JP7214172B2 - Liver function improver - Google Patents

Description

TECHNICAL FIELD The present invention relates to a liver function improving agent.

The liver is the largest organ in the body, responsible for many functions including metabolism, excretion, detoxification, maintenance of fluid homeostasis, and secretion of bile into the duodenum. Liver diseases such as fatty liver, hepatitis, cirrhosis, and liver cancer are known. It is known that representative liver function markers are elevated.
When the liver function declines, it becomes impossible to synthesize the substances necessary for the body, it becomes impossible to decompose waste products produced in the body, and it becomes impossible to decompose harmful substances and drugs from outside the body, and the function of the liver further declines.

Drugs that improve these liver functions include ursodeoxycholic acid and glycyrrhizin. In addition, in order to improve liver function, alcohol intake restriction, dietary therapy such as carbohydrate and lipid restriction, and exercise to prevent obesity are recommended.

In addition, as liver function improving agents, a combination of garlic, plum and oyster (Patent Document 1), maltitol (Patent Document 2), and combinations of various plant extracts and amino acids (Patent Document 3) have been reported. .

JP 2013-241340 A JP 2014-129324 A JP 2017-141199 A U.S. Patent Application Publication No. 2006/0204599 U.S. Patent Application Publication No. 2008/0260881 U.S. Pat. No. 9,526,793

Japan Perfume Manufacturers Association website Toxicol. Res. Vol. 30, No. 3, pp 193-198 (2014) Can. J. Physiol. Pharmacol. 88; 273-284 (2010) Biochimica et Biophysica Acta 1637 (2003) 98-106 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 41; 3527-3536, 2018

However, the effectiveness of the hepatic function-improving agents that have been reported so far is not sufficient, and there has been a demand for new hepatic function-improving agents.
Accordingly, an object of the present invention is to provide a novel liver function-improving agent with high efficacy.

Therefore, the present inventors used various ingredients to examine not only the effects on liver function markers but also the effects on markers such as glycolytic enzymes and oxidative stress. However, with a small amount of oral administration, it reduces liver function markers selected from AST and ALT in blood, reduces oxidative stress, enhances GAPDH (glyceraldehyde-3-phosphate dehydrogenase), short chain The present inventors have completed the present invention based on the finding that it suppresses the production of aldehyde and is useful as a liver function improving agent or a food composition for improving liver function.

That is, the present invention provides the following inventions [1] to [13].
[1] A liver function improving agent containing phenylalkylamine or a salt thereof as an active ingredient.
[2] An agent for lowering a liver function marker selected from blood AST and ALT, containing phenylalkylamine or a salt thereof as an active ingredient.
[3] An oxidative stress reducing agent containing phenylalkylamine or a salt thereof as an active ingredient.
[4] A GAPDH enhancer containing phenylalkylamine or a salt thereof as an active ingredient.
[5] A short-chain aldehyde production inhibitor containing phenylalkylamine or a salt thereof as an active ingredient.
[6] The agent according to any one of [1] to [5], wherein the active ingredient is an algae extract containing phenylalkylamine or a salt thereof.
[7] The agent according to any one of [1] to [6], which is for oral intake.
[8] A food composition for improving liver function containing phenylalkylamine or a salt thereof.
[9] A food composition for lowering a liver function marker selected from AST and ALT in blood, containing phenylalkylamine or a salt thereof.
[10] A food composition for reducing oxidative stress containing phenylalkylamine or a salt thereof.
[11] A GAPDH-enhancing food composition containing a phenylalkylamine or a salt thereof.
[12] A food composition containing a phenylalkylamine or a salt thereof for suppressing formation of short-chain aldehydes.
[13] The composition according to any one of [8] to [12], which contains an algae extract containing phenylalkylamine or a salt thereof.

The liver function-improving agent of the present invention not only reduces AST and ALT in the blood by oral administration of a trace amount of phenylalkylamine or a salt thereof, but also suppresses the production of short-chain aldehydes by enhancing GAPDH. It reduces oxidative stress and enhances antioxidant enzyme activity, resulting in improved liver function.

It is a figure which shows the change of body weight and liver weight. ND: normal diet group, HFD: high fat diet group, WEC: chlorella hot water extract intake group, PL: phenethylamine low dose group, PH: phenethylamine high dose group (same in the following figures). FIG. 4 shows changes in plasma AST and ALT concentrations. FIG. 4 shows changes in TBARS, GSH/GSSG and SOD-like activity. FIG. 4 shows changes in SOD-1. FIG. 3 shows changes in GOX-1. FIG. 4 is a diagram showing changes in GAPDH. FIG. 4 shows changes in β-actin. FIG. 4 is a diagram showing changes in methylglyoxal; It is a figure which shows the change of cysteine.

The active ingredient of the liver function improving agent of the present invention is phenylalkylamine or a salt thereof.
Phenylalkylamines include phenyl-C1-C6 alkylamines, with phenyl C1-C4 alkylamines being more preferred. Specific examples include benzylamine, phenethylamine, 3-phenylpropylamine, 2-phenylpropylamine, 4-phenylbutylamine, 5-phenylpentylamine and the like. Among these, phenethylamine is more preferable.
Further, the phenyl group of phenylalkylamine may be substituted with a halogen atom, a hydroxy group, a C1-C3 alkyl group, an alkoxy group, or the like.

Phenethylamine is a compound present in cheese, fish products, wine, cabbage, cocoa, beer, and the like. In Europe and the United States, it is used as a flavoring agent for various foods for the purpose of reproducing the aroma and improving the flavor. In Japan, it is also approved for use as a food additive flavoring (Non-Patent Document 1).

It is known that acacia exhibits a weight gain suppressing action, and phenethylamine is a component contained in acacia (Patent Document 4). It has been reported that cyanobacteria are useful as diet supplements and that phycocyanin and phenethylamine are contained in the cyanobacteria (Patent Document 5). In addition, it has been reported that phenethylamine nitrate or nitrite can be used as a diet supplement (Patent Document 6).
However, the liver effects of phenylalkylamines such as phenethylamine are completely unknown.

Salts of phenylalkylamine include inorganic acid salts such as hydrochlorides, sulfates, nitrates and phosphates, and organic acid salts such as acetates, tartrates and citrates.

Phenylalkylamine or a salt thereof significantly reduced AST and ALT, which were elevated with a high-fat diet, at oral administration to mice at a low dose of 10 μg/kg/day, as described in the Examples below.
In addition, phenylalkylamine or a salt thereof significantly reduced TBARS (2-thiobarbituric acid-reactive substance), a marker of oxidative stress (lipid peroxidation) that increased with a high-fat diet, at the same low dose as described above. Let It also enhances SOD-like activity, which is an antioxidant enzyme.
Phenylalkylamine or its salt also significantly enhances GAPDH, a glycolytic enzyme that is decreased with a high-fat diet, at the same low doses. In addition, phenylalkylamines or salts thereof, at the same low doses, enhance glutathione and also cysteine, while reducing short-chain aldehydes (methylglyoxal).

Here, it is known that methylglyoxal, which is a short-chain aldehyde, exerts glycative stress and oxidative stress on living organisms and affects the progression of liver diseases (Non-Patent Documents 2 and 3). On the other hand, methylglyoxal is produced from glyceraldehyde 3-phosphate (GAP) or its isomer, dihydroxyacetone phosphate (DHAP), and GAPDH is an enzyme that metabolizes both. Methylglyoxal is also known to react with cysteine. The amount of methylglyoxal is inversely proportional to erythrocyte GAPDH activity and proportional to DHAP. If GAPDH metabolizes GAP/DHAP, methylglyoxal will decrease (Non-Patent Document 4). However, GAPDH is considered to be less variable and is considered as a so-called housekeeping gene. Even when acute hepatitis is induced by administering ethanol to mice, it tends to decrease, but no significant difference is observed (Non-Patent Document 5).
In this study, administration of a high-fat diet to mice resulted in accumulation of fat in the liver and a sharp decrease in free cysteine. It is considered that aldehyde produced by high-fat diet reacted with cysteine to deplete cysteine in the liver. Phenylalkylamines, on the other hand, increase GAPDH slightly less than 2-fold, thereby reducing the short-chain aldehyde methylglyoxal and reducing oxidative stress, thus keeping cysteine reactive with short-chain aldehydes. This increases the activity of antioxidant enzymes that have cysteine active, such as SOD, and maintains the antioxidant system. Thus, phenylalkylamine is thought to improve liver function by reducing oxidative stress caused by a high-fat diet and suppressing the production of short-chain aldehydes by increasing the expression of GAPDH.

Phenylalkylamine or a salt thereof is useful as a liver function improving agent and a food composition for improving liver function due to the various physiological actions described above. Therefore, the liver function-improving agent and the food composition for improving liver function of the present invention may contain phenylalkylamine or a salt thereof, and may contain various plant extracts containing phenylalkylamine or a salt thereof. may
The plant extract containing such phenylalkylamine or its salt is preferably an algae extract containing phenethylamine or its salt, more preferably a chlorella extract containing phenethylamine or its salt. As chlorella, Chlorella pyrenoidosa is preferred.
Plant extracts include water, alcohol, and polyhydric alcohol extracts, with water extracts being preferred. Here, the water may be cold water or hot water. The extraction method may be an immersion method, a Soxhlet method, or the like.

The agent for improving liver function or the food composition for improving liver function of the present invention is preferably in a form for oral intake. Forms for oral intake include, for example, tablets, capsules, granules, sugar-coated tablets, pills, fine granules, powders, powders, sustained-release preparations, suspensions, emulsions, syrups, emulsions, freeze-dried preparations, liquid preparations, elixirs, oral jelly preparations, drinks, gums, confectioneries and the like.

In addition, the form for oral intake of the liver function improving agent or the food composition for improving liver function can be produced by a conventional method, by adding phenylalkylamine or its salt or a plant extract containing phenylalkylamine or its salt alone. It may be used in combination with a pharmaceutically or food acceptable carrier. The pharmaceutically or food acceptable carriers include, for example, excipients, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, flavoring agents, diluents, bactericidal agents, agents, osmotic pressure adjusters, pH adjusters, emulsifiers, preservatives, stabilizers, absorption aids, antioxidants, UV absorbers, wetting agents, thickeners, brighteners, activity enhancers, anti-inflammatory agents, Tonicity agents, soothing agents, corrigents and the like can be mentioned.

Examples of binders include starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol and the like.

Examples of disintegrants include starch, hydroxypropyl starch, sodium carboxymethylcellulose, calcium carboxymethylcellulose, carboxymethylcellulose, low-substituted hydroxypropylcellulose and the like.

Examples of surfactants include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid esters, polysorbate 80 and the like.
Examples of lubricants include talc, waxes, hydrogenated vegetable oils, sucrose fatty acid esters, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol and the like.
Fluidity promoters include, for example, light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate and the like.

Diluents include, for example, distilled water for injection, physiological saline, aqueous glucose solution, olive oil, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol and the like.

In addition, there is no strict limitation on the dose when using phenylalkylamine or a salt thereof as an active ingredient of the liver function improving agent or the food composition for improving liver function of the present invention. Since the effect to be obtained differs depending on various usage modes such as subjects and symptoms, it is desirable to set the dosage as appropriate. It is preferably 1 mg to 200 mg, more preferably 0.5 mg to 150 mg, and more preferably 1 mg to 100 mg.

EXAMPLES Next, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these.

Example 1

(1) Materials and methods a. Materials A hot water extract of Chlorella pyrenoidosa (WEC) was prepared and supplied by Sun Chlorella (Kyoto, Japan).
Phenethylamine, proteinase inhibitor cocktail, butylhydroxytoluene (BHA), diethylenetriaminepentaacetic acid (DTPA), 1,1,3,3-tetraethoxypropane (TEP), thiobarbituric acid (TBA), glutathione (GSH), glutathione Disulfide (GSSG), N-ethylmaleimide (NEM), and enhanced chemiluminescence (ECL) reagents were obtained from Nacalai Tesque (Kyoto, Japan).
Heparin sodium was obtained from Nipro (Osaka, Japan).
Cell lysis reagent was obtained from Sigma-Aldrich (St. Louis, MO, USA). Rabbit immunoglobulin G (IgG) against mouse superoxide dismutase (SOD)-1 and mouse glutathione peroxidase (GPX)1 was obtained from Abcam (Cambridge, UK).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin are available from Proteintech Group (Chicago, Ill., USA) and Santa Cruz Biotechnology (Dallas, Tex., USA). USA).
Goat IgG-horseradish peroxidase (HRP) conjugates for rabbit and mouse IgG were obtained from Cell Signaling Technology (Danvers, Mass., USA).

stomach. Animal Experiments Male C57bl/6J mice (7 weeks old, 21-23 g) were purchased from Japan SLC (Shizuoka, Japan). All mice were randomly divided into 6 groups (n=6) as follows.
Mice were fed normal diet and tap water (ND group), high-fat diet (60% on a calorie basis, CLEA Japan) and tap water (HFD group), high-fat diet and WEC (WEC group) in 100 mg/kg body weight tap water. ), and 10 and 100 μg/kg body weight of high-fat diet and phenethylamine in tap water (PL and PH groups), respectively.
All mice were sacrificed under isoflurane anesthesia without fasting after 12 weeks.
Blood was collected from the inferior vena cava with sodium heparinized syringes.
Plasma was prepared by centrifugation at 3500 rpm for 5 minutes.
The liver was harvested and blood in the liver was purged by injecting cold PBS into the portal vein. Plasma and liver were stored at -30°C.

Methylglyoxal was quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) after derivatization with 2,3-diaminonaphthalene (DAN).
The liver was ground in the same amount of phosphate buffer, ethanol was added in an amount 6 times the weight of the liver (v/w), and the mixture was stirred well. The suspension was centrifuged at 14200g to obtain the supernatant. 50 μL of 0.1% DAN solution was added to 10 μL of the supernatant or the standard substance solution and reacted at 50° C. for 1 hour. After the reaction, 500 μL of ethyl acetate was added and well stirred. 400 μL of the ethyl acetate layer was taken and dried. The residue was dissolved in 30% acetonitrile aqueous solution, filtered and analyzed by LC-MS/MS. 0.1% formic acid solution (solution A) and 0.1% formic acid at a flow rate of 0.2 mL/min using an Inertsil ODS-3 column (2.1 mm × 250 mm, GL Science) equilibrated with 0.1% formic acid was separated by gradient elution using 80% acetonitrile (liquid B) containing Gradient conditions were as follows; 0-15 min; 0-10% B solution, 15-18 min; 100% B solution, 18.1-25 min; 0% B. The column was kept at 40°C. Detection was performed in multi reaction monitoring mode.

For cysteine, the thiol group was labeled with 4-vinylpyridine (4-VP) and the amino group was labeled with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ) and quantified by LC-MS/MS. 22.5 μL of pure water was added to 75 μL of the aforementioned mouse liver extract, and 2.5 μL of 4-VP was further added and reacted at 37° C. for 2 hours. 500 μL of ethyl acetate and 100 μL of pure water were added to the reaction solution and well stirred, and 50 μL of the aqueous phase was collected and dried. 80 μL of 50 mM sodium borate buffer pH 8.8 was added thereto, and 20 μL of 0.3% AccQ acetonitrile solution was further added and reacted at 50° C. for 10 minutes.
The reaction solution was quantified by the aforementioned LC-MS/MS in MRM mode.

hare. Liver SOD-like Activity Liver tissue was washed with 5% by volume (w/v) of 10 mM Tris-HCl buffer containing 0.25 M sucrose and 1 mM EDTA by using BioMasher II (Nippi, 24 Tokyo, Japan). homogenized in pH 7.4).
Homogenates were centrifuged at 10000 g for 60 min and supernatants were collected for SOD-like activity assays of soluble compounds in liver. In addition, liver tissue was homogenized in the same volume (w/v) of PBS and then 6% by volume (w/v) of ethanol was added. The resulting solution was centrifuged at 10,000 g for 10 minutes, and the supernatant was collected and used as the low molecular weight compound fraction. SOD-like activity in the supernatant was assayed using WST-1 SOD assay kit (Dojindo, Kumamoto, Japan).

workman. Plasma Biochemical Analysis Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured by Oriental Yeast.
Thiobarbituric Acid Reactive Substances (TBARS) Assay DTPA was dissolved in 1 M sodium acetate buffer (pH 3.5) to give a 2 mM solution. BHA was dissolved in methanol to give a 10% (w/v) solution. Five milliliters of DTPA solution, 45 mL of hot water, and 100 μL of BHA solution were added to 0.1 grams of TBA to obtain a 0.2% TBA solution.
This solution was used as the TBA reagent.
Ten milliliters of methanol was added into 4.8 μL of TEP, malondialdehyde precursor, to obtain a 2 mM TEP solution. The TEP solution was diluted with methanol to 2-50 mM and used as a standard solution. Liver tissue was homogenized in 9 volumes (w/v) of 1.15% KCl solution. Homogenates were centrifuged at 2000 g for 1 minute.
Supernatants or standard solutions (25 μL) were mixed with TBA reagent (100 μL), vortexed, and then heated at 95° C. for 60 minutes. The reaction was stopped by cooling on ice for 5 minutes. The reactants were centrifuged at 14200 g for 10 minutes. The absorbance at 515 nm of the supernatant was measured. TBARS values were expressed as μM malondialdehyde.
Glutathione and Glutathione Disulfides Measurement of glutathione and its disulfides was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) following derivatization with NEM and AccQ by the method of Yamada et al.

E. Western Blotting Aliquots of liver (approximately 50 mg) were homogenized in 300 μL of cell lysis reagent containing 1% proteinase inhibitors in a BioMasher II. After centrifugation at 12000 g for 15 min (4° C.), the protein concentration in the supernatant was measured by BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) and adjusted to 10 μg/15 μL with the same buffer. Proteins were separated by SDS-PAGE using 12.5% gels, then PVDF membranes (0.45 μm pores, GE healthcare) by using a semi-dry blotting apparatus (WSE 4020, ATTO, Tokyo, Japan). Life Sciences, Chicago, Ill., USA).
The membrane was then blocked with 4% block Ace (Megmilk Snow Brand, Sapporo, Japan) for 30 min at room temperature followed by 2.68 mM KCl containing 0.05% (v/v) Tween 20 (TBST). 5 minutes (5 times) at room temperature with 50 mM Tris-HCl buffer, pH 7.5, containing 137 mM NaCl.
Primary antibodies against SOD-1, GPX-1, β-actin, and GAPDH were 1:5000, 1:5000, respectively, in 0.4% Block Ace solution containing 0.05% (v/v) Tween 20; Diluted 1:1000 and 1:8000. After overnight incubation with primary antibody, the membrane was washed with TBST for 5 minutes (5 times). The HRP-secondary antibody conjugate was diluted 1:10000 with 0.4% Block Ace containing 0.05% (v/v) Tween 20. Membranes were incubated with HRP-secondary antibody conjugate for 1 hour, followed by washing with TBST for 5 minutes (5 times) at room temperature. The membrane was immersed in ECL reagent for 1 minute and bands were detected by using Luminograph I (ATTO).

power. Statistical Analysis Differences in band intensity of proteins detected by Western blotting on the same membrane between one group and the HFD group were analyzed by t-test.
Statistical differences in other parameters between the HFD group and other groups were analyzed using one-way ANOVA followed by Dunnett's test for multiple comparisons.
This difference was considered significant at p<0.05 and trended constant at 0.05<p<0.1.
GraphPad Prism 7 software (GraphPad Software, CA, USA) was used for statistical analysis.

(2) Results a. Body weight and liver weight As shown in Figure 1, the body weight of the groups fed with high-fat diet (HFD, WEC, PL, PH groups) was significantly higher than that of the group fed normal diet (ND group) after 4 weeks. rice field. However, administration of WEC and phenethylamine had no significant effect on weight gain. The liver weight of the HFD group (Fig. 1C) was significantly higher than that of the ND group. The HFD group's liver/body weight ratio (Fig. 1B) tended to be higher than the ND group, but there was no statistical difference. This ratio tended to decrease in mice treated with phenethylamine (PL and PH groups) compared to the HFD group (p<0.1).

stomach. Plasma Biochemical Parameters Plasma AST and ALT activities (FIGS. 2A and B) were significantly higher in the HFD group than in the ND group, indicating that high-fat diet induced liver injury.
In contrast, administration of a low dose of phenethylamine (10 μg/kg/day, PL group) significantly reduced plasma AST and ALT activity compared to the HFD group, whereas administration of a high dose of phenethylamine (100 μg/kg/day) significantly reduced plasma AST and ALT activity compared to the HFD group. , PH group) showed only a tendency to decrease ALT.

hare. Oxidative stress in the liver. High-fat diet intake (HFD group) significantly increased thiobarbituric acid-reactive substances (TBARS), indicators of lipid peroxidation in liver extracts, compared to normal diet (ND group), Fig. 3A. ) value and decreased SOD-like activity (Fig. 3C).
In addition, the high-fat diet group tended to reduce the oxidative stress marker glutathione to glutathione disulfide ratio (GSH/GSSG, FIG. 3B). These facts indicate that a high-fat diet increases oxidative stress in the liver and decreases endogenous antioxidant systems.
In contrast, administration of WEC, both low and high doses of phenethylamine, significantly inhibited high-fat diet-induced lipid oxidation.
Administration of WEC and low dose phenethylamine significantly increased SOD-like activity in liver extracts compared to the HFD group.
Hepatic GSH/GSSG was also significantly increased in the PL group compared to the HFD group (Fig. 3B).

workman. Antioxidant enzyme levels of endogenous antioxidant enzymes, SOD-1 (Fig. 4) and GPX-1 (Fig. 5), in liver were assessed by Western blot analysis.
Surprisingly, high-fat diet intake and/or administration of WEC and phenethylamine significantly affected GAPDH and β-actin levels (Figs. 6, 7).
Moreover, the band intensity of α-tublin was much lower than that of the target protein (data not shown).
The intensity of protein bands in each group was compared with that of the HFD group split on the same membrane by t-test.
SOD-like activity was significantly increased by WEC and low doses of phenylethylamine (Fig. 4). High-fat diet intake significantly decreased GPX-1 and β-actin protein levels in the liver compared to the ND group (Figs. 5, 7).
Administration of low dose phenethylamine tended to increase GPX-1 compared to the HFD group (p=0.058, Figure 5) and significantly increased β-actin levels (Figure 7).
Although there was no significant difference in GAPDH levels between the HFD and ND groups, oral administration of phenethylamine and WEC significantly increased GAPDH levels compared to the HFD group (Figure 6).

Short-chain aldehyde glyoxal and methylglyoxal levels in liver were significantly reduced by oral administration of WEC, phenethylamine (Fig. 8).
Also, free cysteine levels in liver were significantly increased by oral administration of WEC, phenethylamine (Fig. 9).

Claims (5)

A GAPDH enhancer containing phenethylamine or a salt thereof as an active ingredient (excluding prophylactic or therapeutic agents for non-alcoholic fatty liver disease). A methylglyoxal or glyoxal production inhibitor containing phenethylamine or a salt thereof as an active ingredient (excluding prophylactic or therapeutic agents for non-alcoholic fatty liver disease). 3. The agent according to claim 1, which is for oral ingestion. A GAPDH-enhancing food composition containing phenethylamine or a salt thereof (excluding food compositions for the prevention or treatment of non-alcoholic fatty liver disease). A food composition containing phenethylamine or a salt thereof for inhibiting the production of methylglyoxal or glyoxal (excluding a food composition for preventing or treating non-alcoholic fatty liver disease).

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