JP2013103900A - Hepatopathy inhibitor - Google Patents

Abstract

（式中、Ｒ^１及びＲ^２は各々独立に水素原子、水酸基、カルボキシル基、アミノ基、ハロゲン原子、未置換または置換された炭素原子数１〜２０のアルキル基、未置換または置換された炭素原子数１〜２０のアルケニル基、未置換または置換された炭素原子数１〜２０のアルコキシ基、未置換または置換された炭素原子数６〜２０のアリール基、未置換または置換された炭素原子数７〜２０のアルキルアリール基を表す）
で表されるレンチオニンもしくはその誘導体またはそれらの薬理学的に許容され得る塩を含有する肝障害抑制剤。
【選択図】なしA novel liver injury inhibitor is provided.
The following formula I:
[Chemical 1]

Wherein R ¹ and R ² are each independently a hydrogen atom, hydroxyl group, carboxyl group, amino group, halogen atom, unsubstituted or substituted alkyl group having 1 to 20 carbon atoms, unsubstituted or substituted carbon An alkenyl group having 1 to 20 atoms, an unsubstituted or substituted alkoxy group having 1 to 20 carbon atoms, an unsubstituted or substituted aryl group having 6 to 20 carbon atoms, an unsubstituted or substituted carbon atom number Represents an alkylaryl group of 7 to 20)
Or a derivative thereof or a pharmacologically acceptable salt thereof.
[Selection figure] None

Description

  The present invention relates to a liver disorder inhibitor.

  In the treatment of liver diseases, particularly chronic hepatitis and cirrhosis, it is particularly important to suppress liver damage such as degeneration and necrosis of hepatocytes and prevent progression to liver cancer. Various drugs such as liver function improving drugs, interferons, antiviral drugs, etc. are used as therapeutic drugs for liver diseases, but the current situation is that no definitive drugs have been obtained in terms of drug efficacy and side effects. For this reason, various plant material components have been proposed as components for the prevention and treatment of liver damage. For example, a liver fibrosis inhibitor composed of a polyphenol-containing fraction of an aqueous ethanol solution of shiitake mycelium extract has been proposed (Patent Document 1). Further, diallyl sulfide and diallyl trisulfide, which are garlic components, have been proposed as active ingredients for suppressing liver damage (Non-patent Document 1).

JP 2005-239699 A

T. Fukao, T. Hosono, S. Misawa, T. Seki, T. Ariga., The effects of allyl sulfides on the induction of phase II detoxification enzymes and liver injury by carbon tetrachloride, Food Chem Toxicol, 42, 743-9 , 2004.

However, in the invention described in Patent Document 1, it is described that the components in the extract are polyphenol, protein, and saccharide, but the active component is not specified as a single compound.
Further, diallyl disulfide and diallyl trisulfide in Non-Patent Document 1 have a disadvantage that exhaled breath produces a so-called garlic odor after taking.
Therefore, development of a hepatic disorder inhibitor capable of expecting high preventive / therapeutic effects with few side effects is desired.
The present inventors have found that lenthionine, among the organic solvent extract components of shiitake mushroom, has a liver injury inhibitory action and a second-phase detoxification enzyme-inducing action, thereby completing the present invention.

1stly, this invention relates to the following liver disorder inhibitor.
(1) The following formula I:

Wherein R ¹ and R ² are each independently a hydrogen atom, hydroxyl group, carboxyl group, amino group, halogen atom, unsubstituted or substituted alkyl group having 1 to 20 carbon atoms, unsubstituted or substituted carbon An alkenyl group having 1 to 20 atoms, an unsubstituted or substituted alkoxy group having 1 to 20 carbon atoms, an unsubstituted or substituted aryl group having 6 to 20 carbon atoms, an unsubstituted or substituted carbon atom number Represents an alkylaryl group of 7 to 20)
Or a derivative thereof or a pharmacologically acceptable salt thereof.
(2) The hepatopathy inhibitor according to (1), wherein R ¹ and R ^{2 in the} above formula I represent a hydrogen atom.
(3) A hepatic disorder inhibitor characterized by containing a lentionine-containing organic solvent extract of shiitake mushroom.
(4) The liver disorder inhibitor of (3), wherein the extract is an extract of fruit bodies of shiitake mushroom.
(5) The hepatopathy inhibitor according to (3) or (4), wherein the extract is extracted after crushing shiitake mushroom and left standing at a temperature of 3 to 70 ° C. for 1 minute or more.

  The present invention also relates to a composition for preventing, ameliorating and / or treating a liver disease containing any of the above-mentioned hepatic disorder inhibitors.

  The hepatic disorder inhibitor and composition of the present invention are effective for the prevention, improvement and treatment of liver diseases. In addition, the composition of the present invention is induced by the induction of the second phase detoxification enzyme, detoxification of chemical substances, prevention of diseases caused by oxidative stress, such as cancer, inflammation, aging, neurodegenerative disorders, cataracts, retinal degeneration, It is also effective for improvement and treatment, reduction of side effects of drugs, etc.

It is a figure which shows the result of the GC-MS analysis of the lenthionine containing organic solvent extract of the shiitake mushroom of this invention. It is a graph which shows the influence which lenthionine administration has on aspartate aminotransferase (AST) activity. Values are mean ± standard error of 5 mice, and different alphabets in the graph indicate significant differences (p <0.05) in Duncan's multiple test. It is a graph which shows the influence which lenthionine administration has on alanine aminotransferase (ALT) activity. Values are mean ± standard error of 5 mice, and different alphabets in the graph indicate significant differences (p <0.05) in Duncan's multiple test. It is a graph which shows the influence which lenthionine administration has on a cytochrome P450 (CYP450) specific content. Values are the mean ± standard error of 5 mice, and different alphabets in the graph indicate significant differences (p <0.01) in Duncan's multiple test. It is a graph which shows the influence which lenthionine administration has on glutathione-S-transferase (GST) activity. Values are mean ± standard error of 5 mice, and different alphabets in the graph indicate significant differences (p <0.05) in Duncan's multiple test. It is a graph which shows the influence which lenthionine administration has on quinone reductase (QR) activity. Values are the mean ± standard error of 5 mice, and different alphabets in the graph indicate significant differences (p <0.01) in Duncan's multiple test. It is a graph which shows the influence which lenthionine administration has on the amount of lipid peroxides (TBARS). Values are the mean ± standard error of 5 mice, and different alphabets in the graph indicate significant differences (p <0.01) in Duncan's multiple test.

In the present invention, liver damage includes hepatocellular damage, cholestasis, fatty liver, liver fibrosis, hepatocyte necrosis, and the like. Liver diseases include acute hepatitis, fulminant hepatitis, chronic hepatitis, cirrhosis, liver cancer and the like. Furthermore, liver diseases are classified into viral hepatitis, alcoholic hepatitis, nonalcoholic hepatitis, autoimmune hepatitis and the like.
The composition for suppressing, improving and / or treating liver diseases according to the present invention is particularly suitable for the degeneration of hepatocytes, such as fatty liver and liver fibrosis, particularly when hepatocytes are attacked by oxidative stress. As well as preventing hepatocyte necrosis.

  In the present invention, it was also found that the lenthionine and shiitake lenthionine-containing organic solvent extracts induce a second phase detoxification enzyme. Therefore, according to another aspect of the present invention, the present invention provides a phase II detoxification enzyme inducer comprising lenthionine or a derivative thereof or a pharmacologically acceptable salt thereof, or a lentionine-containing organic solvent extract of shiitake mushroom. Also provide. The phase II detoxification enzymes induced are in particular glutathione-S-transferase (GST) and quinone reductase (QR). Phase II detoxification enzyme induces, for example, detoxification of chemical substances, etc., prevention, improvement and / or treatment of diseases caused by oxidative stress such as cancer, inflammation, aging, neurodegenerative disorders, cataracts, retinal degeneration, etc. Can be expected to reduce side effects. Accordingly, the present invention relates to a composition for detoxification of a chemical substance, cancer, inflammation, aging, nerve, comprising lenthionine or a derivative thereof or a pharmacologically acceptable salt thereof, or a lenthionine-containing organic solvent extract of shiitake mushroom. The present invention also relates to a composition for preventing, improving and / or treating degenerative disorders, cataracts, retinal degeneration, and the like, and a composition for reducing side effects.

  The above-described hepatic disorder inhibitor, second detoxification enzyme inducer and composition of the present invention can be used for humans and non-human mammals, for example, domestic animals such as cows, horses and pigs, and pets such as dogs and cats. it can.

(Lentionine or its derivatives)
In the present invention, lenthionine may be obtained by chemical synthesis or may be contained as an ingredient in an extract or a fraction or purified product thereof.
Lentionine can be synthesized, for example, by the method described in Chem. Pharm. Bull. 988-993 15 (1967).
Hydrogen sulfide gas was blown into 600 g of sodium sulfide nonahydrate, 120 g of sulfur, and 2000 mL of water to adjust the pH to 8. Next, 2000 mL of methylene chloride was added, and the mixture was stirred at room temperature for 2 to 3 hours. The methylene chloride layer was separated, washed with water, and dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain 80 g of an oily substance. This was allowed to stand at room temperature to precipitate crystals, and then centrifuged to remove oil. The residue was recrystallized from dioxane to obtain 12 g of lenthionine crystals.

A compound in which R ¹ and R ² represent a substituent other than hydrogen in Formula I can be produced by introducing a substituent into lenthionine by a conventional method.

  Lentionine is known as an aroma component of shiitake mushroom, and the γ-glutamyl group of the precursor lentinic acid is eliminated by the action of γ-glutamyltransferase to produce desglutamyl lentinic acid, which has C-S lyase. By acting, sulfenic acid or thiosulfinate is generated, and a sulfur-containing fragment generated by non-enzymatic cleavage of thiosulfinate is generated by repeating condensation polymerization. At this time, in addition to lenthionine, 1,2,4,6-tetrathiepan, 1,2,4,5,7,8-hexathionan, 1,2,4-trithiolane, 1,2,4,5-tetrathiane , 1,2,4,5,7-pentathiocan, 1,3,5-trithiane, 1,2,3,5-tetrathiane, and the like. The induction effect of the second phase detoxification enzyme is expected. The cyclic sulfide is, for example, a hydroxyl group, a carboxyl group, an amino group, a halogen atom, an unsubstituted or substituted alkyl group having 1 to 20 carbon atoms, an unsubstituted or substituted alkenyl group having 1 to 20 carbon atoms. An unsubstituted or substituted alkoxy group having 1 to 20 carbon atoms, an unsubstituted or substituted aryl group having 6 to 20 carbon atoms, an unsubstituted or substituted alkylaryl group having 7 to 20 carbon atoms, May be substituted.

  Accordingly, the present invention provides a liver disorder inhibitor, a composition for the prevention, amelioration and / or treatment of liver disease, which contains the above-mentioned cyclic sulfide other than lenthionine or a derivative thereof or a pharmacologically acceptable salt thereof. , Phase 2 detoxification enzyme inducer, composition for chemical detoxification, composition for prevention, improvement and / or treatment of cancer, inflammation, aging, neurodegenerative disorder, cataract, retinal degeneration, etc. It also relates to a relief composition.

  Of these compounds, 1,2,4,6-tetrathiepane is, for example, Phosphorus and Sulfur and the Related Elements, 15 (1), 27-32; 1983, Phosphorus and Sulfur and the Related Elements, 8 (2), 157-9; can be synthesized by the method described in 1980 or the like.

  Lentithionin, 1,2,4,6-tetrathiepan, 1,2,4,5,7,8-hexathionan, 1,2,4-trithiolane, 1,2,4,5-tetrathiane used in the present invention, 1,2,4,5,7-pentathiocan, 1,3,5-trithiane, 1,2,3,5-tetrathiane or a derivative thereof, or a pharmacologically acceptable salt thereof (hereinafter referred to as a compound such as lenthionine) May be included by cyclodextrin or the like. Inclusion can be performed, for example, by adding 100 μl of a solution of a compound such as lenthionine to 1 ml of an α-cyclodextrin solution, incubating at 40 ° C. for 3 hours, and centrifuging at 1200 × g for 10 minutes after the incubation. it can. Inclusion can improve the absorbability in the body, bioavailability and the like, and can impart properties such as sustained release.

(Organic solvent extract of shiitake mushroom)
In the present invention, lenthionine may be contained as an organic solvent extract of shiitake mushroom. Therefore, the present invention also relates to a hepatic disorder inhibitor containing an extract containing lenthionine, a fraction thereof, or a purified product of shiitake mushroom, preferably a fruit body of shiitake mushroom. In the present specification, the term “an organic solvent extract of shiitake mushroom” is used in a sense including a fraction and a purified product of an organic solvent extract.

  In the production of such an organic solvent extract, the raw shiitake may be raw shiitake or dried shiitake. The dried shiitake mushroom is produced by, for example, sun drying or mechanical drying using a drying furnace or the like. Shiitake is a fruit body or mycelium, preferably a fruit body.

  The extraction solvent is an organic solvent or a mixture of an organic solvent and water, preferably an organic solvent. Examples of organic solvents include methanol, ethanol, n- or isopropanol, n-, iso, secondary or tertiary butanol, n-, iso, secondary or tertiary pentanol, n-, iso, secondary or secondary. Alcohols such as hexanol, ketones such as acetone and methyl ethyl ketone, esters such as methyl acetate, ethyl acetate and propyl acetate, halogenated hydrocarbons such as chloroform and methylene chloride, ethers such as diethyl ether, hexane, pentane, etc. Aliphatic hydrocarbons, aromatic hydrocarbons such as benzene, toluene and xylene, or a mixture thereof, preferably methylene chloride or ethanol, but not limited thereto.

  The extraction temperature is preferably 70 ° C. or lower, more preferably 40 ° C. or lower, for example, 5 to 30 ° C. This is because the component to be extracted may decompose when heated at a temperature higher than 70 ° C. for a long time.

  The extraction method can be immersion in an organic solvent or a mixture of water and an organic solvent, digestion, heating under reflux cooling, and the like. Extraction may be carried out continuously or batchwise, for example, at a temperature ranging from room temperature to the boiling point of the solvent, under pressure, normal pressure or reduced pressure. The extraction time can be appropriately determined according to the extraction method, the extraction solvent, and the like. For example, in the case of extraction at 5 to 30 ° C., it is 1 minute to 24 hours, preferably 15 minutes to 30 minutes.

  Prior to the extraction, pretreatment such as shiitake crushing and isothermal treatment is preferably performed. This is because the enzymatic reaction for producing a sulfur-containing compound such as lenthionine starting from lentinic acid contained in shiitake mushrooms is efficiently advanced.

The crushing is performed by crushing using a device such as a homogenator in a buffer solution having a pH of 3 to 12, for example.
The isothermal treatment is performed, for example, at a temperature of about 3 to 70 ° C., preferably 15 to 50 ° C., more preferably 25 to 40 ° C. for 1 minute to 24 hours, preferably 10 minutes to 12 hours, more preferably 30 minutes to 3 This is done by maintaining time.

  The organic solvent extract of shiitake mushroom may be further purified by performing an extraction operation with an organic solvent, followed by filtration, drying, fractionation and the like. Filtration, drying and fractionation can be carried out by conventional methods well known to those skilled in the art.

  The extract used for the liver injury inhibitor of the present invention and the composition of the present invention is extracted, purified as desired, concentrated, dried or freeze-dried, and further sterilized as necessary. It may be. Concentration or drying is carried out under normal pressure or reduced pressure, preferably at a temperature of 150 ° C. or lower, more preferably at 30 ° C. or lower. It is because the component which should be extracted may decompose | disassemble when it exceeds 150 degreeC.

  The dosage form of the hepatic disorder inhibitor of the present invention and the composition of the present invention is not particularly limited, but capsules, granules, powders, tablets, liquids, soaking agents, decoction, troches, extracts (soft extract, dried) Extract), flow extract, tincture, eye drops, nasal solution, ointment, cream, lotion, injection, suppository, fragrance preparation, food and drink, and the like.

The amount of compound such as lenthionine varies depending on the dosage form. Moreover, although an active ingredient dosage per day is based on a patient's age, weight, sex, symptom, etc., it is 0.1-10 g, for example, Preferably it is 1-5 g.
When a compound such as lenthionine is contained in the form of an organic solvent extract of shiitake mushroom, the active ingredient dose per day depends on the patient's age, body weight, sex, symptoms, etc., but as the lyophilized weight of the organic solvent extract For example, the amount is 0.1 to 15 g, preferably 1 to 10 g.

These preparations are pharmaceutical preparation techniques such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, solubilizers, suspension agents, coating agents, diluents etc. It is formulated with known auxiliaries that can usually be used in the field.
The hepatic disorder inhibitor and composition of the present invention may contain adjuvants known to be usable in the pharmaceutical formulation technical field.
Furthermore, although the above-mentioned form is an example of the suitable form of this invention, it is not limited to this, In the range which does not deviate from the summary of this invention, various deformation | transformation implementation is possible. For example, in a solvent such as water, ethanol, propylene glycol and glycerin, in combination with various natural and synthetic fragrances, or lenthionine alone to make a fragrance composition, as a fragrance preparation, food and drink, food and drink materials (fragrance composition, etc. ).

  Solid preparations are, for example, powders, granules, tablets, capsules, sugar-coated tablets and the like. The compound of the present invention as an active ingredient and a diluent (for example, lactose, dextrose, sucrose, cellulose, corn starch, potato starch, etc. ), Lubricants (eg silica, talc, stearic acid, magnesium stearate, calcium stearate, polyethylene glycol, etc.), binders (eg starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, polyvinylpyrrolidone etc.), discrete agents (For example, starch, alginic acid, alginates, etc.), saturants, colorants, sweeteners, wetting agents (for example, lecithin, polysorbate, lauryl sulfate, etc.) and the like can be contained. These can be formulated by known methods such as mixing, granulating, tableting, sugar coating and the like.

Liquid preparations can be in the form of, for example, syrups, solutions, emulsions and suspensions.
The suspension and emulsion can contain, for example, natural rubber, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, polyvinyl alcohol and the like as a carrier.
Intramuscular suspensions or solutions contain, as a pharmacologically acceptable carrier, for example, sterile water, olive oil, ethyl oleate, glycols such as propylene glycol, and as appropriate an appropriate amount of lidocaine hydrochloride can do. Intravenous injection or infusion solutions can contain, for example, sterile water as a carrier, but are preferably in the form of sterile isotonic saline solution.
The composition of the present invention may be a pharmaceutical composition, a supplement preparation, or the like. Moreover, this invention can also provide food-drinks, such as the food for specific health containing the said liver disorder inhibitor, the said 2nd phase detoxification enzyme inducer, or the said composition.
The present invention also provides a method for preventing, ameliorating and / or treating a liver disease, which comprises administering the above-mentioned liver disorder inhibitor or a composition containing the same. In addition, the present invention provides a chemical substance detoxification comprising administering the second phase detoxification enzyme inducer or a composition containing the same; prevention of cancer, inflammation, aging, neurodegenerative disorder, cataract, retinal degeneration, etc. Also provided are methods for ameliorating and / or treating; or reducing side effects.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, these do not limit the present invention.

Example 1: Preparation of shiitake extract 500 ml of 0.2 M Tris-HCl buffer (pH 3.0, 5.0, 7.0 or 9.0) was added to 300 g of fresh shiitake fruiting body, and homogenizer (manufactured by Matsubara). For 3 minutes. Then, it incubated at 37 degreeC for 30 minutes.
To this, 50 g of celite was added and filtered. The filtrate and the residue were extracted 5 times with 150 ml of methylene chloride, and the combined extracts were dried over sodium sulfate and filtered. The filtrate was concentrated and lipids were removed by passing through silica gel column chromatography.
The obtained oily substance was analyzed by GC-MS. A chart in the case of incubation at pH 9 is shown in FIG. The components obtained when incubated at pH 3.0, 5.0, 7.0, and 9.0 are shown in Table 1 below.

Example 2 Preparation of Shiitake Mushroom Extract 50 g of dried shiitake fruiting bodies were immersed in 900 ml of 0.2 M Tris-HCl buffer (pH 9.0) at 4 ° C. for 30 minutes, and then 3 using a homogenizer (manufactured by Matsubara). Homogenized for minutes and then incubated at 37 ° C. for 3 hours.
To this, 50 g of celite was added and filtered. The filtrate and the residue were extracted 5 times with 150 ml of methylene chloride, and the combined extracts were dried over sodium sulfate and filtered. The filtrate was concentrated and lipids were removed by passing through silica gel column chromatography.
The resulting oily substance was analyzed by GC-MS. The obtained components are shown in Table 1 above.

Test Example 1:
ICR-type SPF male mice (7 weeks old) were preliminarily raised for 1 week on a normal diet (CE-2) for acclimatization to the environment, and then divided into 6 groups with 5 mice per group to be experimental groups. Each group of mice was placed in an individual gauge made of wire mesh and bred for 5 days in a 12-hour light-dark cycle breeding room. CE-2 was freely ingested as feed and tap water as drinking water. After preliminary breeding, lenthionine dissolved in olive oil was administered orally intragastrically for 5 days under anesthesia at 200 μmol / ml / kg body weight per day. On the other hand, olive oil was administered to the control group.
On day 5 of this breeding, carbon tetrachloride (0.05 ml / kg) was intraperitoneally administered to cause acute liver injury. Fasted from 8 hours after the administration of carbon tetrachloride, and after 16 hours, 0.5 ml of blood was collected from the inferior vena cava under Nembutal anesthesia. Thereafter, the liver was perfused with physiological saline, and the liver was removed after blood removal. After washing the liver with physiological saline, the buffer was added 4 times the weight of the liver and homogenized. The resulting homogenate solution was centrifuged at 10,000 × g and 4 ° C. for 15 minutes to remove the precipitate. The supernatant was ultracentrifuged at 105,000 × g and 4 ° C. for another 60 minutes, and the supernatant as the cytoplasm fraction and the precipitate as the microsome fraction were collected. 1 ml of buffer solution was added to the precipitate and used for the following experiments.

1) Measurement of Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) Activity AST and ALT are liver injury parameters whose values increase with liver injury. After the collected blood was centrifuged (150 × g, 4 ° C., 15 minutes), the AST and ALT activities were measured on the serum fraction of the obtained supernatant using transaminase CII / Test Wako. . The results are shown in the graphs of FIGS. 2 and 3, respectively.
Administration of carbon tetrachloride increased AST and ALT activities, which are liver injury parameters, and induced liver injury. However, oral administration of lenthionine decreased AST and ALT activity.

2) Measurement of specific content of cytochrome P450 (CYP450) CYP450 hydroxylates foreign substances that have entered the body and converts them into water-soluble substances that are easily excreted. Some CYP450s should not increase.
In the measurement of CYP450, a double beam type spectrophotometer was used to measure the spectrum between 510 and 400 nm, and the difference between the sample cell and the control cell was determined. To each of the control cell and the sample cell, 0.8 ml of a microsomal fraction diluted 10-fold with a buffer solution (pH 7.4) was added, and the mixture was sufficiently incubated at 37 ° C., and then a spectrum between 510 to 400 nm was recorded. Next, 2 mg of sodium nitrite was added to both cells while slowly mixing to reduce the heme iron site of the hemoprotein in the microsome. Further, the reduced heme protein and carbon monoxide were bound to the sample cell through 2 ml of carbon monoxide. The spectrum value before passing through carbon monoxide was subtracted from the spectrum between 510 and 400 nm of this solution, and the content of CYP450 was determined from the absorbance difference near 490 to 450 nm by the following formula.
The differential absorption coefficient at 450 nm to 490 nm = 91 mM ⁻¹ cm ⁻¹ , the spectrophotometer cell optical path width = 1 cm, and the amount of CYP450 (nmol / ml microsome solution) is obtained by the following equation according to the Lambert-Beer law.

The CYP450 specific content was calculated by dividing this value by the amount of protein (mg) in the sample solution obtained by the absorbance method (measurement of absorbance at 280 nm). The results are shown in the graph of FIG.
Administration of carbon tetrachloride decreased the activity of CYP450, a phase 1 detoxification enzyme. However, the administration of lenthionine suppressed the decrease in CYP450 activity. In the absence of carbon tetrachloride, lenthionine had no effect on CYP450 activity.

3) Measurement of glutathione-S-transferase (GST) activity The GST activity is determined from the increase in absorbance at 340 nm of S-2,4-dinitrophenylglutathione produced by the conjugation reaction of dinitrochlorobenzene, a foreign substance, with GST. It was. GST is considered to have higher activity because it conjugated and detoxifies foreign substances such as carcinogens.
60 μl of 30 mM glutathione (GSH) solution was mixed with 1.48 ml of a buffer solution (pH 7.4), kept at 37 ° C., and then the absorbance at 340 nm was measured, which was taken as the initial value. Next, 60 μl of 30 mM dinitrochlorobenzene and 300 μl of cytoplasmic fraction containing 100-fold diluted GST were added simultaneously, dinitrochlorobenzene was conjugated to GST, and S-2,4-dinitrophenylglutathione produced by the reaction (dinitrochlorobenzene by GST The absorbance at 340 nm of the conjugation reaction product was measured every minute for 5 minutes. In Blank, buffer was added instead of the cytoplasmic fraction. From the extinction coefficient of the produced S-2,4-dinitrophenyl glutathione = 9.6 mM ⁻¹ cm ⁻¹ , S-2,4-dinitrophenyl glutathione was calculated to determine GST activity. The results are shown in the graph of FIG.
GST activity, which is a phase 2 detoxification enzyme, was decreased by administration of carbon tetrachloride, but lenthionine suppressed the decrease in activity. In addition, lenthionine induced the activity of this phase 2 detoxification enzyme even without administration of carbon tetrachloride.

4) Measurement of quinone reductase (QR) activity The QR activity is 650 nm of oxidized dichloroindophenol when oxidized dichloroindofinol is reduced and detoxified by QR and reduced dichloroindophenol is produced by electron transfer. It calculated | required from the reduction | decrease in the light absorbency in. QR is also considered to have higher activity in order to reduce and detoxify foreign substances such as carcinogens.
800 μl of the reaction mixture was kept at 25 ° C., and the absorbance at 600 nm was measured (initial value). After adding 150 μl of a cytoplasmic fraction containing QR diluted 100 times, a decrease in absorbance at 600 nm was measured every minute for 5 minutes. The value of Blank was determined by adding a buffer instead of the cytoplasm fraction. Using the extinction coefficient of oxidized dichloroindophenol = 21 mM ⁻¹ cm ⁻¹ , the amount of decrease in oxidized dichloroindophenol was calculated to determine the activity of QR. The results are shown in the graph of FIG.
QR activity, which is a phase 2 detoxification enzyme, was decreased by administration of carbon tetrachloride, but lenthionine suppressed the decrease in activity. In addition, lenthionine induced the activity of this phase 2 detoxification enzyme even without administration of carbon tetrachloride.

5) Measurement of lipid peroxide amount (TBARS) TBARS reacts with thiobarbituric acid (TBA) to form an aldehyde-TBA ₂ adduct by reacting aldehyde generated by decomposition of lipid peroxide. This is a method for spectroscopically measuring the amount of aldehyde derived from lipid peroxide from absorption at 535 nm. In this method, a calibration curve is obtained using malondialdehyde (MDA), and the amount of lipid peroxide is expressed as MDA equivalent.
Samples such as urine, plasma, and tissue homogenates have high background absorption. Therefore, the MDA-TBA ₂ adduct can be accurately quantified by measuring the absorbance at 520 nm and subtracting the value. Yes.
Add 0.5 ml of 1% phosphoric acid and 1.0 ml of 0.67% TBA to 0.5 ml of homogenate solution, 0.5 ml of MDA standard solution (for calibration curve), or 0.5 ml of physiological saline (for blind test). And stirred at 95 ° C. for 45 minutes. After the reaction, the mixture was immediately cooled to room temperature, 4 ml of butanol was added, the lid was closed and shaken vigorously. After centrifugation (2650 × g, 10 minutes), the butanol layer (upper layer) was taken, the absorbance at 535 nm and 520 nm was measured using the blind as a control, and the difference (A535-520) was determined. The results are shown in the graph of FIG.
The TBARS value increased with the administration of carbon tetrachloride, but lenthionine suppressed the increase.

  According to the present invention, there are provided a composition for use in a liver disorder inhibitor, prevention, improvement and treatment of liver diseases.

Claims (6)

Formula I:

Wherein R ¹ and R ² are each independently a hydrogen atom, hydroxyl group, carboxyl group, amino group, halogen atom, unsubstituted or substituted alkyl group having 1 to 20 carbon atoms, unsubstituted or substituted carbon An alkenyl group having 1 to 20 atoms, an unsubstituted or substituted alkoxy group having 1 to 20 carbon atoms, an unsubstituted or substituted aryl group having 6 to 20 carbon atoms, an unsubstituted or substituted carbon atom number Represents an alkylaryl group of 7 to 20)
Or a derivative thereof or a pharmacologically acceptable salt thereof. The liver disorder inhibitor according to claim ¹ , wherein R ¹ and R ^{2 in the} formula I represent a hydrogen atom. A hepatic disorder inhibitor comprising a renthionine-containing organic solvent extract of shiitake mushroom. The liver disorder inhibitor according to claim 3, wherein the extract is an extract of a fruit body of shiitake mushroom. The hepatic disorder inhibitor according to claim 3 or 4, wherein the extract is extracted after crushing shiitake mushrooms and left standing at a temperature of 3 to 70 ° C for 1 minute or more. A composition for the prevention, amelioration and / or treatment of a liver disease comprising the liver disorder inhibitor according to any one of claims 1 to 5.

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