Telomerase expression enhancer

Abstract

To provide a telomerase expression enhancer having excellent telomerase expression enhancing effect.SOLUTION: A telomerase expression enhancer contains at least one material selected from a kudzu flower, a black ginger, Lagerstroemia speciosa, a Terminalia plant, an oyster, Citrus unshiu peel and Matteuccia struthiopteris.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a telomerase expression enhancer containing a natural material. [Background technology]

[0002] In recent years, against the backdrop of growing health and safety consciousness, products using natural materials have become popular in the food and cosmetics industries, and there is a desire to make greater use of the useful functions of such natural materials.

[0003] For example, young barley leaves are commonly used as an ingredient in health foods and green juice, and are being widely studied for their functionality, such as their laxative and antihypertensive effects (Patent Documents 1 to 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-151951 [Patent Document 2] Japanese Patent Application Laid-Open No. 2001-314170 [Patent Document 3] Patent No. 4183886 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a telomerase expression enhancer that contains natural materials and has an excellent effect of enhancing telomerase expression. [Means for solving the problem]

[0006] The present inventors have investigated the functions of various natural materials and have found that certain natural materials have an excellent effect of enhancing telomerase expression, leading to the completion of the present invention.

[0007] That is, the present invention is as follows. [1] A telomerase expression enhancer characterized by containing at least one material selected from pine bark, young barley leaves, kudzu flowers, black ginger, banaba, terminalia, oysters, dried orange peel, and cycad. [2] The telomerase expression enhancer according to [1], which is an oral agent. [3] The telomerase expression enhancer according to [1], which is an external preparation. [Effects of the Invention]

[0008] The telomerase expression enhancer of the present invention exhibits an excellent effect of enhancing telomerase expression. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows the relative expression level of the human telomere reverse transcriptase (hTERT) gene when the telomerase expression enhancer of the present invention is applied. DETAILED DESCRIPTION OF THE INVENTION

[0010] The telomerase expression enhancer of the present invention is characterized by containing at least one material selected from pine bark, young barley leaves, kudzu flowers, black ginger, banaba, terminalia, oyster, dried persimmon peel, and cycad. The telomerase expression enhancer of the present invention may contain one material, two materials, or three or more materials. When two or more materials are contained, it is preferable to combine materials that are highly effective individually.

[0011] The telomerase expression enhancer of the present invention enhances the expression of telomerase (telomere synthesis enzyme), thereby lengthening cellular telomeres and preventing cellular senescence.

[0012] Each material contained in the telomerase expression enhancer of the present invention (hereinafter, sometimes referred to as the material of the present invention) will be described below.

[0013] [pine bark] Examples of raw pine species for the pine bark used in the present invention include French maritime pine (Pinus Martima), larch, black pine, red pine, Japanese white pine, Japanese white pine, Korean pine, Japanese stone pine, Ryukyu pine, Japanese silver pine, and white pine. Of these, it is preferable to use French maritime pine, which has a high effect of enhancing telomerase expression.

[0014] In the present invention, pine bark can be processed and used. Examples of processed pine bark products include chips, crushed products, squeezed products, extracts, and dried powders thereof. Considering formulation feasibility, the pine bark used in the present invention is preferably crushed, squeezed, extract, or dried powder thereof, as these are easy to apply, and more preferably an extract or dried powder thereof, from the viewpoint of the telomerase expression enhancing effect. The processed pine bark product may be produced by a method commonly known to those skilled in the art, or may be a product available on the market. For example, a pine bark extract manufactured by Toyo Shinyaku Co., Ltd. can be used.

[0015] Examples of extraction solvents used to obtain a pine bark extract include water, organic solvents, and aqueous organic solvents (e.g., aqueous alcohols such as aqueous ethanol). Examples of organic solvents include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, butane, acetone, hexane, cyclohexane, propylene glycol, aqueous ethanol, aqueous propylene glycol, ethyl methyl ketone, glycerin, methyl acetate, ethyl acetate, diethyl ether, dichloromethane, edible oils and fats, 1,1,1,2-tetrafluoroethane, and 1,1,2-trichloroethene. These water and organic solvents may be used alone or in combination. Water is preferably used as the extraction solvent. The temperature of the solvent used during extraction is not limited as long as it is below the boiling point of the solvent.

[0016] The method for obtaining a pine bark extract is not particularly limited, and examples thereof include a heat extraction method, a supercritical fluid extraction method, a liquid carbon dioxide batch method, a liquid carbon dioxide reflux method, and a supercritical carbon dioxide reflux method. A combination of multiple extraction methods may also be used. By combining multiple extraction methods, it is possible to obtain pine bark extracts with various compositions.

[0017] Supercritical fluid extraction is a method of extraction using a supercritical fluid, which is a fluid in a state exceeding the gas-liquid critical point (critical temperature, critical pressure) of a substance. Examples of supercritical fluids that can be used include carbon dioxide, ethylene, propane, and nitrous oxide (laughing gas), with carbon dioxide being preferred.

[0018] The supercritical fluid extraction method includes an extraction step in which a target component is extracted with a supercritical fluid, and a separation step in which the target component is separated from the supercritical fluid. The separation step may involve extraction and separation by pressure change, extraction and separation by temperature change, or extraction and separation using an adsorbent or absorbent.

[0019] Alternatively, supercritical fluid extraction may be performed using the entrainer addition method. In this method, approximately 2-20 w / v% of an extracting fluid, such as ethanol, propanol, n-hexane, acetone, toluene, or other aliphatic lower alcohols, aliphatic hydrocarbons, aromatic hydrocarbons, or ketones, is added to the extracting fluid, and supercritical fluid extraction is performed using this fluid. This method dramatically increases the solubility of the target extract, such as oligomeric proanthocyanidins (OPCs) or catechins, in the extracting solvent or enhances the separation selectivity, resulting in an efficient method for obtaining pine bark extracts.

[0020] Supercritical fluid extraction has the advantages of being able to operate at relatively low temperatures, making it applicable to substances that change or decompose at high temperatures, leaving no residual extracted fluid, and simplifying the process by eliminating steps such as desolvation, since the solvent can be recycled.

[0021] From the viewpoint of safety, it is preferable to purify the pine bark extract obtained by the above extraction using a column method or a batch method. Examples of the column method include purification methods using an adsorptive carrier such as Diaion HP-20, Sephadex-LH20, or chitin.

[0022] The pine bark extract used in the present invention contains proanthocyanidins as one of its main components. Proanthocyanidins are a group of compounds consisting of condensation polymers with a degree of polymerization of 2 or more, whose constituent units are flavan-3-ol and / or flavan-3,4-diol.

[0023] The pine bark extract used in the present invention preferably contains, as proanthocyanidins, condensation polymers having a degree of polymerization of 2 or more. Proanthocyanidins containing a large amount of condensation polymers having a low degree of polymerization are particularly preferred. Examples of condensation polymers with a low degree of polymerization include condensation polymers (dimers to tridecamers) having a degree of polymerization of 2 to 30, preferably condensation polymers (dimers to decamers) having a degree of polymerization of 2 to 10, and more preferably condensation polymers (dimers to tetramers) having a degree of polymerization of 2 to 4. In this specification, polymers having a degree of polymerization of 2 to 4 are referred to as OPCs (oligomeric proanthocyanidins). The pine bark extract used in the present invention preferably contains 20% by mass or more of OPC, more preferably 30% by mass or more, and even more preferably 40% by mass or more.

[0024] [Young barley leaves] Barley (Hordeum vulgare L.) is an annual or biennial herb belonging to the Poaceae family, believed to be native to Central Asia, and is broadly classified into two-row barley, six-row barley, and the like, depending on the panicle shape. The barley leaves used in the present invention are not particularly limited as long as they are commonly available, and any barley leaves, such as two-row barley or six-row barley, may be used. Furthermore, any variety may be used. The barley leaves may contain barley leaves, and may also contain other parts such as stems.

[0025] The form of the young barley leaves is not particularly limited, and may be, for example, the leaves as they are harvested, or the leaves may be further processed after harvesting. Examples of processing of young barley leaves include dried powder processing, shredded processing and subsequent dried powder processing, juice extraction and subsequent dried powder processing, and extracting and subsequent dried powder processing, and these can be processed using methods employed in the art. That is, processed products include barley leaf chips, pulverized products, juice extraction, extracts, etc., and pulverized products include powders, granules, etc. Methods for producing pulverized products include, for example, a method in which barley leaves and / or stalks are dried, coarsely pulverized, heated at 110°C or higher, and further finely pulverized (see JP 2003-033151 A), and a method in which barley leaves and / or stalks are blanched, dried, and then pulverized (see JP 2002-065204 A).

[0026] The barley leaf juice or extract may be in liquid form, or may be used as a paste or dry powder. The method for extracting barley leaves is not particularly limited, and any juice extraction method commonly used by those skilled in the art can be applied. The extraction / separation method and synthesis method from barley leaves are also not particularly limited, and can be appropriately selected depending on the purpose. For example, a method in which an extraction solvent commonly used by those skilled in the art, such as ethanol, water, or aqueous ethanol, is added and, if necessary, heated for extraction can be used.

[0027] The barley leaves used in the present invention are preferably in the form of a pulverized product, squeezed product, extract, or dried powder thereof, because they are easy to apply in consideration of formulation properties, and are more preferably in the form of a pulverized product, squeezed product, or dried powder thereof, from the viewpoint of the effect of enhancing telomerase expression. The processed barley leaves may be produced by a method commonly known to those skilled in the art, or may be those available on the market.

[0028] [Kudzu flower] Kudzu is a perennial climbing plant of the genus Pueraria in the family Fabaceae. Kudzu flowers may be harvested at any stage, from bud to fully opened flower, or a mixture of flowers harvested at each stage may be used. There are no particular limitations on the type of kudzu, but examples include Pueraria thomsonii, Pueraria lobata, and Pueraria thunbergiana.

[0029] In the present invention, harvested kudzu flowers can be processed and used, and examples of processed kudzu flowers include chips, crushed products, squeezed products, extracts, and dried powders thereof. Considering formulation feasibility, the kudzu flowers used in the present invention are preferably crushed products, squeezed products, extracts, or dried powders thereof, as these are easier to apply, and more preferably extracts or dried powders thereof, in terms of the effect of enhancing telomerase expression. Kudzu flower processed products may be those produced by methods commonly known to those skilled in the art, or may be those available on the market.

[0030] An extract of kudzu flowers can be obtained by extraction using an appropriate solvent, such as water; lower alcohols such as ethanol, methanol, isopropanol, and butanol; lower esters such as ethyl acetate and methyl acetate; acetone; and mixtures of these with water. The temperature of the extraction solvent can be appropriately set between room temperature and the boiling point depending on the solvent used. In the present invention, a mixed solvent of an organic solvent and water can be used, such as a mixed solvent of ethanol, methanol, isopropanol, butanol, ethyl acetate, methyl acetate, or acetone with water. The temperature of the extraction solvent can be appropriately set between room temperature and the boiling point depending on the solvent used. In the present invention, a hot water extract is preferred in terms of its telomerase expression enhancing effect.

[0031] [Black ginger] The black ginger (Kaempferia parviflora) used in the present invention is not particularly limited as long as it is a plant of the genus Kaempferia of the Zingiberaceae family that is known to grow wild in Southeast Asia and the like. For example, parts such as the roots, stems, leaves, flowers, and branches can be used, with the roots and stems being preferred.

[0032] Black ginger may be raw as harvested, or may be processed by subjecting it to a predetermined treatment after harvesting. Examples of processed products include chips, crushed products, squeezed products, extracts, and dried powders thereof. Considering formulation feasibility, the black ginger used in the present invention is preferably crushed, squeezed, extract, or a dried powder thereof, as this is easy to apply. From the viewpoint of the telomerase expression enhancing effect, an extract or a dried powder thereof is more preferable. Furthermore, from the viewpoints of ease of processing, storage, transportation, etc., and versatility of use, a dried powder is preferred. The processed black ginger may be produced by a method commonly known to those skilled in the art, or may be a product distributed on the market.

[0033] The black ginger extract is not particularly limited as long as it is an extract of black ginger components, but examples thereof include an extract obtained by extracting the components contained in black ginger with a solvent according to a conventional method, a diluted solution or concentrated solution thereof, or a dried product or powder thereof. For example, components contained in black ginger include, but are not limited to, polymethoxyflavonoids such as 5,7-dimethoxyflavone.

[0034] Examples of solvents used to obtain black ginger extract include water (e.g., room temperature water, warm water, hot water, etc.); lower alcohols (e.g., ethanol, methanol, isopropanol, butanol); lower esters (e.g., ethyl acetate, methyl acetate); acetone; and mixed solvents of these with water. Examples of mixed solvents include acetone / water (volume ratio: 2 / 8 to 8 / 2) and ethanol / water (volume ratio: 2 / 8 to 8 / 2).

[0035] The method for obtaining black ginger extract is not particularly limited, but examples include adding a solvent in an amount 2 to 20 times the mass of black ginger, and extracting under any conditions, such as standing, shaking, stirring, or refluxing, at temperatures ranging from 0°C to the reflux temperature of the solvent for several minutes to several tens of hours. After the extraction, it is preferable to perform solid-liquid separation procedures such as filtration and centrifugation to remove insoluble solids. An extract can be obtained by further performing dilution, concentration, or other procedures as necessary. Furthermore, the same procedures can be repeated to extract insoluble matter, and the resulting extract can be used together with the previous extract. These extracts can also be further purified using purification methods commonly used by those skilled in the art.

[0036] The method for obtaining a dried product from the extract is not particularly limited, and examples thereof include a method in which the extract or a concentrate thereof is subjected to a drying process commonly used by those skilled in the art, such as spray drying, freeze drying, vacuum drying, fluidized bed drying, etc. Furthermore, the dried product thus obtained can be powdered using a method known to those skilled in the art and used.

[0037] [Banaba] Banaba (Lagerstroemia Speciosa, Linn. or Pers.) is a plant belonging to the family Lythraceae in the order Myrtaceae. It is also commonly known as "crape myrtle" and is widely grown in the Philippines, as well as in Southeast Asia, including India, Malaysia, and southern China, and in Australia.

[0038] In the present invention, banaba flowers, leaves, bark, roots, or seeds can be used, but from the viewpoint of the effect of enhancing telomerase expression, it is preferable to use leaves. Banaba leaves may also include stems. Because banaba leaves are prone to decay, they are preferably dried immediately after harvesting to obtain a dried product. Fresh leaves can be dried either naturally or by air drying, but forced drying by air drying is preferred. Drying is performed so that the moisture content is 20% by mass or less, preferably 10% by mass or less, to prevent microbial decay and stably maintain the components in the banaba leaves.

[0039] In the present invention, banaba can be processed and used, and examples of processed banaba products include chips, crushed products, squeezed products, extracts, and dried powders thereof. Considering formulation feasibility, the banaba used in the present invention is preferably crushed, squeezed, extract, or dried powder thereof, as these are easy to apply, and more preferably extract or dried powder thereof, from the viewpoint of the effect of enhancing telomerase expression. The banaba processed product may be one produced by a method commonly known to those skilled in the art, or may be one that is commercially available.

[0040] There are no particular limitations on the method for obtaining ground banaba, and for example, the ground banaba can be obtained by washing, drying in the sun or using a dryer, and then either cutting the treated product into an appropriate shape and size, or grinding it in a grinding device. A wide range of commonly used grinding devices can be used, including, for example, a grinder consisting of a raw material hopper, grinder, classifier, product holder, etc.

[0041] The method for obtaining banaba juice is not particularly limited, and examples thereof include squeezing banaba or its shredded material, and centrifuging or filtering the shredded banaba. Specific examples of methods for producing squeezed juice include a method in which the juice is extracted using a mechanical crushing means such as a mixer or juicer, and then, if necessary, removing coarse solids by means such as sieving or filtration to obtain a squeezed juice. The squeezed juice may be concentrated as needed, or may be subjected to processes such as freeze-drying, hot air drying, or spray drying to produce a dry powder.

[0042] The method for obtaining a banaba extract is not particularly limited, and examples thereof include an extract obtained by extracting components contained in banaba leaves with a solvent according to a conventional method, a diluted or concentrated solution thereof, or a dried product or powder thereof.

[0043] Examples of solvents used for extraction include water; lower alcohols such as ethanol, methanol, isopropanol, and butanol; lower esters such as ethyl acetate and methyl acetate; acetone; and mixed solvents of these with water. In the present invention, a mixed solvent of an organic solvent and water can be used. Examples of the mixed solvent include a mixed solvent of ethanol, methanol, isopropanol, butanol, ethyl acetate, methyl acetate, or acetone with water. Preferred examples include an acetone / water (volume ratio: 2 / 8 to 8 / 2) mixture and an ethanol / water (volume ratio: 2 / 8 to 8 / 2) mixture. The temperature of the extraction solvent can be set appropriately between room temperature and the boiling point depending on the solvent used.

[0044] Examples of extraction methods include adding a solvent in an amount 2 to 20 times, preferably 5 to 15 times, and more preferably 8 to 10 times the mass of banaba, and performing extraction under any conditions, such as standing, shaking, stirring, or refluxing, at temperatures preferably above 50°C, more preferably between about 50 and 85°C, for several minutes to several tens of hours, preferably 30 minutes to 2 hours. Extraction at high temperatures may result in evaporation of the organic solvent (e.g., ethanol), reducing the extraction efficiency. Therefore, extraction by heating and refluxing is preferred. After extraction, solid-liquid separation procedures such as filtration and centrifugation are preferably performed to remove insoluble solids. Extracts can be obtained by diluting and concentrating the extract as needed. Furthermore, the same procedures may be repeated to extract insoluble matter, and the resulting extract may be used together with the previous extract. These extracts may be further purified using purification methods commonly used by those skilled in the art.

[0045] The method for obtaining a dried product from the extract is not particularly limited, and examples include subjecting the extract or its concentrate to drying processes commonly used by those skilled in the art, such as spray drying, freeze drying, vacuum drying, and fluidized bed drying. Furthermore, the dried product thus obtained can be powdered and used using methods known to those skilled in the art. For example, a banaba extract can be obtained according to the method described in Japanese Patent Application Laid-Open No. 2005-263650. According to this method, a banaba extract rich in corosolic acid can be obtained by subjecting banaba leaves to (a) extraction with an aqueous ethanol solution, (b) treatment with activated carbon, and (c) concentration and recovery of the precipitate.

[0046] [Terminaria] Terminalia is a broad-leaved tree of the genus Terminalia in the family Combretaceae. In the present invention, examples include Terminalia bellirica (belerica), Terminalia catappa, Terminalia tomentosa, Terminalia citrina, Terminalia phellocarpa, Terminalia copelandii, Terminalia brassi, Terminalia ivorensis, Terminalia superba, Terminalia arjuna, and Terminalia chebula. Among these, Terminalia bellirica (belerica) and Terminalia chebula are preferred, and Terminalia belliricca (belerica), which has a high telomerase expression enhancing effect, is particularly preferred. In addition, in the present invention, it is preferable to use the fruit of Terminalia in terms of the telomerase expression enhancing effect.

[0047] In the present invention, harvested Terminalia can be processed and used, and examples of processed Terminalia products include chips, crushed products, squeezed products, extracts, and dried powders thereof. Considering formulation properties, the Terminalia used in the present invention is preferably crushed products, squeezed products, extracts, or dried powders thereof, as these are easy to apply, and more preferably extracts or dried powders thereof from the viewpoint of the telomerase expression enhancing effect. The processed Terminalia product may be produced by a method commonly known to those skilled in the art, or may be one that is available on the market.

[0048] Examples of extracts of Terminalia include extracts obtained by extracting components contained in Terminalia with a solvent according to conventional methods, diluted or concentrated extracts thereof, or dried products or powders thereof.

[0049] Examples of solvents used for extraction include water; lower alcohols such as ethanol, methanol, isopropanol, and butanol; lower esters such as ethyl acetate and methyl acetate; acetone; and mixtures of these with water. In the present invention, a mixture of an organic solvent and water can be used. Examples of such a mixture include a mixture of ethanol, methanol, isopropanol, butanol, ethyl acetate, methyl acetate, or acetone with water. Preferred examples include an acetone / water (volume ratio: 2 / 8 to 8 / 2) mixture and an ethanol / water (volume ratio: 2 / 8 to 8 / 2). In the present invention, water, ethanol, methanol, or a mixture thereof is preferred from the perspective of the telomerase expression enhancing effect. The temperature of the extraction solvent can be appropriately set between room temperature and the boiling point depending on the solvent used.

[0050] [Oysters] The oysters used in the present invention are not particularly limited as long as they belong to the Ostrich family, and examples thereof include oysters belonging to the genus Crassostrea and Ostrich. In the present invention, it is preferable to use oyster meat (oyster meat) from the viewpoint of the effect of enhancing telomerase expression.

[0051] In the present invention, oyster meat can be processed and used. Examples of processed oyster products include crushed oysters, crushed oysters, squeezed oysters, extracts (including enzyme-treated oysters), and dried powders thereof. Considering formulation feasibility, the oysters used in the present invention are preferably crushed oysters, squeezed oysters, extracts, or dried powders thereof, as these are easier to apply. Extracts or dried powders thereof are more preferable in terms of the effect of enhancing telomerase expression. The extraction method using a solvent is similar to that for Terminalia. An example of enzyme treatment is hydrolysis of oyster meat with an enzyme. The hydrolase may be any enzyme capable of hydrolyzing food materials (proteins).

[0052] These solvent extraction treatments and enzyme treatments can be carried out under heating and / or pressure as needed. Furthermore, in order to obtain a larger amount of active ingredients, it is preferable to crush the oyster meat before the extraction or enzyme treatment. For example, crushed oyster meat can be extracted with water at about 40 to 90°C for about 1 to 5 hours, and after removing the oyster meat residue, the extract concentrate (oyster extract) can be obtained in liquid or powder form. Furthermore, crushed oyster meat can be hydrolyzed with an enzyme in water at about 40 to 80°C, and after inactivating the enzyme, unnecessary substances such as oyster meat residue can be removed, and the concentrate can be subjected to spray drying, freeze drying, or the like as needed to obtain the extract concentrate (oyster extract) in liquid or powder form.

[0053] The extract of the present invention may be the extract itself, but is preferably a liquid, semi-solid, or solid concentrate obtained by concentrating the extract. For example, a commercially available product containing oyster extract can be used.

[0054] [Chen Pi] In the present invention, "citrus peel" refers to the peel of a citrus fruit. Generally, dried ripe mandarin peel is used in China, and dried ripe Satsuma mandarin peel is used in Japan. However, in the present invention, the present invention is not limited to these and any citrus peel may be used. Citrus fruits may be, for example, domestic or imported. In the present invention, Kanpei, a type of citrus fruit developed in Ehime Prefecture, is preferred in terms of its telomerase expression enhancing effect.

[0055] In the present invention, processed persimmon peel can be used, and examples of processed persimmon peel include chips, crushed products, squeezed products, extracts, and dried powders thereof. In consideration of formulation, the persimmon peel used in the present invention is preferably crushed, squeezed, extract, or dried powder thereof, as these are easy to apply, and more preferably an extract or dried powder thereof from the viewpoint of the telomerase expression enhancing effect. The processed persimmon peel may be produced by a method commonly known to those skilled in the art, or may be a product available on the market.

[0056] The extract of Citrus unshiu peel can be obtained by extraction with a suitable solvent. The extraction solvent, extraction method, and purification method can be the same as those for pine bark.

[0057] [Cycad] The cycad is a perennial fern of the family Lycopersicaceae or Melastomataceae. The young shoots of the cycad are called "kogomi" and are a type of wild vegetable. In the present invention, it is preferable to use kogomi in view of its effect of enhancing telomerase expression.

[0058] In the present invention, harvested cycads can be processed and used. Examples of cycad processed products include chips, crushed products, squeezed products, extracts, and dried powders thereof. Considering formulation feasibility, the cycads used in the present invention are preferably crushed products, squeezed products, extracts, or dried powders thereof, as these are easier to apply. From the perspective of the telomerase expression enhancing effect, extracts or dried powders thereof are more preferred. Cycad processed products may be produced by methods commonly known to those skilled in the art, or may be commercially available products. The extraction solvent, extraction method, etc. may be the same as for Terminalia.

[0059] The telomerase expression enhancer of the present invention can be used as an oral agent or an external agent to be applied to the skin or scalp. For example, it can be used as a pharmaceutical product (including quasi-drugs), a so-called health food such as a food for specified health uses, a food with nutrient function claims, or a food with functional claims whose efficacy has been approved by a designated organization, as well as cosmetics and toiletries.

[0060] The telomerase expression enhancer of the present invention is not particularly limited as long as it contains the material of the present invention and can be distinguished from other products in that it is used to enhance telomerase expression. For example, the scope of the present invention includes products that display the telomerase expression enhancing function and various functions resulting therefrom on the product itself, packaging, instructions, or promotional materials (advertising media) of the present invention. Furthermore, the telomerase expression enhancer of the present invention is not limited to products that display the material of the present invention as an active ingredient on the product packaging, etc. For example, products that do not specify the active ingredient may also be included in the scope of the present invention. Furthermore, even general foods that are manufactured and sold with a suggested use are included in the scope of the present invention.

[0061] Specifically, examples of so-called health foods include those labeled with claims such as "enhances telomerase expression," "prevents cellular aging," "prevents telomere shortening," "lengthens telomeres," "anti-aging," "anti-aging," and "hair growth." Subjects who may ingest the telomerase expression enhancer of the present invention are not particularly limited as long as they are people who require enhancement of telomerase expression, but preferred examples include people who are aiming for anti-aging or anti-aging.

[0062] When the telomerase expression enhancer of the present invention is an oral preparation, its form may be, for example, tablet, capsule, powder, granule, liquid, granule, rod, plate, block, solid, round, paste, cream, caplet, gel, chewable tablet, stick, etc. Among these, tablet, capsule, powder, granule, round, and chewable tablet forms are preferred, and tablet, capsule, round, and chewable tablet forms are more preferred.

[0063] When the composition of the present invention is made into a tablet, pill, or chewable tablet, it is preferable to add one or more of an excipient, a lubricant, and a fluidizing agent, because this improves moldability and improves the storage stability of the resulting tablet, pill, or chewable tablet. In particular, the use of an excipient and a lubricant can further improve storage stability.

[0064] An excipient is added to improve the handling or molding of the composition or to make it easier to take. The excipients that can be used in the present invention are not particularly limited, and examples thereof include starch or its derivatives such as starch, pregelatinized starch, partially pregelatinized starch, and starch hydrolysates, crystalline cellulose, sugar alcohol, lactose, brewer's yeast, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, refined sucrose, light anhydrous silicic acid, calcium silicate, titanium oxide, precipitated calcium carbonate, etc. These may be used alone or in combination of two or more.

[0065] Lubricants are used to reduce friction between the tablet press punches and dies and the tablet when compressing powder for tablets, and to prevent tableting problems such as sticking. Lubricants that can be used in the present invention are not particularly limited as long as they are components that can achieve the above-mentioned purpose, and examples include stearic acid or salts thereof such as stearic acid, calcium stearate, and magnesium stearate, sodium stearyl fumarate, sucrose fatty acid esters, talc, polyethylene glycol, vegetable oils and fats, and hydrogenated oils. These may be used alone or in combination of two or more.

[0066] A fluidizer is used to improve the fluidity of mixed powders or granules. There are no particular limitations on the fluidizers that can be used in the present invention, and examples include silicon dioxide, aluminum silicate, magnesium aluminosilicate, calcium phosphate, magnesium carbonate, magnesium oxide, etc. These may be used alone or in combination of two or more. In the present invention, commercially available excipients, lubricants, and fluidizers can all be used.

[0067] When the telomerase expression enhancer of the present invention is used as an external preparation, its form may be, for example, a lotion, emulsion, gel, cream, ointment, powder, granules, etc. Specific examples include lotions, cosmetic creams, emulsions, creams, packs, hair tonics, hair creams, shampoos, hair rinses, treatments, body shampoos, facial cleansers, soaps, foundations, face powders, lipsticks, lip glosses, blushers, eye shadows, hair styling products, hair growth agents, aqueous ointments, oily ointments, eye drops, eye washes, patches, gels, etc. Furthermore, when applied to the oral cavity, examples include dentifrices, mouthwashes, sprays, etc.

[0068] The content of the material of the present invention in the telomerase expression enhancer of the present invention may be appropriately determined within the range in which the effect is exhibited.

[0069] Specifically, when the telomerase expression enhancer of the present invention is in the form of a tablet, pill, capsule, or chewable tablet, the material of the present invention is preferably contained in an amount of 0.000001 to 60% by mass of the total, calculated as dry mass, more preferably 0.00001 to 50% by mass, even more preferably 0.0001 to 40% by mass, and particularly preferably 0.01 to 30% by mass from the viewpoint of enhancing telomerase expression.

[0070] When the telomerase expression enhancer of the present invention is in powder or granular form, the material of the present invention preferably accounts for 0.000001 to 100% by mass of the total mass, calculated as dry mass, more preferably 0.00001 to 90% by mass, even more preferably 0.0002 to 80% by mass, and particularly preferably 0.01 to 70% by mass from the viewpoint of enhancing telomerase expression.

[0071] When the telomerase expression enhancer of the present invention is a liquid beverage, the material of the present invention preferably accounts for 0.0000001 to 50% by mass of the total, calculated as dry mass, more preferably 0.00001 to 25% by mass, even more preferably 0.0002 to 10% by mass, and particularly preferably 0.001 to 5% by mass from the viewpoint of enhancing telomerase expression.

[0072] When the telomerase expression enhancer of the present invention is an oral agent, there is no particular limit to the amount of intake, but the daily intake of the material of the present invention for an adult is preferably 10 mg / day or more, more preferably 20 mg / day or more, and even more preferably 30 mg / day or more from the viewpoint of telomerase expression enhancement. The upper limit is, for example, 100,000 mg / day, preferably 80,000 mg / day, and more preferably 60,000 mg / day.

[0073] When the telomerase expression enhancer of the present invention is an oral agent, it can be appropriately designed so that the daily intake is the above-mentioned intake amount, and it can be taken in one dose or in multiple doses. For example, in the case of tablets, capsules, pills, or chewable tablets, it is sufficient to take them 1 to 4 times per day, so that the total amount is sufficient to ingest the above-mentioned intake amount, and in the case of beverages, it is sufficient that the above-mentioned intake amount is blended into the daily intake amount. The telomerase expression enhancer of the present invention can be stored as a daily dose in a single container or divided into multiple containers, for example, 2 to 3, so that the daily intake is the above-mentioned intake amount.

[0074] When the telomerase expression enhancer of the present invention is an external preparation, the material of the present invention is preferably contained in an amount of 0.0000001 to 50% by mass of the total, calculated as dry mass, more preferably 0.000001 to 20% by mass, even more preferably 0.00001 to 10% by mass, and particularly preferably 0.0001 to 5% by mass from the viewpoint of enhancing telomerase expression.

[0075] When the telomerase expression enhancer of the present invention is an external preparation, the amount used is not particularly limited and can be appropriately selected taking into consideration various factors such as the age, weight, and constitution of the individual to be used.

[0076] The telomerase expression enhancer of the present invention can be produced by a known method, adding other components in addition to the material of the present invention, if necessary. When the telomerase expression enhancer of the present invention is an oral preparation, other ingredients can include, for example, vitamins such as water-soluble vitamins (vitamins B1, B2, B3, B5, B6, B7, B9, B12, B13, B15, B17, vitamin C, vitamin P, choline, inositol, PABA) and fat-soluble vitamins (vitamin A, vitamin D, vitamin E, vitamin K); minerals such as magnesium, phosphorus, zinc, and iron; sulfur-containing compounds contained in taurine, garlic, etc.; flavanoids or flavonoids such as hesperidin and quercetin; proteins such as collagen; peptides; amino acids; animal fats and oils; vegetable fats and oils; and crushed products or extracts of animals and plants.

[0077] When the telomerase expression enhancer of the present invention is used as an external preparation, examples of ingredients that can be added include various medicinal ingredients (such as active oxygen scavengers, antioxidants, anti-inflammatory agents, cell activators, vitamins, hormones, and animal and plant-derived extracts that have these effects), other oils (such as unsaturated fatty acids and derivatives thereof, such as linoleic acid, linolenic acid, palmitic acid, DHA, and EPA, and oils extracted from animals and plants, such as linseed oil, coconut oil, jojoba oil, olive oil, squalane, squalene, horse oil, rice bran oil, and castor oil, and derivatives thereof), moisturizers (such as collagen or its hydrolyzates, collagen-like peptides contained in carrot extract, soybean peptides, amino acids, mucopolysaccharides such as hyaluronic acid, amino sugars such as chondroitin, sugars such as trehalose, seaweed, alginic acid, glucomannan, and water-soluble dietary fibers such as pectin), surfactants (such as lecithin, fatty acid esters, and amino acid derivatives), ultraviolet absorbers (such as zinc oxide and titanium oxide), and ultraviolet absorption enhancers. [Example]

[0078] [Sample (test substance) preparation] The pine bark extract used was a dried powder of French maritime pine bark, which was prepared according to the following method. First, water was added to the pine bark and extraction was carried out for at least 1 hour at 95°C or higher. The extract obtained by filtration was then purified and dried, and the resulting dry powder was used as the pine bark extract.

[0079] As the young barley leaves, squeezed barley leaf juice (dried powder) was used. The squeezed barley leaf juice was prepared according to the following method. First, young barley leaves were made into a paste, then pressed, and the barley leaf juice was dried, and the resulting dry powder was used as the barley leaf juice.

[0080] The kudzu flower extract (dried powder) was used as the kudzu flower extract, which was prepared according to the following method. First, water was added to the dried kudzu flowers, followed by hot water extraction. Then, a solid-liquid separation was performed to remove insoluble solids, yielding a kudzu flower extract. The extract was then dried, and the resulting dry powder was used as the kudzu flower extract.

[0081] The black ginger used was an extract (dried powder) of black ginger rhizomes, which was prepared according to the following method. First, black ginger rhizome was extracted with aqueous ethanol. Then, solid-liquid separation was performed to remove insoluble solids, and an extract was obtained. After that, dextrin was added to the extract, and the extract was dried. The obtained dried powder was used as the black ginger extract.

[0082] The banaba leaf extract (dried powder) used was prepared according to the following method. First, dried banaba leaves were crushed to obtain a crushed product. Next, aqueous ethanol was added to the crushed product to extract the product. The extract was then dried and crushed, and the resulting dry powder was used as the banaba leaf extract.

[0083] Terminalia is a plant called Terminalia bellirica. The water extract of Terminalia bellirica fruit (dried powder) was used. The water extract of Terminalia bellirica fruit was prepared according to the following method. First, the remaining part of the fruit of Terminalia bellirica after removing the seeds was mixed with water to prepare a mixture, which was then extracted with hot water. After extraction, the mixture was filtered and the filtrate was freeze-dried, and the resulting dry powder was used as the Terminalia bellirica fruit extract.

[0084] The oysters used were oyster meat extract (dried powder), which was prepared according to the following method. First, oyster meat was heated, cooled, added with enzymes, and pressurized. It was then heat-sterilized and dried. The resulting dried powder was used as the oyster meat extract.

[0085] The dried powder of the peel of Kanpei was used as the dried orange peel. The Kanpei peel extract was prepared according to the following method. First, water was added to the Kanpei peel, and the mixture was heated to perform hot water extraction. The mixture was then filtered to recover the filtrate, which was then further filtered to recover the filtrate, yielding an extract of the Kanpei peel. The filtrate was dried, and the resulting dry powder was used as the Kanpei peel extract.

[0086] The dried powder of kogomi (young shoots of the cycad) was used as the cycad. The dried powder of kogomi was prepared according to the following method. Young shoots of the cycad were washed, sterilized, steamed, and then dried. The dried material was then crushed, and the resulting powder was used as the dried powder of kogomi.

[0087] Each material was dissolved in DMSO at a concentration of 10 mg / mL, and diluted to 10 μg / mL with DMEM medium containing 10% FBS serum to prepare a sample (test substance).

[0088] [Quantitative reverse transcriptase-PCR (RT-PCR) reaction] 1. Total RNA Preparation Total RNA was prepared using the High Pure RNA Isolation Kit (Roche) according to the manufacturer's protocol. All reagents and equipment used from total RNA preparation to the completion of the reverse transcription reaction were RNase-free.

[0089] First, 5.0 x 10 cells were placed in a 5 mL cell culture dish. 4Cells were seeded at 1000 cells / well and cultured at 37°C in DMEM medium containing 10% FBS serum. The following day, various samples (test substances) were added and cultured at 37°C for 48 hours in DMEM medium containing 10% FBS serum. For comparison (control), DMEM medium containing 0.1% DMSO and 10% FBS serum was added. After 48 hours, the medium was completely removed and the dish was washed with 200 μL of 1x PBS. 400 μL of cell lysis solution included in the High Pure RNA Isolation Kit was added on top, and the cell lysis solution was thoroughly dispersed throughout the dish to dissolve the cells. The entire cell lysate was then collected into a 1.5 mL tube.

[0090] The collected sample was thoroughly suspended in a vortex mixer for 60 seconds and briefly spun down. The High Pure filter tube and collection tube included in the kit were assembled, and the cell lysate solution was added to the filter tube. The tube was centrifuged at 10,000 × g for 15 seconds at room temperature. The liquid discharged into the collection tube was discarded, and the filter tube and collection tube were reassembled. To each 1.5 mL tube, 90 μL of DNase incubation buffer and 10 μL of DNase I were added and mixed. This mixture was added to the filter tube and incubated at room temperature for 15 minutes. After 15 minutes, 500 μL of Wash Buffer I included in the kit was added to the filter tube, and the tube was centrifuged at 10,000 × g for 15 seconds at room temperature. After centrifugation, the liquid discharged into the collection tube was discarded, and the filter tube and collection tube were reassembled.

[0091] 500 μL of Wash Buffer II was added to the filter tube and centrifuged at 10,000 × g for 15 seconds at room temperature. After centrifugation, the liquid discharged into the collection tube was discarded, and the filter tube and collection tube were reassembled. 200 μL of Wash Buffer II was added, and the filter was washed by centrifugation at 15,000 × g for 2 minutes at room temperature. After centrifugation, the filter tube was inserted into a new 1.5 mL tube, and 100 μL of elution buffer was added to the center of the filter tube. The tube was then left to stand at room temperature for 3 minutes. The tube was then centrifuged at 10,000 × g for 1 minute at room temperature. The eluate was used as the RNA solution. The RNA concentration in the solution was calculated based on the absorbance at 260 nm using a NanoDrop 2000 / 2000c spectrophotometer (Thermo Fisher Scientific) and used in subsequent experiments.

[0092] 2. cDNA Synthesis 5 pmol of Oligo(dT) per 1.0 μg of total RNA extracted from cells 20 Primers (TOYOBO) were added, and RNase-free water was added to bring the total volume to 13 μL. The reaction was performed at 65°C for 5 minutes in a Thermal Cycler PTC 200 (MJ Research) and then immediately transferred to ice for rapid cooling. During this time, the reverse transcriptase reaction program was advanced to the 42°C step and then paused. After cooling the samples on ice for 5 minutes, a mixture of 4 μL of reverse transcriptase reaction buffer, 2 μL of 10 mM dNTPs (GE Healthcare), and 0.5 μL of reverse transcriptase ReverTra Ace (TOYOBO) was added per sample and mixed gently. cDNA was then synthesized by reacting at 42°C for 20 minutes, 99°C for 5 minutes, and 4°C for 5 minutes. This cDNA was used as a template for quantitative PCR.

[0093] 3. Primer Design The target genes whose expression levels were measured by RT PCR were searched for in NCBI (http: / / www.ncbi.nlm.nih.gov / gene / ), and the primer sequences were determined and synthesized based on the sequences. Primer synthesis was outsourced to Sigma. The primers used to detect the internal controls β-actin and hTERT are shown in Table 1.

[0094] [Table 1]

[0095] 4. Quantitative Reverse Transcriptase-PCR (RT-PCR) Reaction The prepared cDNA was used as a template. A 0.2 mL PCR tube was filled with 49 μL of sterile water, 3.5 μL each of forward and reverse primers diluted to 10 pmol / mL, 7.0 μL of template cDNA, and 24.5 μL of THUNDERBIRD SYBR qPCR Mix (TOYOBO), a master mix for high-efficiency real-time PCR. The mixture was then added in 25 μL portions to three wells of a 96-well plate, and quantitative real-time PCR was performed using a Thermal Cycler Dice Real-Time System (TAKARA BIO). The PCR reaction consisted of denaturation at 95°C for 5 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 20 seconds. This cycle was repeated 45 times (3 steps), and detection was performed by FAM. β-actin was used as a primer for the calibration curve. Relative gene expression levels were calculated by dividing the measured values ​​by the expression levels of β-actin.

[0096] Table 2 and Figure 1 show the relative expression level of the human telomerase reverse transcriptase (hTERT) gene. Human telomerase reverse transcriptase (hTERT) is an enzyme contained in telomerase. As shown in Figure 1, the expression of the human telomerase reverse transcriptase (hTERT) gene was enhanced by adding various materials of the present invention.

[0097] [Table 2]

[0098] Examples of various aspects of the present invention will be given below, but the technical scope of the present invention is not limited to these.

[0099] <Production Examples 1 to 8> At least one ingredient selected from pine bark, young barley leaves, kudzu flowers, black ginger, banaba, terminalia, oyster, dried orange peel, and cycad was blended and mixed uniformly according to the composition shown in Table 3 below, and then formed into tablets using a tableting machine (250 mg per tablet). The resulting dietary composition is expected to have a high effect of enhancing telomerase expression.

[0100] [Table 3]

[0101] <Production Examples 9 to 16> At least one ingredient selected from pine bark, young barley leaves, kudzu flowers, black ginger, banaba, terminalia, oyster, dried orange peel, and cycad was blended and mixed uniformly according to the composition shown in Table 4 below, and then filled into a coating containing gelatin to produce hard capsules (350 mg per capsule). The resulting dietary composition is expected to have a high effect of enhancing telomerase expression.

[0102] [Table 4]

[0103] <Production Examples 17 to 24> At least one ingredient selected from pine bark, young barley leaves, kudzu flowers, black ginger, banaba, terminalia, oyster, dried orange peel, and cycad was mixed and mixed uniformly according to the composition shown in Table 5 below, and then coated with a film containing gelatin and glycerin to produce soft capsules (300 mg per capsule). The resulting dietary composition is expected to have a high effect of enhancing telomerase expression.

[0104] [Table 5]

[0105] <Production Examples 25 to 32> A powdered beverage was produced by blending at least one ingredient selected from pine bark, young barley leaves, kudzu flowers, black ginger, banaba, terminalia, oysters, dried orange peel, and cycad according to the composition shown in Table 6 below, and mixing them uniformly. For example, by mixing 3 g of the resulting composition with 150 mL of water and drinking it, a high telomerase expression enhancing effect can be expected.

[0106] [Table 6]

[0107] <Production Examples 33 to 40> At least one ingredient selected from pine bark, young barley leaves, kudzu flowers, black ginger, banaba, terminalia, oysters, dried orange peel, and cycad was mixed according to the composition shown in Table 7 below, placed in a fluidized bed granulator, mixed with air for several minutes, and granulated by spraying 60 L of water at 2000 mL per minute. The resulting granules were then sieved through a 30-mesh sieve to produce granules. The resulting dietary composition is expected to have a high effect of enhancing telomerase expression.

[0108] [Table 7]

[0109] <Production Examples 41 to 56> Lotions were produced by blending at least one ingredient selected from pine bark, young barley leaves, kudzu flower, black ginger, banaba, terminalia, oyster, dried persimmon peel, and cycad according to the formulations shown in Tables 8 to 11. The obtained topical preparations are expected to have a high effect of enhancing telomerase expression.

[0110] [Table 8]

[0111] [Table 9]

[0112] [Table 10]

[0113] [Table 11]

[0114] <Production Examples 57 to 64> Creams were produced by blending at least one ingredient selected from pine bark, young barley leaves, kudzu flowers, black ginger, banaba, terminalia, oyster, dried orange peel, and cycad, in the formulations shown in Tables 12 and 13. The obtained topical preparations are expected to have a high effect of enhancing telomerase expression.

[0115] [Table 12]

[0116] [Table 13]

[0117] <Production Examples 65 to 72> Emulsions were produced by blending at least one ingredient selected from pine bark, young barley leaves, kudzu flowers, black ginger, banaba, terminalia, oyster, dried orange peel, and cycad, in the formulations shown in Tables 14 and 15. The obtained topical preparations are expected to have a high effect of enhancing telomerase expression.

[0118] [Table 14]

[0119] [Table 15]

[0120] <Production Examples 73 to 80> Gels were produced by blending at least one ingredient selected from pine bark, young barley leaves, kudzu flowers, black ginger, banaba, terminalia, oyster, dried orange peel, and cycad according to the formulations shown in Tables 16 and 17. The resulting topical preparations are expected to have a high effect of enhancing telomerase expression.

[0121] [Table 16]

[0122] [Table 17]

[0123] <Production Examples 81 to 88> Aerosol preparations were manufactured by blending at least one material selected from pine bark, young barley leaves, kudzu flowers, black ginger, banaba, terminalia, oyster, dried orange peel, and cycad according to the formulations shown in Tables 18 and 19. The obtained topical preparations are expected to have a high effect of enhancing telomerase expression.

[0124] [Table 18]

[0125] [Table 19] [Industrial Applicability]

[0126] The telomerase expression enhancer of the present invention can be used as a health food or the like and is therefore industrially useful.

Claims

1. A telomerase expression enhancer characterized by containing at least one material selected from kudzu flowers, black ginger, banaba, terminalia, oysters, dried persimmon peel, and cycad.

2. 2. The telomerase expression enhancer according to claim 1, which is an oral agent.

3. 2. The telomerase expression enhancer according to claim 1, which is an external preparation.

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