AMPK activation composition, hepatic steatosis inhibition composition, hepatic cirrhosis inhibition composition, use of compound for producing AMPK activation composition, use of compound for producing hepatic steatosis inhibition composition, and use of compound for producing hepatic cirrhosis inhibition composition

Abstract

This AMPK activation composition comprises teadenol A.

Description

Composition for activating AMPK, composition for inhibiting fatty liver, composition for inhibiting liver cirrhosis, use of a compound for producing a composition for activating AMPK, use of a compound for producing a composition for inhibiting fatty liver, and use of a compound for producing a composition for inhibiting liver cirrhosis

[0001] The present invention relates to a composition for activating AMPK, a composition for inhibiting fatty liver, a composition for inhibiting cirrhosis, the use of a compound for the preparation of a composition for activating AMPK, the use of a compound for the preparation of a composition for inhibiting fatty liver, and the use of a compound for the preparation of a composition for inhibiting cirrhosis.

[0002] AMPK (5'adenosine monophosphate-activated protein kinase) controls the metabolism of sugars and lipids and plays an important role in the energy metabolism of living organisms. For example, AMPK affects fatty acid oxidation in mitochondria by controlling the activity of acetyl-CoA carboxylase (ACC). Carnitine palmitoyltransferase (CPT-1), which imports long-chain fatty acids into mitochondria, is the rate-limiting enzyme of fatty acid oxidation in mitochondria and is inhibited by malonyl-CoA, a product of ACC. Therefore, it is said that AMPK suppresses ACC activity by phosphorylating ACC, reducing the amount of malonyl-CoA, thereby enhancing CPT-1 activity and promoting fatty acid oxidation.

[0003] As disclosed in Non-Patent Document 1, AMPK is involved in fatty liver disease, and enhancing AMPK activity is effective for fatty liver disease. Patent Document 1 discloses that lysine has an AMPK activating effect and is useful for suppressing fatty liver. Patent Document 2 discloses an AMPK activator and a fatty liver suppressor containing resveratrol as an active ingredient.

[0004] JP 2008-247856 A JP 2006-273834 A

[0005] Chunqiu Fang, 6 others, “The AMPK pathway in fatty liver disease”, 2022, Frontiers in Physiology, 13:970292

[0006] Fatty liver is a condition in which triglycerides accumulate excessively inside liver cells. Fatty liver can lead to serious liver diseases such as liver fibrosis and cirrhosis, so preventing fatty liver is effective in preventing liver diseases.

[0007] The present invention has been made in consideration of the above-mentioned circumstances, and aims to provide a new composition for activating AMPK, a composition for inhibiting fatty liver, a composition for inhibiting cirrhosis, use of a compound for producing a composition for activating AMPK, use of a compound for producing a composition for inhibiting fatty liver, and use of a compound for producing a composition for inhibiting cirrhosis.

[0008] The composition for activating AMPK according to the first aspect of the present invention contains teadenol A.

[0009] The composition for inhibiting fatty liver according to the second aspect of the present invention contains teadenol A.

[0010] A composition for inhibiting liver cirrhosis according to a third aspect of the present invention comprises teadenol A.

[0011] A use according to a fourth aspect of the present invention is the use of teadenol A for the manufacture of a composition for activating AMPK.

[0012] A use according to a fifth aspect of the present invention is the use of teadenol A for the production of a composition for inhibiting fatty liver.

[0013] A use according to a sixth aspect of the present invention is the use of teadenol A for the manufacture of a composition for inhibiting liver cirrhosis.

[0014] According to the present invention, there are provided new compositions for activating AMPK, compositions for inhibiting fatty liver, compositions for inhibiting cirrhosis, use of compounds for the manufacture of compositions for activating AMPK, use of compounds for the manufacture of compositions for inhibiting fatty liver, and use of compounds for the manufacture of compositions for inhibiting cirrhosis.

[0015] 1 shows the results of column fractionation of a 4.5% acetic acid extract of fermented tea in Example 1. 2 shows the results of column fractionation of an ethyl acetate extract from an acetic acid extract of fermented tea in Example 1. 3 shows an adsorption chromatogram of fraction 5 in FIG. 2. 4 shows a high-performance liquid chromatography (HPLC) chart of a fraction containing teadenol A in fraction 5 in FIG. 2. 5 shows an HPLC chart of teadenol A purified by HPLC in Example 1. 6 shows an ultraviolet absorption spectrum of teadenol A purified by HPLC in Example 1. 7 shows the results of Western blotting to detect phosphorylated AMPK (p-AMPK) in Example 2. 8 shows the results of quantitative analysis of p-AMPK expression levels in cells treated with adipolone in Example 2. 9 shows the results of quantitative analysis of p-AMPK expression levels in cells treated with teadenol A in Example 2. 10 shows bands in Western blotting to detect p-AMPK in cells treated with adipolone or teadenol A in Example 3. 5 is a diagram showing the results of quantitative analysis of p-AMPK expression levels in Example 3. FIG. 6 is a diagram showing the relative expression levels of IL-1β in cells treated with lipopolysaccharide (LPS) in Example 4. FIG. 7 is a diagram showing an image of Oil Red O stained liver tissue sections in Example 5. FIG. 8 is a diagram showing the percentage of cells stained with Oil Red O in Example 5. FIG. 9 is a diagram showing the percentage of CD11b-positive macrophages in Example 5. FIG. 10 is a diagram showing the percentage of CD16-positive macrophages in Example 5. FIG. 11 is a diagram showing bands obtained by Western blotting in Example 6. FIG. 12 is a diagram showing the results of quantitative analysis of p-AMPK expression levels in Example 6. FIG. 13 is a diagram showing the relative amount of IL-1β in Example 7. FIG. 14 is a diagram showing the relative expression level of GPR21 in mouse liver sections in Example 8. FIG. 15 is a diagram showing the relative expression level of STAT5 in mouse liver sections in Example 8. FIG. 16 is a diagram showing the relative expression level of NOS2 in mouse liver sections in Example 8. FIG. 17 is a diagram showing the relative expression level of p-AMPK in mouse liver sections in Example 8. FIG. 18 is a diagram showing the relative amount of nuclear phosphorylated STAT5 in HepG2 cells in Example 8. FIG. 10 shows the relative expression level of GPR21 in HepG2 cells in Example 8.27 is a diagram showing the relative expression levels of MARCO, RPS23, and PRF1 mRNA in HepG2 cells in Example 9. 28 is a diagram showing the relative expression levels of DPYD, SYF2, and PARK7 mRNA in Example 9. 29 is a diagram showing an Oil Red O stained image of HepG2 cells in Example 9. 30 is a diagram showing the relative expression level of MARCO mRNA in HepG2 cells treated with a fatty acid mixture in Example 9. 31 is a diagram showing the relative expression level of RPS23 mRNA in HepG2 cells treated with a fatty acid mixture in Example 9. 32 is a diagram showing the relative expression level of PRF1 mRNA in HepG2 cells treated with a fatty acid mixture in Example 9. 33 is a diagram showing the relative expression level of DPYD mRNA in HepG2 cells treated with a fatty acid mixture in Example 9. 34 is a diagram showing a stained image of HepG2 cells in Example 9. 35 is a diagram showing the relative intensity of GPR21 based on the stained image of Figure 27. 36 is a diagram showing the relative intensity of STAT5 based on the stained image of Figure 27. 9 is a diagram showing bands obtained by Western blotting for nuclear extracts in Example 9. FIG. 10 is a diagram showing the nuclear abundance of STAT5 in Example 9. FIG. 11 is a diagram showing bands obtained by Western blotting for HepG2 cells in which the human GPR21 gene is expressed in Example 9. FIG. 12 is a diagram showing the relative expression level of MARCO in total RNA extracted from HepG2 cells in which the human GPR21 gene is expressed in Example 9. FIG. 13 is a diagram showing the relative expression level of RPS23 in total RNA extracted from HepG2 cells in which the human GPR21 gene is expressed in Example 9. FIG. 14 is a diagram showing the relative expression level of PRF1 in total RNA extracted from HepG2 cells in which the human GPR21 gene is expressed in Example 9. FIG. 15 is a diagram showing the relative expression level of DPYD in total RNA extracted from HepG2 cells in which the human GPR21 gene is expressed in Example 9.

[0016] Hereinafter, embodiments of the present invention will be described with reference to the drawings. In each drawing, the same or equivalent parts are designated by the same reference numerals. It should be noted that the present invention is not limited to the following embodiments and drawings. It should be noted that in the following embodiments, the expressions "have," "include," or "contain" also include the meaning of "consisting of" or "consisting of."

[0017] The composition according to the present embodiment contains theadenol A (4,4aα,10,10aα-tetrahydro-7,9-dihydroxy-4-methylenepyrano[3,2-b][1]benzopyran-2-carboxylic acid), which is represented by the following formula (I):

[0018]

[0019] Teadenol A can be synthesized by known methods (for example, JP 2013-151488 A), or can be obtained by fermenting tea leaves with koji mold as shown in the examples below.

[0020] The composition according to the present embodiment may contain other pharmacologically acceptable ingredients in addition to theadenol A as an active ingredient. Examples of other pharmacologically acceptable ingredients include excipients, lubricants, binders, disintegrants, solvents, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, etc. Furthermore, the composition according to the present embodiment may contain additives such as preservatives, antioxidants, colorants, sweeteners, etc., as needed.

[0021] Examples of excipients include lactose, sucrose, D-mannitol, starch, crystalline cellulose, light anhydrous silicic acid, etc. Examples of lubricants include magnesium stearate, calcium stearate, talc, colloidal silica, etc. Examples of binders include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc. Examples of disintegrants include starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethylstarch sodium, etc.

[0022] Examples of solvents include water for injection, alcohol, propylene glycol, macrogol, etc. Examples of solubilizing agents include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, etc. Examples of suspending agents include surfactants, hydrophilic polymers, etc., such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, etc.

[0023] Examples of isotonic agents include sodium chloride, glycerin, D-mannitol, etc. Examples of buffers include phosphate, acetate, carbonate, citrate buffer solutions, etc. Examples of soothing agents include benzyl alcohol, etc. Examples of preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, etc. Examples of antioxidants include sulfites, ascorbic acid, etc.

[0024] As shown in the examples below, the composition according to the present embodiment activates AMPK. Furthermore, the compound has an anti-inflammatory effect. Therefore, applications of the composition include AMPK activation, inhibition of fatty liver, and inhibition of cirrhosis. Here, inhibition of fatty liver and inhibition of cirrhosis include the prevention or treatment of fatty liver and cirrhosis, respectively. Inhibition of fatty liver not only includes the inhibition of triglyceride accumulation in hepatocytes, but also the reduction of excess triglycerides accumulated in hepatocytes. Furthermore, the composition is effective for suppressing elevation of blood glucose levels, reducing triglycerides, improving liver function, suppressing elevation of blood pressure, improving cholesterol metabolism or renal function, improving brain function, and preventing and treating Alzheimer's disease, particularly for improving liver function, cholesterol metabolism, or renal function.

[0025] For example, the composition of this embodiment may be a medicine, which contains 0.1 to 99% by weight, 1 to 50% by weight, and preferably 1 to 20% by weight of teadenol A.

[0026] The pharmaceutical composition is administered to humans or animals other than humans. The animals are preferably mammals, more specifically, dogs, cats, cows, pigs, horses, sheep, deer, etc. The route of administration of the composition to humans, etc. is not particularly limited. The composition is preferably used as an external preparation, an injection, or an oral preparation.

[0027] The medicine is provided in the form of, for example, a liquid, tablet, granule, fine granule, powder, tablet, capsule, or the like.

[0028] The dosage of the pharmaceutical is determined appropriately depending on the age, weight, symptoms, etc. of the human subject. The pharmaceutical is administered so that the amount of teadenol A is effective. Here, the effective amount refers to the amount necessary to treat, delay, inhibit, or prevent the progression of diseases in which activation of AMPK is effective, such as fatty liver and cirrhosis.

[0029] For example, the composition according to the present embodiment may be an oral composition such as an oral composition for activating AMPK, an oral composition for inhibiting fatty liver, or an oral composition for inhibiting liver cirrhosis. Specific examples of such oral compositions include supplements, food compositions, foods and beverages, functional foods, and food additives. The composition according to the present embodiment may be used as a supplement, or may be added to foods and beverages and functional foods.

[0030] The form of the supplement is not particularly limited and may be any form such as tablets, powders, granules, capsules, sugar-coated tablets, films, lozenges, chewable tablets, solutions, emulsions, suspensions, etc. The supplement may contain any ingredient typically used as a supplement.

[0031] The term "functional food" refers to food or beverages taken for the purpose of maintaining health, and includes foods with health claims, foods with specified health uses, foods with functional claims, foods with nutrient functions, health foods, and nutritional supplements. Functional foods are preferably foods with health claims or foods with nutrient functions. When commercializing a functional food, various additives used in foods, specifically colorants, preservatives, thickening agents, antioxidants, bleaching agents, antibacterial and antifungal agents, acidulants, sweeteners, seasonings, emulsifiers, strengthening agents, manufacturing agents, flavorings, etc., may be added to the oral composition.

[0032] Functional foods may be either foods or beverages, and are not particularly limited as long as they can be taken orally. Examples of functional foods include beverages, confectioneries, processed grain products, paste products, dairy products, seasonings, etc. Examples of beverages include nutritional drinks, soft drinks, black tea, green tea, etc. Examples of confectioneries include candy, cookies, tablet candy, chewing gum, jelly, etc. Examples of processed grain products include noodles, bread, cooked rice, biscuits, etc. Examples of paste products include sausages, ham, kamaboko, etc. Examples of dairy products include butter, yogurt, etc.

[0033] The oral composition may be added to food as a food additive, in which case the food additive may be in the form of a paste, gel, powder, liquid, suspension, emulsion, granule, or the like, to facilitate its addition to food.

[0034] The oral composition may contain water, vitamins, minerals, organic acids, organic bases, fruit juice, flavors, functional ingredients, food additives, etc., within the range of maintaining the AMPK activation activity, fatty liver inhibitory activity, or liver cirrhosis inhibitory activity. The oral composition can be produced by a known method, adding ingredients other than theadenol A as needed.

[0035] The oral composition may be contained in one or more containers so that the daily intake amount is the above-mentioned intake amount, and in this case, it is preferable that one container contains one day's worth of oral composition.

[0036] The oral composition is provided in a form that can be distinguished from other products in that it is used for activating AMPK, inhibiting fatty liver, or inhibiting cirrhosis. For example, at least one of the packaging, instructions, and promotional materials for the oral composition product indicates that it has the effect of activating AMPK, inhibiting fatty liver, or inhibiting cirrhosis.

[0037] When the oral composition is provided as a food or beverage composition, it may be provided or sold as a food or beverage labeled with the uses (including health uses) of AMPK activation, suppression of fatty liver, or suppression of liver cirrhosis. "Labeling" includes all acts intended to inform consumers of the above uses. Any expression that can recall or infer the above uses is considered a "labeling" act, regardless of the purpose, content, object, or medium of the labeling.

[0038] It is preferable that the "labeling" be done in an expression that allows consumers to directly recognize the intended use. Specifically, this includes acts of transferring, delivering, displaying for the purpose of transferring or delivering, or importing food and beverage products or product packaging that lists the intended use, displaying or distributing advertisements, price lists, or transaction documents that list the intended use, or providing information containing the above-mentioned uses by electromagnetic means (such as the Internet).

[0039] "Labeling" includes labeling as health food, functional food, enteral nutritional food, special dietary food, health functional food, food for specified health uses, food with nutrient functions, food with functional claims, quasi-drugs, etc. Among these, labeling approved under systems related to foods for specified health uses, foods with nutrient functions, or foods with functional claims, or similar systems, is particularly included. Specific examples include labeling as a food for specified health uses, labeling as a conditional food for specified health uses, labeling that indicates an effect on the structure or function of the body, labeling that reduces disease risk, and labeling of functionality based on scientific evidence. More specifically, labeling as a food for specified health uses (especially labeling of health uses) and similar labeling are typical examples.

[0040] In another aspect of the present embodiment, teadenol A is provided for use in producing a composition for activating AMPK. In another aspect of the present embodiment, use of teadenol A is provided for use in producing a composition for inhibiting fatty liver. In another aspect of the present embodiment, a method for activating AMPK, inhibiting fatty liver, or inhibiting cirrhosis is provided, the method comprising administering teadenol A to a subject. In yet another aspect of the present embodiment, teadenol A is provided for use in activating AMPK, inhibiting fatty liver, or inhibiting cirrhosis.

[0041] Furthermore, as shown in Example 8 below, the composition according to this embodiment increases the expression of GPR21 and STAT5 in a mouse model of fatty liver. Therefore, the composition according to this embodiment is also useful as a composition for enhancing GPR21 expression, activating GPR21, enhancing STAT5 expression, or activating STAT5. Furthermore, as shown in Example 9 below, the composition according to this embodiment can suppress the decrease in the expression levels of MARCO, RPS23, PRF1, and DPYD, genes whose expression is controlled by STAT5, due to fat accumulation in hepatocytes. Therefore, the composition according to this embodiment is also useful as a composition for enhancing MARCO expression, activating MARCO, enhancing RPS23 expression, activating RPS23, enhancing PRF1 expression, activating PRF1, enhancing DPYD expression, or activating DPYD.

[0042] Furthermore, in another aspect of the present embodiment, there is provided teadenol A for producing a composition for enhancing GPR21 expression, a composition for activating GPR21, a composition for enhancing STAT5 expression, a composition for activating STAT5, a composition for enhancing MARCO expression, a composition for activating MARCO, a composition for enhancing RPS23 expression, a composition for activating RPS23, a composition for enhancing PRF1 expression, a composition for activating PRF1, a composition for enhancing DPYD expression, or a composition for activating DPYD.

[0043] Also, another aspect of the present embodiment provides a method for enhancing GPR21 expression, a method for activating GPR21, a method for enhancing STAT5 expression, a method for activating STAT5, a method for enhancing MARCO expression, a method for activating MARCO, a method for enhancing RPS23 expression, a method for activating RPS23, a method for enhancing PRF1 expression, a method for activating PRF1, a method for enhancing DPYD expression, or a method for activating DPYD, the method comprising the step of administering to a subject teadenol A. Furthermore, another aspect of the present embodiment provides teadenol A for enhancing GPR21 expression, GPR21 activation, enhancing STAT5 expression, STAT5 activation, enhancing MARCO expression, MARCO activation, enhancing RPS23 expression, RPS23 activation, enhancing PRF1 expression, PRF1 activation, enhancing DPYD expression, or DPYD activation.

[0044] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0045] [Example 1: Preparation of Teadenol A] Teadenol A was prepared as follows by the method described in JP 2013-151488 A. <Preparation of Fermented Black Tea> 80 kg of raw tea leaves (crude tea) were placed in a drum-type automatic koji-making device, and 80 kg of water was added while stirring by rotating the drum to swell the raw tea leaves. After that, seed koji separately prepared from the raw tea leaves (powdered tea) and black koji was added to the koji-making device, and 100 spores of black koji mold were added. ７ / g (23 kg of seed koji), and fermentation was carried out for 6 days at a set temperature of 35°C. An air-cooling fan was used to suppress temperature rise during fermentation, and efforts were made to maintain the set temperature. The fermented tea leaves were steamed and sterilized at 100°C, and then dried at 55°C to produce fermented black tea.

[0046] Extraction and Purification of Teadenol A The extraction and purification of teadenol A were carried out according to the following procedure. First, 200 g of fermented black tea was extracted with 4.5% acetic acid as the extraction medium, followed by vacuum concentration and freeze-drying to obtain 72.6 g of black tea extract (yield: 36.3%). The black tea extract was fractionated using a Bio-Gel P-10 column (2 x 28 cm) with a 1% aqueous acetic acid solution as the developing solution. The results are shown in Figure 1 and Table 1. HPLC analysis of each fraction revealed that teadenol A was contained in fraction 5 together with brown catechin polymers.

[0047]

[0048] Fraction 5 was extracted with ethyl acetate, and it was confirmed that theadenol A was extracted into the ethyl acetate layer, while the catechin polymer remained in the aqueous layer. The ethyl acetate solution containing theadenol A was concentrated and then diluted with deionized water. The solution was applied to a Bio-Gel P-10 column (3 x 30 cm) and developed with 1% aqueous acetic acid. The elution pattern is shown in Figure 2.

[0049] Methanol was added to the lyophilized fraction 5, and the mixture was stirred and centrifuged. The resulting supernatant was applied to a Sephadex LH column (2 x 27 cm) pre-equilibrated with methanol and developed with methanol. The elution pattern is shown in Figure 3. The HPLC pattern of the fraction with absorption at 300 nm is shown in Figure 4.

[0050] This fraction was purified by HPLC using a TFA-MeCN system to obtain purified crystals of teadenol A, as shown in Figure 5. The ultraviolet absorption spectrum of teadenol A is shown in Figure 6. The obtained teadenol A was used in the following experiments.

[0051] Example 2: Study of AMPK activation 1 C2C12 cells were cultured for 12 hours in Dulbecco's modified Eagle's medium (DMEM) containing 2% fetal bovine serum (FBS). After incubation, the cells were treated with 5 μM or 20 μM Teadenol A or 5 μM or 20 μM Adipolone (AG-CR1-0154-M010, AdipoGen) for 5 minutes. Adipolone is an adiponectin analog and serves as a positive control. The control group was treated with DMSO. After treatment, the cells were harvested and protein was extracted. Western blotting was performed to detect p-AMPK expression (Cell Signaling antibody, catalog number 2535, Cell Signaling Technology).

[0052] Western blot analysis showed that teadenol A increased p-AMPK expression in C2C12 cells, similar to the effect observed with adipolone, as shown in Figure 7. Quantitative analysis of p-AMPK expression levels showed that low concentrations of teadenol A were more effective than adipolone, as shown in Figures 8 and 9.

[0053] Example 3: AMPK activation study 2 C2C12 cells were cultured in 2% FBS-containing DMEM medium for 18 hours and then treated with adipolone or teadenol A at a given concentration for 15 minutes. Control cells were treated with DMSO (0 μg / ml). After treatment, cells were harvested, and an equal amount of protein (20 μg) was extracted from the whole cell lysate. Western blotting was performed (Cell Signaling Antibody, Catalog No. 2535, Cell Signaling Technology) to detect p-AMPK expression.

[0054] Figure 10A shows the bands obtained by Western blotting. The band intensity of p-AMPK shown in Figure 10A was quantified, and as shown in Figure 10B, teadenol A increased p-AMPK expression in C2C12 cells at low concentrations. Adipolone did not increase p-AMPK expression at low concentrations.

[0055] Example 4: Evaluation 1 in an LPS-Induced Inflammation Model. Microglial MG6 cells cultured in 10% FBS-containing high-glucose DMEM (08458-45, Nacalai Tesque) were treated with 5 μg / ml of Teadenol A or Adipolone for 12 hours, followed by 1 μg / ml of LPS for 6 hours to obtain cell extracts (50 μg). The control group was treated with DMSO. To evaluate the expression level of IL-1β, an LPS-induced cytokine, in MG6 cells, ELISA analysis was performed using the Mouse IL-1 beta ELISA kit (catalog number: DY401, R&D Systems) (n = 5). Statistical analysis of p values ​​was performed using ANOVA.

[0056] As shown in Figure 11, pretreatment with Teadenol A suppressed IL-1β expression in MG6 cells. Teadenol A showed superior anti-inflammatory effects compared to Adipolone in an LPS-induced inflammation model using MG6 cells.

[0057] Example 5: Evaluation 1 using a fatty liver mouse model. Eight-week-old male C57BL / 6J mice were fed a high-fat diet (HFD) for 6 weeks to induce hepatic fat accumulation. Subsequently, the mice were switched to a standard diet and allowed to drink water containing 0.01% teadenol A or water without teadenol A ad libitum for 4 weeks. Liver samples were collected from the mice at the end of treatment.

[0058] Figure 12 shows the Oil Red O stained image of liver tissue sections. HFD induced significant fat deposition (red areas). Administration of Teadenol A clearly reduced fat deposition. As shown in Figure 13, the percentage of cells stained with Oil Red O was suppressed by administration of Teadenol A (n = 5, mean ± standard deviation, statistical analysis was performed using ANOVA with Bonferroni's post-hoc test). ＊＊ p<0.01, ＊＊＊ p<0.001), indicating a reduction in fat deposition due to the administration of Teadenol A.

[0059] Liver tissue sections were immunostained using an anti-CD11b antibody (Invitrogen) against CD11b, a macrophage marker. CD11b-positive macrophages were quantified and compared based on the stained images. As shown in Figure 14, HFD increased the infiltration of CD11b-positive macrophages, while administration of Teadenol A reduced their infiltration.

[0060] Liver tissue sections were immunostained using an anti-CD16 antibody (BD Biosciences) against CD16, a macrophage marker. The anti-CD16 antibody was dissolved in TBST buffer before use. CD16-positive macrophages were quantified and compared based on the stained images. As shown in Figure 15, HFD increased CD16-positive macrophages, while administration of Teadenol A decreased them.

[0061] Example 6: Study of AMPK activation 3 HepG2 cells were cultured in 2% FBS-containing DMEM medium for 18 hours and then treated with 1 μM adipolone or teadenol A for 30 minutes. The control group was treated with DMSO. After treatment, the cells were collected, and p-AMPK expression was detected as in Example 3 above. The expression level of p-AMPK was normalized with the expression level of β-actin.

[0062] Figure 16A shows the bands obtained by Western blotting. The p-AMPK band intensities shown in Figure 16A were normalized to the AMPK band intensity and quantified. As shown in Figure 16B, Teadenol A showed stronger p-AMPK induction than adipolone.

[0063] Example 7: Evaluation 2 in an LPS-induced inflammation model. IL-1β expression in macrophage cells (Raw264.7) was measured. Raw264.7 cells were treated with 5 μM Adipolone or Teadenol A for 12 hours, followed by treatment with 1 μg / ml LPS for 6 hours to obtain cell extracts (20 μg). The control group was treated with DMSO. The cell extracts were analyzed by ELISA using an IL-1β ELISA kit (catalog number DY401, R&D Systems) (n=5). Statistical analysis of p values ​​was performed using ANOVA.

[0064] As shown in FIG. 17, pretreatment with teadenol A or adipolone significantly suppressed IL-1β induction, demonstrating an anti-inflammatory effect.

[0065] Example 8: Evaluation 2 using a fatty liver mouse model. Eight-week-old male C57BL / 6J mice were fed a HFD for 6 weeks to induce hepatic fat accumulation. Subsequently, they were switched to a standard diet and allowed to drink water containing 0.01% teadenol A or water without teadenol A ad libitum for 4 weeks. Liver samples were collected from the mice at the end of treatment.

[0066] Mouse liver sections were subjected to immunohistochemical analysis using an anti-NOS2 antibody against NOS2 (Invitrogen), an anti-p-AMPK antibody against p-AMPK (Cell Signaling Technologies), an anti-GPR21 antibody against GPR21, a Class A orphan G protein-coupled receptor (GPCR) (Invitrogen), and an anti-STAT5 antibody against STAT5 (BD Biosciences).

[0067] The relative expression levels of GPR21, STAT5, NOS2, and p-AMPK quantified based on staining images are shown in Figures 18, 19, 20, and 21, respectively. HFD significantly reduced the expression of GPR21 and STAT5, suggesting impaired GPR21-STAT5 signaling associated with the progression of fatty liver. Teadenol A administration increased the expression of both GPR21 and STAT5. Furthermore, the expression levels of NOS2 and p-AMPK were also regulated, indicating beneficial effects on liver inflammation and energy metabolism.

[0068] HepG2 cells were treated with 1 μM or 5 μM teadenol A for 6 hours. DMSO was used as a control. After treatment, whole cell lysates were prepared, and equal amounts of protein (20 μg) were subjected to SDS-PAGE and transferred to a PVDF membrane. Western blot analysis was performed using anti-p-STAT5 antibody (BD Biosciences), anti-GPR21 antibody (Invitrogen), and anti-β-actin antibody. Bands were detected by ECL™ and quantified by densitometry. The expression levels of p-STAT5 and GPR21 were normalized to those of β-actin for comparison.

[0069] As shown in Figure 22, teadenol A increased the amount of nuclear p-STAT5 in a concentration-dependent manner. As shown in Figure 23, teadenol A increased GPR21 expression. This result is consistent with the increase in GPR21 expression observed in the HFD-induced fatty liver mouse model in this example, which was observed with teadenol A administration. These results suggest that while a high-fat diet suppresses the expression of GPR21 and STAT5 in the liver, teadenol A effectively restores their expression, potentially playing a protective role against fatty liver via the GPR21-STAT5 signaling axis.

[0070] Example 9: Identification of STAT5-regulated genes in cirrhosis by analysis using single-cell RNA-seq data. Single-cell RNA-seq data from liver samples from healthy individuals and cirrhotic patients were subjected to UMAP clustering, and a total of 14 transcriptionally distinct clusters were identified. Healthy samples and cirrhotic patient samples were classified using four independent models: XGBoost, Random Forest, SVM, and CNN. Bioinformatics analysis of the gene set obtained by machine learning suggested that MARCO, RPS23, PRF1, DPYD, SYF2, and PARK7 are cirrhosis-related genes whose expression is regulated by STAT5.

[0071] HepG2 cells were treated with 1 μM or 5 μM teadenol A for 12 hours. The mRNA expression levels of MARCO, RPS23, PRF1, DPYD, SYF2, and PARK7 in total RNA extracted after treatment were measured by reverse transcription PCR (RT-PCR) using human gene-specific primers. The nucleotide sequences of the forward and reverse primers for detecting MARCO are shown in SEQ ID NOs: 1 and 2, respectively. The nucleotide sequences of the forward and reverse primers for detecting RPS23 are shown in SEQ ID NOs: 3 and 4, respectively. The nucleotide sequences of the forward and reverse primers for detecting PRF1 are shown in SEQ ID NOs: 5 and 6, respectively. The nucleotide sequences of the forward and reverse primers for detecting DPYD are shown in SEQ ID NOs: 7 and 8, respectively. The nucleotide sequences of the forward and reverse primers for detecting SYF2 are shown in SEQ ID NOs: 9 and 10, respectively. The nucleotide sequences of the forward primer and reverse primer for detecting PARK7 are shown in SEQ ID NOs: 11 and 12, respectively. The band intensities obtained by RT-PCR were quantified by densitometry analysis using ImageJ and shown as relative expression levels normalized with GAPDH (n = 5 independent samples per group).

[0072] As shown in Figures 24A and 24B, the expression levels of MARCO, RPS23, PRF1, and DPYD increased in a teadenol A concentration-dependent manner. On the other hand, the expression level of PARK7 decreased in a teadenol A concentration-dependent manner.

[0073] HepG2 cells were treated with a fatty acid mixture (FA; 100 μM oleic acid and 100 μM palmitic acid) in the presence or absence of 5 μM teadenol A for 22 hours and stained with Oil Red O.

[0074] As shown in Figure 25, FA increased the accumulation of lipid droplets, whereas teadenol A decreased the accumulation of lipid droplets.

[0075] HepG2 cells were treated with FA in the presence or absence of 5 μM teadenol A for 12 hours. The mRNA expression levels of MARCO, RPS23, PRF1, and DPYD in total RNA extracted after treatment were analyzed by RT-PCR using the human gene-specific primers described above. Data were normalized to GAPDH (n = 5).

[0076] As shown in Figures 26A to 26D, the expression levels of MARCO, RPS23, PRF1, and DPYD were reduced by FA, ​​but the reduction in expression levels was suppressed in the presence of teadenol A.

[0077] HepG2 cells were treated with FA for 22 hours in the presence or absence of 5 μM teadenol A. After treatment, HepG2 cells were subjected to immunohistochemical analysis for GPR21 and STAT5. In the immunohistochemical analysis, nuclei were stained with DAPI.

[0078] The stained images of the immunohistochemical analysis are shown in Figure 27. The relative image intensities measured for the stained images were quantified using ImageJ. As shown in Figures 28 and 29, respectively, GPR21 and STAT5 were decreased by FA, ​​but the decrease by FA was suppressed in the presence of teadenol A.

[0079] HepG2 cells were treated with FA for 6 hours, and STAT5 was detected in nuclear extracts by Western blotting. Lamin B was used as a loading control for nuclear proteins. Anti-lamin B antibody (#12586, Cell Signaling Technologies) was used for lamin B. STAT5 band intensity was normalized to Lamin B and quantified.

[0080] The bands obtained by Western blotting are shown in Figure 30A. As shown in Figure 30B, the nuclear abundance of STAT5 was reduced by FA, ​​but this reduction was suppressed in the presence of teadenol A.

[0081] HepG2 cells were transfected with pcDNA3 (empty vector) or pcDNA3 incorporating the human GPR21 gene (pcDNA3-hGPR21) by electroporation. After 24 hours, GPR21, p-AMPK, and p-STAT5 were detected by Western blotting, and the mRNA expression levels of MARCO, RPS23, PRF1, and DPYD in extracted total RNA were measured by RT-PCR using the human gene-specific primers described above. The band intensities obtained by RT-PCR were quantified by densitometry analysis using ImageJ and compared.

[0082] As shown in Figure 31A, expression of GPR21 in HepG2 cells increased the expression levels of GPR21, p-AMPK, and p-STAT5. As shown in Figures 31B, 31D, and 31E, respectively, expression of GPR21 significantly increased the expression levels of MARCO, PRF1, and DPYD. As shown in Figure 31C, RPS23 also tended to increase with expression of GPR21.

[0083] The above-described embodiments are intended to explain the present invention and are not intended to limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not by the embodiments. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

[0084] This application is based on Japanese Patent Application No. 2024-153616, filed on September 6, 2024. The entire specification, claims, and drawings of Japanese Patent Application No. 2024-153616 are incorporated herein by reference.

[0085] The present invention is useful for medicines or foods.

Claims

1. A composition for activating AMPK, comprising teadenol A.

2. A composition for inhibiting fatty liver, comprising teadenol A.

3. A composition for inhibiting liver cirrhosis, comprising teadenol A.

4. Use of teadenol A for the preparation of a composition for activating AMPK.

5. Use of teadenol A for the manufacture of a composition for inhibiting fatty liver.

6. Use of teadenol A for the preparation of a composition for inhibiting liver cirrhosis.

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