JP2021075501A - Composition for enhancing sirtuin 1 (sirt1) expression - Google Patents

Abstract

To provide a composition for enhancing expression of sirtuin 1 (SIRT1).SOLUTION: A composition for enhancing expression of sirtuin 1 (SIRT1) contains Eurycoma longifolia extract as an active ingredient. The Eurycoma longifolia extract is extract obtained from Eurycoma longifolia using water and/or organic solvent, and contains eurycomanone.SELECTED DRAWING: None

Description

The present invention relates to a composition having an expression enhancing action of SIRT1, which is one of sirtuins.

The sirtuin gene is also called a longevity gene, and its activation is said to extend the lifespan of an organism. Sirtuin, a protein synthesized by activation of the sirtuin gene, is a histone deacetylase, which is thought to act on the binding of histones to DNA and prolong life by genetic regulation.

Sirtuin 1 (SIRT1) is activated by calorie restriction while its expression decreases with aging, and it can be activated by resveratrol, which is a polyphenol contained in grape skin, red wine, peanuts, etc. It is known (Non-Patent Document 1).

Sirtuin 1 (SIRT1) plays various roles in each tissue, and studies on its association with aging-related diseases such as Alzheimer-type senile dementia, osteoporosis, arteriosclerosis, and skeletal muscle hypoplasia are also being conducted.

For example, as an action in the central nervous system, in a study using Alzheimer's disease model mice, activation of sirtuin 1 (SIRT1) by resveratrol suppressed neurodegeneration in the hippocampus and improved cognitive function. There is a report (Non-Patent Document 2). On the other hand, as an action on the peripheral nervous system, an example in which a blueberry extract or the like is provided as a composition for treating eye diseases such as refractive error through activation of sirtuin 1 (SIRT1) (Patent Document 1), diabetic retina, etc. There is a report (Non-Patent Document 3) that it is effective in suppressing the disease.
As described above, the activator of sirtuin 1 (SIRT1) is highly expected for its use, and there is a strong demand for a further activator of sirtuin 1 (SIRT1).

On the other hand, the extract of Tonkat Ali (Japanese name: Simaroubaceae Nagaekasa, scientific name: Eurycoma longifolia), which is a kind of plant belonging to Simaroubaceae, has been used as an antipyretic, malaria treatment and gastric disease folk medicine since ancient times in Southeast Asia such as Malaysia. Has been used. In recent years, this extract has been found to have an improving effect on male menopause (Patent Document 2). Further, Patent Document 3 describes that the activity of Tonkat Ali for male function lies in glycosides such as quasinoids and coumarins, and can be extracted with a polar organic solvent. In addition, it was found that the Yuri peptide contained in Tonkat Ali is involved in the biosynthesis of male hormones starting from cholesterol, and the biosynthesized testosterone is found in the male reproductive organs, brain, skin, muscles, bones, kidneys, etc. It has been reported to show activity at various target sites such as the liver. Further, Patent Document 4 describes that a substance having a testosterone action can be isolated by extracting water from the roots of Tonkat Ali and then fractionating by a gel filtration method. In addition, there is a report showing that the immunity was improved by ingestion of Tonkat Ali extract for both men and women. As described above, Tonkat Ali is a plant with many ingestion experiences that has been reported to have various physiological effects on humans, but the expression-enhancing effect of sirtuin 1 (SIRT1) has not been known so far.

Japanese Unexamined Patent Publication No. 2006-298876 Japanese Unexamined Patent Publication No. 2009-051765 Special Table 2010-500342 U.S. Pat. No. 7,132117

Nature. 2003 Sep 11; 425 (6954): 191-6. EMBO J 2007; 26: 3169-3179 Invest Ophthalmol Vis Sci. 2011 Nov 25; 52 (12): 9142-8.

An object of the present invention is to provide a composition for enhancing the expression of sirtuin 1 (SIRT1), which contains a Tonkat Ali extract as an active ingredient.

In the process of studying the effects of a large number of natural products and compounds on retinal-derived Muller cells, the present inventors have found a substance having an effect of acting on Muller cells and enhancing the expression of sirtuin 1 (SIRT1). I did.

That is,
The present invention has the following configuration.
(1) A composition for enhancing the expression of sirtuin 1 (SIRT1), which contains an extract of Tonkat Ali as an active ingredient.
(2) The composition according to (1), wherein the Tonkat Ali extract is a Tonkat Ali water and / or organic solvent extract.
(3) The composition according to any one of (1) and (2), wherein the Tonkat Ali extract is an extract containing yuricomanone.
(4) A composition for enhancing the expression of sirtuin 1 (SIRT1), which contains yuricomanone as an active ingredient.

INDUSTRIAL APPLICABILITY The present invention provides a composition containing an extract of Tonkat Ali as an active ingredient and enhancing the expression of sirtuin 1 (SIRT1).

It is a graph which shows that the extract of Tonkat Ali enhances the expression of sirtuin 1 (SIRT1). It is a graph which shows that the yuricomanone contained in the extract of Tonkat Ali enhances the expression of sirtuin 1 (SIRT1).

The present invention will be specifically described.
The "composition" as used in the present invention refers to a composition containing an extract of Tonkat Ali and enhancing the expression of sirtuin 1 (hereinafter, "SIRT1"). The "composition" includes foods and drinks, pharmaceuticals, and health foods.

The extract of Tonkat Ali used in the present invention is not particularly limited, but an extract of the trunk or root of a tree growing in Southeast Asia is preferably used. Among them, an aqueous solvent extract of the root of Tonkat Ali is preferably used.

The Tonkat Ali extract is preferably extracted by the hot water extraction method equipped with a reflux condenser disclosed in Patent Document 4. Further, extraction with alcohol or a mixed solution of water and alcohol may be used. The extract may be used as an extract by removing the solvent by a drying means such as freeze-drying. Such an extract is commercially available as a dried toncat ant extract, and this may be used in the present invention. As a commercially available product, "Tonkat Ali (Physta: registered trademark) Fista: Ask Pharmaceutical Co., Ltd." can be exemplified.

Further, the activity of the extract can be enhanced by appropriately combining known separation and purification methods.
When the extract of Tonkat Ali is administered to humans, oral administration is preferable, and specifically, it can be used as a health food such as food and drink, pharmaceuticals, and supplements.

When the Tonkat Ali extract is used as food / beverage, for example, fruit juice beverages, carbonated beverages, tea-based beverages, dairy beverages, alcoholic beverages, soft beverages and other beverages, jelly-like foods and various snacks, baked goods, etc. It can be in any food / beverage form such as cakes, chocolates, gums, candy, soups, etc.

When the composition containing the extract of Tonkat Ali is used as a supplement or a pharmaceutical product, for example, it is used as an oral solid molding agent such as tablets and granules, and an oral liquid preparation such as an oral liquid preparation and a syrup preparation. be able to.

When the Tonkat Ali extract is used as a pharmaceutical or supplement formulation, the Tonkat Ali extract is mixed and dispersed directly or with a pharmaceutically acceptable carrier by a general manufacturing method, and then in the desired form. It can be easily obtained by processing. In this case, in addition to the extract used in the present invention, oily bases such as vegetable oils and animal oils commonly used in such forms, pH adjusters, preservatives, thickeners, pigments, fragrances and the like are used in the present invention. It can be appropriately blended within a range that does not interfere with the effect.

The composition containing the extract of Tonkat Ali of the present invention is not particularly limited, but is preferably contained as a dried product of the extract in the range of 0.00001 to 99.5% by mass of the total composition. The composition to be used.

Examples and test examples will be shown, and the present invention will be further described.
<Example 1. SIRT1 expression enhancing effect>
(1) Preparation of primary Muller cells The primary Muller cells were prepared by the following method.
Retinas were isolated from 10 7-day-old SD rats. The isolated retina was allowed to stand at 37 ° C. for 30 minutes in 10 ml of a solution of trypsin-EDTA (Sigma T3924) diluted 5-fold with PBS (−) and DNase I (Roche) added at a final concentration of 100 ng / ml. The retinal tissue was dispersed by a pipetting operation and centrifuged at 2,400 rpm at room temperature for 5 minutes. The supernatant was removed by suction and then suspended in 25 ml of DMEM-F12 medium (Gibco) containing 10% FBS (biosera) and 1% penicillin-streptomycin (Sigma). The suspension was passed through a 70 μM cell strainer (FALCON), then the number of cells was counted and diluted to a concentration of ^{4.2x10 6 cells / ml.} 10 ml each was seeded in a T-75 cell culture flask (Sumitomo Bakelite Co., Ltd.) and _{cultured at 37 ° C. under 5% CO 2} (culture day 0). On the 3rd day of culturing, the medium was removed by suction, replaced with 20 ml of new medium, and cultured _{again at 37 ° C. under 5% CO 2.} On day 7 of culture, Muller cells were purified according to the method of Wei-Tao Song et al. (Ophthermol 2013; 6 (6): 778-784). On the 8th day of culturing, Muller cells were peeled off, ^{suspended to a concentration of 4x10 5} cell / ml, seeded in 100 μL each on a 96-well cell culture plate (FALCON), and cultured at _{37 ° C. under 5% CO 2.}

(2) Confirmation of SIRT1 expression enhancing effect of Tonkat Ali extract The SIRT1 expression enhancing effect of Tonkat Ali extract was tested by the following method.
1) Test sample As the Tonkat Ali extract, obtained from Ask Pharmaceutical Co., Ltd. (Product No. 012.105 "Tonkat Ali Dry Extract Physta") was used for the test. This extract is water-extracted from the dry roots of Eurycoma longifolia Jack, and contains 0.8 to 1.5% by mass of yuricomanone, 35% by mass or more of glycosaponin, and 30% by mass of total polysaccharide as standard components. It is a powder containing% or more and 22% by mass or more of total protein. In the following tests, it was dissolved in DMSO and used.

2) Enhancement of SIRT1 expression The whole medium of the primary Muller cells on the 9th day of culture prepared by the method described in Example 1 was removed and replaced with 100 μL of DMEM-F12 medium containing 1% FBS at 37 ° C. and 5% CO. _The cells were placed in a serum starvation state by culturing at 2 for 90 minutes.
The above-mentioned toncat ant root extract was added to 0, 50, 100 μg / ml at a concentration, and after culturing for 90 minutes, the supernatant was removed, and RNA was used according to the attached instructions using RNeasy 96 kit (QIAGEN). Was prepared. When preparing RNA, genomic DNA was removed using DNaseIKit (QIAGEN). About 40 ng of prepared RNA was prepared using PrimeScript RT reagent kit (Takara) according to the attached instructions.
The expression level of SIRT1 was quantified by the following method using Applied Biosystems TaqMan Gene Expression Assay and rat Gapdh Taqman probe for endogenous control. 1 μL of cDNA was mixed with rat GAPDH Taqman probe, rat SIRT1 Taqman probe, and Taqman Multiplex Master Mix at a predetermined ratio, and PCR grade water (Invitrogen) was added to make a total of 10 μL.
The prepared reaction solution was measured using Quant Studio 5 (Applied Biosystems) under the reaction conditions of [(95 ° C., 20 seconds) → (95 ° C., 1 second) → (60 ° C., 20 seconds)] x 45 cycles. It was.
From the Ct value obtained by the measurement, the relative gene expression level of each sample was determined by the ΔΔCt method using Gapdh as an internal standard.
The product information and specifications of the Taqman probe used are as follows.
Gapdh: Applied biosystems Assay ID: Rn01462662_g1 (Dye: VIC, Quencher: MGB)
SIRT1: Applied biosystems Assay ID: Rn01428096_m1 (Dye: FAM, Quencher: MGB)
3) Results The measurement results are shown in FIG. From FIG. 1, in rat primary Muller cells, the Tonkat ant root extract enhanced the expression of SIRT1 in a concentration-dependent manner.

<Example 2. Expression enhancing effect of SIRT1 by Yuricomanone>
The SIRT1 expression-enhancing effect of Yuricomanone contained in the Tonkat ant root extract was tested by the following method.
(1) Test method 1) Test sample Yuricomanone was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. and used for the test. In the test, it was dissolved in DMSO and used.

2) Enhancement of SIRT1 expression The whole medium of the primary Muller cells on the 9th day of culture prepared by the method described in Example 1 was removed and replaced with 100 μL of DMEM-F12 medium containing 1% FBS at 37 ° C. and 5% CO. _The cells were placed in a serum starvation state by culturing at 2 for 90 minutes.
The above uricomanone was added to a concentration of 0, 10, 30 μM, the supernatant was removed after culturing for 90 minutes, and RNA was prepared using RNeasy 96 kit (QIAGEN) according to the attached instructions. Then, the relative gene expression level of SIRT1 was quantified by the method described in Example 1.

(2) Results The measurement results are shown in FIG.
As shown in FIG. 2, in rat primary Muller cells, Yuricomanone enhanced the expression of SIRT1 in a concentration-dependent manner. Therefore, it was clarified that Yuricomanone is one of the active ingredients of SIRT1 expression enhancing action shown by Tonkat Ali.

From the above test results, Tonkat Ali extract enhances the expression of SIRT1.

Claims (4)

A composition for enhancing the expression of sirtuin 1 (SIRT1), which contains an extract of Tonkat Ali as an active ingredient. The composition according to claim 1, wherein the Tonkat ant extract is a Tonkat ant water and / or organic solvent extract. The composition according to any one of claims 1 and 2, wherein the extract of Tonkat Ali is an extract containing yuricomanone. A composition for enhancing the expression of sirtuin 1 (SIRT1), which contains Yuricomanone as an active ingredient.

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