JP7598664B2 - Cosmetic composition, beauty composition, joint protection composition, composition - Google Patents

Description

The present invention relates to a cosmetic composition, a beauty composition, a joint protection composition, and a composition that contain amino sugars and a plant that contains flavonoids.

In addition to the effects of ultraviolet rays, various factors such as the loss of oil and collagen can lead to skin aging, including wrinkles and sagging. The skin is structured into three layers, from the top down: the epidermis, dermis, and subcutaneous tissue. The stratum corneum, the top layer of the epidermis, is the outermost layer of the skin and is exposed to various external stimuli such as the outside air and ultraviolet rays. Skin problems and aging phenomena such as wrinkles, age spots, dullness, sagging, and rough skin can appear due to various factors such as a weakened immune system due to aging, changes in the external environment such as temperature, ultraviolet rays, external stimuli, and excessive intake of foreign substances.

Recent research has revealed that wrinkles are caused by a decrease in and denaturation of collagen, elastin, and other dermal matrix fibers. Matrix metalloproteinases (MMPs) have been identified as a factor inducing this.

MMPs is a general term for extracellular matrix degrading enzymes that have a zinc (II) ion at the active site. In addition, due to differences in structure and substrate specificity, MMPs are divided into subfamilies such as collagenase (MMP-1 and 13), gelatinase (MMP-2 and 9), and stromelysin (MMP-3 and 10). Of these, the main substrates for MMP-13 are fibrous collagens (types I, II, and III), gelatins, proteoglycans, cytokines, and other components of the extracellular matrix.

It is known that external stimuli such as ultraviolet rays increase the expression of MMPs such as MMP-13, which breaks down components of the skin's extracellular matrix (Patent Document 1, Non-Patent Document 1). For this reason, inhibiting the production of MMP-13 is effective in preventing wrinkles and improving the elasticity of the skin.

It is also known that cartilage degradation by MMPs is closely related to osteoarthritis (sometimes called degenerative osteoarthropathy, hypertrophic osteoarthropathy, etc.), and MMP-13 in particular has high decomposition activity against type II collagen and is considered to be an important matrix decomposition enzyme in cartilage degeneration in osteoarthritis (Non-Patent Document 2). It is known that in chondrocytes in joints, etc., expression of MMP-13 is enhanced by various inflammatory cytokines such as interleukin 1 beta (hereinafter also referred to as "IL1b") (Non-Patent Document 3). MMP-13 is also known to be overexpressed in rheumatoid arthritis and osteoarthritis. Therefore, suppressing the expression of MMP-13 is useful for protecting joints, such as preventing the reduction of collagen in joint cartilage, and is effective in preventing and improving diseases related to cartilage collagen degradation, including osteoarthritis.

In recent years, it has been proposed to use naturally derived materials as substances that suppress the expression of MMP-13, taking into consideration the safety of oral or transdermal ingestion. As such materials, for example, olive leaf extract is described in Non-Patent Document 1. Furthermore, Patent Document 3 describes agar, agarose, and/or agaro-oligosaccharides.

JP 2008-156349 A Special Publication No. 2011-528013 JP 2011-213707 A

Yoshiyuki Kimura, "Anti-carcinogenic effect of natural drugs by UVB irradiation and its mechanism of action", [online], [Retrieved November 21, 2014], Internet <URL: http://kaken.nii.ac.jp/pdf/2010/seika/jsps/16301/20590700seika.pdf> Masaru Okazaki et al., "Regulation of gene expression in cartilage degeneration in rheumatoid arthritis and osteoarthritis" Fukuoka Medical Journal 103(9): 175-181, 2012 Takehiko Matsushita, "The role of SIRT1 in chondrocytes", [online], [Retrieved November 21, 2014], Internet <URL: http://kaken.nii.ac.jp/pdf/2010/seika/mext/14501/21791393seika.pdf>

As described above, various materials are known to have an inhibitory effect on the expression of MMP-13.
However, the effects of these materials are not sufficiently strong, and there is a demand for technology that exerts a stronger effect of suppressing the expression of MMP-13.
Therefore, an object of the present invention is to provide a composition which is highly safe and effective in preventing skin aging, protecting joints, etc. by effectively suppressing the expression of MMP-13.

The present invention provides a cosmetic composition containing an amino sugar and a plant containing flavonoids.
The present invention provides a cosmetic composition containing an amino sugar and a plant containing flavonoids.
The present invention provides a joint protection composition containing an amino sugar and a plant containing flavonoids.
The present invention provides a composition containing an amino sugar and a plant containing flavonoids.

According to the present invention, a composition that is highly safe and effective in suppressing the expression of MMP-13 and is effective in at least one of the actions selected from the action of preventing skin aging and the action of protecting joints can be obtained. Furthermore, the composition of the present invention can contribute to the prevention and prevention of locomotive syndrome (musculoskeletal dysfunction syndrome), such as a decrease in the body's ability to balance, physical strength, and mobility, by preventing joint pain and limited range of motion of the joints.

FIG. 1 is a graph showing the results of evaluating the inhibitory effect on MMP-13 gene expression by the samples of Example 1 and Comparative Examples 1 and 2. FIG. 2 is a graph showing the results of evaluating the MMP-13 gene expression inhibitory effect of the samples of Example 2 and Comparative Examples 3 and 4. FIG. 3 is a graph showing the results of evaluating the inhibitory effect on MMP-13 gene expression by the samples of Example 3 and Comparative Examples 5 and 6.

The present invention will be described below based on its preferred embodiments. The "cosmetic composition", "beauty composition", "joint protection composition", and "composition" of the present invention all contain amino sugars and flavonoid-containing plants. The following description applies to any of the "cosmetic composition", "beauty composition", "joint protection composition", and "composition" unless otherwise specified.
Hereinafter, these compositions are collectively referred to as "the composition of the present invention."

(Amino sugar)
The amino sugar in the present invention means a compound having a structure in which a hydroxyl group of a sugar is replaced with an amino group, a derivative thereof, and a salt thereof. The amino sugar used in the present invention is not limited to a specific chemical structure, purity, properties, or preparation method, as long as it exerts the effect of solving the problems of the present invention when applied to mammals including humans in a state of a composition in combination with a flavonoid-containing plant (described later). Amino sugars relatively desirable in carrying out the present invention are those that exist in a free state in nature or as building blocks of polysaccharides, glycoproteins, glycolipids, etc., and specifically include, for example, glucosamine, mannosamine, neuraminic acid, galactosamine, and derivatives of these amino sugars. Examples of such derivatives that have been confirmed to exist in nature include acylated derivatives such as N-acetylated derivatives, sulfated derivatives such as N-sulfated derivatives and O-sulfated derivatives, and glycolylated derivatives such as N-glycolylated derivatives. These can be used alone or in combination. Of these amino sugars, glucosamines are particularly useful because they are highly effective in the present invention. The glucosamines referred to here include glucosamine (C ₆ H ₁₃ NO ₅ ) or its derivatives and salts thereof. In particular, among the glucosamines, acylated derivatives of glucosamine are preferred, and N-acetylglucosamine (C ₈ H ₁₅ NO ₆ ) is particularly preferred, in order to enhance the effects of the present invention. The amino sugars used in the present invention can be prepared by collecting polysaccharides or glycoproteins containing the above-mentioned amino sugars as structural units from, for example, mammals, fish, mollusks, arthropods, fungi, etc. in a conventional manner, hydrolyzing them under acidic conditions or by the action of an appropriate enzyme, and then subjecting them to a conventional method for separating and purifying sugars, such as chromatography. For example, N-acetylglucosamine can be prepared by partially hydrolyzing polysaccharide chitin prepared from the shells of crustaceans such as crabs and shrimps with an acid, and further decomposing it by the action of an enzyme such as chitinase. In addition, commercially available preparations (for example, commercially available glucosamine) can also be used.

(Plants containing flavonoids)
Flavonoids in flavonoid-containing plants include secondary plant metabolites derived from chalcones formed by polymerization of coumaric acid coenzyme A and malonyl coenzyme A. Examples of flavonoids include flavones, flavonols, flavanols, and proanthocyanidins.

Flavones include 2,3-didehydroflavan-4-one (also called 2-phenylchromone) and its derivatives, and preferred examples thereof include those in which a hydroxyl group or a methoxy group is bonded to one or more of the 3', 4', 5 and 7 positions of the 2,3-didehydroflavan-4-one skeleton.
Flavonols include 3-hydroxyflavone (also called 3-hydroxy-2-phenylchromen-4-one) and its derivatives, preferably those having a hydroxy group or a methoxy group bound to one or more of the 3', 4', 5 and 7 positions of the 3-hydroxyflavone skeleton. These may be glycosides in which a sugar is bound to the hydroxy group at the 3-position of the skeleton.
Flavanols include flavan-3-ol and flavan-3,4-diol, as well as derivatives thereof, preferably those in which a hydroxyl group or a methoxy group is bonded to one or more of the 3', 4', 5 and 7 positions of the flavan-3-ol or flavan-3,4-diol skeleton.
Examples of proanthocyanidins include compounds consisting of condensation polymers of flavan-3-ol or flavan-3,4-diol or derivatives thereof condensed via a 4-8 bond, with a degree of polymerization of 2 or more. Preferred examples include those in which a hydroxyl group or a methoxy group is bonded to one or more of the 3', 4', 5 and 7 positions of the constituent flavan-3-ol or flavan-3,4-diol unit.

Among the flavonoids used in the present invention, particularly preferred are flavans such as 5-hydroxy-7-methoxyflavone, 5,7-dimethoxyflavone, 5-hydroxy-3,7-dimethoxyflavone, 4',5,7-trimethoxyflavone, 5-hydroxy-3,7,4'-trimethoxyflavone, 3',4',5,7-tetramethoxyflavone, 5-hydroxy-3,7,3',4'-tetramethoxyflavone, and 3,5,7,3',4' -pentamethoxyflavone, 3,5,7-trimethoxyflavone, 3,5,7,4'-tetramethoxyflavone, etc., flavonols include quercetin, quercetin-3-rutinoside, etc., flavanols include catechin, epicatechin, etc., proanthocyanidins include polymerized catechin or epicatechin, etc., anthocyanidins include pelargonidin, cyanidin, delphinidin, aurantidin, luteolinidin, peonidin, malvidin, petunidin, europinidin, rosinidin, etc., and derivatives such as glycosides, esters, and polymers of the compounds listed above can also be used.

Flamonoid-containing plants used in the present invention include, for example, citrus fruits such as black ginger and Unshu mandarin oranges that contain flavans, Japanese knotweed and ginkgo trees that contain flavonols, tea that contains flavanols, and pine, grapes, apples, cranberries, and the like that contain proanthocyanidins. The composition of the present invention may contain only one of these flavonoid-containing plants, or may contain a mixture of two or more of them. In the present invention, it is particularly preferable to use one or more of flavonoid-containing plants selected from pine, black ginger, and Japanese knotweed. These preferred materials will be described below.

(Pine)
Examples of pines include plants of the genus Pinus and the family Pinaceae, and specifically include French maritime pine (Pinus Martima), New Zealand pine, Finland pine, Ekinata pine, Japanese larch, Japanese black pine, Japanese red pine, Japanese red pine, Japanese red pine, Korean pine, Japanese stone pine, Japanese white pine, Sugar pine, Ryukyu pine, Slash pine, Caribbean pine, Pinus rigida, Loblolly pine, Pinus grandiflorus, Taiwan red pine, Banks pine, Japanese white pine, White pine, and Aneda in the Quebec region of Canada. Among them, French maritime pine, New Zealand pine, and Finland pine are preferred, and French maritime pine is particularly preferred. French maritime pine refers to a maritime pine that grows in part of the Atlantic coast of southern France.

The parts of the pine tree that can be used include the bark, leaves, and pine cones, with the bark being particularly preferred. As for the pine tree, the bark of French maritime pine is particularly preferred. The bark of French maritime pine contains the flavonoid proanthocyanidin as its main component, as well as organic acids and other physiologically active components. This main component, proanthocyanidin, is known to have a strong antioxidant effect that removes active oxygen.

As for the pine, a processed product obtained by subjecting the pine plant body to one or more processes selected from crushing, extraction, and drying is preferred. A preferred processed product is a crushed product of the plant body obtained by crushing the plant body, or an extract obtained by extracting the plant body with water and/or an organic solvent either as is or after processing. The processing referred to here includes at least one treatment selected from heating, drying, crushing, and squeezing. These processed products can be used alone or in combination. Among them, it is preferable to use an extract, especially an extract of pine bark (hereinafter referred to as "pine bark extract"), from the viewpoint of the remarkable effect of the above-mentioned proanthocyanidins and other physiologically active ingredients when used and the stability of quality. Water or an organic solvent is selected as the solvent when obtaining an extract of the pine plant body. When water is used, warm water or hot water is used. The organic solvent used for extraction may be any commonly used organic solvent, and preferably used are 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, 1,1,1,2-tetrafluoroethane, 1,1,2-trichloroethene, etc. These water and organic solvents may be used alone or in combination of two or more. In particular, hot water, aqueous ethanol, aqueous propylene glycol, etc. are preferably used.

The pine bark used in the above extraction process is one that, when 10 parts by volume of a 50-80% by volume aqueous ethanol solution is added to 1 part by mass of pine bark and extracted at 80-85°C for 1 hour, gives a solid matter (hereinafter referred to as hydrous ethanol soluble components) of 7% by mass or more, preferably 13% by mass or more, and more preferably 13% by mass to 30% by mass, calculated as the dry mass. By using pine bark that contains 7% by mass or more of hydrous ethanol soluble components, it is possible to efficiently obtain the highly effective pine bark extract of the present invention.

The content of the hydrous ethanol soluble components in the pine bark can be measured, for example, as follows. First, 100 g of pine bark with a dry mass is powdered using, for example, a cutter, slicer, mill, or a crusher such as a mixer, juicer, blender, or mass colloider, and 1 L of 50 to 80% by volume hydrous ethanol is added to 100 g of this powder and heated under reflux at 80 to 85°C for 1 hour for extraction. After extraction, separation procedures such as centrifugation and filtration are performed to remove insoluble components to obtain an extract. From the viewpoint of accurately measuring the hydrous ethanol soluble components in the pine bark, it is preferable to perform a re-extraction process in which the extraction and separation procedures are repeated one or more times on the extraction residue. The obtained extract is freeze-dried or concentrated to dryness under reduced pressure to obtain a dried product, and the mass of the dried product is measured. The ratio of the mass of the dried product to the dry mass of the pine bark before extraction is then calculated to determine the content of the hydrous ethanol soluble components.

The above extraction is carried out by maintaining the bark at a predetermined temperature as necessary. From the viewpoint of extraction efficiency, it is preferable to increase the surface area per volume of the pine bark, and crushed bark is particularly suitable. The crushing process of the pine bark is not particularly limited, and for example, the crusher used in measuring the content of the hydrous ethanol soluble components in the pine bark described above can be used. To increase the crushing efficiency, the pine bark may be crushed by adding water or an organic solvent such as ethanol, methanol, or ethyl acetate. The size of the crushed bark is preferably 0.1 to 10 mm, more preferably 0.1 to 5 mm in small pieces.

As mentioned above, in the extraction, at least one of water and an organic solvent, i.e., water, an organic solvent, or a mixed solvent of water and an organic solvent (hereinafter, these are collectively referred to as the extraction solvent), is used. In order to increase the extraction efficiency, a higher extraction temperature is preferable. For example, when water is used, hot water extraction is preferably performed at 50 to 120°C, preferably 70 to 100°C. Hot water may be added to the pine bark, or water may be added to the pine bark and then heated. The extraction time can be appropriately determined depending on the extraction temperature. It is generally 10 minutes to 24 hours.

The amount of extraction solvent may be set taking into consideration the extraction efficiency. For example, when water, ethanol, or hydrous ethanol is used as the extraction solvent, the amount of extraction solvent is set to be preferably 3 to 100 parts by mass, more preferably 10 to 50 parts by mass, or preferably 3 to 100 parts by volume, more preferably 10 to 100 parts by volume per 1 part by mass of the dry mass of pine bark. Note that when crushing is performed by adding water and/or an organic solvent, the amount of extraction solvent added may be adjusted taking into consideration the amount of solvent used for crushing.

There are no particular limitations on the extraction method for obtaining an extract from pine plant matter, but examples include a heated extraction method and a supercritical fluid extraction method. For example, a water-ethanol mixed solvent (hydrous ethanol) with a mass ratio of water to ethanol of 1:1 to 1:9 is used as the extraction solvent for crushed pine bark in an amount 1 to 20 times the amount of pine bark, and the mixture is refluxed at 70 to 85°C while stirring for 0.5 to 6 hours. If reflux is not used, the extraction efficiency can also be increased by, for example, using the above mixed solvent once to perform heated extraction, recovering the supernatant by filtration, etc., and adding the above mixed solvent to the residue again and heating it.

Supercritical fluid extraction is a method of extraction using a supercritical fluid, which is a fluid in a state that exceeds 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.

In the supercritical fluid extraction method, an extraction process is performed in which the target component is extracted with a supercritical fluid, and a separation process is performed in which the target component is separated from the supercritical fluid. In the separation process, extraction and separation may be performed by pressure change, temperature change, or using an adsorbent or absorbent.

Supercritical fluid extraction may also be performed using the entrainer addition method. In this method, for example, ethanol, propanol, n-hexane, acetone, toluene, or other aliphatic lower alcohols, aliphatic hydrocarbons, aromatic hydrocarbons, or ketones are added to the extraction fluid at about 2 to 20 W/V %, and supercritical fluid extraction is performed using this fluid, dramatically increasing the solubility of the target extract in the extraction solvent or enhancing the separation selectivity, making it an efficient method for obtaining a pine bark extract.

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.

In addition, in order to obtain an extract from pine plant bodies, in addition to the above-mentioned extraction methods, extraction methods such as the liquid carbon dioxide batch method, the liquid carbon dioxide reflux method, and the supercritical carbon dioxide reflux method may also be used.

The extraction method for obtaining an extract of pine plant matter may be a combination of multiple extraction methods. By combining multiple extraction methods, it is possible to obtain extracts with a variety of compositions.

For extraction, any extraction apparatus may be used, such as a batch, semi-continuous, or continuous type.

Extracts from pine plants, particularly bark, are preferably purified by ultrafiltration or a column method or batch method using an adsorbent carrier (Diaion HP-20, Sephadex-LH20, chitin, etc.) from the standpoint of safety. In addition, they may be concentrated or dried by methods such as vacuum concentration and freeze-drying to form liquid, paste, powder (extract powder), fine particles, or granules, as necessary. Pine processed products such as pine bark extracts are preferably in powder, fine particles, or granule form. The pine bark extract may be sterilized, and may be further concentrated, dried, and powdered after sterilization. Sterilization, concentration, drying, and powdering are performed by methods commonly used in the art, and among them, freeze-drying, vacuum drying, spray drying, drum drying, shelf drying, and microwave drying are preferred as drying methods. Regarding filtration, in order to complete the filtration process in a short time, it is preferable to add a filter aid, such as diatomaceous earth, to the extract before filtering. In this case, there are no particular restrictions on the amount of filter aid added, and it is added so that the amount of diatomaceous earth in the extract is, for example, about 0.001 g/mL to 0.1 g/mL.

When pine is used in the present invention, it is preferable that the pine contains proanthocyanidin as one of the main components of flavonoids. As described above, proanthocyanidin refers to a group of compounds consisting of condensation polymers with a degree of polymerization of 2 or more, whose constituent units are at least one selected from flavan-3-ol, flavan-3,4-diol, and derivatives thereof. Proanthocyanidin is a powerful antioxidant produced by plants, and is contained intensively in the leaves, bark, fruit skin, and seeds of plants. Note that this proanthocyanidin is a substance that cannot be produced in the human body. It is more preferable that the processed pine product contains proanthocyanidin in an amount of 10% by mass or more, 20% by mass or more, 30% by mass or more, or 40% by mass or more.

As the proanthocyanidin contained in processed pine products, proanthocyanidins containing a large amount of condensation polymers with a low degree of polymerization are preferable. As the condensation polymers with a low degree of polymerization, condensation polymers with a degree of polymerization of 2 to 30 (dimers to 30-mers) are preferable, condensation polymers with a degree of polymerization of 2 to 10 (dimers to 10-mers) are more preferable, and condensation polymers with a degree of polymerization of 2 to 4 (dimers to tetramers) are even more preferable. Proanthocyanidins of condensation polymers with a degree of polymerization of 2 to 4 (dimers to tetramers) are particularly easily absorbed in the body. In this specification, polymers with a degree of polymerization of 2 to 4 are called oligomeric proanthocyanidins (hereinafter referred to as "OPCs").

The pine processed products used in the present invention should contain OPC in a proportion of 10% by mass or more, preferably 20% by mass or more, and more preferably 30% by mass or more.

Pine plants, especially pine bark, are rich in OPCs among proanthocyanidins, and are therefore preferably used as a source of proanthocyanidins. However, pine plants, especially pine bark, contain not only proanthocyanidins but also other components. For example, when a processed pine product is an extract, various organic acids and flavonoids are also extracted by the process of extracting proanthocyanidins from the plant. Such organic acids and flavonoids are components other than proanthocyanidins. Examples of such organic acids and flavonoids include caffeic acid, fumaric acid, quinic acid, gallic acid, vanillic acid, ferulic acid, and catechins.

(Black ginger)
The black ginger (Kaempferia Parviflora) used in the present invention is a plant of the Zingiberaceae and Kaempferia genus that grows naturally in Southeast Asia, and has been reported to promote sexual energy, revitalize, lower blood sugar levels, restore physical strength, improve the digestive system, and alleviate vaginal discharge, hemorrhoids, nausea, stomatitis, joint pain, stomach pain, etc. Thus, black ginger is a natural plant that has been taken by humans for a long time and has a proven track record, and is highly safe.

The parts of black ginger used in the present invention include roots, leaves, stems, flowers, or branches. Among them, rhizomes are preferred. The black ginger used in the present invention includes processed products obtained by subjecting black ginger to at least one treatment selected from heating, drying, crushing, squeezing, and extraction. Specific examples of processed products of black ginger include dried products obtained by subjecting black ginger to drying treatment, dried powder obtained by subjecting black ginger to drying treatment and crushing treatment, cut products obtained by cutting black ginger or powder obtained by drying the cut products, squeezed juice, and extracts. Here, the extract means an extract obtained by extracting the above black ginger or a processed product thereof with a solvent, a diluted solution or concentrated solution thereof, or a dried product thereof and a powder thereof. In the present invention, the processed product includes all of these.

The dried powder obtained by drying and crushing black ginger can be obtained, for example, by washing the black ginger, slicing it, drying it in the sun or using a dryer, and then crushing the processed product obtained as is or cut into an appropriate shape and size using a crushing device. A wide range of commonly used crushing devices can be used, but for example, a crushing device consisting of a raw material hopper, crusher, classifier, product holder, etc. can be used.

Black ginger extract is obtained by extracting black ginger or a processed product thereof with a solvent. The extract is preferably obtained by subjecting a processed product of black ginger that has been subjected to a drying process to an extraction process, and more preferably by subjecting a processed product that has been subjected to a drying process after slicing to an extraction process. Examples of solvents used for extraction include 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. It is also effective to use only water. Among these, it is preferable to use water alone, ethanol alone, or a mixed solvent of water and ethanol (so-called hydrous ethanol). When hydrous ethanol is used, the ethanol concentration in the hydrous ethanol is preferably 20% by mass or more and 80% by mass or less, and more preferably 40% by mass or more and 60% by mass or less.

When using a mixed solvent as the solvent, for example, a mixture of acetone/water (2/8 to 8/2, volume ratio) or a mixture of ethanol/water (2/8 to 8/2, volume ratio) can be used. In the case of ethanol/water, it is preferable to add 2 to 20 times the mass of the black ginger rhizome with the solvent and extract for about 10 minutes to 48 hours at room temperature or under heat.

There are no particular limitations on the extraction method used, but from the standpoint of safety and convenience, it is preferable to perform the extraction under as mild conditions as possible. For example, the raw plant part or its dried matter is crushed, crushed or shredded, and 2 to 20 times the mass of the solvent is added to this, and extraction is performed under any conditions such as standing, shaking, stirring or refluxing at a temperature ranging from 0°C to the reflux temperature of the solvent for 10 minutes to 48 hours. After the extraction operation, a separation operation such as filtration or centrifugation is performed to remove insoluble matter. An extract is obtained by diluting and concentrating the extract as necessary. Furthermore, the same operation may be repeated to extract insoluble matter, and the extract may be used together with the previous extract. These extracts may be further purified by a purification method commonly used by those skilled in the art before use.

The obtained extract can be used as it is or after concentration in the form of a liquid, concentrate, or paste, or can be further dried to form a dried product. Drying can be carried out by methods commonly used by those skilled in the art, such as spray drying, freeze drying, vacuum drying, and fluidized bed drying. Furthermore, the dried products obtained by the above methods can be powdered using well-known methods for use. Processed products such as black ginger extracts are preferably in the form of powder, fine grains, or granules.

The black ginger used in the present invention preferably contains one or more flavonoids selected from 5-hydroxy-7-methoxyflavone, 5,7-dimethoxyflavone, 5-hydroxy-3,7-dimethoxyflavone, 4',5,7-trimethoxyflavone, 5-hydroxy-3,7,4'-trimethoxyflavone, 3',4',5,7-tetramethoxyflavone, 5-hydroxy-3,7,3',4'-tetramethoxyflavone, 3,5,7,3',4'-pentamethoxyflavone, 3,5,7-trimethoxyflavone, 3,5,7,4'-tetramethoxyflavone, etc. In particular, the black ginger used in the present invention preferably contains 0.01 to 50% by mass of 5,7-dimethoxyflavone, more preferably 0.1 to 30% by mass, and even more preferably 0.5 to 20% by mass.

(Japanese Knotweed)
Giant knotweed is a perennial plant of the Polygonaceae family, with the scientific name Polygonum sachalinense.

The parts of Japanese knotweed that are used include the leaves, stems, buds, roots, and rhizomes, with the buds being particularly preferred. The buds are also called young buds or new shoots.

As the Japanese knotweed, a processed product obtained by subjecting the Japanese knotweed plant body to one or more processes selected from crushing, extraction, and drying is preferred. Preferred processed products include a crushed product of the plant body obtained by crushing the plant body, or an extract obtained by extracting the plant body as it is or after processing with water and/or an organic solvent. The processing referred to here includes at least one treatment selected from heating, drying, crushing, and squeezing. These processed products can be used alone or in combination. Among them, it is preferable to use an extract, particularly an extract of Japanese knotweed buds, from the viewpoint of the remarkable effect during use and the stability of quality. Water or an organic solvent is selected as the solvent when obtaining an extract of the Japanese knotweed plant body. When water is used, warm water or hot water is used. The organic solvent used for extraction may be any commonly used organic solvent, and preferably used are 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, 1,1,1,2-tetrafluoroethane, 1,1,2-trichloroethene, etc. These water and organic solvents may be used alone or in combination of two or more kinds. In particular, water, aqueous ethanol, aqueous propylene glycol, etc. are preferably used.

As described above, in the extraction, at least one of water and an organic solvent is used, i.e., water, an organic solvent, or a mixed solvent of water and an organic solvent (hereinafter, these are collectively referred to as the extraction solvent). In order to increase the extraction efficiency, a higher extraction temperature is preferable. For example, when water is used, hot water extraction is preferably performed at 50 to 120°C, preferably 70 to 100°C. Alternatively, hot water may be added to the Japanese knotweed, or water may be added to the Japanese knotweed and then heated. The extraction time can be appropriately determined depending on the extraction temperature. It is generally 10 minutes to 24 hours.

Extracts from the plant body of Japanese knotweed are preferably purified by ultrafiltration or a column method or batch method using an adsorbent carrier (Diaion HP-20, Sephadex-LH20, chitin, etc.) from the viewpoint of safety. In addition, they may be concentrated or dried by a method such as vacuum concentration or freeze-drying to be in a liquid, paste, powder (extract powder), fine granules, or granules, if necessary. Processed products of Japanese knotweed, such as Japanese knotweed extracts, are preferably in a powder, fine granule, or granule form. The Japanese knotweed extract may be sterilized, and may be further concentrated, dried, and powdered after sterilization. Sterilization, concentration, drying, and powderization are performed by methods commonly used in the technical field, and among them, freeze-drying, vacuum drying, spray drying, drum drying, shelf drying, and microwave drying are preferred as drying methods.

When using Japanese Knotweed in the present invention, the processed product preferably contains quercetin-3-rutinoside as a flavonoid. In particular, the Japanese Knotweed used in the present invention preferably contains 0.1% by mass or more of quercetin-3-rutinoside, more preferably 1% by mass or more, and even more preferably 10% by mass or more.

The compositions of the present invention are preferably intended for animals, particularly mammals, and most particularly humans.

The composition of the present invention may consist essentially of plants containing amino sugars and flavonoids. "Consisting essentially of plants containing amino sugars and flavonoids" refers to a case where the composition does not contain any components other than those that are inevitably mixed in during the processing of the plants containing amino sugars and flavonoids, specifically, a case where the total mass of the plants containing amino sugars and flavonoids in the composition of the present invention is 95% by mass or more. However, the composition of the present invention can contain various other components described below in addition to the plants containing amino sugars and flavonoids, as long as the effects of the present invention are not impaired.

(Content)
From the viewpoint of further enhancing the effects of the present invention, when the composition of the present invention is orally ingested, the content of the amino sugar in the composition of the present invention is preferably 10 to 2000 mg, more preferably 100 to 1000 mg, and even more preferably 200 to 800 mg. When the composition of the present invention is orally ingested, the content of the amino sugar in the composition of the present invention is preferably 1 to 80% by mass, more preferably 5 to 50% by mass, and even more preferably 10 to 30% by mass.

From the viewpoint of further enhancing the effect of the present invention, when the composition of the present invention is orally ingested, the content of the flavonoid-containing plant in the composition of the present invention is preferably 1 to 500 mg, more preferably 5 to 200 mg, even more preferably 10 to 100 mg, and particularly preferably 20 to 80 mg, in terms of dry mass. Furthermore, when the composition of the present invention is orally ingested, the content of the flavonoid-containing plant in the composition of the present invention is preferably 0.001 to 50% by mass, more preferably 0.01 to 40% by mass, and even more preferably 0.1 to 30% by mass, in terms of dry mass.

From the viewpoint of further enhancing the effects of the present invention, when the composition of the present invention is taken parenterally, the content of the amino sugar in the composition of the present invention is preferably 0.000001 to 100 mg, more preferably 0.00001 to 10 mg, even more preferably 0.00001 to 1 mg, and particularly preferably 0.0001 to 0.1 mg. Furthermore, when the composition of the present invention is taken parenterally, the content of the amino sugar in the composition of the present invention is preferably 0.000001 to 20% by mass, more preferably 0.00001 to 1% by mass, and even more preferably 0.0001 to 0.1% by mass.

In order to further enhance the effects of the present invention, when the composition of the present invention is taken parenterally, the content of the flavonoid-containing plant in the composition of the present invention is preferably 0.000001 to 100 mg, more preferably 0.00001 to 10 mg, even more preferably 0.00001 to 1 mg, and particularly preferably 0.0001 to 0.1 mg, in terms of dry mass. When the composition of the present invention is taken parenterally, the content of the flavonoid-containing plant in the composition of the present invention is preferably 0.000001 to 20% by mass, more preferably 0.00001 to 1% by mass, and even more preferably 0.0001 to 0.1% by mass, in terms of dry mass.

From the viewpoint of further enhancing the effects of the present invention, whether the composition of the present invention is taken orally or parenterally, the mass ratio of the amino sugar to the dry mass of the flavonoid-containing plant (former:latter) is preferably 1:0.0001-1000, more preferably 1:0.001-100, even more preferably 1:0.01-10, and particularly preferably 1:0.01-1.

The composition of the present invention can be used for cosmetics, in which case it becomes a cosmetic composition. The cosmetic composition of the present invention is administered parenterally, such as by transdermal administration.

In addition to the amino sugars and flavonoid-containing plants, the cosmetic composition of the present invention may contain other ingredients that are commonly used in quasi-drugs or cosmetics, to the extent that the effects of the cosmetics are not impaired. Examples of such ingredients include water, other medicinal ingredients, other oils, bases, moisturizing agents, powders, gelling agents, thickeners, surfactants, emulsifiers, anti-inflammatory agents, antioxidants, pH adjusters, chelating agents, thickeners, pigments, fragrances, preservatives, skin aging prevention and improvement agents such as collagen, hair growth inhibitors, preservatives, fungicides, ultraviolet absorbers, absorption promoters, and ultraviolet dispersion agents. Examples of other medicinal ingredients include active oxygen removers, antioxidants, anti-inflammatory analgesics, antihistamines, antipruritics, disinfectants, vitamins, hormones, carotenoids such as vitamin A, vitamin B, ascorbic acid, vitamin E, and derivatives or salts thereof.

The cosmetic composition of the present invention can be prepared by mixing with other ingredients other than amino sugars and flavonoid-containing plants by a commonly used method, and can be used as a medicine, quasi-drug, cosmetic, or toiletry product. The specific form of the cosmetic is not particularly limited, but may be a skin cosmetic such as a cream, cosmetic cream, milky lotion, skin toner, beauty essence, lotion, face wash, cleansing, body soap, body shampoo, pack, soap, foundation, lipstick, lip gloss, blusher, water-based ointment, oil-based ointment, patch, gel, bath agent, etc.; a hair cosmetic such as a shampoo, rinse, conditioner, treatment, styling agent, hair tonic, hair cream, hair styling agent, hair growth agent, soap, perm/color pre-treatment agent, etc.; or a product (so-called miscellaneous goods) that is similar in formulation to a cosmetic and is not subject to the Pharmaceutical Affairs Law. It can also be administered locally for a long time by a method such as holding and absorbing it in a carrier or crosslinking agent such as a patch or gel and applying it to the local area. It can also be administered as eye drops or nasal drops.

The composition of the present invention can be used for cosmetic purposes or joint protection purposes, in which case it becomes a cosmetic composition or a joint protection composition. The administration form of these compositions may be parenteral or oral. When the cosmetic composition or joint protection composition of the present invention is administered parenterally, the examples of ingredients other than the amino sugar and flavonoid-containing plants of these compositions and the dosage form may be the same as those of the cosmetic composition of the present invention described above. In addition, the examples of ingredients other than the amino sugar and flavonoid-containing plants of the cosmetic composition and joint protection composition of the present invention when administered orally may be the same as those of the oral composition described below. The parenteral administration method for these compositions includes intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, etc. Intraperitoneal administration, which is one type of subcutaneous administration, may also be used. In addition, as enteral administration, it may be administered directly to internal organs using an injection or the like.

When the composition of the present invention is intended to be taken orally, the oral composition may contain, in addition to the amino sugars and flavonoid-containing plants, other commonly used ingredients within a range that does not impair the effects of the oral composition. Examples of such ingredients include various excipients, binders, gloss agents, lubricants, stabilizers, diluents, bulking agents, thickeners, emulsifiers, antioxidants, pH adjusters, colorants, flavorings, additives, etc. The content of other ingredients can be appropriately selected depending on the form of the composition of the present invention, etc.

The composition of the present invention can be formulated into various dosage forms by known formulation methods, adding pharma- ceutically acceptable base materials or carriers as necessary. Examples of dosage forms include powder, granules, tablets, rods, plates, blocks, solids, rounds, liquids, pastes, capsules such as hard capsules and soft capsules, caplets, tablets, gels, chewable tablets, syrups, sticks, etc.

The composition of the present invention is derived from natural products and can be safely taken parenterally and/or orally. As will be clear from the description of the examples below, the composition of the present invention contains amino sugars and flavonoid-containing plants, and thus has the effect of suppressing MMP-13 gene expression. In particular, the composition of the present invention can suppress the production of MMP-13 in various tissues and cells by taking it parenterally and/or orally. For example, by suppressing the production of MMP-13 in the skin, it is possible to prevent a decrease in the amount of collagen in the skin and prevent or improve skin aging, such as preventing wrinkles and improving elasticity. Furthermore, the composition of the present invention is useful for protecting joints, such as reducing collagen in cartilage of joints, by suppressing the production of MMP-13 in chondrocytes of joints, and is effective in preventing and improving diseases related to cartilage collagen degradation, such as osteoarthritis, rheumatoid arthritis, and osteoarthritis. The composition of the present invention is useful as a cosmetic composition or oral composition that can exert any one or more of these effects, and is also useful as a beauty composition, joint protection composition, etc., by exerting any one or more of these effects.

In particular, the composition of the present invention is useful for protecting cartilage in joints and the like and maintaining a healthy state by inhibiting the degradation of cartilage collagen through the suppression of the expression of MMP-13. Protecting cartilage prevents and improves joint pain, prevents restrictions on joint range of motion, and facilitates smooth joint movement. Joint pain and restrictions on range of motion are said to cause a decrease in balance ability, physical strength, and mobility, and are the cause of so-called locomotive syndrome (musculoskeletal function syndrome). Therefore, the composition of the present invention can prevent and prevent locomotive syndrome (musculoskeletal function syndrome) associated with joint pain and restrictions on joint range of motion, prevent a decrease in balance ability, physical strength, and mobility, and contribute to maintaining posture (preventing the back from becoming rounded) and keeping muscles flexible and youthful.

The present invention will be described in more detail below with reference to examples. However, the scope of the present invention is not limited to these examples. Unless otherwise specified, "%" means "mass %" and "parts" means "mass parts". Of the following Examples 1 to 3, Example 1 is an example in which an amino sugar is combined with pine, and Example 2 is an example in which an amino sugar is combined with black ginger. Example 3 is an example in which an amino sugar is combined with Japanese knotweed.

[Example 1, Comparative Examples 1 and 2, and Reference Examples 1 and 2]
Various solutions (1) to (4) were prepared according to the <Preparation of test solutions I> below, and the obtained test solutions were subjected to the <MMP-13 expression test I> below.

<Preparation of test solution I>
As the liquid (1), 10% FBS-DMEM medium was used.
(2) Amino sugar-containing solution N-acetylglucosamine (manufactured by Nacalai Tesque, Inc., hereinafter also abbreviated as "NAG") was used as the amino sugar. This was dissolved in 10% FBS-DMEM medium to a concentration of 10 mg/mL, and after 1.5 hours of end-over-end stirring, the solution was filtered and further serially diluted with the same medium to prepare 10% FBS-DMEM media containing NAG at concentrations 3 and 6 times the desired final concentration (see Table 1 below).
(3) Pine-containing liquid Pine bark extract (trade name "Flavangenol" (registered trademark, manufactured by Toyo Shinyaku Co., Ltd.), powder form, containing 30% by mass or more of OPC) was used as the pine. This was dissolved in 10% FBS-DMEM medium to a concentration of 10 mg/mL, and after 1.5 hours of end-over-end stirring, the solution was filtered and further diluted stepwise with the same medium to prepare 10% FBS-DMEM media containing the pine bark extract at concentrations 3 and 6 times the desired final concentration (see Table 1 below).
(4) Expression inducer-containing solution An aqueous solution of interleukin-1 beta (hereinafter also referred to as "IL1b") (manufactured by Pepro Tech, IL1b concentration 10 μg/mL, hereinafter also referred to as "IL1b stock solution") was used as an MMP-13 gene expression inducer. 3 μL of this IL1b stock solution was added to 15 mL of 10% FBS-DMEM medium to prepare an expression inducer-containing solution.

(Pine bark hydrous ethanol soluble fraction)
The hydrous ethanol soluble content of the pine bark used to prepare the flavangenol was measured according to the following method and found to be 13.2% by mass. First, 2 kg of dried pine bark was ground to a size of 1 to 5 mm using a mill to obtain a ground product, and 10 g of the ground product was taken out to obtain a sample. 100 mL of an aqueous solution containing 80% by volume (v/v) of ethanol was added to the sample, and reflux extraction was performed at 80°C for 1 hour. Then, the sample was filtered and filtrate 1 was collected. The filtration residue was further extracted with an aqueous ethanol solution and filtered in the same manner as above to obtain a filtrate twice (referred to as filtrates 2 and 3, respectively). The obtained filtrates 1 to 3 were combined to obtain an extract, which was then concentrated to dryness under reduced pressure to obtain a dried product (hereinafter referred to as hydrous ethanol soluble component). The mass of the dried product was measured and found to be 1.32 g.

(Method of producing Flavangenol)
Flavangenol was also prepared according to the following method.
First, 0.5 L of water was added to 100 g of pine bark, and reflux extraction was performed at 95° C. or higher for 1 hour. Then, 0.5 L of filtrate was collected by filtration (referred to as filtrate 4). Furthermore, 0.5 L of water was added to the insoluble matter after filtration, and reflux extraction was performed in the same manner as above, and 0.5 L of filtrate was obtained by filtration (referred to as filtrate 5). Filtrate 4 and filtrate 5 were combined to obtain 1 L of pine bark extract. The filtrate was concentrated and dried, and the obtained solid was used as flavangenol.

<MMP-13 Expression Test I>
Normal human articular chondrocytes (NHAC, Lonza) were seeded in a 24-well plate at 5.4×10 ⁴ cells/well and pre-cultured overnight. The above solutions (1), (2), (3) and (4) were added to the cells in the order listed in the amounts in Table 1 below. After addition, the cells were cultured at 37°C in a 5% CO ₂ concentration CO ₂ incubator for 18 hours. Total RNA was extracted from the cultured cells using Sepasol RNA (Nacalai Tesque) according to the attached protocol. Using the obtained total RNA as a template, cDNA was synthesized using a cDNA creation kit (QIAGEN, product name: Quantitect Reverse Transcription Kit). The obtained cDNA-containing reaction solution was diluted 20-fold with Nuclease-free water (QIAGEN), and 2 μL of the diluted solution was mixed with 8 μL of the reaction solution included in the RT-PCR kit (QIAGEN, product name: Rotor-Gene Green PCR Kit), and PCR was performed under the following conditions. The above 8 μL reaction solution was a mixture of 5 μL of 2x Rotor-Gene SYBR Green PCR Master Mix, 1 μL of Primer, and 2 μL of Nuclease-free water. PCR was performed using Rotor-Gene Q (QIAGEN), and the amount of PCR product was measured by monitoring in real time. In the above RT-PCR, GAPDH (housekeeping gene) was used as an internal standard. Specifically, the amount of PCR product of MMP-13/the amount of PCR product of GAPDH was used as the gene expression value of MMP-13. The experiment was performed in triplicate, and the average gene expression value was calculated. For the gene expression values of Example 1, Comparative Examples 1 and 2, and Reference Example 1, the ratio was calculated as a relative value of gene expression when the average value of the gene expression value of MMP-13 in Reference Example 2 was set to 1. The obtained average values of the relative values are shown in FIG.

(RT-PCR conditions)
Hold: 95℃・5min
Cycle: 95℃・5sec → 60℃・10sec →72℃・10sec × 45cycle
Melt: 72- 99℃

(Primer)
・GAPDH: Hs_GAPDH_2_SG QuantiTect Primer Assay (QT01192646)
・MMP-13: Hs_MMP13_1_SG QuantiTect Primer Assay (QT00001764)

As is clear from the results shown in Figure 1, Example 1, which combines amino sugars and pine, significantly suppressed the gene expression of MMP-13 induced by the addition of IL1b. In contrast, Comparative Examples 1 and 2, which combined amino sugars and pine, respectively, did not show a significant inhibitory effect. From these results, it is clear that by combining amino sugars and pine, the composition of the present invention exerts an excellent inhibitory effect on MMP-13 expression. It is also clear that the composition of the present invention can effectively prevent a decrease in the amount of collagen accumulated in various connective tissues such as cartilage and skin through this inhibition of MMP-13 expression, and is effective for beauty and joint protection, etc.

[Example 2, Comparative Examples 3 and 4, and Reference Examples 5 and 6]
Various solutions (i) to (iv) were prepared according to the procedure described in <Preparation of test solutions II> below, and the obtained test solutions were subjected to the <MMP-13 expression test II> below.

<Preparation of test solution II>
As the liquid (i), 10% FBS-DMEM medium was used.
(ii) Amino sugar-containing liquid The procedure was the same as in (2) of <Preparation of test solution I>, except that the concentration of the prepared amino sugar-containing liquid was four times the desired final concentration (see Table 2 below).
(iii) Black ginger-containing liquid Black ginger was used as the powdered extract (hereinafter, also referred to as "BG") produced by the <Production method of black ginger extract> described below. The obtained powdered extract was dissolved in 10% FBS-DMEM medium to a concentration of 10 mg/mL, and after 1.5 hours of end-over-end stirring, the mixture was filtered and further diluted stepwise with the same medium to prepare a 10% FBS-DMEM medium containing black ginger extract at a concentration 4 times the desired final concentration (see Table 2 below).
(iv) Expression inducer-containing solution The same procedure as in (4) in <Preparation of test solution I> was repeated except that 2.67 μL of IL1b stock solution (10 μg/mL) was added to 10 mL of 10% FBS-DMEM.

(Manufacturing method of black ginger extract)
The black ginger rhizome was cut into small pieces and then dried to obtain chip-like rhizome. 300 g of the rhizome chips were weighed and placed in a triangular flask with 3 L of 60% aqueous ethanol. The mixture was left to stand at room temperature for 24 hours while stirring several times during the process. After filtration under reduced pressure, the remaining chips were immersed again in 3 L of 60% aqueous ethanol and left to stand at room temperature for 24 hours to perform extraction. This was filtered under reduced pressure to obtain a second extract. The first and second extracts were combined and concentrated under reduced pressure to about 1/6 of their volume to obtain a stock solution. The resulting stock solution was dried to obtain a powdered extract. The content of 5,7-dimethoxyflavone in the obtained extract was 6% by mass.

<MMP-13 Expression Test II>
Gene expression values were obtained in the same manner as in <MMP-13 Expression Test I>, except that instead of adding the above solutions (1) to (4), the above solutions (i), (ii), (iii) and (iv) were added to normal human articular chondrocytes in the amounts shown in Table 2 below in the order shown. For the gene expression values of Example 2, Comparative Examples 3 and 4, and Reference Example 3, the ratios to the average expression value of Reference Example 4, which was set to 1, were calculated as relative gene expression values. The average relative values obtained are shown in FIG. 2.

As is clear from the results shown in Figure 2, Example 2, which combines amino sugars and black ginger, significantly suppressed the gene expression of MMP-13 induced by the addition of IL1b. In contrast, no significant inhibitory effect was observed in Comparative Examples 3 and 4, in which amino sugars and black ginger were each formulated alone. From these results, it is clear that by combining amino sugars and black ginger, the composition of the present invention exerts an excellent inhibitory effect on MMP-13 expression. It is also clear that the composition of the present invention can effectively prevent a decrease in the amount of collagen accumulated in various connective tissues such as cartilage and skin through this inhibition of MMP-13 expression, and is effective for beauty and joint protection, etc.

[Example 3, Comparative Examples 5 and 6, and Reference Examples 7 and 8]
Various solutions (a) to (d) were prepared according to the following <Preparation of test solutions III>, and the obtained test solutions were subjected to the following <MMP-13 expression test III>.

<Preparation of Test Solution III>
As the liquid (a), a 10% FBS-DMEM medium was used.
(b) Amino sugar-containing liquid The same procedure as in (2) of <Preparation of test solution I> was followed, except that the concentration of the prepared amino sugar-containing liquid was four times the desired final concentration (see Table 3 below).
(c)
As the Japanese Knotweed, a powdered extract produced by the method described below (Production method of Japanese Knotweed extract) was used. This was dissolved in 10% FBS-DMEM medium so that the concentration of Japanese Knotweed extract was 10 mg/mL, and after 1.5 hours of end-over-end stirring, the mixture was filtered and further diluted stepwise with the same medium to prepare a 10% FBS-DMEM medium containing the Japanese Knotweed extract at a concentration 4 times the desired final concentration (see Table 3 below).
(d) Expression inducer-containing solution This was prepared by adding 1.335 μL of IL1b stock solution (10 μg/mL) to 5 mL of 10% FBS-DMEM.

(Method of manufacturing Japanese knotweed extract)
The young shoots of Japanese knotweed were extracted with hot water and purified to obtain an extract. This extract was freeze-dried and made into a powder. The extract thus obtained contained quercetin-3-rutinoside.

<MMP-13 Expression Test III>
Gene expression values were obtained in the same manner as in <MMP-13 Expression Test I>, except that instead of adding the above solutions (1) to (4), the above solutions (a), (b), (c) and (d) were added to normal human articular chondrocytes in the amounts and in the order shown in Table 3 below. For the gene expression values of Example 3, Comparative Examples 5 and 6, and Reference Example 5, the ratios to the average gene expression value of Reference Example 6, which was taken as 1, were used as relative gene expression values. The average values of the obtained relative values are shown in FIG.

As is clear from the results shown in Figure 3, Example 3, which combines amino sugars and Japanese knotweed, significantly suppressed the gene expression of MMP-13 induced by the addition of IL1b. In contrast, Comparative Examples 5 and 6, which combined amino sugars and Japanese knotweed, respectively, showed a significantly inferior effect of suppressing gene expression of MMP-13. From these results, it is clear that by combining amino sugars and Japanese knotweed, the composition of the present invention exerts a synergistic effect of suppressing MMP-13 expression. It is also clear that the composition of the present invention can effectively prevent a decrease in the amount of collagen accumulated in various connective tissues such as cartilage and skin through this suppression of MMP-13 expression, and is effective for beauty and joint protection, etc.

(Formulation Example 1: Lotion)
The composition of the present invention in the form of a lotion was prepared by mixing 0.005 parts by mass of pine bark extract, 0.005 parts by mass of N-acetylglucosamine, 10 parts by mass of glycerin, 3 parts by mass of diglycerin, 12 parts by mass of 1,3-butylene glycol, 3 parts by mass of pentylene glycol, 0.1 parts by mass of sodium hyaluronate, 0.01 parts by mass of citric acid, 0.02 parts by mass of sodium citrate, 0.1 parts by mass of xanthan gum, 0.15 parts by mass of methylparaben, 0.2 parts by mass of carbomer, 0.03 parts by mass of sodium hydroxide, and the remainder of the mixture being 100 parts by mass.

(Formulation example 2: Shampoo)
The composition of the present invention in the form of a shampoo was prepared by mixing 0.001 part by mass of pine bark extract, 0.001 part by mass of N-acetylglucosamine, 7.5 parts by mass of sodium laureth sulfate, 4.2 parts by mass of cocamidopropyl betaine, 3 parts by mass of cocamide DEA, 0.1 part by mass of 1,3-butylene glycol, 0.225 parts by mass of polyquaternium-10, 0.15 parts by mass of citric acid, 0.05 part by mass of sodium citrate, 0.9 parts by mass of phenoxyethanol, and the remainder, water, making the total 100 parts by mass.

(Formulation Example 3: Soap)
The composition of the present invention in the form of a soap was prepared by mixing and solidifying 1 part by mass of black ginger powder, 1 part by mass of N-acetylglucosamine, 2 parts by mass of glycerin, 1 part by mass of olive oil, 0.1 part by mass of EDTA-4 sodium, 0.2 parts by mass of tetrasodium etidronate, and the remainder of the soap base, making the total 100 parts by mass.

(Formulation Example 4: Milk Lotion)
The composition of the present invention was prepared in the form of an emulsion by mixing 0.05 parts by mass of Japanese knotweed extract, 0.001 parts by mass of N-acetylglucosamine, 3 parts by mass of sucrose fatty acid ester, 12 parts by mass of glycerin, 6 parts by mass of squalane, 24 parts by mass of dimethyl silicone oil, 1 part by mass of polypropylene glycol, 0.06 parts by mass of thickener, 0.2 parts by mass of phenoxyethanol, 5 parts by mass of ethanol, 0.01 parts by mass of sodium hydroxide, and the remainder, purified water, making the total 100 parts by mass.

(Formulation Example 5: Cosmetic cream)
Based on a total of 100 parts by mass, the following ingredients are added: Black ginger extract 0.03 parts by mass, N-acetylglucosamine 0.1 parts by mass, squalane 15.0 parts by mass, octyldodecyl myristate 4.0 parts by mass, hydrogenated soybean phospholipid 0.2 parts by mass, butyl alcohol 2.4 parts by mass, hardened oil 1.5 parts by mass, stearic acid 1.5 parts by mass, lipophilic glycerin monostearate 1.5 parts by mass, polyglyceryl monostearate 0.5 parts by mass, behenyl alcohol 0.8 parts by mass, polyglyceryl monomyristate 0.7 parts by mass, white beeswax 0.3 parts by mass, d-δ-tocopherol 0.1 parts by mass, methylparaben 0.3 parts by mass, C10-30 alkyl-modified carboxyvinyl polymer 0.2 parts by mass, carboxyvinyl polymer 0.1 parts by mass, 1,3-butanediol A composition of the present invention in the form of a cosmetic cream was prepared by mixing 18.0 parts by mass of glycerin, 0.1 parts by mass of sodium hydroxide, and the remainder being purified water.

(Formulation Example 6: Pack Agent)
The composition of the present invention was prepared in the form of a pack by mixing 0.1 part by mass of Japanese knotweed extract, 0.01 part by mass of N-acetylglucosamine, 20.0 parts by mass of polyvinyl alcohol, 5.0 parts by mass of glycerin, 20.0 parts by mass of ethanol, 6.0 parts by mass of kaolin, 0.2 parts by mass of preservative, 0.1 part by mass of fragrance, and the remainder being purified water, making the total 100 parts by mass.

(Formulation Example 7: Tablets)
The composition of the present invention was prepared in the form of tablets by mixing and compressing 5 parts by weight of black ginger powder, 10 parts by weight of N-acetylglucosamine, 5 parts by weight of vitamin _B1 , 12 parts by weight of vitamin _B6 , 4 parts by weight of calcium stearate, 30 parts by weight of maltose, and the remainder (total 100 parts by weight).

(Formulation Example 8: Granules)
The composition of the present invention was prepared in the form of granules by mixing and granulating 5 parts by mass of pine bark extract, 8 parts by mass of Japanese knotweed extract powder, 5 parts by mass of N-acetylglucosamine, 5 parts by mass of black ginger extract, 10 parts by mass of lactose, 1 part by mass of calcium stearate, and the remainder of crystalline cellulose, making the total amount 100 parts by mass.

(Formulation Example 9: Capsules)
The composition of the present invention was prepared in the form of a capsule by mixing 10 parts by mass of Japanese knotweed extract, 10 parts by mass of N-acetylglucosamine, 10 parts by mass of black ginger extract, 8 parts by mass of lecithin, and the remainder of olive oil (total of 100 parts by mass). The mixture was used as the content liquid, and this was encapsulated in a capsule shell.

(Formulation Example 10: Liquid) With the total amount being 100 parts by weight, pine bark extract 1 part by weight, N-acetylglucosamine 1 part by weight, vitamin B1 ₁ part by weight, fructose glucose liquid sugar 10 parts by weight, citric acid 1 part by weight,
A composition of the present invention in the form of a liquid was prepared by mixing 0.02 parts by mass of sodium benzoate, 2 parts by mass of a flavor preparation, 0.05 parts by mass of sucralose, 0.03 parts by mass of acesulfame potassium, and the remainder, purified water.

Claims (3)

A composition for protecting or maintaining joints or cartilage containing N-acetylglucosamine and Japanese knotweed (excluding the following (A) to (D): (A) a composition containing proteoglycan, N-acetylglucosamine, Japanese knotweed, and Japanese knotweed; (B) a composition containing N-acetylglucosamine, Japanese knotweed, chondroitin, hyaluronic acid, and collagen; (C) a composition containing non-denatured type II collagen, hyaluronic acid, N-acetylglucosamine, Japanese knotweed, and Chinese knotweed; and (D) a jelly containing N-acetylglucosamine, Japanese knotweed, and Chinese knotweed). A cosmetic composition containing N-acetylglucosamine and Japanese Knotweed (excluding the following (A) to (D): (A) a composition containing proteoglycan, N-acetylglucosamine, Japanese Knotweed, and Japanese Knotweed; (B) a composition containing N-acetylglucosamine, Japanese Knotweed, chondroitin, hyaluronic acid, and collagen; (C) a composition containing non-denatured type II collagen, hyaluronic acid, N-acetylglucosamine, Japanese Knotweed, and Chinese Knotweed; and (D) a jelly containing N-acetylglucosamine, Japanese Knotweed, and Chinese Knotweed). An oral composition containing N-acetylglucosamine and Japanese Knotweed (excluding the following (A) to (D): (A) a composition containing proteoglycan, N-acetylglucosamine, Japanese Knotweed, and Japanese Knotweed; (B) a composition containing N-acetylglucosamine, Japanese Knotweed, chondroitin, hyaluronic acid, and collagen; (C) a composition containing non-denatured type II collagen, hyaluronic acid, N-acetylglucosamine, Japanese Knotweed, and Chinese Knotweed; and (D) a jelly containing N-acetylglucosamine, Japanese Knotweed, and Chinese Knotweed).