Glutathione function enhancer and method thereof

Abstract

To provide methods for enhancing the function of glutathione.SOLUTION: Surprisingly, the inventors of the present application have found that the presence of cysteine, which is a constituent amino acid of glutathione, enhances the function of glutathione, and completed the present invention.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to promoting the detoxification function of glutathione.

Background Art

[0002] Glutathione is a tripeptide composed of cysteine, glutamic acid, and glycine, and is also called γ-L-glutamyl-L-cysteinyl-glycine. This glutathione is widely distributed in the living body, and the liver, spleen, adrenal glands, etc. contain particularly large amounts.

[0003] Glutathione is known to exert various functions in the living body. For example, antioxidant action by eliminating various reactive oxygen species (ROS), suppression of melanin production, whitening and beautifying effects by promoting collagen production, therapeutic effect on non-alcoholic fatty liver, detoxifying action on carbonyl compounds, etc.

[0004] Regarding the detoxifying action, the mechanism of the detoxifying action of carbonyl compounds has been elucidated. For example, carbonyl compounds such as methylglyoxal and glyoxal react non-enzymatically with the thiol group of glutathione and are metabolized by glyoxalase. NAD(P)H activates glutathione reductase and increases the glutathione concentration. It is known that a decrease in the concentration of redox coenzymes such as glutathione and NAD(P) results in a decrease in the detoxifying action of carbonyl compounds (Patent Document 1).

[0005] Thus, since glutathione has various functions, substances that promote glutathione production in the living body have been reported. For example, extracts of Portulaca oleracea (Patent Document 2), triterpenic acids or their derivatives (Patent Document 3), low molecular weight substances such as agar and agarose (Patent Document 4), etc. have been reported. Furthermore, methods have been proposed not only to promote glutathione production but also to enhance the function of glutathione. For example, methods utilizing rice bran or hydrolyzed protein products derived from rice (Patent Document 5), and methods utilizing yeast extracts containing glutathione (Patent Document 6). In addition, cysteine ​​is a type of amino acid and is used not only as a food additive, pharmaceutical, and cosmetic, but also as a synthesis precursor for coenzyme A glutathione and other compounds.

[0006] In recent years, the diversification of dietary habits has led to an increase in the consumption of high-fat foods. This can cause hyperlipidemia, hypertension, and hyperglycemia, which are thought to be caused by the accumulation of visceral fat, and research is underway to develop treatments or preventative measures for these metabolic syndromes (Patent Document 7). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO2001 / 045733 [Patent Document 2] Japanese Patent Publication No. 2010-195744 [Patent Document 3] Japanese Patent Publication No. 2010-105937 [Patent Document 4] WO2008 / 047663 [Patent Document 5] Japanese Patent Publication No. 2015-166327 [Patent Document 6] WO2015 / 151867 [Patent Document 7] WO2007 / 037438 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] In this context, the present invention aims to find a method for enhancing the function of glutathione. [Means for solving the problem]

[0009] The inventors of the present application surprisingly found that the function of glutathione can be enhanced by the presence of cysteine, which is a constituent amino acid of glutathione, and thus completed the present invention. The present invention is the following invention. (1) A composition containing glutathione and a sulfur-containing amino acid (2) A composition in which the sulfur-containing amino acid in (1) is selected from one or more of cysteine, cystine, and methionine (3) A lifestyle disease-improving agent containing the composition described in (1) or (2) above.

Effect of the Invention

[0010] According to the present invention, there can be provided pharmaceuticals, foods, feeds, etc. that enhance the function of glutathione. More specifically, it can be used for the prevention, treatment, improvement, etc. of lifestyle diseases such as metabolic syndrome.

Brief Explanation of the Drawings

[0011] [Figure 1] Structure example [Figure 2] Blood biochemistry test results [Figure 3] Quantification of glutathione in the liver [Figure 4] Amount of glutathione peroxidase in the liver [Figure 5] Quantification of glutathione in the liver [Figure 6] Amount of glutathione-cysteine disulfide [Figure 7] NAD / NADH ratio in the liver [Figure 8] Example of the mechanism of action of the present invention [Figure 9] Quantification of DTCA, etc. [Figure 10] Quantification of DTCA, etc. [Figure 11] Quantification of DTCA in the liver [Figure 12] Amount of selenocysteine-glyceraldehyde complex in the liver

Modes for Carrying Out the Invention

[0012] As used in the present invention, glutathione refers to reduced glutathione, oxidized glutathione, or a mixture thereof. Reduced glutathione refers to a tripeptide having the structure of γ-L-Glu-L-Cys-Gly, and oxidized glutathione refers to a compound in which two molecules of reduced glutathione are linked by an S-S bond. The form of glutathione may be anything as long as it contains glutathione as an active ingredient.

[0013] The glutathione of the present invention is not particularly limited as long as it is obtained by a method that can be used as a pharmaceutical or food. Usually, there are methods such as obtaining from microorganisms such as yeast (Japanese Patent Laid-Open No. 59-151894), methods produced by synthesis using enzymes, etc. (Japanese Patent Laid-Open No. 2020-72), methods produced by chemical synthesis methods (Bull. Chem., Soc. Jpn., 1980, Vol. 53, p. 2529), etc., and there is no limitation on the glutathione used in the present invention. Also, glutathione and oxidized glutathione can be purchased, and as long as they can be used as foods, pharmaceuticals, etc., the purchased ones may also be used.

[0014] In the present invention, yeast containing glutathione can also be used. Examples of yeast containing glutathione include "Hightion Corbo MG" (manufactured by Kojin Life Science Co., Ltd.), and examples of yeast extract containing glutathione include "Hightion Extract YH" (manufactured by Kojin Life Science Co., Ltd.).

[0015] Examples of sulfur-containing amino acids in the present invention include cysteine, cystine, and methionine. There are no restrictions on the manufacturing methods of these sulfur-containing amino acids; any method used for food or pharmaceutical applications, such as fermentation or synthesis, is acceptable. More specifically, for L-cysteine, methods such as isolation and extraction from acid hydrolysates of hair and feathers, fermentation from Corynebacterium bacteria (JP 2010-22215), and enzyme-based methods (WO / 2018 / 056305) are known, and these manufacturing methods can be used without restriction in the present invention. These sulfur-containing amino acids may be used individually, or two or more may be used in combination. Furthermore, the present invention exerts its effects when it has the structure shown in Figure 1. Since the present invention exerts its function through the bonding of the sulfur atom of glutathione with the sulfur atom of an amino acid, the function of the present invention can be exerted as long as it is a sulfur-containing amino acid. For this reason, the sulfur-containing amino acid may be in the form of an amino acid, or it may be in the form of a dipeptide that can form an SS bond with glutathione.

[0016] There are no restrictions on the amount or ratio of glutathione and sulfur-containing amino acids used in this invention. As mentioned above, glutathione and sulfur-containing amino acids form disulfide bonds, so it is desirable to include equal amounts of glutathione and sulfur-containing amino acids. However, since glutathione and sulfur-containing amino acids are substances that exist in living organisms, the function of this invention will be exhibited even in compositions that contain a large amount of glutathione or a large amount of sulfur-containing amino acids. Therefore, there are no restrictions on the ratio of glutathione to sulfur-containing amino acids. The formulation method is also not restricted; formulation methods used in pharmaceuticals and foods can be used as appropriate.

[0017] The composition of the present invention is a composition containing glutathione and sulfur-containing amino acids, but other substances can be added as long as they do not inhibit the disulfide bond between the glutathione and sulfur-containing amino acids. These can include excipients commonly used in supplements, animal feed, and pharmaceuticals, various vitamins, inorganic substances such as calcium, magnesium, iron, and zinc, organic acids such as lactic acid, citric acid, and malic acid, proteins, lactic acid bacteria extracts, polyphenols, plant extracts, and other organic substances. Acidulants, sweeteners, flavorings, and stabilizers commonly used in food products can also be added. The method of administering the present invention is not particularly limited and may include oral administration, intravenous administration, intraperitoneal administration, or subcutaneous administration. Specifically, it may be any oral preparation such as tablets, powders, granules, pills, suspensions, emulsions, infusions / decoctions, capsules, syrups, liquids, elixirs, extracts, tinctures, or fluid extracts, or any parenteral preparation such as injections, drips, creams, or suppositories. Conventional methods can be used to prepare these tablets, etc., and there are no limitations in the present invention.

[0018] The dosage of the present invention is not particularly limited as long as it is an amount that produces the aforementioned functions. The dosage and frequency of administration to humans will vary depending on the form of administration, the age and weight of the recipient, etc., but for adults, glutathione and sulfur-containing amino acids are usually administered once to several times a day in amounts of 50 mg to 30 g, preferably 100 mg to 10 g, and particularly preferably 200 mg to 3 g per day.

[0019] The composition of the present invention can be taken not only as a pharmaceutical but also as a functional food and nutritional supplement, in which case it should be taken in the amount specified above. The composition of the present invention can be used as a composition for improving, preventing, and treating lifestyle-related diseases such as metabolic syndrome.

[0020] The present invention will be described in more detail below. However, the present invention is not limited by the following description. (Animal experimentation) Seven-week-old male C57BL / 6J mice were purchased from SLC Japan, and after a one-week acclimatization period, they were divided into four groups of six mice each and reared under 12-hour light / dark conditions for 12 weeks, from eight to twenty weeks of age. Water and food were provided freely. Normal diet (MF pellets) was purchased from Oriental Yeast, and 60% high-fat diet (High Fat Diet 32) was purchased from CREA Japan. L-glutathione for oral administration to the mice was provided by Kojin Life Science. Each group was given (B) normal diet, (HF) 60% high-fat diet, (LG) 60% high-fat diet + glutathione 6 mg / kg body weight / day, or (HFG6) 60% high-fat diet + glutathione 60 mg / kg body weight / day (HFG60). Glutathione was dissolved in drinking water and allowed free access. After the rearing experiment, mice were inhaled with isoflurane (Fujifilm Wako Pure Chemical Industries) and then laparotomy was performed. Blood was collected from the inferior vena cava using a syringe and needle (23G, 0.60×32 mm, TERUMO) treated with an anticoagulant (heparin sodium, Nipro). The blood was centrifuged (3,000 rpm, 4℃, 10 min), and the supernatant (plasma) was transferred to a new tube and stored at -30℃. Immediately after collection, chilled phosphate-buffered saline pH 7.4 (PBS) was injected arterially into the liver to thoroughly flush out the blood inside, and the liver was weighed. It was then stored after appropriate processing as described later for use in subsequent experiments. Various adipose tissues were also collected, weighed, and stored at -80℃.

[0021] (Blood biochemistry test) AST, ALT, γ-GT, total cholesterol, triglycerides (TG), free fatty acids (FAA), and total ketones were measured in the collected plasma (measurements were requested from Oriental Yeast Co., Ltd.). (Figure 2)

[0022] (Quantitative determination of glutathione in the liver) The glutathione content in the liver was quantified using the method of Yamada et al (1). The standard reagent, glutathione (reduced form), was purchased from Nacalai Tesque. (Figure 3)

[0023] (Extraction of proteins from the liver) Cell Lysis Reagent (CelLytic MT Cell Lysis Reagent, Sigma), a phosphatase inhibitor cocktail (Nacalai Tesque), and a protease inhibitor cocktail (Sigma) were pre-mixed in a 200:2:1 ratio and prepared in 400 μL portions in tubes. 30-100 mg of liver was added to each, and the mixture was thoroughly crushed using a biomasseur until no liver fragments were visible. The mixture was mixed well every 10 minutes and allowed to stand on ice for 60 minutes. The mixture was then centrifuged (12,000 g, 4°C, 15 min), the supernatant was transferred to a new tube, and stored at -30°C until use.

[0024] (Extraction of nuclear proteins) Nuclear proteins were extracted using a nuclear extraction kit (NE-PER Nuclear and Cytoplasmic Extraction Reagents, Thermo Fisher). The CER I and protease inhibitor cocktail from the kit were pre-mixed 200:1 and placed in 400 μL tubes. 20-100 mg of liver tissue collected immediately after dissection was added to each tube, and the mixture was mashed using a biomassher until no tissue fragments were visible. These steps were performed on ice. Afterward, the mixture was thoroughly mixed for 15 seconds and allowed to stand on ice for 10 minutes. 22 μL of ice-collapsed CER II from the kit was added, thoroughly mixed for 5 seconds, and allowed to stand on ice for 1 minute. Further mixing for 5 seconds and centrifugation (16,000 g, 4°C, 5 min) were performed. The cytoplasmic supernatant was transferred to a new tube and stored at -30°C. The precipitate fraction containing nuclei was pre-mixed with NER and the protease inhibitor cocktail 200:1 and added in 200 μL tubes, thoroughly mixed for 15 seconds. The mixture was left to stand on ice for 40 minutes, during which time it was mixed thoroughly for 15 seconds every 10 minutes. Then, it was centrifuged (16,000 g, 4°C, 10 min), and the supernatant fraction containing the nuclear extract was transferred to a pre-chilled tube and stored at -30°C until use.

[0025] (Western blot) Reaction solutions A and B from the protein assay kit (Pierce BCA Protein Assay Kit, Thermo Fisher) were mixed in a 50:1 ratio and 80 μL each was placed in a microplate for absorption measurement. The included albumin standard solution and sample solution were mixed and added to the microplate in 4 μL portions, which were then mixed. After standing at 37°C for 30 minutes, absorbance analysis was performed at a wavelength of 570 nm using an absorbance analyzer (Model 680 Microplate Reader, Bio Rad), and the sample volume for the following electrophoresis was prepared to be 10 μg / 15 μL. 3 μL of 6×SDS Buffer (360 mM Tris-HCl (pH 6.8), 0.15% bromophenol blue, 12% sodium lauryl sulfate, 60% glycerin) containing 1% 2-mercaptoethanol was added to 15 μL of the sample. The mixture was reacted at 100°C for 5 minutes, cooled on ice, and then added to a 10% polyacrylamide gel (Super Sep™ Ace, Fujifilm Wako Pure Chemical Industries) for electrophoresis. After completion, a semi-dry blotting apparatus (WSE-4020, ATTO) was used, and semi-dry blotting buffer (EzFastBlot, ATTO) was used as the electrode solution. The samples were then transferred to nitrocellulose membranes (Amersham Protran Premium 0.45 μm NC, GE Healthcare Life Sciences) at a rate of 0.5 mA / membrane for 20 minutes. After blocking for 30 minutes (EzBlock Chemi, ATTO), the membranes were washed three times for 5 minutes each with 0.45 mM Tween 20 (Polyoxyethylen (20) Sorbian Monolaurate, Fujifilm Wako Pure Chemical Industries)-TBS (Tris buffered saline, Bio-Rad) (hereinafter, TBS-T). The primary antibodies Anti-Glutathione Peroxidase 1 Antibody (Abcam), Anti-Glutathione Reductase Antibody (Abcam), and Anti-Nrf2 Antibody (Abcam) were diluted 1000-fold in 10 mL of TBS-T containing 0.5 mL of blocking solution, and the membranes were immersed in this solution and left to stand in the refrigerator overnight.The following day, the primary antibody reaction solution was removed, the membrane was lightly rinsed three times with TBS-T, and the secondary antibody reaction solution was added. The secondary antibodies used were a 5000-fold dilution of anti-mouse-goat secondary antibody (Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP, Thermo Fisher) and a 10000-fold dilution of anti-rabbit-goat secondary antibody (Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP, Thermo Fisher). One hour after adding the secondary antibody reaction solution, the solution was removed, the membrane was washed three times for 5 minutes each, and a chemiluminescent reagent (Chemiluminescent One Super, Nacalai Tesque) was added dropwise to the membrane. After 5 minutes, the excess reaction solution was removed, and the bands were detected using an image analysis system (WSE-6100 LuminoGraph I, ATTO) and quantified using ImageJ. To detect other antibody-specific bands on the same membrane, it was incubated with stripping solution (Fujifilm Wako Pure Chemical Industries) for 10 minutes, followed by washing with TBS-T for 5 minutes. Subsequent steps were the same as those from the blocking stage. In this study, measurements are presented as mean ± standard error. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software) with a one-way ANOVA test. When statistically significant differences were found between groups, Dunnett's multiple comparison test was used to compare the differences between the HF group and each other group in a post-hoc comparison.

[0026] As shown in Figure 2, high-fat diet intake significantly increased plasma AST and ALT levels, leading to liver damage. Administration of glutathione at doses of 6 and 60 mg / kg body weight significantly decreased AST and ALT levels, resulting in improved liver function. Furthermore, plasma triglycerides and free fatty acids were significantly reduced in a dose-dependent manner with glutathione administration. These results replicate those of human trials. Human trials: Honda, Y., Kessoku, T., Sumida, Y., Kobayashi, T., Kato, T., Ogawa, Y. Tomeno, W., Imajo, K., Fujita, K., Yoneda, M., Kataoka, K., Taguri, M., Yamanaka, T., Seko, Y., Tanaka, S., Saito, S., Ono, M., Oeda, S., Eguchi, Y., Aoi, W., Sato, K., Itoh, Y., Nakajima, A. (2017) Efficacy of glutathione for the treatment of nonalcoholic fatty liver disease: an open-label, single-arm, multicenter, pilot study. BMC Gastroenterology 17-96, 1-8.

[0027] On the other hand, there was no significant difference in total glutathione levels in the liver between any of the groups (Figure 3). However, as shown in Figure 4, glutathione peroxidase levels in the liver were significantly reduced in the high-fat diet group and increased in a dose-dependent manner with glutathione administration. Nuclear translocation of Nrf2, a transcription factor for glutathione peroxidase, was significantly increased with glutathione administration but not decreased with the high-fat diet group, which cannot explain the decrease in glutathione peroxidase levels with the high-fat diet group. Furthermore, there was no significant difference in the amount of glutathione reductase protein, which is transcribed by Nrf2, between the groups.

[0028] Glutathione quantification was performed according to the following literature. Yamada, H., Ono, S., Wada, S., Aoi, W., Park, EY, Nakamura, Y., Sato, K. (2018) Status of food-derived glutathione in intestine, blood, and liver of rat. Science of Food, 2:3; DOI:10.1038 / s441538-018-0011-y.

[0029] Seven-week-old Wistar rats (weighing 200-220g) were acclimatized for one week under a 12-hour light-dark cycle. Food and water were provided free of charge. 13 Rats were orally administered 1 mL of PBS aqueous solution containing 14C-labeled GSH at a dose of 50 mg / kg body weight. Before administration, and at 30, 60, and 120 minutes after administration, rats were sacrificed by collecting blood from the inferior vena cava under anesthesia with pentobarbital sodium solution (40-50 mg / kg body weight). Liver blood was collected after flushing the portal vein with cold PBS. Portal vein blood and peripheral blood were collected using heparinized syringes. Plasma and blood cells were collected after centrifugation of the blood (8,000 g × 10 min 4 °C). Samples were stored at -80°C until use.

[0030] (Sample preparation) Plasma was mixed with three times its volume of 100% ethanol, centrifuged (14,200 g × 10 min 4 °C), and separated into supernatant and precipitate. The supernatant was used for total glutathione quantification.

[0031] (Quantification of total glutathione and glutathione) 300 μL of plasma supernatant soluble in 75% ethanol was dried under vacuum, and a 2% (v / v) 2-mercaptoethanol solution containing 50 μL of 5% TCA (v / v) was added. The mixture was reacted at room temperature for 30 minutes and then dried under vacuum. 20 μL of methanol:triethylamine:water = 7:2:1 was added to the dried sample and dried under vacuum. 20 μL of 20 mM HCl, 60 μL of borate buffer (pH=8.8), and 20 μL of AccQ acetonitrile solution were added, and the mixture was reacted at 50 °C for 10 minutes. The AccQ derivative was diluted with 5 mM sodium phosphate buffer containing 10% acetonitrile (v / v) (hereinafter referred to as Pico-tag buffer, pH=7.4) and subjected to LC-MS / MS.

[0032] (Quantitative determination of protein-bound glutathione) The aforementioned plasma precipitate insoluble in 75% ethanol was reacted at room temperature for 30 minutes after adding 100 μL of 2% 2-mercaptoethanol solution containing 5% TCA. 50 μL was allowed to dry under vacuum. The dried sample was derivatized with AccQ, diluted with Pico-tag buffer, and subjected to LC-MS / MS.

[0033] (Synthesis and quantification of glutathione-cysteine ​​disulfide) L-cysteine ​​(Fujifilm Wako Pure Chemical Industries) and glutathione were dissolved in equal molar amounts in 5% triethylamine and reacted at 50°C for 3 days. The mixture was then centrifuged under reduced pressure to dryness and purified twice by HPLC. The HPLC system consisted of LC-10AD, SIL-10AD, CBM-20A, CTO-10AC, and SPD-10A. A COSMOSIL 5C18-MS-II column (10 ID × 250 mm, Nacalai Tesque) was used, and the column temperature was maintained at 40°C. Mobile phase (A) was 0.1% formic acid, and mobile phase (B) was 0.1% formic acid-80% acetonitrile, with a flow rate of 1.0 mL / min. The gradient was set to 0-35 min, B 0-20%; 35.1-45 min, B 100%; 45.1-60 min, B 0%. The column was pre-equilibrated with solution A before analysis. Detection was performed using absorbance at 254 nm and 214 nm. Peaks with high intensity were isolated and confirmed to be the target disulfide compound by LC-MS / MS. Subsequently, the concentration of the disulfide compound was determined by the aforementioned amino acid analysis and used as a standard.

[0034] (result) As shown in Figure 5, when glutathione was administered orally, the total glutathione (glutathione released by reduction treatment) in the low molecular weight fraction (75% ethanol soluble fraction) was significantly higher in the portal vein than in the abdominal vena cava. On the other hand, no difference was observed in protein-bound glutathione between the portal vein and the abdominal vena cava. Therefore, it was revealed that glutathione in the low molecular weight fraction was taken up by the liver. Furthermore, it was revealed that the glutathione in the low molecular weight fraction in the portal vein was glutathione-cysteine ​​disulfide.

[0035] Next, we investigated how glutathione-cysteine ​​disulfide behaves in response to glutathione administration.

[0036] (Animal experimentation) Eleven-week-old male Wistar / ST rats were purchased from Nippon SLC. After a one-week acclimatization period, they were divided into four groups of four rats each. After 18 hours of fasting, glutathione was dissolved in 1 mL of water to a concentration of 60 mg / kg body weight and administered via a gastric tube. Dissections were performed at 0, 30, 60, and 120 minutes. (The group administered at 0 minutes was administered water as a control and dissected immediately.) For dissection, the rats were anesthetized by inhalation with isoflurane, and the abdomen was opened. Blood was collected from the portal vein and inferior vena cava using a syringe and needle (23G, 0.60 × 32 mm, TERUMO) treated with an anticoagulant (heparin sodium, Nipro). After centrifugation (3,000 rpm, 4℃, 10 min), the supernatant (plasma) was transferred to a new tube and stored at -80℃. The liver was thoroughly washed with chilled PBS immediately after collection to remove any blood, and stored at -80℃ until use. The quantitative analysis of glutathione-cysteine ​​disulfide in portal blood, peripheral blood, and liver was performed using the method described above.

[0037] (Quantitative determination of NAD, NADH, NADP, and NADPH in the liver) β-nicotinamide adenine dinucleotide (NAD), β-nicotinamide adenine dinucleotide disodium (reduced form, NADH), β-nicotinamide adenine dinucleotide sodium phosphate (NADP), and β-nicotinamide adenine dinucleotide tetrasodium phosphate (reduced form, NADPH) were purchased from Nacalai Tesque. 50-200 mg of liver was homogenized in a biomassier with an equal volume of PBS, three times the volume of ethanol was added, and the liver was thoroughly ground until no more liver fragments were visible. After centrifugation (14,200 g, 4°C, 10 min), the supernatant was transferred to new tubes in 150 μL portions and centrifuged to dryness under reduced pressure. After complete drying, 50 μL of ultrapure water was added and mixed well. After centrifugation (14,200 g, 4°C, 10 min), the supernatant was subjected to HPLC. A Scherzo SM-C18 (150 × 2 mm, 3 μm, IMTAKT) and a COSMOSIL 5C18-MS-II (2.0 ID × 150 mm, Nacalai Tesque) column was used in combination, and the column temperature was maintained at 45°C. Mobile phase (A) was 100 mM formic acid, and mobile phase (B) was 200 mM ammonium formate-30% acetonitrile, with a flow rate of 0.175 mL / min. The gradient was 0-30 min, B 0-35%; 30-38 min, B 35-100%; 38.1-45 min, B 0%. The column was pre-equilibrated with solution A before analysis. Detection was performed by absorbance at 260 nm and 214 nm. Statistical processing In this study, measurements are presented as mean ± standard error. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software) with a one-way ANOVA test. When statistically significant differences were found between groups, Dunnett's multiple comparison test was used to compare the differences between the HF group and each other group in a post-hoc comparison.

[0038] As shown in Figure 6, glutathione-cysteine ​​disulfide levels increased in the portal vein 60 minutes after glutathione administration, but no increase was observed in the liver or peripheral vena cava. The NAD / NADH ratio in the liver did not change significantly after glutathione administration, but the NADP / NADPH ratio increased significantly between 30 and 60 minutes after administration (Figure 7). NADPH is a coenzyme of glutathione reductase. Furthermore, it has been confirmed that glutathione reductase reduces not only glutathione disulfide but also glutathione-cysteine ​​disulfide in the presence of NADPH. From these results, it was found that the glutathione-cysteine ​​disulfide increased by glutathione administration is taken up by the liver and reduced to glutathione and cysteine ​​(Figure 8).

[0039] Next, we investigated the functionality of glutathione-cysteine ​​disulfide. Male C57BL / 6J mice, aged 10 weeks, were purchased from SLC Japan. For diet, a standard diet (MF mouse, rat, and hamster feed) was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan), and a high-fat diet (60 kcal% / fat, D12492) was purchased from Research Diets, Inc. (New Brunswick, NJ, USA). The mice were divided into a standard diet group (Group B) and a 60% high-fat diet group (Group HF), and were reared for 10 weeks from 4 to 14 weeks of age. After fasting, a single intraperitoneal administration of 2.0% GA-PBS solution was given at a concentration of 10 μL / g BW (PBS was administered at 0 minutes). After euthanasia with isoflurane at 0, 30, 60, and 180 minutes post-administration, the mice were dissected. Peripheral blood was collected using a heparinized 1 mL syringe (Terumo, Tokyo, Japan) and a 23G injection needle (Terumo, Tokyo, Japan), and injected into a NaF-treated microtube. The collected peripheral blood was rapidly centrifuged at 3000 rpm for 10 minutes at 4°C to obtain plasma. In addition, after blood collection from mice, the liver was collected after removing systemic blood by cardiac perfusion with PBS.

[0040] (Quantitative determination of glyceraldehyde (GA)) 10 μL of the sample and 10 μL of standard GA as an internal standard were added to a microtube. Then, 60 μL of a mixed solution of equal volumes of 0.5 M PMP (methanol solution) and 7% aqueous ammonia was added, and the mixture was reacted at 70°C for 30 minutes. After the reaction, it was allowed to cool for 20 minutes, and the reactants were completely dried by centrifugation under reduced pressure. 200 μL of 20 mM sodium acetate buffer was added and vortexed, then 200 μL of chloroform was added and vortexed for 30 seconds. The mixture was then centrifuged at 14200 g for 5 minutes at 20°C. 100 μL of the aqueous phase was filtered using an aqueous filter (Cosmonice Filter, diameter 0.45 μm, Nacalai Tesque, Kyoto, Japan), and 10 μL was injected into LC-MS / MS for analysis. PMP-derived standards or plasma samples were injected into an electrospray ionization triple quadrupole mass spectrometer (LC-MS / MS: LCMS-8040, Shimadzu Corporation, Kyoto, Japan) and positive-mode MRM (Multiple Reaction Monitoring) analysis was performed.12, 15) The HPLC section consisted of LC-20AD, CMB-20A, SIL-20AC, SPD-20A, and CTO-20AC. PMP-GA was quantified by MRM by measuring the precursor ion m / z = 421.0, product ion m / z = 175.0, and 373.1. The column used was COSMOSIL 5C18-MS-II (2.0 mm ID × 150 mm, 5 μm, Nacalai Tesque, Kyoto, Japan), and the eluents used were 0.1% formic acid (Solution A) and 80% acetonitrile-0.1% formic acid (Solution B). A 100 mM ammonium formate solution was used for needle washing. The flow rate was 0.2 mL / min, the column temperature was 40°C, and the gradient was set as follows: 0-3 min, B 20%; 3-13.5 min, B 20-45%; 13.5-18.5 min, B 100%; 18.5-30 min, B 20%.

[0041] (Determination of 2-(1,2-dihydroxyethyl)-4-thiazolidinedcarboxylic acid (DTCA)) Liver extracts were converted to AccQ using the method described above, and the resulting AccQ-DTCA was quantified by LC-MS / MS. An Inertsil ODS-3 column (2.1 mm × 250 mm, 4.0 μm, GL Sciences, Tokyo, Japan) was used, with 0.1% formic acid (Solution A) and 80% acetonitrile-0.1% formic acid (Solution B) as eluents. The flow rate was set to 0.2 mL / min and the column temperature to 40°C. The gradient was set to 0.0-4.0 min, B 0%; 4.0-15 min, B 0-60%, 15-20 min, B 100%, and 20-30 min, B 0%. Detection was performed by MRM.

[0042] As shown in Figure 9, glyceraldehyde and glucose concentrations were significantly higher in the high-fat diet group before glyceraldehyde administration, while DTCA, a metabolite of glyceraldehyde, was significantly lower. Glyceraldehyde administration significantly increased hepatic DTCA in the normal diet group, but no increase in DTCA was observed in the high-fat diet group despite high glyceraldehyde levels. Therefore, it is thought that chronically high glyceraldehyde levels in the high-fat diet group led to a decrease in cysteine, resulting in no DTCA production even after glyceraldehyde administration. The results so far suggest that glutathione administration suppresses the toxicity of glyceraldehyde by allowing cysteine ​​derived from glutathione-cysteine ​​disulfide to act as a link.

[0043] Next, we further investigated the functionality of cysteine ​​and glutathione derived from glutathione-cysteine ​​disulfide. The experimental animals were 7-week-old C57BL / 6J male mice purchased from Nippon SLC. After a one-week acclimatization period, they were reared for 15 weeks from 8 to 23 weeks of age at 24±1℃ under 12-hour light / dark conditions. For the regular diet, Certified Diet MF solid (12 mmφ pellets, 359 kcal / 100 g) for mice, rats, and hamsters was purchased from Oriental Yeast Co., Ltd., and for the 60% high-fat diet, High Fat Diet 32 ​​(12.5 mmφ pellets, 507.6 kcal / 100 g) for mice and rats was purchased from Nippon CREA. Three mice were placed in each cage. Each group consisted of six mice, and they were given either a regular diet (B), a 60% high-fat diet (HF), or a 60% high-fat diet + glutathione 60 mg / kg / day (HF+GSH). Glutathione was dissolved in drinking water, and the mice were allowed to drink freely. The glutathione concentration was determined from the amount of water they drank the previous week. Fasting was not performed before dissection. The mice were anesthetized by inhalation using isoflurane (Fujifilm Wako Pure Chemical Industries), then the abdomen was opened, and after cardiac perfusion, the liver was collected.

[0044] (Formation and quantification of reaction products between selenocysteine ​​and glyceraldehyde) 100 μL of 1 μM selenocystin (Sec-Sec), 10 μL of 100 μM dithiothreitol (DTT), and 10 μL of 100 μM GA were mixed and incubated at 37°C. After 0, 30, 60, and 120 minutes, 20 μL samples were taken, 20 μL of 0.3% AccQ-acetonitrile solution and 60 μL of borate buffer (pH: 8.8) were added, and after thorough vortexing, the mixture was incubated at 50°C for 10 minutes. For comparison, we also prepared 100 μL of 1 μM Sec-Sec + 20 μL of ultrapure water, 100 μM GA + 110 μL of ultrapure water, 100 μM DTT + 110 μL of ultrapure water, and 100 μL of 1 μM Sec-Sec + 100 μM DTT + 10 μL of ultrapure water, and similarly incubated them at 37°C for 120 minutes to derivatize them. After adding 100 μL of Pico-Tag Buffer and filtering using an aqueous filter, 10 μL was used for LC-MS / MS analysis. AccQ derivatized compounds were injected into a triple quadrupole mass spectrometer. The HPLC section consisted of LC-20AD, CBM-20A, SIL-20AC, SPD-20A, and CTO-20AC. An Inertsil ODS-3 column (2.1 mm × 250 mm, 4.0 μm, GL Sciences, Tokyo, Japan) was used as the column, with 0.1% formic acid (Solution A) and 80% acetonitrile-0.1% formic acid (Solution B) as the eluents. The flow rate was set to 0.2 mL / min and the column temperature to 40°C. Ultrapure water was used as the needle washing solution. The gradient was set to 0-15.0 min, B 0-30%; 15.1-20.0 min, B 30-100%; 20.1-25.0 min, B 100%; and 25.1-35 min, B 0%. In the MS department, positive-mode Q3 scan analysis was used to detect compounds in the m / z ranges of 150-175, 175-200, 200-225, 225-250, 250-300, 300-350, 350-400, 400-500, and 500-1000 (+). Furthermore, positive-mode precursor ion scan analysis was used to comprehensively detect compounds producing product ions with a m / z of 171.0. The quantification of the selenocysteine-glyceraldehyde complex was performed in MRM mode using standards prepared and optimized as described above. The quantification of DTCA was as described above.

[0045] As shown in Figure 10, when selenocystin was reduced with DTT to release selenocysteine, a cyclic compound similar to DTCA was produced when it was reacted with glyceraldehyde. In rats fed a high-fat diet, DTCA levels in the liver were significantly reduced, similar to the previous study. Glutathione administration tended to increase DTCA levels (Figure 11). Furthermore, the selenocysteine-glyceraldehyde complex in the liver increased with the high-fat diet and decreased with glutathione administration (Figure 12). DTCA is a stable complex, and selenocysteine-glyceraldehyde is also a stable complex; therefore, the selenium within this complex is considered unusable by the body. This suggests that glutathione peroxidase, a selenium enzyme, decreased with the high-fat diet (Figure 4). Glutathione administration sent cysteine ​​to the liver (Figures 6 and 7), which then metabolized glyceraldehyde, ultimately protecting selenocysteine ​​and increasing the bioavailability of selenium. As a result, glutathione peroxidase levels increased, reducing oxidative stress and improving liver function.

[0046] As shown in Figure 1, a high-fat diet impairs liver function, but it was found that this liver function can be improved by glutathione-cysteine ​​disulfide, which is produced by glutathione administration.

Claims

[Claim 1] A composition for improving impaired liver function caused by a high-fat diet, comprising a compound in which glutathione and cysteine ​​are linked by a disulfide bond.

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