JP2015160841A - Hepatopathy inhibitor induced by ethanol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a hepatopathy inhibitor induced by ethanol containing as an active ingredient a natural compound obtained from a safe food material which has been eaten for a long time.SOLUTION: The hepatopathy inhibitor induced by ethanol of the invention contains as an active ingredient at least one kind of compounds selected from the group consisting of Bisacurone, ar-turmerone, α-turmerone, and β-turmerone contained in Curcuma domestica Valeton and the like.

Description

  The present invention relates to an inhibitor of hepatocyte damage and liver damage induced by ethanol, which comprises a natural ingredient as an active ingredient.

  Turmeric is a medicinal plant belonging to the genus Turmeric belonging to the ginger family that is cultivated in tropical and subtropical regions around the world, mainly in Southeast Asia.

  Turmeric rhizomes contain 3-5% curcumin (yellow pigment). Various utilities are known for turmeric extract and curcumin. For example, Non-Patent Document 1 suggests that a turmeric extract-containing beverage has an action of preventing sickness while appropriately expressing the “sickness” inherent in alcohol by ingesting together with alcohol.

  Patent Document 1 describes that curcumin is effective as a medicament for the treatment of liver diseases.

  There are few reports on the usefulness of curcumin other than curcumin contained in the rhizomes of turmeric, compared with the report on the usefulness of curcumin. It is disclosed that.

  On the other hand, hepatocyte damage caused by excessive intake of ethanol causes many liver diseases, and its suppression is desired. Moreover, it is desired to suppress hepatocyte damage induced by ethanol using a naturally derived ingredient with abundant dietary experience and high safety as an active ingredient.

  In Patent Document 2, it has been confirmed that the decrease in liver function induced by galactosamine is suppressed by bisaborane-type sesquiterpenes derived from turmeric or the like. However, this test system cannot confirm the effect of suppressing ethanol-induced hepatocyte damage and liver damage.

JP-A-5-262659 JP 2004-331539 A

Takuya Hamano et al. “Examination of the effect of turmeric extract on alcohol metabolism in healthy adults”, Applied Pharmacology, 72 (1/2), 31-38 (2007)

  An object of the present invention is to provide an inhibitor of hepatocyte injury and hepatic injury induced by ethanol, which comprises a natural compound obtained from a highly safe food material rich in dietary experience as an active ingredient.

  The present inventors have at least one compound selected from the group consisting of bisaclone, ar-turmerone, α-turmerone, and β-turmerone contained in turmeric to suppress hepatocyte damage induced by ethanol. As a result, the present invention has been completed.

The present invention includes the following inventions:
(1) An inhibitor of liver damage induced by ethanol, comprising as an active ingredient at least one compound selected from the group consisting of bisaclone, ar-turmerone, α-turmerone, and β-turmerone.
(2) The inhibitor according to (1), containing bisaclone as an active ingredient.
(3) An inhibitor of hepatocyte damage induced by ethanol, comprising as an active ingredient at least one compound selected from the group consisting of bisaclone, ar-turmerone, α-turmerone, and β-turmerone.
(4) The inhibitor according to (3), containing bisaclone as an active ingredient.

  In the present invention, the “inhibitor of liver injury induced by ethanol” is mainly taken by the subject in advance (or administered to the subject) before the onset of ethanol-induced liver injury. , Preventive purpose to prevent the onset of the liver disorder, or to reduce the degree even if the liver disorder develops later compared to when not ingested or not administered Can be used in

  Similarly, the “inhibitor of hepatocyte injury induced by ethanol” in the present invention is mainly ingested by the subject in advance (or is given to the subject before the hepatocyte injury induced by ethanol occurs). Administration) in order to prevent the occurrence of injury of the hepatocytes in advance, or even if the damage of the hepatocytes occurs later, the degree is compared with that at the time of not ingesting or not administering It can be used for preventive purposes to alleviate.

  ADVANTAGE OF THE INVENTION According to this invention, the inhibitor of the hepatocyte damage | damage | damage and liver damage induced by ethanol which use a natural compound as an active ingredient is provided.

FIG. 1 shows an example of cell viability change by ethanol administration. FIG. 2 shows the inhibitory activity of ethanol-induced hepatocyte injury by curcumin. FIG. 3 shows a comparison result of the curative activity of curcuminoids such as curcumin and turmeric extract for suppressing ethanol-induced hepatocellular injury. FIG. 4 shows a comparison result of ethanol-induced hepatocellular injury inhibitory activity of fractions A to E (see FIG. 8) having a polarity lower than that of curcuminoid. FIG. 5 shows a comparison result of ethanol B-induced hepatocellular injury inhibitory activity of fractions B-1 to B-5 (see FIG. 8) from which fraction B was separated. FIG. 6 shows the comparison results of the ethanol-induced hepatocellular injury inhibitory activity of fractions F to J (see FIG. 8) that are more polar than curcuminoids. FIG. 7 shows the comparison results of the inhibitory activity of ethanol-induced hepatocellular injury with ar-turmerone, bisaclone, and curcumin alone. FIG. 8 is a diagram showing an outline of the acquisition path of each fraction shown in the embodiment.

<Active ingredient>
The compound used as an active ingredient in the present invention will be described.

In the present invention, bisaclone refers to a compound represented by the following formula:

In the present invention, ar-turmerone refers to a compound represented by the following formula:

In the present invention, α-turmerone refers to a compound represented by the following formula:

In the present invention, β-turmerone refers to a compound represented by the following formula:

  ar-turmerone, α-turmerone and β-turmerone are also called ar-turmerone, α-turmerone and β-turmerone, respectively.

  The above four compounds are compounds classified as bisaborane-type sesquiterpenes. Only one of the four compounds may be used, or two or more may be used in combination. Of the four compounds, bisaclone has particularly high activity of suppressing hepatocellular injury induced by ethanol, and therefore the inhibitor of the present invention preferably contains at least bisaclone as an active ingredient.

  Bisaclone, ar-turmerone, α-turmerone, or β-turmerone may be concentrated or separated from plant materials, or may be artificially synthesized, but from the viewpoint of safety. It is preferable to use what was concentrated or separated from plant raw materials.

The plant raw material is preferably a ginger family plant, particularly Curcuma longa (Curcuma), Curcuma aromatica , Curcuma zedoaria , Curcuma phaeocaulis , Curcuma kwangsiensis , Curcuma wenyujin , Curcuma xanthorrhiza . It can be used as a raw material for the production of an active ingredient in an appropriate part such as a rhizome of a plant raw material as it is, in a form cut into an appropriate size or shape, or in the form of a pulverized product. These raw materials may be appropriately dried.

  The concentration or separation of the active ingredient from the plant material is not particularly limited. For example, the above active ingredients such as water, ethanol, methanol, isopropanol, propylene glycol, diethyl ether, petroleum ether, hexane, acetone, acetonitrile, ethyl acetate, animal and vegetable fats and oils, or a mixture of two or more thereof can be dissolved. A solvent-soluble component is extracted from a plant material using a suitable solvent, and further, if necessary, separation such as solvent fractionation, chromatography (column chromatography, high performance liquid chromatography (HPLC), etc.) and / or recrystallization The active ingredient can be separated or concentrated using a concentrating means and obtained.

  At least one active ingredient selected from bisaclone, ar-turmerone, α-turmerone and β-turmerone need not be used in an isolated and purified form, and may be provided as a mixture with other components. Preferably, the total content of the active ingredients is 0.2% by weight or more, preferably 0.5% by weight or more, more preferably 1% by weight or more, and most preferably 10% by weight or more based on the dry matter. Provided as a concentrate or purified product. In particular, a concentrate or purified product in which the content of any one active ingredient is within the above content range is preferable. In addition, each said active ingredient may be used with the form isolated and refined, for example, the content of the said active ingredient is a total of 4 types, and is 80 weight% or more on a dry matter basis.

<Inhibitor of hepatocyte injury induced by ethanol and inhibitor of liver injury induced by ethanol>
In the present invention, “hepatocyte injury induced by ethanol” is caused by ingestion of alcoholic beverages.

  It is known that the damage of hepatocytes induced by ethanol causes various liver disorders, specifically liver disorders such as fatty liver, liver fibrosis, alcoholic hepatitis, alcoholic cirrhosis and the like.

  That is, the active ingredient is useful as an active ingredient of an inhibitor of these liver disorders induced by ethanol.

  The inhibitor of the present invention can be used in the form of foods and drinks, pharmaceuticals (including those classified as quasi-drugs, and the same below). In particular, it is preferably an oral ingestion agent that is orally ingested in the form of foods and drinks, pharmaceuticals, etc., but is not limited thereto, and other routes such as intravenous administration, intrathecal administration, subcutaneous administration, tongue It may be an agent administered by routes such as pre-administration, rectal administration, vaginal administration, eye drop, nasal injection, inhalation, transdermal administration, and transdermal absorption.

  The inhibitor of the present invention may consist entirely of the active ingredient or a material containing the active ingredient (for example, a concentrate or purified product of the active ingredient obtained from a plant). The composition containing the raw material containing it and the other component accept | permitted in the objective of this invention may be sufficient. Other components are not particularly limited as long as they are components allowed in the final form of foods and drinks, pharmaceuticals and the like.

  The shape of the inhibitor of the present invention is not particularly limited and can be liquid, solid, or semi-solid.

  The inhibitor of the present invention can be produced by combining the active ingredient or a material containing the active ingredient and, if necessary, the other ingredients as described above in combination by an ordinary method.

  The aspect of the said food / beverage products is not limited, It can be aspects, such as a health food, a dietary supplement, a nutritional functional food, a food for specified health. The food and drink is the active ingredient or a material containing the active ingredient and other ingredients acceptable as food and drink, for example, fructose glucose liquid sugar, cyclic oligosaccharide, acidulant, thickener, inositol, flavor, niacin, Antioxidants, vitamins, sweeteners, water and the like can be included. The form of the food or drink is not particularly limited, and may be various forms such as beverages, powders, granules, and tablets.

  The pharmaceutical dosage form is not particularly limited, and examples thereof include capsules, tablets, granules, injections, suppositories, patches, ointments, fine granules, caplets, powders, liquids, and troches. The pharmaceutical is the active ingredient or a material containing the same, other pharmaceutically acceptable formulation materials such as excipients, disintegrants, lubricants, binders, antioxidants, colorants, agglomerates. It can be produced by preparing a combination of an inhibitor, an absorption accelerator, a solubilizer, a stabilizer and the like as appropriate.

The individual to which the inhibitor of the present invention is applied is preferably a mammal, particularly a human.
The amount of intake (or dose) of the inhibitor of the present invention is not particularly limited, and is an amount that can substantially suppress hepatocyte damage induced by ethanol when ingested (or administered to an individual). There is no particular limitation as long as the active ingredient can be ingested (or administered). What is necessary is just to determine suitably the specific value of the effective amount of the said active ingredient suitably according to the biological parameter | index (age, sex, weight, etc.) etc. of a subject individual | organism | solid. The intake (or administration) of the inhibitor of the present invention can be performed once to several times a day.

1. Evaluation of ethanol-induced hepatocellular injury inhibitory activity
Using SD rats, hepatocytes were isolated by collagenase perfusion.

The separated hepatocytes were suspended in William's Medium E (10% FBS, 0.1 μM insulin, 1 μM dexamethasone added) at 2 × 10 ⁵ cells / mL, and seeded at 300 μL / well on a collagen-coated 48-well plate.

After culturing at 37 ° C. under 5% CO ₂ for 4 hours, the medium was replaced with William's Medium E (10% FBS added) containing a sample (dissolved in DMSO, final concentration of DMSO 0.1%).
After culturing at 37 ° C. under 5% CO ₂ for 16 hours, the plate was replaced with William's Medium E (1% FBS, 25 mM HEPES added) containing 1 to 4% (v / v) ethanol, and the plate was sealed.
After culturing at 37 ° C. for 24 hours, cell viability was measured using CellQuanti-Blue Cell Viability Assay Kit (BioAssay Systems).

  The cell viability is shown as a relative value when the average value of the fluorescence intensity in the sample-free group (added only with DMSO) is 100%.

FIG. 1 shows an example of changes in cell viability due to ethanol administration in a sample-free group.
The ethanol-induced hepatocellular injury inhibitory activity of the sample is relative to the average value of the cell viability in the sample-free group (Control) when 2% to 3% (v / v) ethanol is administered. Shown as a value.

  Dunnet's method was used for the significant difference test, and the non-added group (Control) was used as a control, and Control <sample added group was tested as an alternative hypothesis. Significant differences are shown as **: 1% significance, *: 5% significance.

(Effect of curcumin)
FIG. 2 shows the results when curcumin is used as a substance exhibiting an effect of suppressing ethanol-induced hepatocyte injury.

When curcumin was added at final concentrations of 6 μM, 12 μM, and 24 μM, cytotoxic activity was observed at 6 μM and 12 μM.
The decrease in activity at 24 μM was predicted due to cell injury.

2. Identification of active ingredients
2-1. Curcumin and turmeric extract powder 50 mg of turmeric extract powder was extracted by adding 1 mL of dimethyl sulfoxide (DMSO) and centrifuged at 15000 rpm for 5 minutes to obtain a “turmeric extract powder” sample.

  Curcuminoid (mixture of curcumin, demethoxycurcumin, and bisdemethoxycurcumin) 2.2 mg (the amount of curcuminoid contained in 50 mg of turmeric extract powder) dissolved in 1 mL of DMSO was used as a “curcuminoid” sample.

  The ethanol-induced hepatocellular injury inhibitory activity of turmeric extract powder and curcuminoids was evaluated by the method described in 1.

  The results are shown in FIG. Turmeric extract has higher hepatocellular injury-inhibiting activity than curcuminoid, suggesting the possibility that active ingredients other than curcuminoid exist in turmeric extract.

2-2. Identification of active ingredients (turmerones) 10 g of turmeric extract powder was extracted with 200 mL of methanol to obtain 1.9 g of an extract. The obtained extract was fractionated using the alcohol-induced hepatocellular injury inhibitory activity as an index.

  The extract was eluted on a silica gel column with ethyl acetate and n-hexane to obtain fractions A, B, C, D and E having a polarity less than that of curcuminoid. When the alcohol-induced hepatocyte injury inhibitory activity was evaluated, the activity was detected in fraction B (FIG. 4).

  In FIGS. 4 to 6, “L” is the result of an evaluation test of “low concentration” using a predetermined fraction in an amount corresponding to 50 mg of turmeric extract powder as a sample, and “H” is a sample. The results of an evaluation test of “high concentration” using a predetermined fraction of 5 times or 10 times the amount corresponding to 50 mg of turmeric extract powder (magnification varies depending on the fraction).

  The active fraction B was fractionated with methanol and water using a preparative HPLC column to obtain fractions B-1, B-2, B-3, B-4, B-5. . The results are shown in FIG. In these fractions, activity was detected in fractions B-2 and B-4.

The active fractions B-2 and B-4 were purified to a single component using a preparative HPLC column, and the structure was determined by NMR analysis. As a result, fraction B-2 was mainly composed of ar-turmerone. As a component, it was found that fraction B-4 was mainly composed of α-turmerone and β-turmerone. The obtained ¹ H-NMR and ¹³ C-NMR data (however, only ¹ H-NMR for α-turmerone and β-turmerone) is shown in 3.

  It was confirmed that ar-turmerone, α-turmerone and β-turmerone purified to a single component were also active.

2-3. Identification of active ingredient (bisaclone) A methanol extract of turmeric extract powder was eluted with a silica gel column using methanol and chloroform to obtain fractions F, G, H, I, and J, which are more polar than curcuminoids. Alcohol-induced hepatocellular injury inhibitory activity was evaluated. The results are shown in FIG. The strongest activity was detected in fraction G.

The active fraction G was purified to a single component using a preparative HPLC column and the structure was determined by NMR analysis. As a result, it was found that the fraction G was mainly composed of bisaclone. The ¹ H-NMR and ¹³ C-NMR data of the obtained bisaclone are shown in 3.

2-4. Effect of purified active ingredients (ar-turmerone, bisaclone) The effects of ar-turmerone, bisaclone, and curcumin (reagent) purified to a single ingredient were confirmed. The results are shown in FIG.

  At 1 μM treatment, only bisaclone was active, and at 6 μM, curcumin, ar-turmerone, and bisaclone were all active.

3. Compound specific data
ar-turmeron
¹ H-NMR (CDCl ₃ ) δ; 7.10 (4H, m), 6.02 (1H, d, J = 1.2 Hz), 3.28 (1H, ddd, J = 6.9, 6.9, 16.1 Hz), 2.70 (1H, dd , J = 5.7, 15.5 Hz), 2.60 (1H, dd, J = 6.9, 16.1 Hz), 2.30 (3H, s), 2.10 (3H, s), 1.85 (3H, s), 1.23 (3H, d, (J = 6.9 Hz)

α-turmerone
¹ H-NMR (CDCl ₃ ) δ; 6.05 (1H, d, J = 1.7 Hz), 5.78 (1H, m), 5.64 (1H, m), 5.43 (1H, m), 2.51 (1H, dd, J = 14.8, 4.0 Hz), 2.25-1.97 (5H, m), 2.13 (3H, s), 1.87 (3H, d, J = 1.1 Hz), 1.70 (3H, d, J = 1.7 Hz), 0.89 (3H , d, J = 6.3 Hz).

β-turmerone
¹ H-NMR (CDCl ₃ ) δ; 6.14 (1H, dd, J = 9.8, 2.9 Hz), 6.06 (1H, d, J = 1.1 Hz), 5.66 (1H, d, J = 9.8 Hz), 4.76 ( 1H, s), 4.74 (1H, s), 2.50-2.40 (2H, m), 2.35-2.15 (3H, m), 2.13 (3H, d, J = 1.2 Hz), 2.03 (1H, m), 1.88 (3H, d, J = 1.2 Hz), 1.72 (1H, m), 1.39 (1H, m), 0.88 (3H, d, J = 6.3 Hz).

Bisacron
¹ H-NMR (CDCl ₃ ) δ; 6.07 (1H, dd, J = 1.2, 1.2 Hz), 5.64 (2H, s), 3.79 (1H, dd, J = 3.2, 7.0 Hz), 2.44 (1H, dd , J = 5.2, 14.9 Hz), 2.30 (1H, m), 2.26 (1H, m), 2.20 (1H, m), 2.14 (3H, d, J = 1.2 Hz), 1.89 (3H, d, J = 1.2 Hz), 1.82 (1H, ddd, J = 2.8, 7.4, 10.9 Hz), 1.71 (1H, ddd, J = 6.3, 6.3, 12.6 Hz), 1.52 (2H, br s), 1.30 (3H, s) , 0.91 (3H, d, J = 6.7 Hz).

Claims (2)

  An inhibitor of liver damage induced by ethanol, comprising as an active ingredient at least one compound selected from the group consisting of bisaclone, ar-turmerone, α-turmerone, and β-turmerone.   The inhibitor according to claim 1, comprising bisaclone as an active ingredient.