JP2013133279A - EXPRESSION ENHANCER OF Sirt1-RELATED GENE - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an expression enhancer of a Sirt1-related gene selected from a group comprising Sirt1, Pgc-1α, Lpin1, Igfbp1 and Ucp2.SOLUTION: It is found that expression of a gene of Sirt1, Pgc-1α, Lpin1, Igfbp1 and Ucp2 is enhanced in the level of mRNA expression and/or protein expression, by administration of black vinegar produced from rice, malt and water, or a composition containing a component derived from black vinegar as an effective component.

Description

  The present invention relates to a Sirt1-related gene expression enhancer comprising black vinegar or a black vinegar-derived component as an active ingredient. The present invention also relates to a method for enhancing the expression of a Sirt1-related gene using the expression enhancer.

The Sirtuin gene family, a homologue of Silent information regulator 2 (Sir2), is a class III histone deacetylase (HDAC) that deacetylates histones and other specific proteins in a NAD ⁺ -dependent manner. It is an enzyme that performs. Sirtuin1 (Sirt1) is known to be upregulated by calorie restriction, and is thought to play a central role in prolonging life caused by calorie restriction. Sirt1 also plays a role in a variety of biological functions that control aging and longevity, including metabolism, cancer prevention, genome stability, endocrine signaling system, stress responsiveness, cognitive function, direct or It is considered to have a function of indirectly extending the life of a living individual (Non-patent Document 1). These are the reasons why Sirt1 is often called a longevity gene.

  Pgc-1α interacts with Sirt1 to control sugar metabolism and lipid metabolism (Non-patent Documents 2 and 3). In addition, there is a report that enhanced expression of a homologue of Pgc-1α causes an increase in the life of an individual in insects (Non-patent Document 4).

  Igfbp1 is an insulin-like growth factor binding protein and regulates the disappearance of insulin-like growth factor Igf. Igf has been suggested to be involved in cancer, diabetes, and aging, and it has been reported that high serum Igfbp1 lowers fasting blood glucose, fasting insulin, and obesity (non-patented) Reference 5). It has been suggested that Sirt1 controls Igf signaling via Igfbp1 (Non-patent Documents 6 and 7).

  Lpin1 is related to the metabolism of triglycerides and has been suggested to be associated with dyslipidemia and insulin resistance (Non-patent Documents 8 and 9). Lpin1 is linked to Sirt1 by forming a complex with the aforementioned Pgc-1α and functioning, and the Lpin1 gene itself is also regulated by Sirt1 through a protein called LXR (Non-patent Document 9).

  Ucp2 is a gene involved in the regulation of energy metabolism in mitochondria, and it has been reported that its expression correlates with the life of an individual (Non-patent Document 10). Ucp2 is also a target of Sirt1, which is a deacetylase (Non-patent Document 11).

  It can be seen that each of the genes mentioned above has a function closely related to Sirt1, and actually functions cooperatively while affecting Sirt1 at some level. Therefore, in the present specification, these genes including the Sirt1 gene are collectively referred to as Sirt1-related genes.

  Controlling the expression of Sirt1 and other Sirt1-related genes is thought to be useful in preventing or treating so-called adult diseases including metabolic diseases and cancers, and may lead to longevity. It is possible (Non-Patent Document 12).

  As a method for controlling the function of Sirt1, many approaches have been taken in which the enzyme activity itself is regulated rather than the amount of mRNA or protein (for example, Patent Documents 1 to 3 and Non-patent Document 13). On the other hand, Patent Document 4 discloses that when various plant material extracts are added to cultured cells, mRNA expression of Sirt1 is increased up to 1.5 times (Patent Document 4). As for Pgc-1α, it has been disclosed that clenbuterol, which is a drug that stimulates the β2 receptor, enhances mRNA expression of Pgc-1α (Patent Document 5), but clenbuterol may have harmful effects such as poisoning. It is known and its applicability is considered limited. In addition, it has been disclosed that administration of a compound called kaempferol 3-0-glucoside increases the mRNA expression of Lpin1 in a dose-dependent manner (Patent Document 6). As a compound that can increase the mRNA expression of Ucp2, Fucoxanthin (patent document 7) contained in the kelp extract, resveratrol (patent document 8), which is a compound also known as an enzyme activator of Sirt1, and aminoguanidine carboxylic acid (patent document 9) are disclosed.

JP 2009-126799 A JP 2007-326872 A JP 2009-500377 A JP 2009-161494 A JP 2007-217368 A JP 2009-256309 A JP 2010-270021 A JP 2010-024208 A JP-T-2002-508770

Recent progress in the biology and physiology of sirtuins. Finkel T et al., Nature. 2009; 460: 587-91. Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. Rodgers JT et al., Nature. 2005; 434: 113-8. PGC-1 coactivators: inducible regulators of energy metabolism in health and disease.Finck BN and Kelly DP, J Clin Invest. 2006; 116: 615-22. Modulation of longevity and tissue homeostasis by the Drosophila PGC-1 homolog.Rera et al., Cell Metab. 2011; 14: 623-34. Serum insulin-like growth factor-1 binding proteins 1 and 2 and mortality in older adults: the Health, Aging, and Body Composition Study. Hu D et al., J Am Geriatr Soc. 2009; 57: 1213-8. The Sirt1 deacetylase modulates the insulin-like growth factor signaling pathway in mammals. Lemieux et al., Mech Ageing Dev. 2005; 126: 1097-105. FoxO-dependent and -independent mechanisms mediate SirT1 effects on IGFBP-1 gene expression.Gan et al., Biochem Biophys Res Commun. 2005; 337: 1092-6. Lipin expression is attenuated in adipose tissue of insulin-resistant human subjects and increases with peroxisome proliferator-activated receptor gamma activation. Yao-Borengasser A et al., Diabetes. 2006; 55: 2811-8. PPAR control: it's SIRTainly as easy as PGC. Sudgen et al., J Endocrinol. 2010; 204: 93-104. Uncoupling protein-2 regulates lifespan in mice.Andrews and Horvath, Am J Physiol Endocrinol Metab. 2009; 296: E621-7. Metabolic benefits from Sirt1 and Sirt1 activators. Chaudhary and Pfluger, Curr Opin Clin Nutr Metab Care. 2009; 12: 431-7. Sirtuins in aging and age-related disease.Longo VD and Kennedy BK. Cell. 2006; 126: 257-68. The Sirtuin family: therapeutic targets to treat diseases of aging. Milne JC and Denu JM. Curr Opin Chem Biol. 2008; 12: 11-7. The use of body surface area as a criterion of drug dosage in cancer chemotherapy.Pinkel D. Cancer Res. 1958; 18: 853-6.

  An object of the present invention is to provide a method for enhancing the expression of a Sirt1-related gene selected from the group consisting of Sirt1, Pgc-1α, Lpin1, Igfbp1, and Ucp2 in a safe and economical manner, and the method. It is to provide a means for achieving this, namely, an expression enhancer for a Sirt1-related gene.

  The present inventors have administered black vinegar produced from rice, rice bran, water, or a composition containing an ingredient derived from the black vinegar as an active ingredient, so that Sirt1, Pgc-1α, Lpin1, Igfbp1 and Ucp2 genes It has been found that expression is enhanced at the level of mRNA expression and / or protein expression, and the present invention has been completed.

That is, the present invention includes the following items.
(1) Sirt1-related gene expression enhancer containing black vinegar as an active ingredient, wherein the Sirt1-related gene is selected from the group consisting of Sirt1, Pgc-1α, Lpin1, Igfbp1, and Ucp2 to enhance gene expression Is an expression enhancer that appears as an increase in mRNA and / or protein.
(2) A Sirt1-related gene expression enhancer comprising a black vinegar freeze-dried product as an active ingredient, wherein the Sirt1-related gene is selected from the group consisting of Sirt1, Pgc-1α, Lpin1, Igfbp1, and Ucp2, An expression enhancer, wherein gene expression enhancement is manifested as an increase in mRNA and / or protein.
(3) A method for enhancing the expression of mRNA and / or protein of a Sirt1-related gene, comprising the step of administering a composition containing black vinegar as an active ingredient in a mammal, wherein the Sirt1-related gene is Sirt1, Pgc- A method selected from the group consisting of 1α, Lpin1, Igfbp1, and Ucp2.
(4) A method for enhancing the expression of mRNA and / or protein of a Sirt1-related gene in a mammal, comprising a step of administering a composition comprising a freeze-dried black vinegar as an active ingredient, wherein the Sirt1-related gene comprises: A method selected from the group consisting of Sirt1, Pgc-1α, Lpin1, Igfbp1, and Ucp2.
(5) The method according to (3) or (4), wherein the expression of mRNA and / or protein of the Sirt1-related gene is enhanced in the liver.
(6) The method according to any one of (3) to (5), wherein the mammal is a human.
(7) The method according to any one of (3) to (5), wherein the mammal is other than a human.

  In the present invention, “black vinegar” is a kind of rice vinegar prescribed by the JAS method, and means rice black vinegar produced by adding rice cake and water to rice and fermenting and aging. 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, “black vinegar freeze-dried product” 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.

  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 Sirt1-related gene expression enhancer of the present invention may contain black vinegar, black vinegar freeze-dried product or black vinegar-derived component alone, but the effect of black vinegar, black vinegar freeze-dried product or black vinegar-derived component It will be appreciated by those skilled in the art that other components may be included as long as they are not completely impaired. 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 expression enhancer of the present invention, the lyophilized black vinegar (or black vinegar-derived component) may be used in the form of a powder or used in the form of a solution, for example, an aqueous solution, particularly a black vinegar concentrate. May be. The proportion of black vinegar, black vinegar freeze-dried product or black vinegar-derived component in the expression enhancer of the present invention is 1% or more, 3% or more, 5% or more, 10% or more, 30% or more in terms of mass. It is preferably 50% or more, 70% or more, or 90% or more, and may be 100%.

  In general, the term “expression” of a gene refers to the production of the translation product or protein of the gene, but in a broader sense it can also refer to the production of a transcription product or mRNA of the gene. In the present specification, “expression” refers to both expression at the protein level and expression at the mRNA level unless otherwise specified. An increase in the expression level at the protein level reflects an increase in the final product that performs the function of the gene, and an increase in the expression level at the mRNA level reflects a direct result of the activation of the gene. In general, it is usually assumed that an increase in mRNA expression leads to an increase in protein expression, but the increase in mRNA expression and protein expression may not always correlate due to the effect of microRNA on protein translation. Protein expression even if elevated mRNA expression is detected, such as when mRNA is not translated immediately for post-transcriptional control, or when the protein produced escapes detection due to rapid degradation, post-translational modification, etc. It is possible that an increase in is not detected. In any case, the increase in mRNA expression can be the basis for the enhancement of the function of the gene. Conversely, for example, when administration of a drug acts on a translation mechanism or protein stabilization, mRNA expression does not increase, but only protein expression may increase.

  Various methods for detecting or quantifying the expression of a particular protein are well known to those skilled in the art and include, for example, Western blotting, ELISA, immunostaining, and the like. Those skilled in the art can select or design an appropriate detection method or quantification method according to the characteristics of individual proteins. Various methods for detecting or quantifying the expression of specific mRNA are also well known to those skilled in the art, and include, for example, Northern blotting, RT-PCR, microarrays and the like. RT-PCR by TaqMan system using a probe and primer specific to each gene is preferable because of its excellent sensitivity, quantitativeness, and specificity.

  The individual to which the expression enhancer of the present invention is administered may be any species as long as it is a mammal, and a mouse or a human is particularly preferable. In particular, preventing an individual having a disease or disorder caused by decreased or insufficient expression of a Sirt1-related gene selected from the group consisting of Sirt1, Pgc-1α, Lpin1, Igfbp1, and Ucp2, or the disease or disorder Individuals that are particularly required are preferred as subjects to which the expression enhancer of the present invention is administered. Examples of these individuals include patients with metabolic disorders, patients with dyslipidemia, patients with diabetes, patients with cancer, patients with metabolic syndrome, and increased risk for these diseases or disorders, and preventive measures should be taken. Individuals who need it, as well as individuals who are obese and those who are middle-aged or older in age. The tissue in which the expression of the Sirt1-related gene is enhanced by administration of the expression enhancer of the present invention is not particularly limited. For example, liver, adipose tissue, pancreas, kidney, cardiovascular tissue, hematopoietic tissue, blood cell, immune system cell, Examples include nervous system tissue, digestive system tissue, respiratory system tissue, reproductive system tissue, skin tissue, bone tissue, muscle, and the like. Moreover, the aspect which administers the expression enhancer of this invention with respect to a cultured tissue or a cultured cell in vitro is also assumed.

  There are no particular restrictions on the method of administering the expression enhancer of the present invention, but oral administration is particularly preferred. In addition to oral administration, intravascular administration can be considered as a method of delivering black vinegar components substantially throughout the body, and local administration enhances the expression of Sirt1-related genes only in specific parts of the body Is also possible.

  There is no particular limitation on the amount of the expression enhancer of the present invention to be administered. 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 1-1000 ml of black vinegar per square meter of body surface per day, or an amount corresponding to 10-100 ml of black vinegar per square meter of body surface per day can be administered. The expression enhancer of the present invention is preferably administered continuously for several days, for example, 10 days, but the frequency and interval of administration can be appropriately changed.

  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.

Preparation of freeze-dried black vinegar and concentrated black vinegar 1000 ml of the above black vinegar was freeze-dried by lyophilization machine FD-81 of Tokyo Science Instruments and powdered. Distilled water was added thereto, and freeze-drying treatment was performed again. This operation was repeated four times to completely remove acetic acid in black vinegar. The obtained powder was dissolved in 100 ml of distilled water to obtain a black vinegar concentrate.

Preparation of black vinegar concentrate fraction Apply 10 ml of black vinegar concentrate obtained by the above method to Bio Gel P-4 (fractionation range 800-4000 Dalton) (Bio-Rad Laboratories) column, and eluate Was observed at a wavelength of 280 nm. Five peaks were observed, and the eluate containing each peak was collected as a fraction. These fractions were numbered as fractions I, II, III, IV, and V in order of elution. The separation conditions are as follows.

Sample: Black vinegar 10 times concentrated solution 10 ml
Eluent: Ultrapure water Flow rate: 3 ml / min
Gel bed: φ5 × 60 cm
Preparative: 30 ml / volume:
I: 30 ml x 25 bottles (1-25 bottles) = 750 ml
II: 30 ml x 12 bottles (26-37 bottles) = 360 ml
III: 30 ml x 14 bottles (38-51 bottles) = 420 ml
IV: 30 ml x 14 bottles (52-65th bottle) = 420 ml
V: 30 ml x 55 bottles (66-120 bottles) = 1650 ml

The above fractions were collected and powdered by lyophilization, and the amount of the lyophilized product obtained was as follows.
I: 0.335 g, II: 1.3804 g, III: 0.0297 g, IV: 0.0249 g, V: 0.0441 g
Fractions III-V contained only a small dry weight despite showing a peak in magnitude comparable to fractions I and II for absorbance at 280 nm. This suggests that these peaks may not necessarily be separated based on molecular weight. Therefore, the fractionation experiment shows useful information about the molecular weight of the black vinegar component, except that it shows that most of the freeze-dried black vinegar (about 95% by dry weight) was recovered in fractions I and II. Does not provide.
In the administration experiment, the main fractions, fractions I and II, were used.

Animals Male C57BL / 6J mice (weighing 18 ± 2 g (mean ± SD)) were obtained from Inc. Hokudo a standard breeding environment (room temperature 21 ± 2 ° C. for ventilated animal rooms, 12-hour light-dark cycle) Maintained. During the experiment, the mice were given food for the mice and were allowed to drink water freely. Black vinegar, black vinegar-derived components or water was manually orally administered once a day for 10 consecutive days. The day after the completion of administration, the liver was removed from the mouse. For RNA analysis, the extracted organ was permeated into RNAlater (Life technologies, Carlsbad, Calif., USA), which is a storage reagent, in order to stabilize and protect RNA and stored at −30 ° C. For protein analysis, the removed organs were stored at -30 ° C until analysis. All animal experiments were conducted based on the standards for use and breeding of laboratory animals at Hokkaido University.

Primary antibodies used for antibody western blotting and their sources are as follows: rabbit anti-PGC-1α polyclonal antibody (Cayman Chemical, 101707, MI, USA), mouse anti-SIRT1 monoclonal antibody (Abnova, 7c2, Taipei, Taiwan), Rabbit anti-IGFBP1 polyclonal antibody (ProteinTech Group, Inc., No. 13981-1-AP, Chicago, USA), rabbit anti-LPIN1 polyclonal antibody (Abnova, PAB12400, Taipei, Taiwan), mouse anti-UCP2 polyclonal antibody (Abnova, B01P, Taipei, Taiwan), mouse anti-β-actin monoclonal antibody (Sigma-Aldrich, A2228, clone AC-74, St. Louis, USA).
Secondary antibodies used were anti-rabbit IgG antibody and anti-mouse IgG antibody (Nacalai Tesque, Kyoto) with horseradish peroxidase (HRP) conjugate.

RNA extraction from reagents and instrument tissue samples was performed using TRIzol (Life technologies, Carlsbad, CA, USA). RNA reverse transcription was performed using AMV Reverse Transcriptase XL (Takara Bio Inc., Otsu). TaqMan Gene Expression Assay was purchased from Life technologies (Carlsbad, CA, USA). The gene-specific TaqMan probes and primers used here are those provided by the company as part of the assay, and the ID numbers of the reagents used are as follows. beta-actin: Mm00607939_s1, Sirt1: Mm01168521_m1, Pgc1 alpha: Mm00447181_m1, Lpin1: Mm01276800_m1, Igfbp1: Mm00833447_m1, Ucp2: Mm00627599_m1. THUNDERBIRD (registered trademark) Probe qPCR Mix was purchased from Toyobo Co., Ltd. (Osaka). Using these reagents and the Mx3000P system (Agilent technologies, Santa Clara, USA), quantitative real-time PCR was performed by standard methods to quantify each mRNA.
Nitrocellulose membrane, ECL Western blotting detection system, and Hyperfilm ECL were purchased from GE Healthcare UK Ltd. (Buckinghamshire, UK). Protease inhibitor cocktail tablets (Complete®, EDTA free) were purchased from Roche Diagnostics GmbH (Basel, Switzerland). The liver tissue was homogenized in a buffer (pH 7.4) containing 250 mM sucrose and an appropriate amount of protease inhibitor cocktail to extract the protein. Using these reagents and materials and the above-mentioned antibodies, Western blotting experiments were performed by standard methods to quantify each protein. The total amount of protein and the antibody dilution ratio of the total homogenate separated by Western blotting are as follows. Anti-β-actin: 4 μg, 100,000 times, Anti-PGC-1: 4 μg, 500 times, Anti-SIRT1: 4 μg, 500 times, Anti-IGFBP1: 8 μg, 500 times, Anti-LPIN1: 8 μg, 1,000 times, Anti-UCP2 : 8 μg, 1,000 times, secondary antibody: 1,000 times. The target protein was detected by chemiluminescence using ECL Western blotting reagent and exposure to Hyper film ECL. The exposed film was imaged as TIFF data with a scanner, and the expression level was quantified using Image J (http://rsbweb.nih.gov/ij/).

Example 1: Effect of administration of black vinegar or black vinegar concentrate on the expression of Sirt1-related gene mRNA

  The black vinegar and black vinegar concentrate prepared as described above were orally administered to mice manually once a day for 10 days at the doses shown below. For comparison, experiments in which water or 4% acetic acid was administered were performed in parallel. Acetic acid and black vinegar were used after neutralizing by adding 1 equivalent of sodium bicarbonate.

The daily dose in each group is as follows.
Water administration group: 7.5 ml of water per kg of body weight
Acetic acid administration group: 7.5 ml of 4% acetic acid aqueous solution per 1 kg of body weight
Black vinegar administration group: 7.5 ml of black vinegar (undiluted, unconcentrated stock solution) per kg of body weight
Black vinegar concentrate administration group: 10 fold black vinegar concentrate diluted with water per 1 kg body weight (ie, black vinegar lyophilized product dissolved in the same volume of water as the original black vinegar, black 7.5 ml of freeze-dried vinegar solution

The dose of 7.5 ml / kg in mice corresponds to 18 ml / m ² per body surface area, which is 33.3 ml when converted to a human body weight of 70 kg and body surface area of 1.85 m ^2. Corresponds to the daily intake of 30 ml. (Non-patent document 14)

  RNA was extracted from liver extracted from the next day after black vinegar, black vinegar-derived component or water was administered for 10 days continuously, and mRNA expression level was quantified using TaqMan Gene Expression Assay. The results are shown in Table 1. The β-actin gene was used as an endogenous control for normalization, and the data were expressed as relative values with the amount of mRNA in the water administration group being 1 (Table 1).

水投与群を1とした時の相対的mRNA発現量（平均値±標準誤差）を示す。*: p<0.05, **: p<0.01, ***: p<0.001（水投与群との比較）。n=6, Student's t-test法にて検定した。肝臓摘出時の体重はそれぞれ、水投与群：19.9 ± 0.4、酢酸投与群：20.1 ± 0.2、黒酢投与群：19.9 ± 0.7、黒酢濃縮液投与群：19.8 ± 0.2（平均値 ± 標準誤差、ｇ）であり、黒酢投与による体重、摂食量の変化は認められなかった。

The relative mRNA expression level (mean ± standard error) when the water administration group is 1 is shown. *: p <0.05, **: p <0.01, ***: p <0.001 (comparison with water administration group). n = 6, tested by Student's t-test method. The body weights at the time of liver removal were 19.9 ± 0.4 for water administration group, 20.1 ± 0.2 for acetic acid administration, 19.9 ± 0.7 for black vinegar administration, 19.8 ± 0.2 for black vinegar concentrate administration (mean ± standard error, g), and changes in body weight and food intake by administration of black vinegar were not observed.

  When black vinegar was administered, mRNA expression was increased for all Sirt1-related genes of Sirt1, Pgc-1α, Lpin1, Igfbp1, and Ucp2. When black vinegar concentrate was administered, mRNA expression of Sirt1, Pgc-1α, Lpin1, and Ucp2 was increased, but Igfbp1 did not change compared to water administration. With regard to Igfbp1, even when acetic acid is administered, mRNA expression is increased, although not as much as with black vinegar. While acetic acid alone has a certain effect of increasing Igfbp1 mRNA expression, other components of black vinegar can be interpreted as having the effect of further enhancing the effect of acetic acid. The reason why black vinegar concentrate does not show the effect is that the “other ingredients” here are volatile like acetic acid, or they cannot be effective unless they coexist with acetic acid. It is done.

Example 2: Effect of administration of black vinegar or black vinegar concentrate on the expression of Sirt1-related gene protein

  Using the same liver sample as obtained in Example 1, changes in protein expression level were analyzed by Western blotting. Β-actin was used as an endogenous control for normalization, and the data were expressed as relative values, with the amount of protein in the water administration group being 1 (Table 2).

水投与群を１とした時の相対的タンパク質発現量（平均値±標準誤差）を示す。投与の用法・用量は表１と同じ。*: p<0.05, **: p<0.01（水投与群との比較）。n=6, Student's t-test法にて検定した。

The relative protein expression level (average value ± standard error) when the water administration group is 1 is shown. The dosage and administration are the same as in Table 1. *: p <0.05, **: p <0.01 (compared to the water administration group). n = 6, tested by Student's t-test method.

  When black vinegar was administered, an increase in protein expression was observed for all Sirt1-related genes of Sirt1, Pgc-1α, Lpin1, Igfbp1, and Ucp2. As for Pgc-1α, an increase in protein expression was observed even when acetic acid alone was administered, but even in that case, the increase in expression due to administration of black vinegar was greater. In addition, when black vinegar concentrate was administered, protein expression was increased for all Sirt1-related genes of Sirt1, Pgc-1α, Lpin1, Igfbp1, and Ucp2. Overall, the increase in protein expression was more gradual than the increase in mRNA expression, which may be due to biological reasons or due to differences in detection techniques.

Example 3: Comparison of effects of administration of black vinegar-derived components and fasting

  It is a well-established finding in various organisms including mammals that calorie restriction extends life span. It is known that the expression of Sirt1 is enhanced by calorie restriction, and it has been pointed out that Sirt1 may play a central role in the life extension effect of calorie restriction. Therefore, changes in the expression of Sirt1 and Pgc-1α were compared between when the black vinegar-derived component was administered and when calorie restriction (fasting) was performed. Here, as the black vinegar-derived component, the components contained in the above-described black vinegar concentrated liquid fractions I and II were used in the administration experiment.

  In the water administration group, 7.5 ml of water was administered per kg of body weight per day. Fractions I and II were lyophilized and weighed, dissolved and diluted with water to a concentration of 100 mg / ml, and administered at a dose of 150 mg per kg body weight per day. As in Example 1, the liver was excised from the mouse after 10 days of administration, and the expression level of mRNA was measured. As indicated in the column “Preparation of black vinegar concentrate” above, fraction I: 0.335 g and fraction II: 1.3804 g were obtained from 10 ml of black vinegar concentrate. In 7.5 ml of vinegar stock solution, fraction I: 25 mg and fraction II: 104 mg are contained (as the mass of the freeze-dried product), respectively. The fasting group was analyzed as a positive control for increased expression of the Sirt1, Pgc-1α gene. After fasting for 24 hours, the liver was excised and the mRNA expression level was measured (Non-patent Document 2). The results are shown in Table 3.

水投与群を1とした時の相対的発現量（平均値±標準誤差）を示す。肝臓摘出時の体重はそれぞれ、水投与群：19.9 ± 0.2、画分Ι群：20.0 ± 0.3、画分ΙΙ群：19.4 ± 0.1、絶食群：17.1 ± 0.1（平均値 ± 標準誤差、ｇ）であり、黒酢濃縮液画分の投与による体重、摂食量の変化は認められなかった。*: p<0.05, ***: p<0.001, 水群との比較, Student's t-test法にて検定した。

The relative expression level (mean value ± standard error) when the water administration group is 1 is shown. The body weights at the time of liver extraction were: water administration group: 19.9 ± 0.2, fractionation group: 20.0 ± 0.3, fractionation group: 19.4 ± 0.1, fasting group: 17.1 ± 0.1 (mean value ± standard error, g) There was no change in body weight or food intake due to administration of the black vinegar concentrate fraction. *: p <0.05, ***: p <0.001, Comparison with water group, Tested by Student's t-test method.

  Increased mRNA expression levels of the Sirt1 gene and the Pgc-1α gene were observed both when administering fraction I, when administering fraction II, and when fasted (Table 3). In particular, for the Sirt1 gene, administration of fraction I or II showed an increase in mRNA expression that exceeded the effect of fasting. That is, with regard to the enhancement of the expression of the Sirt1 gene, it was shown that the administration of the black vinegar-derived component contained in the lyophilized black vinegar has an effect comparable to or higher than the calorie restriction treatment.

  Based on the above data, administration of black vinegar, black vinegar freeze-dried product, or components derived from black vinegar enhances mRNA expression and / or protein expression of Sirt1-related genes, ie, Sirt1, Pgc-1α, Lpin1, Igfbp1, and Ucp2. It was proved to have the effect of

  Black vinegar has no toxicity or side effects. Accordingly, the present invention provides and achieves a method for safely and economically enhancing the expression of a Sirt1-related gene selected from the group consisting of Sirt1, Pgc-1α, Lpin1, Igfbp1, and Ucp2. Therefore, there is provided an agent for enhancing expression of a Sirt1-related gene. Managing the expression of these genes may be effective in preventing and treating so-called adult diseases and metabolic syndrome, and thus achieving longevity, and thus the present invention can be utilized in related industrial fields of medicine or veterinary medicine. .

Claims (4)

  Sirt1-related gene expression enhancer comprising black vinegar as an active ingredient, wherein the Sirt1-related gene is selected from the group consisting of Sirt1, Pgc-1α, Lpin1, Igfbp1, and Ucp2, the gene expression enhancement is mRNA and An expression enhancer that appears as an increase in protein.   An expression enhancer of a Sirt1-related gene, comprising a freeze-dried black vinegar as an active ingredient, wherein the Sirt1-related gene is selected from the group consisting of Sirt1, Pgc-1α, Lpin1, Igfbp1, and Ucp2, and gene expression An expression enhancer wherein enhancement is manifested as an increase in mRNA and / or protein.   A method for enhancing the expression of mRNA and / or protein of a Sirt1-related gene in a mammal other than a human, comprising the step of administering a composition comprising black vinegar as an active ingredient, wherein the Sirt1-related gene is expressed as Sirt1, Pgc- A method selected from the group consisting of 1α, Lpin1, Igfbp1, and Ucp2.   A method for enhancing the expression of mRNA and / or protein of a Sirt1-related gene in a mammal other than a human, comprising a step of administering a composition comprising a lyophilized product of black vinegar as an active ingredient, wherein the Sirt1-related gene comprises: A method selected from the group consisting of Sirt1, Pgc-1α, Lpin1, Igfbp1, and Ucp2.