Composition for preventing or improving alcoholic liver disease

Abstract

To provide novel means capable of preventing or improving alcoholic liver disease.SOLUTION: A composition for preventing or improving alcoholic liver disease contains Diospyros ferrea Bakhauizen var. buxifolia Bakh. extract.SELECTED DRAWING: None

Description

Technical Field

[0001] It relates to a composition for preventing or improving alcoholic liver disease.

Background Art

[0002] Alcoholic liver disease is a disease caused by excessive and habitual alcohol intake. In this disease, fatty liver develops as an initial symptom, and then progresses to hepatitis, liver fibrosis, liver cirrhosis, and further to hepatocellular carcinoma (Non-Patent Document 1). Therefore, prevention and improvement of alcoholic liver disease are extremely important.

[0003] Normally, alcohol is decomposed into acetaldehyde by alcohol dehydrogenase (ADH), and further, acetaldehyde is decomposed into acetic acid by aldehyde dehydrogenase (ALDH). However, when a large amount of alcohol is ingested, this decomposition pathway alone cannot process all the alcohol, so alcohol is decomposed into acetaldehyde by another decomposition pathway, the microsomal ethanol-oxidizing system (MEOS). In this alternative decomposition pathway, cytochrome P450 2E1 (CYP2E1), which detoxifies drugs, is mainly involved in the metabolism of alcohol. However, with the metabolism by CYP2E1, reactive oxygen species (ROS), which induce cell damage, are excessively produced. ROS mainly produced by CYP2E1 is considered to be involved in the progression of alcoholic liver disease.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a novel means for preventing or improving alcoholic liver disease.

Means for Solving the Problems

[0006] As a result of intensive studies by the present inventors, it was found that the extract of Euphorbia kurokii might be effective in preventing and improving alcoholic liver disease, and further studies were conducted. This disclosure includes inventions represented, for example, by the following. Item 1. A composition for preventing or improving alcoholic liver disease, containing an extract of Euphorbia kurokii. Item 2. The composition according to Item 1, wherein the extract of Euphorbia kurokii is an extract with water, ethanol, or a mixture thereof. Item 3. The composition according to Item 1 or 2, wherein the alcoholic liver disease is fatty liver, hepatitis, liver fibrosis, or cirrhosis caused by alcohol intake. Item 4. The composition according to any one of Items 1 to 3, which is an oral composition. Item 5. The composition according to any one of Items 1 to 4, which is a pharmaceutical composition or a food composition.

Effects of the Invention

[0007] A novel means for preventing or improving alcoholic liver disease is provided.

Brief Description of the Drawings

[0008]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

[0009] The composition for preventing or improving alcoholic liver disease included in the present disclosure contains the extract of *Opuntia tuna*. Hereinafter, the composition may be referred to as "the composition of the present disclosure".

[0010] The Ryukyu Koku-tan (Diospyros ferrea Bakhauizen var. buxifolia Bakh. (also described as Diospyros ferea Bakh., etc.)) is a plant belonging to the genus Diospyros of the Ebenaceae family. It grows in groups on each island of Okinawa Prefecture and is also known by the alias Yaeyama Koku-tan. The part of the Ryukyu Koku-tan used for the extract is not limited as long as the effects of the present disclosure can be obtained, and it may be any of fruits, leaves, stems, trunks, seeds, etc., and preferably fruits are exemplified. The part used may be used alone or in combination of two or more kinds.

[0011] The method for producing the extract (extraction method) and extraction conditions, etc. are not limited, and may follow a conventionally known method. For example, the above-mentioned part of the Ryukyu Koku-tan can be directly used, and after being cut, pulverized or dried as necessary, an extract can be obtained by solvent extraction.

[0012] The solvent used for solvent extraction is not limited as long as the effects of the present disclosure can be obtained. For example, water; lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, isopropanol, propylene glycol, 1,3-butylene glycol, and alcohols such as polyhydric alcohols (regardless of anhydrous or hydrous) can be mentioned. Preferably, the solvent is water; alcohols such as methanol, ethanol, isopropanol, propylene glycol, 1,3-butylene glycol, more preferably water, methanol, ethanol, and still more preferably water, ethanol, hydrous ethanol. The concentration of ethanol when hydrous ethanol is used as the extraction solvent is not limited as long as the effects of the present disclosure can be obtained and can be appropriately determined. However, in hydrous ethanol, the ethanol content is preferably exemplified by 10 to 80% by volume, more preferably 20 to 75% by volume, 30 to 70% by volume. The solvent may be used alone or in combination of two or more kinds.

[0013] The extract (solvent extract) obtained through solvent extraction as described above may be used as it is, or may be used after undergoing treatments such as concentration, drying, separation of highly active fractions, etc. Such treatments can be carried out according to known procedures such as filtration, concentration under reduced pressure, freeze-drying, adsorption treatment, HPLC (High Performance Liquid Chromatography), etc. The extract may be in any form of liquid, semi-solid, or solid.

[0014] The amount of the extract of Opuntia ficus-indica contained in the composition of the present disclosure is not particularly limited as long as it is within the range where the preventive or ameliorative effect on alcoholic liver disease can be obtained. Examples include about 0.1 to 100% by mass, about 1 to 99% by mass, about 2 to 80% by mass, about 5 to 70% by mass, about 10 to 60% by mass, etc.

[0015] In addition to the extract of Opuntia ficus-indica, the composition of the present disclosure may contain, if necessary, any components such as pharmaceutically acceptable components, edible components, etc. Examples of such optional components include solvents (alcohols such as water, methanol, ethanol, lower alcohols such as isopropanol, polyhydric alcohols such as propylene glycol and 1,3-butylene glycol (regardless of anhydrous or hydrated)), excipients, disintegrants, diluents, lubricants, fragrances, colorants, sweeteners, flavoring agents, suspending agents, wetting agents, emulsifiers, solubilizing agents, dispersing agents, buffering agents, binders, penetration enhancers, stabilizers, bulking agents, preservatives, thickening agents, pH adjusters, surfactants, coating agents, absorption promoters, adsorbents, fillers, antioxidants, cooling agents, film-forming agents, gelling agents, amino acids, antioxidant components, anti-fatigue components, and various other pharmacologically active components and various nutritional components. These may be used alone or in combination of two or more, and their contents may also be determined appropriately.

[0016] The usage mode of the composition of the present disclosure is not restricted and may be appropriately set according to the purpose. It can be used as a pharmaceutical composition, food composition (including beverages, health functional foods (including specific health foods, nutritional functional foods, foods with function claims, etc.), supplements, foods for patients, foods for nursing care), feed composition, etc. Also, it can be used as an additive to pharmaceutical compositions, food compositions, feed compositions, etc.

[0017] The form of the composition of the present disclosure is not limited either, and may be appropriately set according to the purpose, and may be in any of solid, semi-solid, and liquid forms. Therefore, the composition may be any of powders, fine granules, granules, tablets, pills, capsules (including hard capsules and soft capsules), troches, chewables, gels, pastes, creams, solutions, suspensions, emulsions, sprays, freeze-dried products in liquid form, etc. Further, for example, when the composition of the present disclosure is in solid form, the composition may be used after being mixed with water or the like.

[0018] As for the administration (intake) method of the composition of the present disclosure, as long as the preventive or ameliorating effect on alcoholic liver disease can be obtained, oral administration (intake) and parenteral administration (intake) are not limited, and oral administration, transvascular administration (especially intravenous), subcutaneous administration, etc. are preferably exemplified, and oral administration is more preferably exemplified. From this viewpoint, the composition of the present disclosure can be preferably used as an oral preparation, an injection, a drip preparation, etc.

[0019] The composition of the present disclosure may be produced according to the usual procedures known in the art in the above-mentioned various forms, usage modes, etc. Using the extract of Calophyllum inophyllum L., and further, if necessary, mixing the above-mentioned optional components within the range not interfering with the effects of the present disclosure, etc., it may be produced.

[0020] The subjects (target animals) of the composition of the present disclosure are not limited, and examples include humans and mammals other than humans. Examples of mammals other than humans include animals such as mice, rats, guinea pigs, rabbits, dogs, cats, monkeys, pigs, cows, etc. Further, the composition of the present disclosure can be preferably used for preventing alcoholic liver disease not only for subjects (target animals) suffering from alcoholic liver disease, but also for non-drinking subjects or subjects (target animals) who drink alcohol daily or sometimes but do not suffer from liver disease. In particular, the composition of the present disclosure is suitable for subjects (more specifically, for example, subjects who are likely to turn red or blush easily when drinking alcohol) with weak activity of alcohol dehydrogenase.

[0021] The dosage of the composition of the present disclosure applied to the subject (target animal) is not particularly limited, and may be appropriately set within the range where the preventive or ameliorating effect on alcoholic liver disease can be obtained according to the physique, age, symptoms, application form, etc. of the subject (target animal). As an example, when applied orally, for an adult (body weight 60 kg), the amount of the extract of Euphorbia Kansui (in terms of dry matter) per day is exemplified to be about 5 to 10,000 mg, preferably about 8 to 8,000 mg, about 10 to 5,000 mg, about 10 to 3,000 mg, or about 15 to 2,500 mg. Here, the dry matter refers to the dry matter of the extract of Euphorbia Kansui obtained by concentrating under reduced pressure according to the test examples described below to remove the solvent and drying. It may be administered (ingested) once a day or multiple times a day. When applied parenterally or applied to non-humans, the dosage, number of applications, etc. may be appropriately determined based on the description of the oral application.

[0022] The alcoholic liver disease is not particularly limited as long as it is a liver disease caused by alcohol intake, and examples include fatty liver, hepatitis, liver fibrosis, or liver cirrhosis. In particular, fatty liver or hepatitis, which are the initial symptoms of alcoholic liver disease, are preferred.

[0023] From these, the present disclosure also includes a method for ameliorating alcoholic liver disease, which comprises administering (ingesting) the composition of the present disclosure to a patient with alcoholic liver disease. The present disclosure also includes a method for preventing alcoholic liver disease, which comprises administering (ingesting) the composition of the present disclosure to a patient with non-alcoholic hepatitis (for example, a healthy person, a person who drinks alcohol daily or occasionally but does not have liver disease). The conditions of the composition of the present disclosure, the extract of Euphorbia Kansui, etc. in these methods are explained in the same manner as described above.

[0024] Hereinafter, the embodiments included in the present disclosure will be described in more detail. In the present disclosure, "comprising" also includes the meanings of "consisting essentially of" and "consisting of". The present disclosure includes all arbitrary combinations of the constituent elements described in this specification.

[0025] In addition, for each of the embodiments of the present disclosure described above, the various characteristics (properties, structures, functions, etc.) can be combined in any manner when specifying the subject matter encompassed by the present disclosure. That is, the present disclosure encompasses all subject matters consisting of any combination of the combinable characteristics described herein.

Example

[0026] Hereinafter, the embodiments of the present disclosure will be described more specifically with examples, but the embodiments of the present disclosure are not limited to the following examples.

[0027] Test Example 1) Preparation of Ryukyu Kokutan Extract The fruits of Psidium cattleianum Sabine were extracted with 50% ethanol to obtain an extract of Psidium cattleianum Sabine fruits. Specifically, the fruits of Psidium cattleianum Sabine were ground and extracted with 50% ethanol (a mixed solution of water and ethanol) at room temperature (25 °C) for 2 hours. Then, it was filtered by exposure to sunlight, filtered under reduced pressure with filter paper, concentrated under reduced pressure to remove the solvent, and dried to make a powder to prepare an extract of Psidium cattleianum Sabine fruits. Hereinafter, the extract of Psidium cattleianum Sabine fruits thus obtained may be referred to as EDF. In the following tests, EDF was dissolved in dimethyl sulfoxide (DMSO) and used.

[0028] 2) Preparation of Hepatocytes (Pre-culture) As hepatocytes, known rat normal hepatocytes (RLN-B2 cells (JCRB Cell Bank, National Institute of Biomedical Innovation, Health and Nutrition, Japan)) were used. RLN-B2 cells were inoculated into Dulbecco's modified Eagle's medium (DMEM, Nissui Pharmaceutical Co., Ltd.) containing 10% fetal bovine serum (FBS) so that the cell number became 1.5×10 5 cells / ml, and cultured in an incubator adjusted to 37 °C and 5% CO 2 for 24 hours.

[0029] 3) Establishment of Alcoholic Hepatocyte Injury Model An alcoholic liver injury model was established using RLN-B2 cells. Specifically, the pre-cultured RLN-B2 cells were changed to a new DMEM medium supplemented with 10% FBS, and at the same time, ethanol was added to the medium to a concentration of 100 - 200 mM, which is equivalent to the blood ethanol concentration of heavy drinkers, and then cultured in an incubator adjusted to 37 °C and 5% CO 2 for 24 hours. After the culture, the cell viability was measured and calculated by the Neutral red method. As a control, cells were cultured in the medium without added ethanol for 24 hours in the same manner.

[0030] The Neutral red method allows Neutral red to pass through the undamaged cell membrane and be taken up by the lysosomes of living cells. By utilizing this property, the amount of Neutral red taken up into the cells was measured, and the cell viability was calculated by measuring the proportion of living cells. Specifically, the following procedure was followed. After culturing for 24 hours as described above, the medium was removed, 1 ml of 0.005% Neutral red solution was added, and the cells were cultured in an incubator (37 °C, 5% CO 2 ) for 2 hours. Then, the cells were washed with a 1% formaldehyde / 1% calcium chloride solution. Further, the solution was replaced with a 1% acetic acid / 50% ethanol solution and left standing at room temperature for 30 minutes to extract the Neutral red taken up by the hepatocytes. The absorbance of the extracted solution was measured at 540 nm using a spectrophotometer (V-530, JASCO Corporation).

[0031] The cell viability was calculated as the ratio to the control, with the control set at 100%.

[0032] As a result, the cell viability decreased in an ethanol concentration-dependent manner. Therefore, the following tests were conducted using the case with 200 mM ethanol added as an alcoholic liver injury model.

[0033] 4) Examination of the Effects of Ryukyu Kokutan Extract on Cell Viability and ROS Production 4-1) Cell Viability 4-1-1) Test Procedure It was investigated whether the reduction in the survival rate of alcohol-induced hepatocytes could be recovered by the extract of *Euphorbia antiquorum* L. Specifically, the RLN-B2 cells pre-cultured as described in 2) above were simultaneously replaced with a new DMEM medium supplemented with 10% FBS, and ethanol (final concentration 200 mM) was added, or ethanol (final concentration 200 mM) and the extract of *Euphorbia antiquorum* L. (EDF) were added simultaneously. After culturing for 24 hours, the cell viability was measured. At this time, EDF was added to the medium at a final concentration of 6.25 μg / ml (Example 1), 12.5 μg / ml (Example 2), 25 μg / ml (Example 3), or 50 μg / ml (Example 4). In addition, when RLN-B2 cells were inoculated into the medium and cultured for 24 hours without adding either ethanol or EDF, it was used as a control. The cell viability was measured and calculated by the Neutral red method as described above.

[0034] In addition, the cytotoxicity of the extract of *Euphorbia antiquorum* L. against hepatocytes was also investigated. Specifically, the RLN-B2 cells pre-cultured as described in 2) above were inoculated into a DMEM medium supplemented with 10% FBS, and then EDF was added at a final concentration of 12.5 μg / ml (Example 5), 25 μg / ml (Example 6), or 50 μg / ml (Example 7) (without adding ethanol). After culturing for 24 hours, the cell viability was measured and calculated in the same manner as above. When RLN-B2 cells were inoculated into the medium and cultured for 24 hours without adding either EDF or ethanol, it was used as a control.

[0035] 4-1-2) Results The results of the recovery of the reduction in the survival rate of alcohol-induced hepatocytes are shown in Figure 1, and the results of the cytotoxicity are shown in Figure 2.

[0036] In Figure 1, "Control" represents control, "200 mM EtOH" represents the addition of only ethanol (200 mM), and "6.25", "12.5", "25", and "50" in "200 mM EtOH + dragon fruit extract (μg / ml)" represent Examples 1 to 4 respectively. As shown in Figure 1, compared with the case of adding only ethanol, in Examples 1 to 4 where dragon fruit extract was added together with ethanol, the cell viability was improved. From this, it was found that the dragon fruit extract can significantly recover the decrease in the viability of hepatocytes induced by alcohol.

[0037] In Figure 2, "Control" represents control, and "12.5", "25", and "50" in "dragon fruit extract (μg / ml)" represent Examples 5 to 7 respectively. As shown in Figure 2, in Examples 5 to 7, no significant increase or significant reduction in cell viability was observed compared with the control. From this, it was found that no cytotoxicity was observed at the addition amount of the dragon fruit extract used in this test example.

[0038] 4-2) ROS Production 4-2-1) Test Procedure In the same manner as in 4-1-1), RLN-B2 cells were cultured in DMEM medium supplemented with 10% FBS for 9 hours in the presence of ethanol (200 mM) under the conditions of Example 3 (in the presence of ethanol and EDF 25 μg / ml) or under the conditions of Example 6 (in the presence of EDF 25 μg / ml without the addition of ethanol). At this time, as described above, the case where RLN-B2 cells were inoculated into the medium and cultured without adding either ethanol or EDF was used as a control. According to the following procedure, the production of intracellular reactive oxygen species (ROS) was observed, measured, and calculated under a fluorescence microscope.

[0039] H 2 O 2The non-fluorescent 2’,7’-dichlorofluorescein diacetate (DCFH-DA), a relatively specific probe, is oxidized to 2’,7’-dichlorofluorescein (DCF) by ROS after being taken up into cells. Utilizing this characteristic, the amount of intracellular ROS production was quantified by measuring the fluorescence intensity of the generated DCF amount and observing it under a fluorescence microscope.

[0040] Specifically, 5 μl of 2.4 mM DCFH-DA was added to the medium (2 ml) 30 minutes before the end of the culture, and the culture was further continued for 30 minutes. After the end of the culture, it was washed twice with phosphate-buffered saline (PBS). After removing the PBS, a cover glass was attached to the bottom of the petri dish, and the fluorescence image of the cells was observed under a fluorescence microscope using an all-in-one fluorescence imaging system (FSX100 Bio Imaging Navigator, Olympus Corporation).

[0041] 4-2-2) Results The fluorescence image of the cells under the fluorescence microscope is shown in Figure 3. As shown in Figure 3, compared with the control (labeled “Control” in the figure), when only ethanol was added (labeled “200 mM EtOH” in the figure), more fluorescence derived from intracellular ROS was observed, and the brightness was also higher. On the other hand, in Example 3 where EDF was added together with ethanol (labeled “+ Lycium barbarum fruit extract (25 μg / ml)” in the figure), the fluorescence derived from intracellular ROS was significantly reduced, and the level was the same as that of the control. Also, it was the same level as in Example 6 where only EDF was added (labeled “Lycium barbarum fruit extract (25 μg / ml)” (without ethanol addition)). From this, it was understood that in Example 3 where EDF was added together with ethanol, the amount of intracellular ROS production was significantly reduced compared to the case where only ethanol was added, and the level was the same as that of the control and also the same as in Example 6. From this, it was found that the Lycium barbarum extract can significantly reduce the increased amount of ROS production in hepatocytes induced by alcohol.

[0042] 5) Examination of the Effects of Ryukyu Kokutan Extract on Alcohol Metabolism-related Enzyme Activity The effects of the Okinawa black sugar extract on the activities of alcohol metabolism-related enzymes (CYP2E1, ADH, and ALDH) were examined. With the promotion of metabolism by CYP2E1, excessive amounts of reactive oxygen species (ROS) that induce cell damage are produced, and the ROS thus produced is considered to contribute to the progression of alcoholic liver disease. From this, it can be said that reducing CYP2E1 activity or suppressing the enhancement of this activity is useful for the prevention and improvement of alcoholic liver disease. Also, alcohol is decomposed into acetaldehyde by ADH, and furthermore, acetaldehyde is decomposed into acetic acid by ALDH. From this, it can be said that enhancing ADH activity is useful for the prevention and improvement of alcoholic liver disease, and also that enhancing ALDH activity is useful for the prevention and improvement of alcoholic liver disease.

[0043] 5-1) Test Procedure Specifically, in the same manner as in the above 4-2-1), RLN-B2 cells were cultured in DMEM medium supplemented with 10% FBS in the presence of ethanol (200 mM) under the conditions of Example 3 (in the presence of ethanol and 25 μg / ml of EDF) or under the conditions of Example 6 (in the presence of 25 μg / ml of EDF, without addition of ethanol). At this time, the case where RLN-B2 cells were inoculated into the medium and cultured without adding either ethanol or EDF was used as a control. The measurement and calculation of the activities of CYP2E1, ADH, and ALDH were performed as follows.

[0044] <CYP2E1 (Cytochrome P450 2E1)> CYP2E1 activity was measured and calculated according to a conventionally known method. p-Nitrophenol (PNP), which is a specific substrate for CYP2E1, is hydroxylated to p-nitrocatechol in the presence of nicotinamide adenine dinucleotide phosphate (NADPH). Utilizing this property, the production amount of p-nitrocatechol was measured as the enzyme activity of CYP2E1. Specifically, after washing twice with PBS at the end of the culture, immediately before collecting the cells, 9.73 ml of recovery buffer stock (10 mM HEPES, 1.5 mM MgCl2 ·6H 2 O, 10 mM KCl, pH 7.4), 1 μl each of leupeptin solution (10 mg / ml) and pepstatin solution (10 mg / ml), 10 μl of sodium fluoride (NaF) solution (41.99 mg / ml), and 10 μl of sodium orthovanadate (Na 3 VO 4 ) solution (36.78 mg / ml) were added, and this was used as the recovery buffer. Cells were recovered with the recovery buffer, and after repeating freeze-thawing using liquid nitrogen twice, the cells were sonicated and centrifuged at 15,000 × g at 4°C for 20 minutes to recover the supernatant. CYP2E1 activity was measured by the hydroxylation rate of p-nitrophenol (PNP) at 546 nm. That is, the recovered supernatant was added to 100 mM KH 2 PO 4 (containing 0.2 mM PNP and 2.0 mM NADPH, pH 6.8), and incubated in a water bath at 37°C for 20 minutes. Subsequently, the reaction was stopped using 0.6 M perchloric acid (250 μl), and 75 μl of 10 M NaOH was added to the remaining supernatant. CYP2E1 activity was expressed as p-nitrophenol (nmol / mg protein / min). For the measurement of the intracellular protein amount, the dye-binding method by Read and Northcote, which is a partially modified Bradford method, was used. Specifically, 5 μl of the cell suspension (already sonicated) or 0 - 20 μl of the standard solution (1 mg / ml bovine serum albumin: BSA), 75 - 95 μl of distilled water, and 2900 μl of the protein dye solution (0.01% serve Blue G solution) were mixed to a total volume of 3000 μl, and the absorbance was measured at a wavelength of 595 nm using a spectrophotometer (JASCO V-730 BIO Spectrophotometer). Figure 4 described later is the result of converting the production amount of p-nitrocatechol (nmol / min) to nmol / mg protein / min based on the result of protein quantification.

[0045] <ADH (alcohol dehydrogenase) and ALDH (aldehyde dehydrogenase)> ADH activity and ALDH activity were measured and calculated according to conventionally known methods. In the process of decomposing alcohol, ADH oxidizes it to NADH using NAD + as a substrate. Utilizing this reaction, in this test example, the UV method was used to measure the increase in NADH, and the ADH activity (U / V) in the cells was measured. Also, since ALDH is a NAD + -dependent enzyme, in the process of metabolizing acetaldehyde, it oxidizes acetaldehyde using NAD + as a substrate to produce NADH. In this test example, by utilizing this reaction, 4-methylpyrazole (4MP), an ADH inhibitor, was added to the measurement buffer, and the ALDH activity was measured in the same manner as the ADH activity.

[0046] Specifically, after culturing, the cells were washed twice and further collected with PBS. After centrifugation at 2600×g for 1 minute at 4°C, buffer (50 mM HEPES, pH 7.5, 0.25 M sucrose, 1 mM EDTA, 1 mM dithiothreitol (DTT), 3 mM MgCl 2 , 1 mM phenylmethylsulphonyl fluoride) was added. After repeating the freeze-thaw cycle using liquid nitrogen twice, the cells were sonicated and centrifuged at 12000×g for 20 minutes at 4°C, and the supernatant was collected. The ADH activity was measured at 25°C in a 1.5 ml volume (50 mM HEPES, pH 8.0, 10 mM MgCl 2 , 1 mM DTT, 300 μM NAD + ) in the presence or absence of ethanol (50 μl). The ALDH activity was measured by inhibiting the ADH activity with 4MP and in a 1.5 ml volume (50 mM HEPES, pH 8.0, 10 mM MgCl 2 , 1 mM DTT, 300 μM NAD +) It was measured at 25 °C. At this time, ethanol or acetaldehyde was added to initiate the reaction, and the absorbance at 340 nm was measured with a spectrophotometer. The specific activity was measured from the initial linear increase in absorbance. Also, for the measurement of the intracellular protein amount, the dye-binding method by Read and Northcote, which is a partial modification of the Bradford method, was used in the same manner as described above.

[0047] According to the above-mentioned procedure, the increase in absorbance at 340 nm per minute (= E / min) was measured, and the ADH activity and ALDH activity per protein amount were calculated from the following equations. In both equations, V: final reaction solution (ml), v: sample volume (ml), d: optical path length (cm), and the molar absorption coefficient of NADH at 340 nm was taken as 6220 (L / mol / cm). In this test example, one with an optical path length of 1 cm was used.

[0048] ADH activity (μmol / mg protein / min) = E / min × V × 10 6 / (6220 × v × d) / protein of sample (mg / 100 μl)

[0049] ALDH activity (μmol / mg protein / min) = E / min × V × 10 6 / (6220 × v × d) / protein of sample (mg / 100 μl)

[0050] 5-2) Results The results regarding CYP2E1 activity are shown in Figure 4, the results regarding ADH activity are shown in Figure 5, and the results regarding ALDH activity are shown in Figure 6. As shown in Figure 4, compared with the case where only ethanol was added (「200 mM EtOH」 in the figure), in Example 3 where EDF was added together with ethanol (「+Dragon Fruit Extract (25 μg / ml)」 in the figure), the CYP2E1 activity significantly decreased to the control level. Moreover, the degree was the same as that in Example 6 where EDF was added without adding ethanol (「Dragon Fruit Extract (25 μg / ml)」 in the figure). From this, it was found that the dragon fruit extract can significantly reduce the increased CYP2E1 activity in hepatocytes induced by alcohol.

[0051] Also, as shown in Figure 5, compared with the case where only ethanol was added (「200 mM EtOH」 in the figure), in Example 3 where EDF was added together with ethanol (「+Dragon Fruit Extract (25 μg / ml)」 in the figure), the ADH activity significantly increased. The degree was the same as that in Example 6 where EDF was added without adding ethanol (「Dragon Fruit Extract (25 μg / ml)」 in the figure). From this, it was found that the dragon fruit extract can significantly improve the ADH activity in hepatocytes induced by alcohol.

[0052] Also, as shown in Figure 6, compared with the case where only ethanol was added (「200 mM EtOH」 in the figure), in Example 3 where EDF was added together with ethanol (「+Dragon Fruit Extract (25 μg / ml)」 in the figure), the ALDH activity significantly increased. The degree was higher than that in Example 6 where EDF was added without adding ethanol (「Dragon Fruit Extract (25 μg / ml)」 in the figure). From this, it was found that the dragon fruit extract can significantly improve the ALDH activity in hepatocytes induced by alcohol.

[0053] From these findings, it was found that the Ryukyu kokutan extract can significantly decrease CYP2E1 activity while enhancing ADH and ALDH activities.

[0054] From the above, it was found that the Ryukyu kokutan extract has a preventive and ameliorating effect on alcoholic liver disease. Also, as its mechanism, it was found that the control of alcohol metabolism-related enzymes contributes.

Claims

1. A composition for preventing or ameliorating alcoholic liver disease, comprising an extract of Ryukyu Kokutan.

2. The composition according to claim 1 , wherein the Ryukyu Kokutai extract is an extract made from water, ethanol or a mixture thereof.

3. The composition according to claim 1, wherein the alcoholic liver disease is fatty liver, hepatitis, liver fibrosis or cirrhosis caused by alcohol intake.

4. The composition of claim 1 , which is an oral composition.

5. The composition according to any one of claims 1 to 4, which is a pharmaceutical composition or a food composition.

Images