JP6183656B2 - Nonalcoholic fatty liver suppressor - Google Patents

Description

  The present invention relates to a non-alcoholic fatty liver inhibitor, a dehydroepiandrosterone-sulfate (DHEA-S) reduction inhibitor, and a weight loss inhibitor in old age, which contain a black vinegar-derived component as an active ingredient. More specifically, as a black vinegar-derived component, a non-alcoholic fatty liver inhibitor, a dehydroepiandrosterone-sulfate (DHEA-S) decrease inhibitor, and a weight loss inhibitor in the elderly, which contains black vinegar excluding acetic acid as an active ingredient .

Black vinegar is a kind of rice vinegar stipulated by the JAS method. Steamed rice, rice bran, and water are put in a potter's pot, and saccharification, alcoholic fermentation, and acetic acid fermentation progress in one pot and further mature. This vinegar has a unique deep taste.
Black vinegar has long been manufactured in Kirishima City, Kagoshima Prefecture, and was used exclusively as a seasoning in the past.In recent years, black vinegar has been shown to be effective in lowering blood pressure and smoothing blood. With the rise of vinegar, black vinegar has been diluted and drunk. In addition, some black vinegar drinks are also approved as food for specified health use.
Recently, black vinegar has been reported to have various effects such as lipid metabolism improving effect, erythrocyte deformability improving effect, blood pressure regulating effect, blood glucose regulating effect, antioxidant effect, obesity suppressing effect and blood adiponectin concentration increasing promoting effect. (Patent Documents 1, 2, and 3). It has also been reported that the expression of a Sirt1-related gene, which is a gene that brings about longevity, is enhanced (Patent Document 4).
Non-alcoholic fatty liver is increasing in advanced Western countries and Japan along with so-called lifestyle-related diseases such as diabetes, dyslipidemia and hypertension. Among these fatty livers, non-alcoholic steatohepatitis (NASH) with inflammation and fibrosis may cause cirrhosis and liver cancer, and is a fatty liver with a poor prognosis. Therefore, preventing the onset of fatty liver is an important issue for preventing the occurrence of NASH.
There is no established pharmacotherapy for nonalcoholic steatohepatitis (NASH). Fatty liver based on obesity and diabetes is known to improve markedly by exercise, caloric restriction, or blood glucose control. ing. As a treatment for NASH, various drug therapies including antioxidants such as vitamin E and ursodeoxycholic acid have been tried, but a certain view has not yet been obtained (Non-patent Document 1). There is a possibility that a drug for improving lipid metabolism abnormalities may be effective for NASH, but there are some reports that even if lipid metabolism abnormalities can be improved, fatty liver can or cannot be improved (Non-patent Document 2). The effectiveness of drugs that improve abnormal lipid metabolism in the liver has not been determined. Development of a therapeutic drug for nonalcoholic steatohepatitis is strongly desired, but the contribution of the drug to the treatment and the satisfaction with the treatment are both 30% or less (Non-patent Document 3), and the development of a new drug is extremely delayed. One of the areas.
Androgen dehydroepiandrosterone (DHEA) is a sex steroid estrogen and testosterone precursor steroid, and DHEA-sulfate (DHEA-s), which can be interconverted with its sulfated form, is anti-obesity, anti-diabetic, carcinogenic, arteriosclerosis It has a beneficial effect on memory maintenance, osteoporosis, etc., and is known as one of biomarkers involved in aging (Non-patent Document 4). Therefore, if the decrease in DHEA-S can be suppressed, the possibility of improving various symptoms associated with aging can be expected. Here, it is known that black vinegar has a cognitive function lowering suppressing action (Patent Document 5). However, cognitive decline is thought to occur due to the complex involvement of various factors, and the literature does not suggest any relationship with DHEA-S.
In addition, many studies on weight and survival in older age have been reported, and obesity in younger age increases the risk of death, whereas conversely, weight loss in older age has been reported to increase mortality. (Non-Patent Documents 7 and 8). It has been suggested that overweight and obesity in the elderly are not harmful or have an effect of reducing the risk of death (Non-patent Document 8). However, the method for suppressing weight loss in the elderly is unknown.

JP 2005-263760 A JP 2007-29020 A JP 2011-168540 A JP 2013-133279 A JP 2013-060380 A

New clinical internal medicine 9th edition 8． Fatty liver, non-alcoholic steatohepatitis Medical School (2009) Lipid-lowering agents in nonalcoholic fatty liver disease and steatohepatitis: human studies.Nseir W. et al., Dig Dis Sci. 2012; 57: 1773-81. National Institute of Pharmaceutical Sciences. “Development and approval status of drugs for unmet medical needs” Policy Research News No.34 (November 2011) Development of a Model of Functional Endocrine Age in Japanese People -Serum Dehydroepiandrosterone-sulfate (DHEA-s) Concentration as an Index of Aging-.Nomoto K. et al., Anti-Aging Medicine, 2011; 8: 69-74. The use of body surface area as a criterion of drug dosage in cancer chemotherapy.Pinkel D. Cancer Res. 1958; 18: 853-6. Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. Rodgers JT et al., Nature. 2005; 434: 113-8. Body mass index, change in body mass index, and survival in old and very old persons.Dahl AK et al., J Am Geriatr Soc. 2013; 61: 512-8. Survival in older men may benefit from being slightly overweight and centrally obese--a 5-year follow-up study in 4,000 older adults using DXA.Auyeung TW et al., J Gerontol A Biol Sci Med Sci. 2010; 65: 99- 104.

  The present invention suppresses the development of non-alcoholic fatty liver by ingesting black vinegar-derived ingredients that have been confirmed to be safe as foods, and dehydroepiandrosterone-sulfate (DHEA-S), which is one of the biomarkers of aging It is an object to suppress a decrease in body weight and / or a decrease in weight in the elderly.

  The present invention provides a non-alcoholic fatty liver onset inhibitor, a DHEA-S reduction inhibitor associated with aging and / or a weight loss inhibitor in old age, comprising black vinegar as an active ingredient. In particular, a non-alcoholic fatty liver onset inhibitor, a DHEA-S reduction inhibitor associated with aging, and / or weight loss in old age, which contains so-called black vinegar-specific components obtained by removing the acetic acid component from black vinegar Provide an inhibitor.

  Since black vinegar has been confirmed to be safe as a food, taking the agent of the present invention containing black vinegar as an active ingredient suppresses the risk of developing non-alcoholic fatty liver without side effects, and aging The accompanying decrease in DHEA-S and / or weight loss in the elderly can be suppressed. Moreover, since the agent of this invention uses black vinegar as an active ingredient, it is also economical.

Macroscopic image of liver and tissue image stained with hematoxylin and eosin a: Macro image of fatty liver developed in the liver of mice fed with high fat diet b: Enlarged tissue image of fatty liver, arrow region a. In fatty liver tissue, large and small vacuoles are seen, the nucleus of hepatocytes is squeezed out at one end, and the endoplasmic reticulum is enlarged. The structure of the hepatic cell cord has become unknown, and the sinusoids have also narrowed. c: Macro image of normal liver in mice administered with black vinegar concentrate d: Micro image of normal liver, arrow region of c was enlarged. e: Macroscopic images of fatty liver and lipoma produced in mice administered with a high fat diet. The top two images show lipoma and the bottom two images show fatty liver lesions. Histological image of hepatocellular carcinoma stained with hematoxylin and eosin a: Macro image of hepatocellular carcinoma that occurred in the liver of mice fed with high-fat diet b: Enlarged tissue image of hepatocellular carcinoma, arrow region a was enlarged. Tumor cells similar to hepatocytes with strong atypia proliferate like islands in normal tissue. c: Metastasis to the small intestine of hepatocellular carcinoma of a. There are similar island-shaped tumor cells observed in b. d: Lipoma and hepatocellular carcinoma found in mice fed the high-fat diet. Lipoma with enlarged vesicles on the right and atypical on the left. Strong hepatocellular carcinoma is observed. Change in body weight over time The average value of each group is shown. Changes in body weight were shown every week. High fat diet administration group: Shown as HC, feed administration group for this study: BV, resveratrol administration group: Res, standard diet administration group: SD. Significant increase suppression was confirmed from the 11th to 23rd week of breeding and from the 25th to 37th week. From the 76th week to the 96th week of breeding, a significant reduction in body weight was observed in the mice fed the test feed compared to mice fed the high fat diet. (Tukey method)

  In the present invention, “black vinegar” as an active ingredient is a kind of rice vinegar defined by the JAS method, and means rice black vinegar produced by adding rice bran and water to fermentation and aging. To do. Rice black vinegar is produced using more than 180 grams of rice per liter of vinegar and has become brown or blackish brown after a long aging period of at least half a year. Differentiated from grain vinegar. In particular, put "mixed rice cake", which is 3% -milled rice with 3% shaved brown rice, seeded rice, "steamed rice", steamed rice for 3 minutes, and water in this order in the pot Finally, rice black vinegar produced by covering the water surface uniformly with straw, covering the straw, fermenting for more than half a year, and further aging for more than half a year is preferable. Black vinegar that can be preferably used in the present invention can be obtained from Sakamoto Brewing Co., Ltd., for example. Black vinegar contains about 4% acetic acid and is thought to contain proteins, oligopeptides, amino acids, organic acids, etc., but the detailed composition is not understood.

  In the present invention, the “black vinegar from which the acetic acid component has been removed” as an active ingredient refers to a product obtained by removing acetic acid and other volatile components from black vinegar by at least one treatment such as freeze-drying. Any means known to those skilled in the art may be selected as the lyophilization treatment means. For example, freeze-drying treatment can be performed by a freeze dryer FD-81 of Tokyo Science Instruments. In order to remove acetic acid more completely, it is preferable to repeat the operation of dissolving the powder obtained by lyophilization of black vinegar in water and lyophilizing the solution again. The operation can be repeated once, twice, three times, or four times, for example. Even if it is obtained by other types of treatments instead of general freeze-drying treatment, it is included in the freeze-dried product of the present invention as long as it is substantially the same in that volatile components are removed. Let's say. As described above, “black vinegar from which acetic acid components have been removed” is obtained by removing acetic acid and other volatile components from black vinegar. Therefore, in this specification, unless otherwise specified, it is synonymous with “black vinegar concentrate”. In addition, “black vinegar freeze-dried product” can be used as an actual use example.

  The black vinegar freeze-dried product is in a powder state immediately after the freeze-drying treatment, but by dissolving the powder in water, it does not contain acetic acid and other volatile components, but contains other components of black vinegar. An aqueous solution is obtained. This aqueous solution of black vinegar freeze-dried product is referred to as black vinegar freeze-dried solution aqueous solution. When the volume of water used for dissolution is smaller than the volume of black vinegar before lyophilization, the resulting aqueous solution is particularly called “black vinegar concentrate”. For example, a solution obtained by freeze-drying 1000 ml of black vinegar as described above and finally dissolving in 100 ml of water is a black vinegar concentrate containing black vinegar freeze-dried product. Can also be referred to as a black vinegar 10-fold concentrate.

  The “black vinegar-derived component” means any component that is contained in black vinegar and can be separated from black vinegar. Especially in this specification, the component contained in the freeze-dried black vinegar or the freeze-dried fraction of the black vinegar concentrate may be referred to as a black vinegar-derived component. The term “component” does not necessarily mean a single compound, and a mixture of a large number of compounds may be called a component.

  The non-alcoholic fatty liver inhibitor, dehydroepiandrosterone-sulfate (DHEA-S) reduction inhibitor and weight loss inhibitor in the elderly of the present invention contain black vinegar, black vinegar freeze-dried product or black vinegar-derived component alone. However, it is obvious to those skilled in the art that other components may be included as long as the effect as an active ingredient of black vinegar, black vinegar freeze-dried product or black vinegar-derived component is not completely impaired. Understood. Excipients, diluents, solvents, thickeners, emulsifiers, pH adjusters, buffers, stabilizers, preservatives, seasonings, flavors, etc. known in the field of pharmaceutical formulations or food processing are additional ingredients. Take as an example. One skilled in the art can select appropriate additional ingredients depending on the formulation and mode of administration. In the production of the non-alcoholic fatty liver inhibitor, dehydroepiandrosterone-sulfate (DHEA-S) decrease inhibitor and weight loss inhibitor in the elderly of the present invention, lyophilized black vinegar (or a component derived from black vinegar) It may be used in the state, or may be used in the state of a solution, for example, an aqueous solution, particularly a black vinegar concentrate.

  The effect of the non-alcoholic fatty liver inhibitor of the present invention refers to the effect of suppressing the occurrence of non-alcoholic fatty liver by the administration of the agent of the present invention, the effect of reducing the risk of occurrence, the effect of preventing steatohepatitis, etc. . It has been confirmed that the non-alcoholic fatty liver inhibitor of the present invention is particularly effective for fatty liver that occurs with intake of a high fat diet. Therefore, if the non-alcoholic fatty liver inhibitor of the present invention is administered, the risk of fatty liver development can be suppressed and fatty liver can be prevented even in an individual who takes a high-fat diet for a long time.

  The effect of the dehydroepiandrosterone-sulfate (DHEA-S) decrease inhibitor of the present invention refers to the effect of suppressing the decrease of DHEA-S by administration of the agent of the present invention. Suppression of the reduction means that the degree of the reduction is less than the original reduction. Specifically, the level of DHEA-S is maintained without being reduced, or the DHEA-S is reduced. Including increase without. Since the decrease in DHEA-S is known to decrease with aging, the agent of the present invention can be suitably used as a DHEA-S decrease inhibitor with aging.

  Moreover, the effect of the weight loss inhibitor in the elderly of the present invention refers to an effect of suppressing the decrease in body weight in the so-called elderly by administration of the agent of the present invention. In the case of human beings, so-called old age is 65 years or older, or 70 years or older. It is known that obesity in the young age is not good for health, but the risk of mortality increases when the old age is conversely thin (Non-patent Documents 7 and 8, etc.). In the present invention, when black vinegar is fed with a high fat diet for a long period of time, it is an ideal weight control that suppresses weight gain in the young and middle age and suppresses weight loss in the elderly. Clarified that there is an effect. That is, the agent of the present invention can be suitably used as an obesity inhibitor in the early age and middle age, as a weight loss inhibitor in the elderly, or as a weight control agent according to the human health condition. it can.

The individual to which the non-alcoholic fatty liver inhibitor of the present invention, dehydroepiandrosterone-sulfate (DHEA-S) decrease inhibitor and weight loss inhibitor in the elderly are administered may be any species as long as it is a mammal, Mouse or human is preferred. Individuals who ingest a high-fat diet such that the proportion of lipids in the total calorie intake is 30% are desirable.
Examples of these individuals include patients with dyslipidemia, patients with diabetes, patients with metabolic syndrome, and individuals who are at increased risk for these diseases or disorders and need preventive measures, as well as obese individuals, Individuals older than middle age can be mentioned.

  There are no particular restrictions on the method of administering the non-alcoholic fatty liver inhibitor, dehydroepiandrosterone-sulfate (DHEA-S) decrease inhibitor and weight loss inhibitor in the elderly, but oral intake is particularly preferred.

  There is no particular limitation on the amount of the active ingredient when administering the non-alcoholic fatty liver inhibitor, dehydroepiandrosterone-sulfate (DHEA-S) decrease inhibitor and weight loss inhibitor in the elderly in the present invention. An example of a preferred dose is an amount equivalent to 18 ml of black vinegar per square meter of body surface per day (for example, an amount equivalent to 7.5 ml of black vinegar per day for mice, for humans). The amount is equivalent to approximately 30 ml of black vinegar per day), and may be increased or decreased as appropriate. For example, an amount corresponding to 10 to 100 ml of black vinegar per square meter of body surface per day can be administered. Black vinegar as an active ingredient of the present invention is preferably administered continuously every day, but can be consumed for a lifetime.

  Examples of specific embodiments for carrying out the present invention will be described below. These are merely examples, and the present invention is not limited to these examples.

“Sakamoto no Kurozu” (trade name) commercially available from Kurozu Sakamoto Brewing Co., Ltd. was used as the black vinegar in the examples.

[Test Example 1]
(1) Preparation of 10 times concentrated black vinegar for this test Black vinegar contains about 4% acetic acid, and several physiological effects have been reported for this acetic acid. In this test, in order to examine the action of components other than acetic acid among black vinegar components, that is, components specific to black vinegar, acetic acid was removed from black vinegar by the following operation.
1000 ml of black vinegar (Sakamoto Brewing Co., Ltd.) was lyophilized in a vacuum state using a vacuum freeze dryer (Tokyo Rika Kikai Co., Ltd. FD-81D). That is, 200 ml of the sample is frozen in the eggplant type flask, and this is attached to a predetermined position of the freeze dryer, and the inside of the container is depressurized. (× 5) [2] Freeze-dry overnight with the vacuum maintained. [3] End when the sample has been drained or no water droplets have been deposited on the container, or the container has become cold by touching. [4] Add 200 ml of ultrapure water to the dried product in the container and dissolve it. This operation was repeated 5 times, and the final total amount of the dried product was made 100 ml with ultrapure water, and acetic acid was almost completely removed. In addition, the removal of acetic acid confirmed that the peak corresponding to an acetic acid was less than 0.01% in HPLC (high performance liquid chromatography).

(2) Preparation of feed For the feed, Quick Fat manufactured by CLEA Japan was used. Quick Fat is a high-calorie diet with increased fat content and sucrose addition in CE-2, a standard diet for growing mice and rats. The nutrient components and energy content of each feed are shown below.
(2-1) Nutritional components and energy content of normal dietary feed (CE-2) 9.1%
Crude protein 25.0%
Crude lipid 4.8%
Crude fiber 4.2%
Coarse ash 6.7%
Soluble nitrogen free 50.1%
Energy 3.442 kcal / g
(2-2) Moisture content of high fat diet (Quick Fat) 7.1%
Crude protein 24.1%
Crude lipid 13.9%
Coarse fiber 2.9%
Coarse ash 5.2%
Soluble nitrogen free 46.8%
Energy 4.087 kcal / g
(2-3) Composition of the test feed The test black vinegar prepared in the above (1) was added to 6 ml of Quick Fat 1 kg as the test feed. This test feed is a high-fat diet containing 0.6% (V / W) black vinegar for this test. If the mouse's daily intake is 2.5 g, it will take 0.015 ml of 10 concentrated black vinegar. In terms of the dose per body surface area, you will consume 20 ml / m ^{2 of} black vinegar stock solution. For adults with a height of 170 cm and 65 kg (body surface area: 1.75 m ² , calculated using the DuBois equation) Will be 35 ml per day. This corresponds to 30 ml, which is a standard for daily intake of black vinegar in humans. (Non-Patent Document 5)
(2-4) Composition of feed for control test
A diet containing 0.4 g resveratrol (0.04% w / w) per 1 kg of Quick Fat was used as a control test feed. (Non-patent document 6)

(3) Test animals and methods (3-1) Test animals
Ten-week-old C57BL6J male mice (Hokudo Co., Ltd.) were used. 15 to 16 animals were reared in each group, and each feed was fed freely and reared for 110 weeks. The breeding room was maintained at a 12 hour light / dark cycle and a temperature of 23 ° C. ± 2 ° C. Hereinafter, the HC (High Calorie) group is the Quick Fat administration group, the BV (Black Vinegar) group is the feed administration group for this test, the Res (Resveratrol) group is the resveratrol administration group, and the SD (Standard Diet) is the CE-2 administration group ) Shown as a group. This experiment was conducted according to the “Regulations on Animal Experiments at Hokkaido University” and was in compliance with ethical standards for animal experiments.
(3-2) Test method (3-2-1) Collection of blood and organ
Mice reared for 110 weeks were anesthetized by intraperitoneal administration of pentobarbital, and then died from blood loss simultaneously with blood collection from the heart. When the liver was collected, fatty liver development and abdominal organ mass formation were observed, and the metastasis was used for histological analysis. The organs obtained at the time of tissue collection were fixed in 10% neutral buffered formalin solution, sections were collected from the refrigerated tissues, stained with hematoxylin and eosin, and used for histological analysis.
(3-2-2) Measurement of aging-related hormones Serum was separated from blood obtained at the time of organ collection and stored frozen. The obtained serum is Mouse Dehydroepiandrosterone sulfate, DHEA-S ELISA Kit (TSZ Scientific, MA, USA), Mouse / Rat IGF-1 Quantikine ELISA (R & D System, Inc. MN, USA), Rat / Mouse Growth Hormone ELISA KIT (Millipore, MA, USA) was used to measure dehydroepiandrosterone sulfate (DHEA-S), insulin-like growth factor 1 (IGF-1) and growth hormone (GH), respectively.

(5) Results (5-1) Effect of suppressing the incidence of fatty liver
Table 1 shows the incidence of fatty liver and liver cancer after 110 weeks of breeding. The number of fatty liver occurrence was examined by visual inspection at the time of organ collection. For fatty liver and hepatocellular carcinoma, sections of the recognized tissue were prepared and histologically evaluated for the occurrence of fatty liver and hepatocellular carcinoma. Representative tissue images of the collected liver are shown in FIG. 1 and FIG.

Fatty liver incidence test: C-BV: p = 0.0170; HC-SD: p = 0.0029, SD-BV: p = 1.0; BV-Res: p = 0.54; HC-Res: p = 0.11; Rate test: HC-BV: p = 0.600; HC-SD: p = 0.3137
(5-2) Aging hormone
Table 2 shows the values of serum aging-related hormones after 110 weeks of breeding. Significant increases in DHEA-S were observed in the mice fed the test feed and resveratrol.

(5-3) Change in body weight FIG. 3 shows the change in body weight every week during the breeding period.
From the 11th week to the 47th week of breeding, there was a tendency to suppress the increase in the body weight of the test feed-fed mice (BV) compared to the high-fat diet-fed mice (HC). Up to 25 to 37 weeks, significant increase suppression was confirmed (FIG. 3). A similar weight gain inhibitory effect was also observed in the resveratrol administration group, which is known as a positive control that prolongs the survival period (Non-Patent Document 6) (Tukey method).
In the period corresponding to the elderly period from the 76th week to the 96th week of breeding, a significant decrease in the body weight of the test feed-fed mice (BV) was observed compared to the high-fat diet feed-fed mice (Hc). It was. Such an effect of suppressing weight loss was not observed in the resveratrol administration group (Res).
Food intake from week 91 to week 96 was examined, but no significant difference was observed (HD group: 2.47 ± 0.24, BV group: 2.50 ± 0.07, Res group: 2.55 ± 0.19, SD group: 3.26 ± 0.11 , Daily intake per animal (g), mean ± standard deviation). Survival rates were not significantly different in each group. (Log-Rank test)

(6) Consideration Based on the above results, components other than acetic acid in black vinegar, that is, black vinegar-specific components suppress the development of fatty liver accompanying high-fat diet, and DHEA-S is one of the indicators of aging. It was found that the decrease was suppressed. Furthermore, when it is known that an increase in fat risk due to weight loss is known in the elderly in terms of body weight (Non-patent Documents 7 and 8), it has been clarified that black vinegar-specific components have an effect of suppressing weight loss. .
Until now, the inventors have reported that black vinegar has an effect of improving lipid metabolism and enhancing the expression of sirtuin gene. In addition to these effects, the incidence of fatty liver associated with a high-fat diet as an effect of long-term administration is reported. It was thought that there was an ideal body weight control effect with aging.

  Black vinegar is used exclusively as a food and is a highly safe food. Therefore, the present invention provides a method for suppressing the generation of fatty liver and the decrease in DHEA-S associated with a high-fat diet and / or the suppression of weight loss in old age, safely and economically. An established method for preventing the development of fatty liver is not known, and the present invention may be effective in preventing the development of fatty liver. Therefore, the present invention is utilized in the related industrial fields of medicine or veterinary medicine. obtain.

Claims (4)

Dehydroepiandrosterone-sulfate (DHEA-S) lowering inhibitor containing black vinegar as an active ingredient (except for Alzheimer's disease improving agent) . Dehydroepiandrosterone-sulfate (DHEA-S) decrease inhibitor (excluding Alzheimer's ameliorating agent ) containing black vinegar as the active ingredient, excluding the acetic acid component. An agent for inhibiting weight loss in the elderly with black vinegar as an active ingredient. An inhibitor of weight loss in the elderly with black vinegar as an active ingredient excluding the acetic acid component.