Mitochondrial function improver

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

[Background technology]

[0001] The present invention relates to a fermented honeybush extract for improving mitochondrial function.

[0002] Mitochondria are one of the organelles in eukaryotes, and play an important role in producing ATP, an energy source necessary for cellular activity. Meanwhile, in the electron transport chain, which is the process of this energy metabolism, superoxide, a type of reactive oxygen, is generated when electrons leak from 1-5% of the oxygen consumed. Therefore, mitochondria are not only a place for energy production for cells, but also a place for reactive oxygen to be generated.

[0003] Although some of the reactive oxygen species generated in mitochondria are necessary for use in signal transduction, etc., it is known that excessive generation of the reactive oxygen species can damage mitochondrial DNA and proteins. In addition, since mitochondria are organs with a lipid double membrane, the lipids present in the membrane are good targets for reactive oxygen species, and lipid peroxides are easily formed. Lipids present in the mitochondrial membrane are involved in maintaining the membrane structure and signal transduction, and the breakdown of membrane homeostasis due to reactive oxygen damage impairs important functions such as lipid metabolism, inflammation, and induction of apoptosis in mitochondria (Non-Patent Document 1). In addition, mitochondria with impaired function produce even more reactive oxygen species, causing a negative spiral in functional disorders. Therefore, it can be said that suppressing the formation of lipid peroxides in the mitochondrial membrane is effective in maintaining and recovering mitochondrial function.

[0004] Mitochondrial function declines with age and is therefore considered to be a factor in physiological aging, but on the other hand, when mitochondrial function declines significantly in tissues, it can cause illness. For example, when mitochondrial function declines in the central nerve cells of the brain, symptoms such as declines in memory and cognitive function appear. It is believed that Alzheimer's disease is caused by amyloid beta inhibiting mitochondrial function and increasing reactive oxygen, which causes cell apoptosis. It has also been reported that Parkinson's disease and neurodegenerative ALS are caused in part by a decline in the mitophagy function, which removes abnormal mitochondria (Non-Patent Document 2). On the other hand, although not clearly a disease, physical disorders such as muscle weakness, easy fatigue, anemia, and constipation are also known to be symptoms of mitochondrial dysfunction.

[0005] Therefore, maintaining and restoring mitochondrial function is important for preventing and improving diseases caused by physiological aging and mitochondrial dysfunction. As mentioned above, mitochondrial dysfunction is largely caused by active oxygen damage, so the use of compounds with high antioxidant effects (active oxygen scavenging ability), such as vitamin C and vitamin E, is expected to be a method of prevention and improvement.

[0006] However, compounds with high antioxidant effects may be easily oxidized and consumed before reaching mitochondria, or may have low accumulation in mitochondria due to 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 a compound does not have an antioxidant effect, for example, a compound that activates the antioxidant system of the mitochondria itself can be selected as a mitochondrial function improving component.

[0007] In the past, mitochondrial function improving ingredients have been disclosed, such as Phellodendron amurense extract and Coptis japonica extract, which promote mitochondrial transfer between cells (Patent Document 1), and berberine chloride (Patent Document 2) as a MITOL expression promoter that can control mitochondrial dynamics and prevent and improve heart disease caused by aging. However, no ingredients are known that have the effect of inhibiting lipid peroxide formation in the mitochondrial membrane.

[0008] Honeybush is an evergreen shrub of the genus Cyclopia in the family Fabaceae, and is found in the southwestern and southeastern parts of South Africa. There are about 20 known species. Recent research has revealed that honeybush contains isoflavones, flavones, cinnamic acid, coumestans, xanthones, etc. (Non-Patent Document 3).

[0009] Extracts derived from honeybush have been reported to have antidiabetic effects (Patent Document 3), inhibitory effects on the enzyme ceramidase (Patent Document 4), and wrinkle-improving effects (Patent Document 5). On the other hand, Non-Patent Document 4 reports that when three types of unfermented honeybush (C. Subternata, C. genistoides, C. longifolia) were extracted with water, 40% ethanol, and 70% ethanol, all three water extracts and a 70% ethanol extract of a specific species (C. genistoides) had the effect of promoting ATP production and improving mitochondrial membrane potential in neuroblastoma. The results suggest that the water extract is useful, while the 40% ethanol extract and most 70% extracts have low effects. It is stated that unfermented honeybush was selected in the experiment because unfermented honeybush contains more phenols and has a higher antioxidant effect than fermented honeybush. In this context, the effect of improving mitochondrial function in fermented honeybush extracts was not expected. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Special Publication No. 2018-123130 [Patent Document 2] Special Publication No. 2019-178070 [Patent Document 3] Special Publication No. 2010-520914 [Patent Document 4] Japanese Patent Publication No. 2017-124984 [Patent Document 5] KR1020130010250

Non-licensed documents

[0011]

Non-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

[0012] An objective of the present invention is to provide an agent that has the effect of preventing and improving mitochondrial dysfunction, which is the cause of aging and disease. [Means for solving the problem]

[0013] As a result of the inventor's investigation, it was confirmed that the antioxidant effect (active oxygen scavenging ability) of the compound itself does not coincide with the inhibitory effect on the formation of lipid peroxides in the mitochondrial membrane, which is an indicator of mitochondrial dysfunction. Therefore, in order to solve this problem, it is necessary to select a compound that has a high inhibitory effect on the formation of lipid peroxides in the mitochondrial membrane.

[0014] Under these circumstances, the inventors conducted extensive research and discovered that fermented honeybush extract has an excellent effect of inhibiting the formation of lipid peroxides in mitochondrial membranes, leading to the present invention. That is, the present invention provides [1] An agent for inhibiting lipid peroxide formation in mitochondrial membranes, containing fermented honeybush extract as an active ingredient [2] Mitochondrial function improver containing fermented honeybush extract as an active ingredient [3] The agent according to [1] or [2], wherein the fermented honeybush extract is an extract of 30 to 70% (v / v) ethanol or 30 to 70% (v / v) 1,3-butylene glycol. [4] Use of a composition containing a fermented honeybush extract for the purpose of inhibiting lipid peroxide formation in mitochondrial membranes and / or improving mitochondrial function. It is. Effect of the Invention

[0015] The agent for improving mitochondrial function of the present invention suppresses the formation of lipid peroxides in the mitochondrial membrane. Since the formation of lipid peroxides disrupts the homeostasis of the mitochondrial membrane and causes mitochondrial dysfunction, the agent containing the fermented honeybush extract of the present invention as an active ingredient can prevent and improve aging and diseases caused by mitochondrial dysfunction. [Brief description of the drawings]

[0016] [Figure 1] Evaluation of reactive oxygen scavenging ability (DPPH radical scavenging ability) of each test substance [Diagram 2] Evaluation of the inhibitory effect of each test substance on lipid peroxide formation in mitochondrial membranes [Diagram 3] Evaluation of the inhibitory effect of fermented honeybush extracts with different extraction solvents on lipid peroxide formation in mitochondrial membranes BEST MODE FOR CARRYING OUT THEINVENTION

[0017] The present invention relates to an agent for inhibiting lipid peroxide formation in mitochondrial membranes and an agent for improving mitochondrial function, which contains a fermented honeybush extract as an active ingredient.

[0018] The honeybush (genus Cyclopia) used in the present invention is an evergreen tree that grows in South Africa. There are more than 20 species of plants called honeybush, and representative ones include Cyclopia intermedia, Cyclopia genistoides, and Cyclopia subternata, but any species of honeybush may be used in the present invention. Honeybush is mainly popular as tea, and is fermented during processing. Fermentation here refers to the "oxidation" process caused by the action of enzymes in the tea leaves themselves, and is not fermented by microorganisms (fermentation in the original sense is mediated by microorganisms, but in the case of tea, the oxidation process is conventionally called fermentation). In the present invention, honeybush that has undergone an oxidation process (fermentation) is used, not fresh honeybush that has not undergone an oxidation process (unfermented). Specifically, honeybush that is generally distributed on the market as honeybush tea can be used. Therefore, the fermented honeybush of the present application is not honeybush (unfermented) or honeybush that has been artificially fermented by adding bacteria such as lactic acid bacteria (microbial fermentation), and is distinct from these. Hereinafter, when the term "honeybush" is used simply in the examples, it refers to fermented honeybush.

[0019] The honeybush extract used in the present invention may be extracted from the flowers, stems, or a part of the leaves, or from the whole plant. The extraction solvent is not particularly limited, but may be, for example, water; lower monohydric alcohols such as methanol and ethanol; liquid polyhydric alcohols such as glycerin, propylene glycol, and 1,3-butylene glycol; or a mixture of these. Among them, a mixture of water and ethanol (hereinafter EtOH) or 1,3-butylene glycol (hereinafter BG) is particularly suitable.

[0020] When a mixed solvent is used, the ratio of water to EtOH or BG may be any ratio, but the ratio of EtOH or BG to water is preferably about 30 to 70% (v / v), more preferably about 40 to 60% (v / v).

[0021] The extraction method is preferably to use 1 to 100 parts by mass of an extraction solvent per part by mass of dried plant, and extract for several hours to 7 days, particularly several hours to 2 days, at a temperature of 5 to 70°C, preferably 10 to 60°C. After extraction, the extract can be filtered and used as is, but if necessary, it can be further purified by deodorization, decolorization, etc., within a range that does not affect the effect. Furthermore, it can be used in a powdered state by freeze-drying, etc.

[0022] The agent of the present invention can be blended into functional foods, cosmetics, quasi-drugs, and medicines for the purpose of improving mitochondrial function. The types of cosmetics to be blended include lotions, beauty essences, milky lotions, beauty creams, sunscreens, and cosmetics for hair growth and gray hair improvement, but can be blended into any cosmetics that are applied to the skin. It can also be blended into oral medicines as supplements or medicines for health and beauty purposes. The agent of the present invention can be blended with optional ingredients used in cosmetics and medicines, etc., as needed, within a range that does not impair the effects of the agent. Examples of the optional ingredients include oils, surfactants, powders, coloring materials, water, alcohols, thickeners, chelating agents, silicones, antioxidants, ultraviolet absorbers, moisturizers, fragrances, various medicinal ingredients, preservatives, pH adjusters, and neutralizers.

[0023] The agent of the present invention can be used as it is, either as an extract or in powder form, or mixed with a general base. In the case of mixing with a base, it is sufficient to have an effective amount of the component having the effect of improving mitochondrial function, but the concentration of the fermented honeybush extract in the agent for improving mitochondrial function is preferably in the range of 0.0001-1.0% (w / w) in terms of dry residue mass, and is most preferably in the range of 0.001-0.1% (w / w).

[0024] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Furthermore, unless otherwise specified, the blend amounts are indicated as volume percent concentrations (v / v).

[0025] Example 1: Confirmation of active oxygen scavenging ability To confirm the relationship between the antioxidant effect of the test substance itself, i.e., its ability to scavenge reactive oxygen species, and the inhibitory effect on the formation of lipid peroxides in the mitochondrial membrane, the DPPH radical scavenging ability evaluation (a method using the scavenging ability against DPPH (2,2,-Diphenyl-l-picrylhydrazyl), an artificially created radical, as an index), which is commonly used as a method for measuring the reactive oxygen species scavenging ability of a substance itself, was carried out.

[0026] <Preparation of test substance> For the honeybush, the dried product (whole plant) of the Cyclopia Intermedia species, which is commercially available as honeybush tea, was used. The dried raw material 1 was mixed at a mass ratio of 20 times the extraction solvent. The extraction solvent used was 50% BG, and the extraction operation was carried out under the conditions of 60 °C for 5 hours. After extraction, the raw material 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 honeybush extract with a solid content of about 1% (w / w, 10,000 ppm) was obtained. This was diluted 100-fold to obtain the test substance (solid content 100 ppm). Also, as comparative examples, ascorbic acid (25 ppm, dissolved in purified water) and gallic acid (25 ppm, dissolved in EtOH), which are well known for their high antioxidant ability, were prepared. <Preparation of DPPH solution> The DPPH solution was prepared by mixing the following components in a volume ratio of (A):(B):(C) = 1:4:3. (A) 5.52 g of ES (2-(N-morpholino)ethanesulfonic acid) was dissolved in 100 ml of water and adjusted to pH 6.1 with 1N-NaOH. (B) 15.7 mg of DPPH (1,1-diphenyl-2-picrylhydrazyl) was dissolved in 100 ml of ethanol. (C) Purified water <Preparation of Trolox solution> 25 mg of Trolox was dissolved in 10 ml of DMSO (dimethyl sulfoxide) to prepare a 10 mM solution. <DPPH test> 10μl of the test substance was added to one well of a 96-well plate, then 190μl of DPPH solution was quickly added and mixed. After 30 minutes, the absorbance of each well was measured at 540nm. The Trolox equivalent of the test solution was determined by taking 10μl of Trolox solution of 0.156mM, 0.313mM, 0.625mM, 1.25mM, 2.5mM, and 5.0mM, adding 190μl of DPPH solution, mixing, measuring the absorbance at 540nm after 30 minutes, and creating a calibration curve of Trolox concentration and absorbance at 540nm. The results are shown in Figure 1.

[0027] As shown in Figure 1, the active oxygen scavenging ability of honeybush extract (100 ppm solid content) was similar to that of ascorbic acid at 25 ppm. Also, the active oxygen scavenging ability of gallic acid at 25 ppm was significantly higher than that of honeybush extract.

[0028] Example 2: Confirmation of the inhibitory effect on lipid peroxide formation in mitochondrial membrane <Preparation of test substance> Honeybush was used as dried matter (whole plant) of Cyclopia Intermedia and Cyclopia Subternata species, which are commercially available as honeybush tea. They were mixed at a mass ratio of 1:20 of dried raw material to extraction solvent. 50% BG was used as the extraction solvent, and the extraction work was performed at 60°C for 5 hours. After extraction, the raw material was removed from the solution, filtered through a 5 μm filter, and then further filtered through a 0.2 μm filter for sterilization purposes. This operation yielded a honeybush extract with a solid content of about 1% (w / w, 10,000 ppm). In addition, as a comparative example, ascorbic acid (dissolved in purified water) and gallic acid (dissolved in EtOH) were prepared to a solid content of 0.25% (w / w, 2500 ppm), and filtered through a 0.2 μm filter.

[0029] <Cell culture and staining of mitochondrial membrane lipid peroxides> 1.5 × 10 dermal fibroblasts derived from normal adult skin 4The cells were suspended in D-MEM medium (containing 10% FBS) to a concentration of 100 cells / ml, seeded in 200 μl portions on 96-well black plates (CELLSTAR, Greiner), and cultured in a CO2 incubator (37°C, 5% CO2). After 24 hours of culture, the medium was replaced with D-MEM medium containing 1% FBS, and the test substances were added to the medium at 1% of the medium. As controls, the solvent for each test substance was added to the medium at 1% of the medium. After 72 hours of culture, the cells were washed twice with PBS(-), and stained with MitoPeDPP (mitochondrial membrane lipid-soluble peroxide detection reagent, Dojindo Laboratories) and Hoechst33342 (nuclear staining reagent, Dojindo Laboratories) diluted 1000-fold in HBSS(+) for 20 minutes in a CO2 incubator. The cells were then washed three times with PBS(-) and replaced with HBSS(+) and photographed under a fluorescence microscope (Keyence BZ-700) (20x objective lens used; green: lipid peroxides in mitochondrial membranes, blue: nuclei).

[0030] <Image analysis> The obtained green image was binarized by setting a certain threshold value in ImageJ (NIH) to calculate the fluorescence intensity of lipid peroxides in the mitochondrial membrane. In addition, the number of nuclei contained in the blue image was calculated in the same manner, and then the fluorescence intensity in the acquired image was divided by the number of nuclei to calculate the fluorescence intensity per cell, i.e., the amount of lipid peroxides in the mitochondrial membrane per cell. The amount of lipid peroxides in the mitochondrial membrane of cells treated with the extracting solvent of the test substance (control) and the amount of lipid peroxides in the mitochondrial membrane of cells treated with the test substance were substituted into equation 1 to calculate the amount of lipid peroxides in the mitochondrial membrane. In this calculation formula, the amount of lipid peroxides in the mitochondrial membrane is less than 1, and the smaller the value, the higher the ability of the substance to inhibit lipid peroxide formation can be determined. Substances with a value of 0 to 0.5 can be determined to have a high effect of inhibiting lipid peroxides in the mitochondrial membrane.

[0031]

number

[0032] Figure 2 shows the results of measuring 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 amount of lipid peroxides in the mitochondrial membrane was reduced to about 0.2 in the cells to which honeybush extract (Intermedia species, Subternata species) was added to the medium (final concentration of solids: 100 ppm). This result confirmed that the honeybush extract has a high effect of inhibiting lipid peroxide formation in the mitochondrial membrane. On the other hand, ascorbic acid (25 ppm) and gallic acid (25 ppm) used as comparative examples had a lower effect of inhibiting lipid peroxide formation in the mitochondrial membrane compared to the honeybush extract. In Example 1, it was confirmed that the active oxygen scavenging ability of ascorbic acid (25 ppm) and honeybush extract (100 ppm) was about the same, and that gallic acid (25 ppm) was significantly higher. Considering these results together, it was shown that it is difficult to predict the effect of inhibiting lipid peroxide formation in the mitochondrial membrane from the active oxygen scavenging ability of the substance itself.

[0033] Example 3: Confirmation of the inhibitory effect of honeybush extracts with different extraction solvents on the formation of lipid peroxides in mitochondrial membranes <Preparation of test substance> Honeybush was used as dried material (whole plant) of Cyclopia Intermedia species, which is commercially available as honeybush tea. The dry raw material was mixed with 20 parts of the extraction solvent by mass. The extraction solvents used were water, 20% EtOH, 40% EtOH, 60% EtOH, 80% EtOH, and 100% EtOH. The extraction was performed for water, 20% EtOH, 40% EtOH, 60% EtOH, and 80% EtOH at 60°C for 5 hours, and for 100% EtOH at room temperature for 2 days. After extraction, the raw material 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 in

[0029] , but the test substance was added to the medium so that it became 0.5%. The staining and image analysis of lipid peroxides in the mitochondrial membrane were performed in the same manner as described above.

[0034] FIG. 3 shows the results of measuring the amount of lipid peroxide in the mitochondrial membrane. As shown in Figure 3, all extraction solvents were found to have an inhibitory effect on the formation of lipid peroxides in the mitochondrial membrane, but the 40-60% EtOH extract was found to have a particularly high inhibitory effect.

[0035] The formation of lipid peroxides in the mitochondrial membrane disrupts the homeostasis of the mitochondrial membrane, which causes mitochondrial dysfunction. It is known that mitochondrial dysfunction is involved in physical disorders even if it is not a clear disease, and oral and external preparations containing the fermented honeybush extract of the present invention as an active ingredient can be used as agents for improving mitochondrial dysfunction.

Claims

1. An agent for reducing lipid peroxide content in mitochondrial membranes of skin cells, comprising a fermented honeybush extract as an active ingredient, The fermented honeybush extract is 40-60% (v / v) ethanol or 40-60% % (v / v) of 1,3-butylene glycol extract as a lipid peroxide reducing agent.

2. 2. Use of a composition containing a fermented honeybush extract according to claim 1 for the purpose of reducing the amount of lipid peroxide formation in mitochondrial membranes (excluding therapeutic use in humans).

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