[0018] The endoplasmic reticulum stress-suppressing composition and liver function-improving composition contain an effective amount of the endoplasmic reticulum stress-suppressing agent and / or liver function-improving agent. The effective amount can be appropriately determined. For example, it is preferably 1 to 1000 μg / mL, more preferably 1 to 750 μg / mL, and even more preferably 1 to 500 μg / mL. In the endoplasmic reticulum stress-suppressing composition and liver function-improving composition, the content of the endoplasmic reticulum stress-suppressing agent and / or liver function-improving agent is generally in the range of 0.01 to 100% by weight.

[0019] The endoplasmic reticulum stress-suppressing composition and liver function-improving composition can further contain any component, such as a carrier known to be usable in foods, pharmaceuticals, cosmetics, feed, etc. Such components include water, oils and fats, sugars, vitamins, sweeteners, seasonings, acidulants, preservatives, fragrances, colorants, excipients, bulking agents, binders, thickeners, stabilizers, emulsifiers, pH adjusters, etc.

[0020] <Dosage form> The form of the endoplasmic reticulum stress-suppressing composition and the liver function-improving composition is not limited. For example, they may be solids such as powders and granules, liquids, pastes, etc. Preferred dosage forms for each of the applications, such as food, pharmaceuticals, cosmetics, and feed, are described below.

[0021] <Food> In one embodiment, the endoplasmic reticulum stress-suppressing composition and liver function-improving composition are food products. The food may be, for example, a so-called health food, functional food, food with functional claims, nutritional supplement, supplement, food for specified health uses, food for the sick, combined food for the sick (a type of food for special dietary uses, Ministry of Health, Labour and Welfare), or food for the elderly (a type of food for special dietary uses, Ministry of Health, Labour and Welfare), etc. The food product may further contain a suitable carrier and / or additive. Specific examples of the carrier and additive include various nutrients, various vitamins, minerals, dietary fiber, etc. When the endoplasmic reticulum stress-suppressing composition and liver function-improving composition are in the form of food, various dosage forms can be used, such as powder, granules, capsules, tablets, powders, coated tablets, liquids, syrups, and juices. Specific examples of food products include beverages, spreads, dressings, breads, cooked rice, noodles, sauces, and confectioneries. The food product is preferably a food product for human consumption.

[0022] <Pharmaceuticals> In one embodiment, the endoplasmic reticulum stress-suppressing composition and liver function-improving composition are pharmaceuticals. When the endoplasmic reticulum stress-inhibiting composition and liver function-improving composition are pharmaceuticals, the dosage form may be, for example, orally ingestible forms such as tablets, capsules, granular tablets, and syrups; or externally applied forms such as ointments, creams, and topical liquids. The pharmaceutical agent is administered to a subject in need thereof in an effective amount. The subject in need of such treatment is, for example, a subject experiencing ER stress. ER stress, in particular, is a risk factor or cause of diseases in organs such as the liver, pancreas, and nerves. Examples of pathologies caused by ER stress include liver dysfunction such as NASH / NAFLD (non-alcoholic fatty liver disease / hepatitis); diabetes and diseases associated with diabetes such as type 1 diabetes, type 2 diabetes, Wolcott-Rallison syndrome, and Wolfram syndrome; neurodegenerative diseases such as Alzheimer's disease, Creutzfeldt-Jakob disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease; metabolic diseases such as obesity and dyslipidemia; cardiovascular diseases such as arteriosclerosis and myocardial infarction; various cancers; hypertension; renal disorders; and diseases associated with skeletal dysplasia such as Wolcott-Rallison syndrome and Coffin-Lowry syndrome. These subjects are at risk of developing or experiencing pathologies caused by ER stress.

[0023] <Cosmetics> In one embodiment, the endoplasmic reticulum stress-suppressing composition and liver function-improving composition are cosmetics. Cosmetics include cosmetics sold as quasi-drugs. When the endoplasmic reticulum stress-inhibiting composition and liver function-improving composition are in the form of cosmetics, the formulation may be an ointment, cream, lotion, or the like. When the formulation is an ointment, it can be prepared by mixing the above-mentioned endoplasmic reticulum stress inhibitor and / or liver function-improving agent with an oily base. When the formulation is a cream, it can be manufactured by mixing the above-mentioned endoplasmic reticulum stress inhibitor and / or liver function-improving agent with an oil-in-water (O / W) or water-in-oil (W / O) liquid. When the formulation is a lotion, it can be an emulsion lotion or a suspension lotion. An emulsion lotion is an emulsion, and a suspension lotion is a lotion whose main ingredient is alcohol or water. Additives used in cosmetics include sugars, proteins, amino acids, lipids, moisture, vitamins, minerals, etc.

[0024] <Feed> In one embodiment, the endoplasmic reticulum stress-suppressing composition and liver function-improving composition are feed. The feed may be intended for consumption by, for example, non-human mammals, birds, fish, etc. The mammal may include humans or mammals other than humans. Non-human mammals include pigs, cows, horses, sheep, monkeys, and pet animals such as dogs and cats. Birds include meat chickens, laying hens, turkeys, ducks, pigeons, etc. Furthermore, fish include salmonids such as salmon, trout, yamame trout, char, and huchen, as well as carp, crucian carp, tilapia, catfish, sea bass, yellowtail, amberjack, yellowtail, flounder, sea bream, tuna, and eel. [Example]

[0025] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0026] (Medicinal plant extract) Of the medicinal plant extract library (approximately 15,000 items) held by the Medicinal Plant Resources Research Center of the National Institutes of Biomedical Innovation, Health and Nutrition, 4,006 extracts from plants that are collected domestically and have a history of consumption were screened, and medicinal plant extracts that exhibit the effect of suppressing endoplasmic reticulum stress were screened using the method described in Example 1 below. Each medicinal plant extract constituting the library was prepared by dissolving a methanol extract obtained from a plant body in dimethyl sulfoxide (DMSO) at a concentration of 40 mg / mL and then sterilizing the extract through a filter.

[0027] Example 1: UPRE reporter assay study (Materials, etc.) HepG2 cells: purchased from the European Collection of Authenticated Cell Cultures (ECACC). 96-well multiwell plate, flat bottom (Corning, Falcon brand, part number 353072) PBS, pH 7.4 (Thermo Fisher Scientific, Gibco brand, product number 10010023) IBT21 (compound name: 7-Methylsulphonyl-2-phenylimidazo[2,1-b]benzothiazole, manufactured by Butt Park, product number 15\02-80) The compound was dissolved in DMSO (Nacalai Tesque, Inc., product number 13408-64) at a concentration of 10 mM, and filter-sterilized before use. Tunicamycin (Sigma-Aldrich, product number T7765) The compound was dissolved in DMSO at a concentration of 10 mg / mL and sterilized by filter before use. Picagene Dual Sea Pansy Lighting Kit (Toyo B-Net Co., Ltd., product number PD11) TriStar multimode plate reader (Berthold Technologies, part number LB941)

[0028] DMEM medium (1) DMEM medium (Thermo Fisher Scientific, Gibco brand, product number 11885084): basal medium Fetal bovine serum (FBS) (Biowest, product number S1760-500): final concentration 10% (v / v) Penicillin-streptomycin mixed solution (Nacalai Tesque, Inc., product number 26253-84): final concentration 100 μg / mL

[0029] Transfection mixture pGL3-pro-UPRE: 0.1 μg pRuc-Renilla: 0.01 μg PEI-MAX (Polysciences, product number 24765-1): 0.5 μg Opti-MEM (Thermo Fisher Scientific, product number 31985062): 15 μL

[0030] pGL3-pro-UPRE is an endoplasmic reticulum stress reporter construct that expresses firefly luciferase as a reporter gene downstream of the unfolded protein response element (UPRE) sequence and the c-fos minimal promoter. pRuc-Renilla is an internal standard construct that expresses Renilla luciferase as a reporter gene downstream of the SV40 promoter.

[0031] (method) HepG2 cells derived from human liver were cultured in a CO2 incubator using the DMEM medium (1) described above. Then, 5 × 10 HepG2 cells were added to the culture medium. 4 The cells were seeded onto a 96-well multiwell plate at a density of 100 cells / well and cultured in 100 μL / well of DMEM medium (1) for 24 hours. Then, 15 μL / well of the transfection solution (1) was added for transfection. After culturing for 24 hours, the medium was replaced and the cells were further cultured in 100 μL / well of DMEM medium (1) for 7 hours.

[0032] The medicinal plant extract was dissolved in DMEM medium (1) at a concentration of 0.4 mg / mL (DMSO concentration 1%), and 50 μL / well of this solution was added to the HepG2 cells in culture. As a positive control for endoplasmic reticulum stress inhibition, IBT21 was dissolved in DMEM medium (1) at a concentration of 100 μM (DMSO concentration 1%) instead of the medicinal plant extract, and 50 μL / well of this solution was added to cultured HepG2 cells. The medium volume in each well was 150 μL, and the cells were cultured for 1 hour.

[0033] As an endoplasmic reticulum stress inducer, tunicamycin was dissolved in DMEM medium (1) at a concentration of 8 μg / mL (DMSO concentration 0.08%) and added at 50 μL / well to cultured HepG2 cells. The medium volume in each well was 200 μL, and the cells were cultured for 16 hours.

[0034] At this time, the final concentrations of each reagent were as follows: The medicinal plant extract-added groups evaluated contained tunicamycin at 2 μg / mL, medicinal plant extract at 0.1 mg / mL, and DMSO at a concentration of 0.27%. The number of groups was 3 for group α (described below) and 1 for group β. The IBT21-added group, which served as a positive control for endoplasmic reticulum stress inhibition, contained tunicamycin at 2 μg / mL, IBT21 at 25 μM, and DMSO at a concentration of 0.27% (n=8). In the group to which only tunicamycin was added for standardization, the final concentration of each reagent was 2 μg / mL for tunicamycin and 0.27% for DMSO (n=8). The negative control group, which was not treated with tunicamycin, had a final DMSO concentration of 0.27% (n=8).

[0035] After removing the medium and washing with PBS (pH 7.4), firefly luciferase activity and Renilla luciferase activity were measured using the Picagene Dual Sea Pansy Luminescence Kit according to the manufacturer's protocol. Firefly luciferase activity (Firefly) indicates UPRE activity, and Renilla luciferase activity (Rennila) serves as an internal standard. A TriStar multimode plate reader was used for measurement. Firefly luciferase activity (UPRE activity) = A Renilla luciferase activity (internal standard) = B

[0036] For each group, UPRE activity (A / B) corrected with an internal standard was calculated to correct for transfection efficiency in each well. Furthermore, the relative activity (%) (Firefly / Rennila (%) in the figure) of each group evaluated was calculated, with the UPRE activity (A / B) of the group to which only tunicamycin was added being set at 100, and the UPRE activity inhibitory ability of each group was determined. The results are shown in the graph in Figure 1.

[0037] The medicinal plant extracts of the α group listed below reduced UPRE activity in the UPRE reporter assay compared to the tunicamycin group, demonstrating their effectiveness in suppressing endoplasmic reticulum stress. <α group> Mallotus japonicus (seed) Mayumi (fruit) A Deutzia paniculata (leaves) Mayumi (fruit) B Mayumi (branch) B White-spotted grass (branch) Mayumi (branch) A Mitsuba (above ground) Mayumi (leaves) Plantain (above ground)

[0038] "Mayumi (fruit) A" and "Mayumi (fruit) B", as well as "Mayumi (branch) A" and "Mayumi (branch) B", are samples collected from different locations.

[0039] The following β group is shown as an example of a medicinal plant extract that did not change or activated UPRE activity compared to the tunicamycin group in the UPRE reporter assay. <β group> Ardisia crenata (leaves) Zelkova (immature fruit) Hornbeam (leaves) Loosestrife (stem) Kishirifune (whole plant / flower) Fern sieboldii (above ground) Gonzui (fruit) Trachelospermum asiaticum (fully ripe fruit) Manryo (leaves) Honeysuckle (branch)

[0040] Example 2: Western blot study (Materials, etc.) The overlap with Example 1 will be omitted. 6-well multiwell plate, flat bottom (Corning, Falcon brand, part number 353046) Sodium oleate (Nacalai Tesque, Inc., product number 25702-82) Sodium palmitate (Nacalai Tesque, Inc., product number 25919-62) Protease inhibitor cocktail (Nacalai Tesque, Inc., product number 2595524) MG-132 (Peptide Institute, Inc., product number 3175-v) EzApply (ATTO Corporation, product number AE-1430) Luminometer image analyzer LAS-3000 mini (Fujifilm Corporation)

[0041] CHOP protein detection reagent Primary antibody: CHOP (L63F7) Mouse mAb (manufactured by Cell Signaling Technology, product number 2895) Secondary antibody: Anti-Mouse IgG HRP-Linked Whole Ab Sheep (Cytiva, product number GE-NA931-100UL) Detection reagent: Chemiluminescence One Super (Nacalai Tesque, Inc., product number 02230-14) GAPDH protein detection reagent Primary antibody: Anti-GAPDH / G3PDH, Rabbit Poly (R&D Systems, product number FN-2275-PC-100) Secondary antibody: Anti-Rabbit IgG HRP-Linked Whole Ab Donkey (Cytiva, product number GE-NA934-100UL) Detection reagent: Western Blotting Luminol Reagent (Santa Cruz, product number sc-2048)

[0042] (method) As in Example 1, HepG2 cells were used. 5 × 10 HepG2 cells 5 The cells were seeded onto a 6-well multiwell plate at a density of 100 cells / well and cultured in 2 mL / well of DMEM medium (1) for 48 hours. After changing the medium, each of the medicinal plant extracts of group α obtained in Example 1 was added to a concentration of 40 μg / mL (DMSO concentration 0.1%) (n=3 for each). As a positive control for endoplasmic reticulum stress inhibition, sodium oleate was added to a final concentration of 0.5 mM (DMSO concentration 0.1%) (n=3). DMSO was also added to an untreated control group at a concentration of 0.1% and cultured for 1 hour (n=3).

[0043] Sodium palmitate was added to a final concentration of 0.5 mM, and the mixture was further cultured for 6 hours. Thereafter, the medium was removed, and the cells were washed with PBS, pH 7.4, and then harvested using a cell scraper and centrifuged at 5000 rpm for 2 minutes to recover the cells.

[0044] Cell pellets were prepared for Western blot analysis using EzApply supplemented with a protease inhibitor cocktail and MG132 according to the manufacturer's instructions. Electrophoresis and transfer to a PVDF membrane were performed using standard methods.

[0045] CHOP and GAPDH proteins were detected using anti-CHOP and anti-GAPDH antibodies in 5% skim milk-PBST solution. The results were analyzed using a luminometer image analyzer to measure the expression levels of CHOP protein and GAPDH protein. CHOP protein expression level = C GAPDH protein expression level = D

[0046] For each group, the expression level of CHOP protein (C / D) corrected with the expression level of GAPDH protein, which is an internal standard, was calculated. Furthermore, the relative CHOP expression level (%) of each group evaluated was calculated, assuming the CHOP protein expression level (C / D) in the palmitic acid-only group to be 100, and the CHOP expression inhibitory ability of each group was determined. The results are shown in Figure 2, which shows the average ± standard deviation of three measurements taken on separate days. In the figure, ** indicates p<0.01 and * indicates p<0.05. Tests were performed using a one-sample t-test.

[0047] The medicinal plant extract that demonstrated the effect of suppressing endoplasmic reticulum stress in Example 1 demonstrated a statistically significant reduction in CHOP expression levels compared to the palmitic acid-added group in Western blot analysis, demonstrating the effect of suppressing endoplasmic reticulum stress.

[0048] Example 3: Western blot study (3-1) Preparation of plant extract from plantain leaves Fresh plantain leaves (purchased from Charm Co., Ltd.) were freeze-dried. The mass was reduced to 16.98% of the mass before the process (freeze-dried sample). The freeze-dried sample was subjected to methanol extraction to obtain an extract with a solid content of 18.66% of the mass of the freeze-dried sample. The obtained extract was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 40 mg / mL and sterilized by filter to obtain a plant extract derived from plantain leaves (plantain leaf extract sample).

[0049] (3-2) Analysis of oleic acid in plantain-derived samples The presence or absence and content of oleic acid in (i) the freeze-dried sample derived from plantain leaves and (ii) the plantain leaf extract sample obtained in (3-1) above were measured by gas chromatography. The measurement was outsourced to the Food Analysis Center Foundation. The results are shown in Table 1.

[0050] [Table 1]

[0051] (3-3) Western blot test The plantago ovata leaf extract sample obtained in (3-1) above was subjected to a Western blot test in the same manner as in Example 2 above. The results are shown in Figure 3.

[0052] (3-4) Western blot test As in Example 2, HepG2 cells were used. 5 × 10 HepG2 cells 5 The cells were seeded onto a 6-well multiwell plate at a density of 100 cells / well and cultured in 2 mL / well of DMEM medium (1) for 48 hours. After changing the medium, plantago ovata leaf extract was added to a concentration of 100 μg / mL (DMSO concentration 0.25%). As a positive control for endoplasmic reticulum stress inhibition, IBT21 was added to a final concentration of 25 μM (DMSO concentration 0.25%). DMSO was also added to the untreated control group at a concentration of 0.25% and cultured for 1 hour.

[0053] Tunicamycin was added to a final concentration of 0.2 μg / mL, and the cells were cultured for an additional 6 hours. Thereafter, the medium was removed, and the cells were washed with PBS, pH 7.4, and then harvested using a cell scraper and centrifuged at 5000 rpm for 2 minutes to recover the cells. The subsequent steps were carried out in the same manner as in Example 2 and subjected to Western blot testing. The results are shown in Figure 4.

Claims

1. An endoplasmic reticulum stress-suppressing composition and / or liver function-improving composition comprising one or more plant bodies and / or extracts thereof selected from the group consisting of plantain, Mayumi, Hydrangea paniculata, Cordyceps gracilis, Mallotus japonicus, and Mitsuba.

2. 10. The composition of claim 1, which is a tablet, capsule, granular dose, or syrup.

3. The composition of claim 1 which is a food product.

4. The composition of claim 1 which is a pharmaceutical.

5. The composition of claim 1 which is a cosmetic.

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