JP2022056495A - Mitochondrial function improver - Google Patents

Abstract

To provide an agent having preventing/improving effect to mitochondrial dysfunction that could cause aging or disease.SOLUTION: A mitochondrial function improver using fermented honey bush extract. The improver suppresses peroxide lipid formation on a mitochondrial membrane. Because the peroxide lipid formation disintegrates homeostasis of the mitochondrial membrane and causes mitochondrial dysfunction, an agent including the fermented honey bush extract of the present invention as an active ingredient can prevent or improve aging or diseases caused by mitochondrial dysfunction.SELECTED DRAWING: Figure 3

Description

The present invention relates to improving mitochondrial function of fermented honey bush extract.

Mitochondria are one of the organelles in eukaryotes and have an important function of producing ATP, which is an energy source required for cell activity. On the other hand, in the electron transport chain, which is the process of energy metabolism, superoxide, which is a kind of active oxygen, is generated by leakage of electrons from 1 to 5% of the consumed oxygen. Therefore, mitochondria are not only a place of energy production for cells but also a place of generation of active oxygen.

Part of the active oxygen generated in mitochondria is necessary because it is used for signal transduction and the like, but it is known that when it is excessively generated, it damages mitochondrial DNA and protein. In addition, since mitochondria are organs having a lipid double membrane, the lipids present in the membrane are good targets for active oxygen, and lipid peroxides are easily formed. Lipids present in mitochondrial membranes are involved in membrane structure maintenance and signal transduction, and the disruption of membrane homeostasis due to reactive oxygen species impairs important functions such as lipid metabolism, inflammation, and induction of apoptosis in mitochondria. (Non-Patent Document 1). In addition, impaired mitochondria produce more reactive oxygen species, causing a negative spiral in dysfunction. Therefore, it can be said that suppressing the formation of lipid peroxide in the mitochondrial membrane is effective in maintaining and recovering the function of mitochondria.

Mitochondrial function declines with age and is thought to contribute to physiological aging, but on the other hand, when mitochondrial function decline in tissues becomes significant, it causes illness. For example, when mitochondrial dysfunction occurs in central nerve cells of the brain, symptoms such as memory and cognitive dysfunction appear. In Alzheimer's disease, amyloid β inhibits mitochondrial function, and it is thought that the increase in active oxygen causes cell apoptosis. In addition, it has been reported that Parkinson's disease and neurodegenerative ALS have a decrease in mitophagy function for removing abnormal mitochondria, which is one of the causes of the onset of the disease (Non-Patent Document 2). On the other hand, although it is not an obvious disease, physical disorders such as muscle weakness, fatigue, anemia, and constipation are also known as symptoms due to mitochondrial dysfunction.

Therefore, maintaining and restoring mitochondrial function is important for the prevention and improvement of diseases caused by physiological aging and mitochondrial dysfunction. As described above, the decrease in mitochondrial function is largely due to active oxygen damage, so a method using a compound having a high antioxidant effect (active oxygen scavenging ability) such as vitamin C and vitamin E is assumed as a preventive and ameliorating method.

However, a compound having a high antioxidant effect may be easily oxidized and consumed before reaching mitochondria, or may have low accumulation in mitochondria depending on the polarity of the compound. Therefore, it is considered difficult to predict the effect on mitochondria from the antioxidant effect. On the other hand, even if there is no antioxidant effect, for example, a compound that activates the antioxidant system of mitochondria itself can be selected as a mitochondrial function improving component.

In the past technology, as a component for improving mitochondrial function, Kihada extract and Coptis chinensis extract, which promote mitochondrial transfer occurring between cells (Patent Document 1), can control mitochondrial dynamics and prevent or improve heart disease due to aging. Berberine chloride (Patent Document 2) is disclosed as a mitochondrial expression promoter. However, a component having an effect of suppressing the formation of lipid peroxide in the mitochondrial membrane is not known.

Honey Bush is an evergreen shrub of the genus Cyclopia of the family Leguminosae, a plant that inhabits the limited areas of southwestern and southeastern South Africa, and about 20 species are known. Recent studies have revealed that honey bush contains isoflavones, flavones, cinnamic acid, coumestan, xanthones, etc. (Non-Patent Document 3).

The honey bush-derived extract has been reported to have an antidiabetic effect (Patent Document 3), an enzyme ceramidase inhibitory effect (Patent Document 4), and a wrinkle improving effect (Patent Document 5).
On the other hand, in Non-Patent Document 4, unfermented honey bushes of three types (C. Subternata, C. genistoides, C. longifolia) were extracted with water, 40% ethanol, and 70% ethanol, and all three types of water extracts and , 70% ethanol extract of a specific species (C. genistoides) has been reported to have an ATP production promoting effect and a mitochondrial membrane potential improving effect on neuroblastoma. The results suggest that while water extracts are useful, 40% ethanol extracts and most 70% extracts are less effective. The unfermented honey bush has a higher phenol content than the fermented honey bush and has a high antioxidant effect, so it is stated that the unfermented honey bush was selected in the experiment. Against this background, the fermented honey bush extract was not expected to have an effect of improving mitochondrial function.

Japanese Unexamined Patent Publication No. 2018-123130 Japanese Unexamined Patent Publication No. 2019-178070 Special Table 2010-520914 Japanese Unexamined Patent Publication No. 2017-124984 KR1020130010250

O.S.Ademowo, H.K.I.Dias, D.G.A.Burton, H.R.Griffiths, Biogerontology, 2017 Dec; 18 (6): 859-879 Lipid (per) oxidation in mitochondria: an emerging target in the aging process? Shigeru Yanagi, Special Edition of Experimental Medicine Vol.37-No12 2019, Mitochondria and Diseases, Aging E.Joubert, M.E.Joubert, C.Bester, D.de Beer, J.H.De Lange, South African Journal of Botany, 2011 Oct; 77 (4): 887-907 Honeybush (Cyclopia spp.): From local cottage industry to global markets － The catalytic and supporting role of research Anastasia Agapouda, Veronika Butterweck, Matthias Hamburger, Dalene de Beer, Elizabeth Joubert, Anne Eckert, oxdative medicine and cellular longevity, 2020 Aug 7 honeybush Extracts (Cyclopia spp.) Rescue Mitochondrial Functions and Bioenergetics against Oxidative Injury

An object of the present invention is to provide an agent having a preventive and ameliorating effect on mitochondrial dysfunction that causes aging and diseases.

As a result of the examination by the inventor, it was confirmed that the antioxidant effect (active oxygen scavenging ability) of the compound itself and the effect of suppressing the formation of lipid peroxide in the mitochondrial membrane, which is an index of mitochondrial dysfunction, do not match. Therefore, in order to solve this problem, it is necessary to select a substance having a high effect of suppressing the formation of lipid peroxide in the mitochondrial membrane.

As a result of diligent studies by the inventor under such circumstances, the present invention was found to have an excellent effect of suppressing the formation of lipid peroxide in the mitochondrial membrane in the fermented honey bush extract.
That is, the present invention
[1] Inhibitor of lipid peroxide formation of mitochondrial membrane containing fermented honey bush extract as an active ingredient [2] Mitochondrial function improving agent containing fermented honey bush extract as an active ingredient [3] The fermented honey bush extract , 30-70% (v / v) ethanol or 30-70% (v / v) 1,3-butylene glycol extract [1] or [2] agent according to [4] fermentation of mitochondrial membrane. The use of a fermented honey bush extract-containing composition for the purpose of suppressing lipid oxide formation and / or improving mitochondrial function.

The mitochondrial function improving agent of the present invention suppresses the formation of lipid peroxide in the mitochondrial membrane. Since the formation of lipid peroxide disrupts the homeostasis of the mitochondrial membrane and causes mitochondrial dysfunction, the agent containing the fermented honey bush extract of the present invention as an active ingredient prevents aging and diseases caused by mitochondrial dysfunction. It can be improved.

Evaluation of active oxygen scavenging ability (DPPH radical scavenging ability) of each test substance Evaluation of the inhibitory effect of each test substance on the formation of lipid peroxide in the mitochondrial membrane Evaluation of the effect of fermented honey bush extract of different extraction solvents on the formation of lipid peroxide in mitochondrial membrane

The present invention is an agent for suppressing the formation of lipid peroxide in a mitochondrial membrane and an agent for improving mitochondrial function, which contain a fermented honey bush extract as an active ingredient.

The honey bush (genus Cyclopia) used in the present invention is an evergreen tree that inhabits South Africa. A plant called a honey bush contains more than 20 species, and typical ones include Cyclopia intermedia, Cyclopia genistoides, Cyclopia subternata, and the like, but any species of honey bush may be used in the present invention. Honey bush is mainly popular as tea and is fermented during processing. Fermentation here refers to the "oxidation" process by the action of the enzyme of the tea leaves themselves, not by microorganisms (fermentation in the original sense is mediated by microorganisms, but in the case of tea, it is customary. The oxidation process is called fermentation). In the present invention, a honey bush that has undergone an oxidation step (fermentation) is used instead of a fresh one that has not undergone an oxidation step (unfermented). Specifically, honey bush tea that is generally distributed in the market can be used.
Therefore, the fermented honey bush of the present application is not honey bush (unfermented) or artificially fermented by adding bacteria such as lactic acid bacteria (microbial fermentation), and is distinguished from these.
Hereinafter, when the term "honey bush" is simply used in the examples, it refers to a fermented honey bush.

The extraction site of the honey bush extract used in the present invention may be a part of a flower, a stem, a leaf, or a whole plant. The extraction solvent is not particularly limited, but for example, water; lower monohydric alcohols such as methanol and ethanol; liquid polyhydric alcohols such as glycerin, propylene glycol and 1,3-butylene glycol; and a mixed solvent thereof may be used. Can be done. Among them, a mixed solvent of water and ethanol (hereinafter referred to as EtOH) or 1,3-butylene glycol (hereinafter referred to as BG) is particularly suitable.

When a mixed solvent is used, the ratio of water to EtOH or BG may be anything, but the ratio of EtOH or BG to water is preferably 30 to 70% (v / v), more preferably 40 to 60% (v / v). It is near v / v).

As an extraction method, 1 to 100 parts by mass of an extraction solvent is used with respect to 1 part by mass of dried plants, and the temperature is 5 to 70 ° C., preferably 10 to 60 ° C. for several hours to 7 days, particularly several hours to 2 It is preferable to extract for days. After extraction, it can be filtered and used as it is, but if necessary, further purification treatment such as deodorization and decolorization can be performed within a range that does not affect the effect. Further, it can be freeze-dried and used in a powder state.

The agent of the present invention can be added to functional foods, cosmetics, quasi-drugs, and pharmaceuticals for the purpose of improving mitochondrial function. The types of cosmetics to be blended include lotion, beauty essence, milky lotion, beauty cream, sunscreen, hair growth and gray hair improvement cosmetics, etc., but if it is applied to the skin, it can be blended in any cosmetics. be able to. It can also be added to oral drugs as a supplement or drug for health or beauty purposes. The agent of the present invention can appropriately contain any compounding ingredients used in cosmetics, pharmaceuticals, etc., as long as the effects are not impaired. Examples of the optional compounding ingredients include oils, surfactants, powders, coloring materials, water, alcohols, thickeners, chelating agents, silicones, antioxidants, ultraviolet absorbers, moisturizers, fragrances, and various medicinal properties. Ingredients, preservatives, pH regulators, neutralizers and the like can be mentioned.

The agent of the present invention can be used as it is as an extract or in a powder state, but it can also be mixed with a general base and used. When mixed with the base, in principle, it is sufficient to have an effective amount of a component having a mitochondrial function improving effect, but in terms of dry residue mass, the fermented honey bush in the mitochondrial function improving agent It is desirable that the extract has a concentration range of 0.0001 to 1.0% (w / w), and particularly preferably a concentration range of 0.001 to 0.1% (w / w).

Hereinafter, examples of the present invention will be specifically described, but the present invention is not limited to these examples. Unless otherwise specified, the blending amount is indicated by volume percent concentration (v / v).

Example 1: Confirmation of active oxygen scavenging ability In order to confirm the relationship between the antioxidant effect of the test substance itself, that is, the active oxygen scavenging ability and the effect of suppressing the formation of lipid peroxide in the mitochondrial membrane, the activity of the substance itself is generally confirmed. DPPH radical scavenging ability evaluation (a method using the scavenging ability for an artificially created radical DPPH (2,2-Diphenyl-l-picrylydrazyl) as an index), which is often used as a method for measuring oxygen scavenging ability, is carried out. bottom.

<Preparation of test substance>
As the honey bush, a dried product (whole plant) of Cyclopia Intermediaia species commercially available as honey bush tea was used.
The mixture was mixed at a mass ratio of the ratio of the extraction solvent 20 to the dry bulk material 1. The extraction work was carried out under the conditions of 60 ° C. and 5 hours using 50% BG as the extraction solvent. After extraction, the drug substance was removed from the solution, filtered through a 5 μm filter, and then further filtered through a 0.2 μm filter for sterilization purposes. By this operation, a honey bush extract having a solid content of about 1% (w / w, 10,000 ppm) was obtained. This was diluted 100-fold to obtain a test substance (solid content 100 ppm). In addition, ascorbic acid (25 ppm, dissolved in purified water) and gallic acid (25 ppm, dissolved in EtOH), which are well known to have high antioxidant capacity, were prepared as comparative examples.

<Preparation of DPPH solution>
The DPPH solution was prepared by mixing each of the following components in a ratio of (A): (B): (C) = 1: 4: 3 volume.
(A) 5.52 g of ES (2- (N-morpholino) ethanesulfonic acid) was dissolved in 100 ml of water to prepare PH 6.1 with 1N-NaOH.
(B) 15.7 mg of DPPH (1,1-diphenyl-2-picrylhydrazil) was dissolved in 100 ml of ethanol.
(C) Purified water

<Preparation of Trolox solution>
Trollox 25 mg was dissolved in 10 ml of DMSO (dimethyl sulfoxide) to prepare a 10 mM solution.

<DPPH test>
10 μl of the test substance was added to 1 well of the 96 well plate, and then 190 μl of the DPPH solution was rapidly added and mixed. After 30 minutes, the absorbance of each well was measured at 540 nm. The Trolox equivalent of the test solution is 0.156 mM, 0.313 mM, 0.625 mM, 1.25 mM, 2.5 mM, 5.0 mM Trolox solution 10 μl, 190 μl of DPPH solution is added and mixed, and 540 nm after 30 minutes. The absorbance of the solution was measured, and a calibration curve of the Trolox concentration and the absorbance value at 540 nm was prepared and calculated. The results are shown in FIG.

From FIG. 1, the active oxygen scavenging ability of the honey bush extract (solid content 100 ppm) was about the same as that of ascorbic acid 25 ppm. The gallic acid of 25 ppm was significantly higher than that of the honey bush extract.

Example 2: Confirmation of lipid peroxide formation inhibitory effect on mitochondrial membrane <Preparation of test substance>
As the honey bush, dried products (whole plant) of Cyclopia Intermediaa and Cyclopia Subternata, which are commercially available as honey bush tea, were used. The mixture was mixed at a mass ratio of the ratio of the extraction solvent 20 to the dry bulk material 1. The extraction work was carried out under the conditions of 60 ° C. and 5 hours using 50% BG as the extraction solvent. After extraction, the drug substance was removed from the solution, filtered through a 5 μm filter, and then further filtered through a 0.2 μm filter for sterilization purposes. By this operation, a honey bush extract having a solid content of about 1% (w / w, 10,000 ppm) was obtained. As a comparative example, ascorbic acid (dissolved in purified water) and gallic acid (dissolved in EtOH) were prepared so as to have a solid content of 0.25% (w / w, 2500 ppm), and filtered through a 0.2 μm filter. ..

<Cell culture and staining of mitochondrial membrane lipid peroxide>
Dermal fibroblasts from normal adult skin were suspended in D-MEM medium (containing 10% FBS) to 1.5 × 10 ⁴ cell / ml and 96 well black plate (CELLSTAR, Gleiner). 200 μl each was seeded and cultured in a CO ₂ incubator (37 ° C., 5% CO ₂ ). After culturing for 24 hours, the medium was replaced with D-MEM medium supplemented with 1% FBS, and the test substance was added so as to be 1% of the medium. As a control, 1% of the solvent of each test substance was added to the medium. After culturing for 72 hours, it was washed twice with PBS (-) and diluted 1000-fold with HBSS (+) MitoPeDPP (mitochondrial membrane fat-soluble peroxide detection reagent, Dojin Chemical Laboratory) and Hoechst 33342 (nuclear staining reagent, Dojin Chemistry). Staining by the laboratory) was performed in a CO ₂ incubator for 20 minutes. After that, the cells were washed 3 times with PBS (-), replaced with HBSS (+), and photographed with a fluorescence microscope (KEYENCE BZ-700) (using a 20x objective lens, green: lipid peroxide of mitochondrial membrane). , Blue: Nuclear)

<Image analysis>
For the obtained green image, the fluorescence intensity of the lipid peroxide in the mitochondrial membrane was calculated by setting a constant threshold value with ImageJ (NIH) and binarizing it. Further, by calculating the number of nuclei contained in the blue image by the same method and then dividing the fluorescence intensity in the acquired image by the number of nuclei, the fluorescence intensity per cell, that is, the mitochondrial membrane per cell. The amount of lipid peroxide was calculated. By substituting the amount of lipid peroxide in the mitochondrial membrane of the cell treated with the extraction solvent of the test substance (control) and the amount of lipid peroxide in the mitochondrial membrane of the cell treated with the test substance into Equation 1, the mitochondrial membrane can be calculated. The amount of lipid peroxide was calculated. In this calculation formula, it can be determined that the amount of lipid peroxide in the mitochondrial membrane is less than 1, and the smaller the value, the higher the ability to suppress the formation of lipid peroxide. It can be determined that a substance having a value of 0 to 0.5 has a high mitochondrial membrane inhibitory effect on lipid peroxide.

FIG. 2 shows the measurement results of the amount of lipid peroxide in the mitochondrial membrane.
Compared to the control (cells to which the solvent of the test substance was added), the cells to which the honey bush extract (Intermediaa species, Subternata species) was added to the medium (final concentration: solid content 100 ppm) had a higher amount of lipid peroxide in the mitochondrial membrane. It had decreased to about 0.2. From this result, it was confirmed that the honey bush extract has a high mitochondrial membrane lipid peroxide formation inhibitory effect. On the other hand, ascorbic acid (25 ppm) and gallic acid (25 ppm) used as comparative examples had a lower effect of suppressing lipid peroxide on the mitochondrial membrane than the honey bush extract. In Example 1, it was confirmed that the active oxygen scavenging ability of ascorbic acid (25 ppm) and the honey bush extract (100 ppm) were similar, and that the gallic acid (25 ppm) was remarkably high. Considering these results together, it was shown that it is difficult to predict the effect of suppressing the formation of lipid peroxide in the mitochondrial membrane from the active oxygen scavenging ability of the substance itself.

Example 3: Confirmation of lipid peroxide formation inhibitory effect of mitochondrial membrane by honey bush extract of different extraction solvent <Preparation of test substance>
As the honey bush, a dried product (whole plant) of Cyclopia Intermediaia species commercially available as honey bush tea was used.
The mixture was mixed at a mass ratio of the ratio of the extraction solvent 20 to the dry bulk material 1. The extraction solvent used is water, 20% EtOH, 40% EtOH, 60% EtOH, 80% EtOH, 100% EtOH, and water, 20% EtOH, 40% EtOH, 60% EtOH, 80% EtOH is 60 ° C., 5 The extraction work was carried out under the condition of time, and 100% EtOH was carried out at room temperature for 2 days. After extraction, the drug substance was removed from the solution, filtered through a 5 μm filter, and then further filtered through a 0.2 μm filter for sterilization purposes. The cell culture was basically the same as that of [0029], but the test substance was added so as to be 0.5% of the medium. Staining and image analysis of lipid peroxide on the mitochondrial membrane was performed in the same manner as described above.

FIG. 3 shows the measurement results of the amount of lipid peroxide in the mitochondrial membrane.
From FIG. 3, it was confirmed that all the extraction solvents had an effect of suppressing the formation of lipid peroxide in the mitochondrial membrane, but a particularly high effect was confirmed in the 40-60% EtOH extract.

Industrial availability

The formation of lipid peroxides in the mitochondrial membrane disrupts homeostasis of the mitochondrial membrane and causes mitochondrial dysfunction. It is known that mitochondrial dysfunction is involved in physical disorders even if it is not an obvious disease, and oral and external preparations containing the fermented honey bush extract of the present invention as an active ingredient improve mitochondrial dysfunction. It can be used as an agent.

Claims (4)

Mitochondrial membrane lipid peroxide formation inhibitor containing fermented honey bush extract as an active ingredient Mitochondrial function improving agent containing fermented honey bush extract as an active ingredient The agent according to claim 1 or 2, wherein the fermented honey bush extract is 30 to 70% (v / v) ethanol or 30 to 70% (v / v) 1,3-butylene glycol extract. Use of a fermented honey bush extract-containing composition for the purpose of suppressing the formation of lipid peroxide in the mitochondrial membrane and / or improving the mitochondrial function.

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