JP2013224326A - Obesity dissolution agent, lipolysis promoter, cyclic amp-phosphodiesterase activity inhibitor, and lipolysis promoter for rat epididymis adipocyte - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide highly safe natural antioxidants of which the raw material can be easily available having at least any one of a superoxide dismutase (SOD)-like action, an inhibitory action of membrane lipid peroxidation by singlet oxygen, a hydrogen peroxide eliminating action, and a radical eliminating effect and the like.SOLUTION: An obesity dissolution agent, a lipolysis promoter, a cyclic AMP-phosphodiesterase activity inhibitor, and a lipolysis promoter for rat epididymis adipocytes contain an extract of black ginger, and have at least any one of an obesity dissolution action, a lipolysis promoting action, a cyclic AMP-phosphodiesterase activity inhibiting action, and a lipolysis promoting action for a rat epididymis adipocyte lipolysis.

Description

  The present invention relates to an obesity-resolving agent, a lipolysis promoter, a cyclic AMP-phosphodiesterase activity inhibitor containing a black ginger extract, and a lipolysis promoter of rat epididymal fat cells.

In recent years, active oxygen has attracted attention as a factor that oxidizes biological components, and its adverse effect on living organisms has become a problem. Reactive oxygen is generated in the process of energy metabolism in living cells, and superoxide [that is, superoxide anion (· O ₂ ⁻ ) generated by one-electron reduction of oxygen molecules, hydrogen peroxide (H ₂ O ₂ ), Singlet oxygen ( ¹ O ₂ ), hydroxy radical (.OH)], and the like. Such active oxygen is essential for the phagocytic sterilization mechanism and plays an important role in removing viruses and cancer cells.
However, excessive generation of the active oxygen attacks in vivo molecules constituting membranes and tissues in the living body and induces various diseases. Superoxide, which is produced in vivo and is a starting material for other active oxygen, is usually eliminated sequentially by the catalytic action of superoxide dismutase (SOD) contained in cells. However, when the production of superoxide is excessive or when the action of SOD is reduced, the superoxide is insufficiently erased and the superoxide concentration becomes high. This is one of the causes of tissue damage such as rheumatoid arthritis, Behcet's disease, myocardial infarction, stroke, cataract, spots, freckles, wrinkles, diabetes, arteriosclerosis, shoulder stiffness, coldness, skin aging, etc. It is considered.
Among these, since the skin is directly affected by environmental factors such as ultraviolet rays, superoxide is likely to be generated. Therefore, when the superoxide concentration is increased, biological tissues such as collagen are decomposed, denatured, or crosslinked. Or oxidize fats and oils to produce lipid peroxides that damage cells, forming skin wrinkles, causing skin aging, inflammation, and skin pigmentation (See Non-Patent Document 1).
Therefore, attempts have been made to obtain active oxygen scavenging substances, radical scavenging substances, hydrogen peroxide scavenging substances, etc. from natural products advantageous in terms of safety, and extracts of Brassica Brassica plants (see Patent Document 1), Efficacy has been confirmed for extracts of plants belonging to the genus Ryukyubekei (see Patent Document 2), extracts of scallops (see Patent Document 3), extracts of sulfur (see Patent Document 4), and the like.

The epidermis and dermis of the skin are composed of epidermal cells, fibroblasts, and an extracellular matrix such as collagen that is outside these cells and supports the skin structure. In young skin, moisture retention, flexibility, elasticity and the like are ensured by maintaining the constancy of the interaction between these skin tissues, and the skin is maintained in a fresh and fresh state.
However, when there is an influence of certain external factors such as ultraviolet irradiation, drastic air drying, excessive skin washing, etc., or when aging progresses, the production amount of collagen, which is the main component of the extracellular matrix, is reduced. Reduces elasticity due to cross-linking. As a result, the skin's moisturizing function and elasticity are lowered, and the horny layer begins to exfoliate abnormally, so that the skin loses its tension and gloss, and exhibits aging symptoms such as roughness and wrinkles.
Thus, changes accompanying skin aging, that is, wrinkles, dullness, disappearance of texture, decrease in elasticity, and the like are associated with reduction or modification of dermal matrix components such as collagen.

In recent years, as a factor for inducing reduction or denaturation of dermal matrix components, there is degradation and reconstruction of a group of proteolytic enzymes called matrix metalloproteases (hereinafter sometimes referred to as “MMPs”).
The MMPs are classified into (1) collagenase group (MMP-1, MMP-8 and MMP-13), (2) gelatinase group (MMP-2 and MMP-9), (1) due to differences in primary structure and substrate specificity. 3) Stromelysin group (MMP-3 and MMP-10), (4) Membrane-bound matrix metalloprotease group (MMP-14, MMP-15, MMP-16, and MMP-17), (5) Others ( MMP-7, MMP-11, and MMP-12) (see Patent Document 5).
Among the MMPs, MMP-2 and MMP-9 are classified into gelatinase group, but not only gelatin of extracellular matrix but also type IV collagen, type V collagen, type I collagen which are main components of skin basement membrane. It is known as an enzyme that cleaves various substrates such as laminin, fibronectin, proteoglycan and elastin. In addition, the expression is greatly increased by the irradiation of ultraviolet rays, which contributes to decrease or denaturation of collagen by ultraviolet rays, and is considered to be a major factor such as the formation of wrinkles on the skin.

  In addition, there are various causes and onset mechanisms of inflammatory diseases such as contact dermatitis (rash), psoriasis, pemphigus vulgaris, and various other skin diseases with rough skin. As its cause, hexosaminidase (histamine) release inhibitory action and nitric oxide (NO) production inhibitory action are known.

  The histamine release is a phenomenon in which histamine in mast cells is released to the outside of the cells, and the released histamine causes an inflammatory reaction. For this reason, attempts have been made to prevent or treat allergic diseases and inflammatory diseases with substances that inhibit or suppress histamine release. As such histamine release inhibitors, for example, tranilast, sodium cromoglycate, baicalin, baicalein, promethazine hydrochloride and the like have been used. However, all of these substances have side effects and have problems in terms of safety.

Nitric oxide (NO) is a nitrogen oxide that causes air pollution, acid rain, and the like. In recent years, nitric oxide (NO) is a physiologically active substance that exhibits various functions in vivo, such as vascular endothelium-derived relaxing factor (EDRF), neurotransmitters, microorganisms in biological defense, and tumor cell injury factors. It has been reported. As a physiologically active substance, it is known that nitric oxide produced from macrophages protects against bacterial and viral infections. However, when a large amount of nitric oxide produced from the macrophages is biosynthesized, it is not non-toxic to the living body and causes destruction of the self-tissue, which may cause pathological conditions such as worsening inflammation, rheumatism and diabetes. . Also, large amounts of biosynthesized nitric oxide can cause vascular smooth muscle relaxation and excessive permeability, and can cause endotoxin shock due to a significant decrease in blood pressure.
In such inflammatory diseases, it is important to suppress excessive production of nitric oxide (NO). Examples of herbal medicines having an inhibitory effect on the production of nitric oxide include, for example, rosemary extract, carnosol, carnosic acid, coffee bean extract, primrose extract, auren extract, buckwheat extract, licorice extract, Inu rose extract, Senkyu extract, Tonin extract, Peonies extract, Yakuinin extract, Red grape extract (refer to Patent Document 6), Tang Dynasty, Cod root bark, Japanese continuation, Carbago, Toko Grass roots, half-branches, spikelets, flower buds (see Non-Patent Document 2), and the like have been reported.

Inflammation is a complex reaction that exhibits symptoms such as redness, edema, fever, pain, and dysfunction. Microscopically, it consists of common reactions such as blood vessel reaction that causes plasma leakage, leukocyte infiltration, tissue destruction by inflammatory cells, and also causes systemic reactions involving the central nervous system such as fever and hyperalgesia There is. Prostaglandins play an important role in such individual reactions of inflammation, and it has become clear that cyclooxygenase-2, an inducible cyclooxygenase, is mainly involved in the production of prostaglandins during this inflammation. Yes.
For this reason, many cyclooxygenase activity inhibitors represented by aspirin are used for the purpose of preventing and preventing inflammatory reactions (see Non-Patent Document 3). As plant-derived cyclooxygenase activity inhibitors, α-mangostin and γ-mangostin in mangosteen peel extract are known (see Patent Document 7). Further, as a compound having an inhibitory action on cyclooxygenase-2 activity, 2-phenyl-1,2-benzisoselenazol-3 (2H) -one, a salt thereof, or a hydrate thereof is known (patent) Reference 8).

In addition, it is considered effective for prevention of obesity to suppress the action of cyclic AMP phosphodiesterase, which is an enzyme that degrades cyclic AMP involved in promoting fat metabolism. In fact, it is known that when the action of cyclic AMP phosphodiesterase is suppressed, the concentration of intracellular cyclic AMP increases, lipid metabolism becomes active, and obesity is resolved.
Therefore, attempts have been made to extract substances having a cyclic AMP phosphodiesterase inhibitory action from natural products. For example, Fuji tea extract (see Patent Document 9), maple plant extract (see Patent Document 10), Etc. have been reported.

  Many steroid hormones are expressed in the form of molecules secreted from production organs and bind to receptors to exert their effects. In the case of male hormones collectively called androgens, for example, testosterone enters the cells of target organs and becomes testosterone. After being reduced to 5α-dihydrotestosterone (5α-DHT) by 5α-reductase, it binds to the receptor and develops an action as an androgen.

The androgen is an important hormone, but if it acts excessively, it can be affected by various factors such as androgenetic alopecia, hirsutism, seborrhea, acne (such as acne), benign prostatic hyperplasia, prostate tumor, premature male sexuality, etc. Induces undesirable symptoms. Therefore, conventionally, in order to improve these various symptoms, a method of suppressing the action of excess androgen, specifically, by inhibiting the action of testosterone 5α-reductase that reduces testosterone to active 5α-DHT, There have been proposed a method for suppressing the generation of active 5α-DHT and a method for preventing the expression of androgen activity by inhibiting the binding of 5α-DHT generated from testosterone to the receptor.
As such a plant extract having an action of inhibiting the binding between 5α-DHT and its receptor, for example, an extract of at least one of majito and cuta is known (see Patent Document 11).

  Hair repeats growth and loss according to a periodic hair cycle (hair cycle) consisting of a growth period, a regression period, and a rest period. Of these hair cycles, the stage at which new hair follicles are formed from the resting stage to the growing stage is considered to be the most important for hair growth. In this stage, the proliferation and differentiation of hair follicle epithelial cells are considered. It is believed that the dermal papilla cells play an important role. The dermal papilla cell is a cell located inside the hair follicle epithelial cell composed of outer root sheath cells and matrix cells in the vicinity of the hair root, and located in the root portion of the hair root wrapped in the basement membrane. It plays an important role in the growth and differentiation of hair follicle epithelial cells and the formation of hair, such as by acting on epithelial cells to promote their growth (see Non-Patent Document 4). The dermal papilla cell plays the most important role in the growth and differentiation of hair follicle epithelial cells and the formation of hair. The cultured dermal papilla cell is contacted with a target substance, and the presence or absence of the proliferation activity of the cell. A method for testing the hair-growth effect of the target substance by specifying strength is proposed (see Patent Document 12). As herbal medicines having such a dermal papilla cell proliferation promoting action, for example, an oyster extract, an auren extract, an anemone extract of anemone, etc. are known (see Patent Document 13 and Patent Document 14).

  The development of whitening agents so far has been focused on tyrosinase, which is the rate-limiting enzyme for melanin production. Recently, production from epidermal keratinocytes increases after UV-UVB irradiation and activates pigment cells (melanocytes). As cytokines, α-melanocyte-stimulating hormone (α-MSH), endothelin-1 (ET-1), nitric oxide (NO), basic fibroblast growth factor (bFGF), granulocyte / macrophage / colony stimulating factor (GM) -CSF), stem cell factor (SCF), etc. have been reported, and the development of substances that suppress the melanin production and lead to the whitening effect by blocking the information transmission system in which these are involved has become active. ing. As extracts of herbal medicines that inhibit the action of endothelin-1 (ET-1) on pigment cells (melanocytes), for example, chamomile extract and altea extract have been reported (see Non-Patent Document 5).

  However, until now, however, it is a natural product that is easy to obtain, inexpensive, and highly safe, and does not adversely affect the quality of the additive in terms of taste, smell, feeling of use, etc. Antioxidants, anti-aging agents, anti-inflammatory agents, hair-restoring agents, anti-obesity agents, and whitening agents that can be widely used in cosmetics and beauty foods and drinks have not been provided yet, and their prompt provision is strongly demanded. The current situation is.

JP 2003-81848 A JP 2005-29483 A JP 2006-321730 A JP 2007-8902 A JP 2000-344672 A JP 2002-87975 A JP 2002-47180 A JP 2000-16935 A JP 2003-12532 A JP 2003-1113068 A JP 2002-241297 A JP-A-10-229978 JP-A-9-208431 Japanese Patent Laid-Open No. 11-12134

“Fragrance Journal” Special Issue No. 14, p156, 1995 “Wakan Journal of Pharmaceutical Sciences”, Vol. 15, p. 302-303, issued in 1998 Pharmacology Atlas (P184), translated by Takehiko Fukuhara, Bunkodo “Trends Genet”, 1992, Vol. 8, pp. 56-61 “Fragrance Journal”, Vol. 28, no. 9, p65-71, published in 2000

An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the present invention first has at least one of superoxide dismutase (SOD) -like action, membrane lipid peroxidation inhibiting action by singlet oxygen, hydrogen peroxide scavenging action, and radical scavenging action. It is an object to provide a natural antioxidant that has high properties and is readily available as a raw material.
In addition, the present invention secondly provides at least a matrix metalloproteinase-2 (MMP-2) activity inhibitory action, a matrix metalloproteinase-9 (MMP-9) mRNA expression increase inhibitory action, and an epidermal keratinocyte proliferation promoting action. An object of the present invention is to provide a natural anti-aging agent that has any of these, has high safety, and is readily available for raw materials.
In the third aspect of the present invention, at least one of a hexosaminidase release inhibitory action, a nitric oxide (NO) production inhibitory action, a hyaluronidase activity inhibitory action, and a cyclooxygenase-2 (COX-2) activity inhibitory action is provided. It is an object of the present invention to provide a natural anti-inflammatory agent that has high safety and is easily available.
In addition, a fourth object of the present invention is to provide a natural hair restorer that has at least one of an androgen receptor antagonistic action and a hair papillary cell proliferation promoting action, is highly safe, and is readily available for raw materials. And
In addition, the present invention fifthly has at least one of a cyclic AMP-phosphodiesterase activity inhibitory action and a lipolysis promoting action using rat epididymal fat cells, is highly safe, and easy to obtain raw materials. The object is to provide a natural anti-obesity agent.
In addition, the present invention sixthly provides a natural whitening agent having at least one of endothelin-1 mRNA expression increase inhibitory action and SCF mRNA expression increase inhibitory action, high safety, and easy acquisition of raw materials. With the goal.
In addition, the present invention seventhly includes at least one of the antioxidant, the anti-aging agent, the anti-inflammatory agent, the hair-restoring agent, the anti-obesity agent, and the whitening agent of the present invention as an active ingredient. It aims at providing the cosmetics and the food / beverage products for beauty.

  In order to solve the above-mentioned problems, it is a natural product that is easy to obtain, inexpensive, and highly safe, and does not adversely affect the quality of the object to be added in terms of taste, smell, feeling of use, etc. Extraction of black ginger as a result of our extensive research on antioxidants, anti-aging agents, anti-inflammatory agents, hair restorers, anti-obesity agents, and whitening agents that can be widely used in cosmetics and beauty foods and drinks The product has at least one of (1) excellent superoxide dismutase (SOD) -like action, membrane lipid peroxidation inhibiting action by singlet oxygen, hydrogen peroxide scavenging action, and radical scavenging action, and an antioxidant. (2) excellent matrix metalloproteinase-2 (MMP-2) activity inhibitory action, matrix metalloproteinase-9 (MMP-9) mRNA expression increase inhibitory action, and epidermis It has at least one of cell-proliferation-promoting action and is useful as an anti-aging agent, (3) excellent hexosaminidase release inhibitory action, nitric oxide (NO) production inhibitory action, hyaluronidase activity inhibitory action, And / or cyclooxygenase-2 (COX-2) activity inhibitory action, and is useful as an anti-inflammatory agent, and (4) at least one of an excellent androgen receptor antagonistic action and hair papillary cell proliferation promoting action. It has at least one of the excellent cyclic AMP-phosphodiesterase activity inhibitory action and the lipolysis promoting action using rat epididymal fat cells, and is useful as an anti-obesity agent. (6) Excellent endothelin-1 mRNA expression increase inhibitory action and SCF mRNA expression At least one of inhibitory effect to be useful as a whitening agent, and findings, respectively.

The present invention is based on the above findings of the present inventors, and means for solving the above problems are as follows. That is,
<1> An antioxidant containing black ginger extract.
<2> The antioxidant according to <1>, which has at least one of a superoxide dismutase (SOD) -like action, a membrane lipid peroxidation inhibiting action by singlet oxygen, a hydrogen peroxide scavenging action, and a radical scavenging action It is.
<3> An anti-aging agent characterized by containing an extract of black ginger.
<4><3> having at least one of matrix metalloproteinase-2 (MMP-2) activity inhibitory action, matrix metalloprotease-9 (MMP-9) mRNA expression increase inhibitory action, and epidermal keratinocyte proliferation promoting action > Is an anti-aging agent.
<5> An anti-inflammatory agent characterized by containing an extract of black ginger.
<6> The above <5>, which has at least one of hexosaminidase release inhibitory action, nitric oxide (NO) production inhibitory action, hyaluronidase activity inhibitory action, and cyclooxygenase-2 (COX-2) activity inhibitory action It is an anti-inflammatory agent.
<7> A hair restorer comprising an extract of black ginger.
<8> The hair restorer according to <7>, wherein the hair restorer has at least one of an androgen receptor antagonistic action and a dermal papilla cell proliferation promoting action.
<9> An anti-obesity agent characterized by containing an extract of black ginger.
<10> The anti-obesity agent according to <9>, which has at least one of a cyclic AMP-phosphodiesterase activity inhibitory action and a lipolysis promoting action using rat accessory testicular fat cells.
<11> A whitening agent characterized by containing an extract of black ginger.
<12> The whitening agent according to <11>, wherein the whitening agent has at least one of an endothelin-1 mRNA expression increase suppressing action and an SCF mRNA expression increase suppressing action.
<13> A cosmetic comprising the black ginger extract according to any one of <1> to <12> as an active ingredient.
<14> A cosmetic food or drink comprising the black ginger extract according to any one of <1> to <12> as an active ingredient.

According to the antioxidant of the present invention, an excellent superoxide dismutase (SOD) -like action, a membrane lipid peroxidation inhibiting action by singlet oxygen, a hydrogen peroxide scavenging action, and a radical scavenging action Can prevent or improve skin aging.
According to the anti-aging agent of the present invention, excellent matrix metalloproteinase-2 (MMP-2) activity inhibitory action, matrix metalloproteinase-9 (MMP-9) mRNA expression increase inhibitory action, and epidermal keratinocyte proliferation promoting action Through at least one of them, it is possible to prevent or improve skin wrinkles and skin elasticity reduction.
According to the anti-inflammatory agent of the present invention, at least one of an excellent hexosaminidase release inhibitory action, nitric oxide (NO) production inhibitory action, hyaluronidase activity inhibitory action, and cyclooxygenase-2 (COX-2) activity inhibitory action Through this, inflammatory diseases can be prevented or ameliorated.
The hair restorer of the present invention is extremely useful for the prevention or treatment of androgenetic alopecia through at least one of an excellent androgen receptor antagonistic action and hair papillary cell proliferation promoting action.
The anti-obesity agent of the present invention can prevent or prevent obesity through at least one of an excellent cyclic AMP-phosphodiesterase activity inhibiting action and a lipolysis promoting action using rat epididymal fat cells.
The whitening agent of the present invention can exert a whitening effect through at least one of an excellent endothelin-1 mRNA expression increase suppressing action and SCF mRNA expression increase suppressing action.
In addition, the antioxidant, anti-aging agent, anti-inflammatory agent, hair restorer, anti-obesity agent, and whitening agent of the present invention are natural extracts that are excellent in safety, in terms of taste, smell, feeling of use, etc. Since it does not adversely affect the quality of the object to be added, it is suitable for blending into cosmetics or adding to cosmetic foods and drinks.

(Antioxidants, anti-aging agents, anti-inflammatory agents, hair restorers, anti-obesity agents, and whitening agents)
The antioxidant, anti-aging agent, anti-inflammatory agent, hair restorer, anti-obesity agent, and whitening agent of the present invention contain an extract of black ginger and further contain other components as necessary. Become.

The antioxidant has at least one of a superoxide dismutase (SOD) -like action, a membrane lipid peroxidation inhibiting action by singlet oxygen, a hydrogen peroxide scavenging action, and a radical scavenging action as an antioxidant action. Yes.
The anti-aging agent has, as an anti-aging action, a matrix metalloproteinase-2 (MMP-2) activity inhibitory action, a matrix metalloproteinase-9 (MMP-9) mRNA expression increase inhibitory action, and an epidermal keratinocyte proliferation promoting action. Have at least one.
The anti-inflammatory agent has at least one of hexosaminidase release inhibitory action, nitric oxide (NO) production inhibitory action, hyaluronidase activity inhibitory action, and cyclooxygenase-2 (COX-2) activity inhibitory action as an anti-inflammatory action. have.
The hair restorer has at least one of androgen receptor antagonistic action and hair papilla cell proliferation promoting action as a hair restoring action.
The anti-obesity agent has at least one of a cyclic AMP-phosphodiesterase activity inhibitory action and a lipolysis promoting action using rat epididymal fat cells as an anti-obesity action.
The whitening agent has at least one of endothelin-1 mRNA expression increase suppression action and SCF mRNA expression increase suppression action as a whitening action.
The details of the substance having at least one of antioxidative action, anti-aging action, anti-inflammatory action, hair-growth action, anti-obesity action, and whitening action in the black ginger extract are unclear, but extraction of the black ginger It has not been known so far that products have these excellent actions and are useful as antioxidants, anti-aging agents, anti-inflammatory agents, hair restorers, anti-obesity agents, and whitening agents, These are new findings from the inventors' diligent research.

The black ginger (black ginger) is a plant belonging to the genus Van turmeric, and the scientific name is Kaempferia parviflora .
The black ginger is distributed in Southeast Asia such as Thailand and is easily available from this region.

  The black ginger extract can be easily obtained by a method generally used for plant extraction. The black ginger extract includes a black ginger extract, a dried product obtained by drying a diluted solution of the extract, and a crude product or a purified product thereof.

  There is no restriction | limiting in particular as an extraction raw material of the said black ginger, According to the objective, it can select suitably, For example, the rhizome part of a black ginger etc. can be used.

  The rhizome part of black ginger, which is the extraction raw material, can be obtained by drying or pulverizing as it is using a crusher and subjecting it to solvent extraction. Drying may be performed in the sun or using a commonly used dryer. The black ginger may be used as an extraction raw material after pretreatment such as degreasing with a nonpolar solvent such as hexane or benzene. In addition, the extraction process by the polar solvent of black ginger can be performed efficiently by performing pre-processing, such as degreasing.

As the solvent used for the extraction, it is preferable to use water, a hydrophilic organic solvent, or a mixed solvent thereof at a temperature from room temperature to the boiling point of the solvent.
Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as water that has been subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, and the like. The water that can be used as the extraction solvent includes purified water, hot water, ion exchange water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

Examples of the hydrophilic organic solvent include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene glycol, propylene glycol, Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycerin, and a mixed solvent of these hydrophilic organic solvents and water can be used.
In addition, when using the mixed solvent of the said water and a hydrophilic organic solvent, in the case of a lower alcohol, 1 mass part-90 mass parts with respect to 10 mass parts of water, and in the case of a lower aliphatic ketone, 10 masses of water. It is preferable to add 1 to 40 parts by mass with respect to parts. In the case of polyhydric alcohol, it is preferable to add 1 part by mass to 90 parts by mass with respect to 10 parts by mass of water.

  In the present invention, a special extraction method is used for extracting a substance having at least one of an antioxidant action, an anti-aging action, an anti-inflammatory action, a hair-growth action, an anti-obesity action, and a whitening action from black ginger as an extraction raw material. It is not necessary to employ, and extraction can be performed using any extraction device at room temperature or under reflux heating.

  Specifically, in the treatment tank filled with the extraction solvent, the rhizome part of black ginger as an extraction raw material is added, and further, with occasional stirring as necessary, left for 30 minutes to 2 hours to leave soluble components. After elution, the solid matter is removed by filtration, and the extract is obtained by evaporating the extraction solvent from the obtained extract and drying. The amount of the extraction solvent is usually 5 to 15 times (mass ratio) of the extraction raw material. The extraction conditions are usually about 1 to 4 hours at 50 to 95 ° C. when water is used as the extraction solvent. Moreover, when the mixed solvent of water and ethanol is used as an extraction solvent, it is normally 30 minutes-about 4 hours at 40 to 80 degreeC. It should be noted that the extract obtained by extraction with a solvent, as long as the extraction solvent is highly safe, as it is, the antioxidant, anti-aging agent, anti-inflammatory agent, hair restorer, anti-obesity agent, and It can be used as a whitening agent.

  The obtained black ginger extract is diluted, concentrated, dried, purified, etc. according to a conventional method in order to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a crudely purified product or purified product thereof. You may perform the process of.

  The obtained black ginger extract can be used as it is as an antioxidant, anti-aging agent, anti-inflammatory agent, hair restorer, anti-obesity agent, and whitening agent. It is easier to use. In obtaining a dried extract, a carrier such as dextrin or cyclodextrin may be added to improve hygroscopicity. Further, since the black ginger extract has a characteristic odor, it can be purified for the purpose of decolorization, deodorization and the like within a range that does not cause a decrease in its physiological activity. In addition, since it is not used in a large amount when added to a food or drink for beauty, there is no practical problem even if it is not purified. The purification can be performed, for example, by activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, or the like.

  The black ginger extract obtained as described above has superoxide dismutase (SOD) -like action, membrane lipid peroxidation inhibiting action by singlet oxygen, hydrogen peroxide scavenging action, radical scavenging action, matrix metalloprotease-2 (MMP-2) activity inhibitory action, matrix metalloproteinase-9 (MMP-9) mRNA expression increase inhibitory action, epidermal keratinocyte proliferation promoting action, hexosaminidase release inhibitory action, nitric oxide (NO) production inhibitory action , Hyaluronidase activity inhibitory action, cyclooxygenase-2 (COX-2) activity inhibitory action, androgen receptor antagonistic action, hair papilla cell proliferation promoting action, cyclic AMP-phosphodiesterase activity inhibiting action, promotion of lipolysis using rat accessory testicular fat cells Action, endothelin-1mRN It has at least one of an expression increase inhibitory action and an SCF mRNA expression increase inhibitory action, and based on these actions, the antioxidant, anti-aging agent, anti-inflammatory agent, hair restorer, anti-obesity agent of the present invention, And can be used as a whitening agent.

The antioxidant action of the antioxidant of the present invention is based on at least one of superoxide dismutase (SOD) -like action, membrane lipid peroxidation inhibiting action by singlet oxygen, hydrogen peroxide scavenging action, and radical scavenging action. Demonstrated.
The anti-aging action of the anti-aging agent of the present invention includes matrix metalloproteinase-2 (MMP-2) activity inhibitory action, matrix metalloproteinase-9 (MMP-9) mRNA expression increase inhibitory action, and epidermal keratinocyte proliferation promoting action. Based on at least one of the above.
The anti-inflammatory action in the anti-inflammatory agent of the present invention is at least one of hexosaminidase release inhibitory action, nitric oxide (NO) production inhibitory action, hyaluronidase activity inhibitory action, and cyclooxygenase-2 (COX-2) activity inhibitory action. Demonstrated based on
The hair-restoring action in the hair-restoring agent of the present invention is exhibited based on at least one of androgen receptor antagonistic action and hair papilla cell proliferation promoting action.
The anti-obesity action in the anti-obesity agent of the present invention is exhibited based on at least one of a cyclic AMP-phosphodiesterase activity inhibitory action and a lipolysis promoting action using rat epididymal fat cells.
The whitening effect of the whitening agent of the present invention is exhibited based on at least one of endothelin-1 mRNA expression increase suppression action and SCF mRNA expression increase suppression action.
The black ginger extract of the present invention has at least one of excellent antioxidant action, anti-aging action, anti-inflammatory action, hair growth action, anti-obesity action, and whitening action, and when applied to the skin and scalp. Since it is excellent in use feeling and safety, it is particularly suitable for blending into the cosmetic of the present invention described below.
Further, the black ginger extract of the present invention has at least one of excellent antioxidant action, anti-aging action, anti-inflammatory action, hair growth action, anti-obesity action, and whitening action and is digested in the digestive tract. Since it has been confirmed that this is not the case, it is particularly suitable for blending into the cosmetic food and drink of the present invention described below.

(Cosmetics)
The cosmetic of the present invention contains at least one of the antioxidant, the anti-aging agent, the anti-inflammatory agent, the hair restorer, the anti-obesity agent, and the whitening agent of the present invention as an active ingredient. Further, it contains other components appropriately selected as necessary.

  Here, the use of the cosmetic is not particularly limited and can be appropriately selected from various uses. For example, ointments, creams, emulsions, lotions, packs, jellies, lip balms, lipsticks, bath preparations, astringents, Hair tonics, hair creams, hair liquids, shampoos, pomades, rinses, etc.

  The amount of the antioxidant, the anti-aging agent, the anti-inflammatory agent, the hair-restoring agent, the anti-obesity agent, or the whitening agent of the present invention in the entire cosmetic is the type of cosmetic and the physiological condition of the extract. Although it can adjust suitably by activity etc., it is 0.0001 mass%-10 mass% in conversion to the said black ginger extract, and 0.001 mass%-1 mass% are more preferable.

If necessary, the cosmetic may further contain various main ingredients and auxiliaries usually used in the production of cosmetics and other components within a range that does not impair the object and effects of the present invention.
The other components are not particularly limited as long as they do not interfere with the antioxidant action, anti-aging action, anti-inflammatory action, hair growth action, anti-obesity action, and whitening action of the present invention, and are appropriately selected according to the purpose. For example, astringents, bactericides, antibacterial agents, ultraviolet absorbers, humectants, cell activators, fats and oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants , Fragrances, and the like. When used in combination with the black ginger extract, these components may act synergistically to provide superior effects that are normally expected.

  The cosmetic of the present invention has high safety when used on the skin and scalp, has an excellent superoxide dismutase (SOD) -like action, a membrane lipid peroxidation inhibiting action by singlet oxygen, and a hydrogen peroxide scavenging action , Radical scavenging action, matrix metalloproteinase-2 (MMP-2) activity inhibitory action, matrix metalloproteinase-9 (MMP-9) mRNA expression increase inhibitory action, epidermal keratinocyte proliferation promoting action, hexosaminidase release inhibitory action , Nitric oxide (NO) production inhibitory action, hyaluronidase activity inhibitory action, cyclooxygenase-2 (COX-2) activity inhibitory action, androgen receptor antagonistic action, hair papillary cell proliferation promoting action, cyclic AMP-phosphodiesterase activity inhibitory action, rat Lipolysis-promoting action using accessory testicular fat cells, end It effectively exhibits at least one of the inhibitory effect on the increase in phosphorus-1 mRNA expression and the inhibitory effect on the increase in SCF mRNA expression, and is useful for in vivo oxidation prevention, hair growth, whitening, aging prevention, and prevention or treatment of inflammatory diseases. is there.

(Beauty food and drink)
The cosmetic food and drink of the present invention contains as an active ingredient at least one of the antioxidant, the anti-aging agent, the anti-inflammatory agent, the hair restorer, the anti-obesity agent, and the whitening agent of the present invention. And further contains other components appropriately selected as necessary.
Here, the above-mentioned beauty foods and drinks are those that are less likely to harm human health and are taken orally or by digestive tract administration in normal social life. It is not limited to categories such as quasi-drugs, and includes, for example, foods that include a wide range of foods that are taken orally, such as general foods, health foods, health functional foods, quasi-drugs, and pharmaceuticals.

  The cosmetic food and drink according to the present invention may be obtained by blending an extract of black ginger into any food or drink so as not to hinder its activity, or nutrition based on the extract of black ginger. It may be a supplement.

  There is no restriction | limiting in particular as said beauty food-drinks, Although it can select suitably according to the objective, For example, drinks, such as a soft drink, a carbonated drink, a nutrition drink, a fruit drink, a lactic acid drink; Ice cream, ice sherbet , Shaved ice and other frozen desserts; noodles such as buckwheat, udon, harsame, gyoza skin, sushi mai, Chinese noodles, instant noodles; rice cakes, candy, gum, chocolate, tablet confectionery, snacks, biscuits, jelly, jam, cream, Sweets such as baked goods and bread; crab, salmon, clams, tuna, sardines, shrimp, skipjack, mackerel, whale, oyster, saury, squid, red sea bream, scallop, abalone, sea urchin, sea bream, tocobushi, etc .; Processed fishery and livestock products such as ham and sausage; Dairy products such as processed milk and fermented milk; salad oil, tempura oil, margarine, mayonnaise, Fats and processed foods such as yotoning, whipped cream, dressing, etc .; seasonings such as sauces, sauces; curry, stew, oyakodon, rice cake, miscellaneous cooking, Chinese rice cakes, tempura, eel rice cake, hayashi rice, oden, marbodorf, Retort pouch foods such as beef bowl, meat sauce, egg soup, omelet rice, dumplings, shumai, hamburger, meatballs; various forms of health foods and nutritional supplements; pharmaceuticals such as tablets, capsules, drinks, troches, etc. Examples include foreign products.

As said other component, the auxiliary | assistant raw material or additive normally used in manufacturing the said cosmetics food / beverage products is mentioned.
The raw material or additive is not particularly limited and may be appropriately selected depending on the intended purpose.For example, glucose, fructose, sucrose, maltose, sorbitol, stevioside, rubusoside, corn syrup, lactose, citric acid, Tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, gum arabic , Carrageenan, casein, gelatin, pectin, agar, vitamin Bs, nicotinamide, calcium pantothenate, amino acids, calcium salts, pigments, fragrances, preservatives, and the like.

  The addition amount of the antioxidant, the anti-aging agent, the anti-inflammatory agent, the hair-restoring agent, the anti-obesity agent, or the whitening agent of the present invention in the beauty food or drink is that of the target beauty food or drink. Although it differs depending on the type and cannot be specified unconditionally, it may be added within a range that does not impair the original taste of the beauty food and drink, and is 0.001% to 50% by weight with respect to various target beauty foods and drinks. Is preferable, and 0.01 to 20% by mass is more preferable. Further, in the case of a cosmetic food or drink in the form of granules, tablets or capsules, 0.01% by mass to 100% by mass is preferable, and 5% by mass to 100% by mass is more preferable.

  The cosmetic food and drink of the present invention can be taken orally on a daily basis, and the action of black ginger extract, which is an active ingredient, provides an antioxidant action, anti-aging action, anti-inflammatory action, hair growth action, At least one of an obesity effect and a whitening effect can be exhibited extremely effectively.

  In addition, although the antioxidant, anti-aging agent, anti-inflammatory agent, hair-restoring agent, anti-obesity agent, whitening agent, cosmetics, and beauty food and drink of the present invention are suitably applied to humans. As long as each effect is exhibited, it can also be applied to animals other than humans.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Production Example 1)
-Manufacture of black ginger water extract-
As a raw material for extraction, 100 g of pulverized black ginger rhizome was put in 1,000 mL of water, kept at 80 ° C. for 2 hours with gentle stirring, and then filtered. The filtrate was concentrated under reduced pressure at 40 ° C. and further dried with a vacuum dryer to obtain an extract (powder). The yield of the obtained extract is shown in Table 1.

(Production Example 2)
-Production of 50% by weight ethanol extract of black ginger-
As a raw material for extraction, 100 g of pulverized material of black ginger rhizome was put into 1,000 mL of 50 mass% ethanol (mass ratio of water and ethanol of 1: 1), and kept at 80 ° C. for 2 hours with gentle stirring. After that, it was filtered. The filtrate was concentrated under reduced pressure at 40 ° C. and further dried with a vacuum dryer to obtain an extract (powder). The yield of the obtained extract is shown in Table 1.

(Production Example 3)
-Production of black ginger ethanol extract-
As a raw material for extraction, 100 g of a ground product of black ginger rhizome was put into 1,000 mL of ethanol, kept at 80 ° C. for 2 hours with gentle stirring, and then filtered. The filtrate was concentrated under reduced pressure at 40 ° C. and further dried with a vacuum dryer to obtain an extract (powder). The yield of the obtained extract is shown in Table 1.

Example 1
-Superoxide dismutase (SOD) -like action test-
Each extract of Production Examples 1 to 3 was used as a sample, and the superoxide scavenging action was tested by the following test method.
In a test tube, 0.05 mL / L sodium bicarbonate buffer (pH 10.2) 2.4 mL, 2 mmol / L xanthine 0.1 mL, 3 mmol / L EDTA 0.1 mL, 50 μg / mL bovine serum albumin ( BSA) 0.1 mL and 0.75 mmol / L nitro blue tetrazolium 0.1 mL were added. To this, 0.1 mL of each sample solution was added and left at 25 ° C. for 10 minutes.
Next, 0.1 mL of xanthine oxidase solution was added, stirred rapidly, and reacted at 25 ° C. for 20 minutes. Thereafter, 0.1 mL of 6 mmol / L copper chloride was added to stop the reaction, and the absorbance at a wavelength of 560 nm was measured. A blank test was performed and corrected in the same manner.
From each measured absorbance, the superoxide elimination rate was determined by the following formula 1.
<Formula 1>
Superoxide elimination rate (%) =
{1- (St-Sb) / (Ct-Cb)} × 100
Where St is the absorbance of the sample solution at a wavelength of 560 nm, Sb is the absorbance of the sample solution at a wavelength of 560 nm, Ct is the absorbance of the control solution at a wavelength of 560 nm, and Cb is the absorbance of the control solution blank at a wavelength of 560 nm. Represent each.

  Next, the sample concentration was decreased stepwise to measure the superoxide erasure rate, and the sample concentration (μg / mL) at which the superoxide erasure rate was 50% was determined by interpolation. The results are shown in Table 2.

表２の結果から、製造例１〜３のブラックジンジャーの抽出物が、高いスーパーオキサイド消去作用を有し、ＳＯＤ様作用を有することが確認できた。

From the results in Table 2, it was confirmed that the black ginger extracts of Production Examples 1 to 3 had a high superoxide scavenging action and an SOD-like action.

(Example 2)
-Peroxide inhibition test of membrane lipids by singlet oxygen-
Each extract of Production Examples 1 to 3 was used as a sample, and the peroxidation inhibiting action of membrane lipids by singlet oxygen was tested by the following test method.
Transparent sample bottle (made of glass) in 10 mL, 2 mL of 2% erythrocyte suspension, 5 mL of PBS (−), or 5 mL of PBS (−) containing each sample solution, and 10 mmol / L hematoporphyrin—20 mmol / L water 0.01 mL of sodium oxide solution was added and mixed well. This reaction solution was irradiated uniformly on a merry-go-round with a 15 W halogen lamp for 35 minutes to generate ¹ O ₂ and hemolyze erythrocytes.
After completion of the reaction, 1 mL of the reaction solution was collected, and 2 mL of PBS (−) was added and mixed well. After centrifugation (1660 × g, 5 minutes, 4 ° C.), the absorbance of the supernatant at a wavelength of 540 nm was measured. As a control, 2 mL of purified water was added to 1 mL of the reaction solution to prepare a mixed solution in which red blood cells were completely hemolyzed. The absorbance of the mixture at a wavelength of 540 nm was measured. A blank test was performed and corrected in the same manner.
And the peroxidation suppression rate of the membrane lipid by singlet oxygen was computed from the obtained result by the following numerical formula 2. Table 3 shows the inhibition rate of membrane lipid peroxidation at a sample concentration of 50 μg / mL.
<Formula 2>
Permeation inhibition rate of membrane lipid (%) = (1−B / A) × 100
In Formula 2, A represents the absorbance at a wavelength of 540 nm of completely dissolved blood, and B represents the absorbance at a wavelength of 540 nm of the supernatant of the reaction solution.

表３の結果から、製造例１〜３のブラックジンジャーの抽出物が、一重項酸素による膜脂質の過酸化抑制作用を有することが確認できた。

From the results in Table 3, it was confirmed that the black ginger extracts of Production Examples 1 to 3 had a membrane lipid peroxidation inhibitory action by singlet oxygen.

(Example 3)
-Hydrogen peroxide scavenging action test-
Using each extract of Production Examples 1 to 3 as a sample, the hydrogen peroxide scavenging action was tested by the following test method.
After adding 10 μL of each sample solution to 10 μL of 1.5 mmol / L hydrogen peroxide solution and reacting at 37 ° C. for 20 minutes, a coloring solution [100 μmol / L DA-64, 0.5 mass% Triton X-100 2. Add 1 mL of 100 units / mL peroxidase to 100 mL of 0.1 mol / L PIPES buffer solution (pH 7.0)] and add 2.98 mL and react at 37 ° C. for 5 minutes. After completion of the reaction, absorbance at a wavelength of 727 nm was measured. A blank test was performed and corrected in the same manner.
And from the obtained result, the hydrogen peroxide elimination rate was computed by following Numerical formula 3. Table 4 shows the hydrogen peroxide elimination rate at a sample concentration of 100 μg / mL.
<Formula 3>
Hydrogen peroxide elimination rate (%) = {1− (St−Sb) / (Ct−Cb)} × 100
In Formula 3, St is the absorbance of the sample solution at a wavelength of 727 nm, Sb is the absorbance of the sample solution at a wavelength of 727 nm, Ct is the absorbance of the control solution at a wavelength of 727 nm, and Cb is the absorbance of the control solution blank at a wavelength of 727 nm. Represent.

表４の結果から、製造例１〜３のブラックジンジャーの抽出物が、過酸化水素消去作用を有することが確認できた。

From the results in Table 4, it was confirmed that the black ginger extracts of Production Examples 1 to 3 had a hydrogen peroxide scavenging action.

Example 4
-Radical scavenging test for DPPH-
Each extract of Production Examples 1 to 3 was used as a sample, and the radical scavenging action was tested using 1,1-diphenyl-2-picrylhydroxy radical (DPPH), which is a very stable radical, by the following test method.
3 mL of each sample solution was added to 3 mL of 150 μmol / L DPPH ethanol solution, and the container was immediately sealed and shaken and allowed to stand for 30 minutes, and then the absorbance at a wavelength of 520 nm was measured. A blank test was performed and corrected in the same manner.
And DPPH radical elimination rate (%) was computed by following Numerical formula 4 from each measured light absorbency. Table 5 shows the DPPH radical scavenging rate at a sample concentration of 200 μg / mL.
<Formula 4>
DPPH radical scavenging rate (%) = {C− (St−Sb)} / C × 100
In Formula 4, St represents the absorbance of the sample solution at a wavelength of 520 nm, Sb represents the absorbance of the sample solution blank at a wavelength of 520 nm, and C represents the absorbance of the control solution at a wavelength of 520 nm.

表５の結果から、製造例１〜３のブラックジンジャーの抽出物が、ＤＰＰＨに対するラジカル消去作用を有することが確認できた。

From the results in Table 5, it was confirmed that the black ginger extracts of Production Examples 1 to 3 had a radical scavenging action on DPPH.

(Example 5)
-Nitric oxide (NO) production inhibitory action test-
Each extract of Production Examples 1 to 3 was used as a sample, and the nitric oxide (NO) production inhibitory action was tested by the following test method.
Mouse macrophage cells (RAW264.7) were cultured using Dulbecco's MEM containing 10% fetal bovine serum (FBS), and then the cells were collected with a cell scraper. The collected cells are diluted with 10% FBS-containing phenol red-free Dulbecco MEM to a concentration of 3.0 × 10 ⁶ cells / mL, and then seeded at 100 μL per well in a 96-well microplate for 4 hours. Cultured. After completion of the culture, the medium was removed, 100 μL of a sample solution dissolved in 10% by mass of FBS-containing phenol red-free Dulbecco MEM containing 2% by mass of DMSO was added to each well, and 10% at a final concentration of 1 μg / mL. 100 μL of lipopolysaccharide (LPS, E. coli 0111; B4, manufactured by DIFCO) dissolved in FBS-containing phenol red-free Dulbecco MEM was added and cultured for 48 hours. The amount of NO production was measured using the amount of nitrite ion (NO ₂ ⁻ ) as an index. After completion of the culture, the same amount of grease reagent (1% by mass of sulfanilamide, 0.1% by mass of N-1-naphthyl ethylenedihydride in 5% by mass of phosphoric acid solution) was added to the culture solution in each well, Reacted for 10 minutes at room temperature. After the reaction, absorbance at a wavelength of 540 nm was measured. Based on the amount of nitric oxide (NO) produced by the control, the NO production inhibition rate was calculated from the following formula 5.

<Formula 5>
NO production inhibition rate (%) = {1− (A−B) / (C−D)} × 100
In Formula 5, A is the absorbance at a wavelength of 540 nm when the sample is added and stimulated with LPS, B is the absorbance at a wavelength of 540 nm when the sample is added and LPS is not stimulated, C is the absorbance at a wavelength of 540 nm when LPS is stimulated for control, and D Represents the absorbance at a wavelength of 540 nm when LPS was not stimulated as a control.

Next, the concentration of each extract solution in Production Examples 1 to 3 was decreased stepwise to measure the NO production inhibition rate, and the concentration IC _{50 at} which the NO production inhibition rate was 50% was determined by interpolation. (The smaller the IC ₅₀ value, the stronger the NO production inhibitory effect). The results are shown in Table 6.

表６の結果から、製造例１〜３のブラックジンジャーの抽出物が、高い一酸化窒素（ＮＯ）産生抑制作用を有することが認められた。

From the result of Table 6, it was recognized that the extract of the black ginger of manufacture examples 1-3 has a high nitric oxide (NO) production inhibitory effect.

(Example 6)
-Hyaluronidase activity inhibition test-
Each extract of Production Examples 1 to 3 was used as a sample, and the hyaluronidase activity inhibitory action was tested by the following test method.
First, 0.1 mL of 0.1 mol / L acetate buffer solution (pH 3.5) in which each extract was dissolved was added 0.1 mL of hyaluronidase solution [Type IV-S (from bovine tests; SIGMA 400 NF units / mL)]. In addition, the mixture was reacted at 37 ° C. for 20 minutes. Further, 0.2 mL of 2.5 mmol / L calcium chloride was added as an activator and reacted at 37 ° C. for 20 minutes. 0.4 mg / mL sodium hyaluronate solution (from robster comb) was added to this and reacted at 37 ° C. for 40 minutes. Thereafter, 0.2 mL of 0.4 mol / L sodium hydroxide was added to stop the reaction and cool, and then 0.2 mL of boric acid solution was added to each reaction solution and boiled for 3 minutes. After cooling with ice, 6 mL of p-DABA reagent was added and reacted at 37 ° C. for 20 minutes. Thereafter, the absorbance at a wavelength of 585 nm was measured. A blank test was performed and corrected in the same manner.
And from these results, the hyaluronidase activity inhibition rate was calculated by the following formula 6. Table 7 shows the inhibition rate of hyaluronidase activity at a sample concentration of 400 μg / mL.
<Formula 6>
Hyaluronidase activity inhibition rate (%) =
[1- (St-Sb) / (Ct-Cb)] × 100
However, in Formula 6, St represents the absorbance at a wavelength of 585 nm of the sample solution. Sb represents the absorbance of the sample solution blank at a wavelength of 585 nm. Ct represents the absorbance of the control solution at a wavelength of 585 nm. Cb represents the absorbance of the control solution blank at a wavelength of 585 nm.

表７の結果から、製造例１〜３のブラックジンジャーの抽出物が、ヒアルロニダーゼ活性阻害作用を有することが確認できた。

From the results of Table 7, it was confirmed that the black ginger extracts of Production Examples 1 to 3 had a hyaluronidase activity inhibitory action.

(Example 7)
-Hexosaminidase release inhibitory action test-
About each extract of manufacture example 1-3, the hexosaminidase release inhibitory effect was tested with the following test method. Since intracellular histamine is released and hexosaminidase is released at the same time, the histamine release inhibitory action can be evaluated using hexosaminidase release as an index.
Rat basophil leukemia cells (RBL-2H3) were cultured using 15% FBS-added S-MEM, and then cells were collected by trypsin treatment. The collected cells are diluted with a medium to a concentration of 4.0 × 10 ⁵ cells / mL, and DNP-specific IgE (manufactured by SIGMA) is added to a final concentration of 0.5 μg / mL. 100 μL per seed was seeded and cultured overnight. After completion of the culture, the medium was removed, and washing was performed twice with 100 μL of Siraganian buffer. Next, 30 μL of the same buffer solution and 10 μL of the sample solution prepared with the same buffer solution were added, and the mixture was allowed to stand at 37 ° C. for 10 minutes. Thereafter, 10 μL of a 100 ng / mL DNP-BSA (manufactured by LSL Co., Ltd.) solution was added, and the mixture was allowed to stand at 37 ° C. for 15 minutes to release hexosaminidase. Thereafter, the release was stopped by allowing the 96-well plate to stand on ice. 10 μL of the cell supernatant in each well and 10 μL of 1 mmol / L p-NAG (p-nitrophenyl N-acetyl β-D-glucosamine; (manufactured by SIGMA)) solution were added to a new 96-well plate, The reaction was carried out for 1 hour. After completion of the reaction, 250 μL of 0.1 mol / L Na ₂ CO ₃ / NaHCO ₃ was added to each hole, and the absorbance at a wavelength of 415 nm was measured. In addition, as a blank test, the absorbance at a wavelength of 415 nm of a solution obtained by mixing 10 μL of cell supernatant and 250 μL of 0.1 mol / L Na ₂ CO ₃ / NaHCO ₃ was measured and corrected. And the hexosaminidase release suppression rate was computed from following formula 7.
<Formula 7>
Inhibition rate of hexosaminidase release (%) = {1− (BC) / A} × 100
However, in Formula 7, A represents the absorbance at a wavelength of 415 nm when the sample solution is not added. B represents the absorbance at a wavelength of 415 nm when the sample solution is added. C represents the absorbance at a wavelength of 415 nm when the sample solution is added and p-NAG is not added.

  Next, the hexosaminidase release inhibition rate was measured by gradually reducing the sample concentration, and the sample concentration (μg / mL) that inhibits hexosaminidase release by 50% was determined by interpolation. . The results are shown in Table 8.

表８の結果から、製造例１〜３のブラックジンジャーの抽出物が、ヘキソサミニダーゼ遊離抑制（ヒスタミン遊離抑制）作用を有することが確認できた。

From the results of Table 8, it was confirmed that the extracts of black ginger of Production Examples 1 to 3 have a hexosaminidase release inhibition (histamine release inhibition) action.

(Example 8)
-Cyclooxygenase (COX-2) activity inhibitory action test-
Using each extract of Production Examples 1 to 3 as a sample, cyclooxygenase (COX-2) activity inhibitory action was tested by the following test method.
Mouse macrophage cells (RAW264.7) were cultured using Dulbecco's MEM containing 10% fetal bovine serum (FBS), and then the cells were collected with a cell scraper. The collected cells were diluted with Dulbecco MEM containing 10% FBS so as to have a concentration of 2.0 × 10 ⁵ cells / mL, then seeded at 100 μL per well in a 96-well microplate, and cultured for 18 hours. After culturing, in order to acetylate and inactivate COX-1 already present and COX-2 expressed in a small amount, the medium was replaced with a 500 μmol / L aspirin-containing medium and cultured for 4 hours. The cells were washed three times with PBS (−), and 100 μL of a sample solution dissolved in Dulbecco MEM containing 10% FBS containing DMSO having a final concentration of 0.5% by mass was added to each well, and then the final concentration was 1 μg / mL. 100 μL of lipopolysaccharide (LPS, E. coli 0111; B4, manufactured by DIFCO) dissolved in Dulbecco MEM containing 10% FBS was added and cultured for 24 hours. After completion of the culture, the amount of prostaglandin E2 in the culture supernatant of each well was quantified using PGE2 EIA Kit (manufactured by Cayman Chemical).
And from the obtained result, the cyclooxygenase (COX-2) activity inhibition rate was computed by following Numerical formula 8.
<Formula 8>
COX-2 activity inhibition rate (%) = {1− (AC) / (BC)} × 100
In Formula 8, A is the amount of prostaglandin E2 when the sample is added and stimulated with LPS, B is the amount of prostaglandin E2 when no sample is added and when stimulated with LPS, and C is the prostaglandin when no sample is added and when LPS is not stimulated Each represents the amount of gin E2.

Next, the concentration of each extract solution of Production Examples 1 to 3 was decreased stepwise to measure the COX-2 activity inhibition rate, and the concentration IC _{50 at} which the COX-2 activity inhibition rate was 50% was adjusted. It was determined by the insertion method. The results are shown in Table 9.

表９の結果から、製造例１〜３のブラックジンジャーの抽出物が、極めて強いシクロオキシゲナーゼ（ＣＯＸ−２）活性阻害作用を有することが確認できた。

From the results in Table 9, it was confirmed that the black ginger extracts of Production Examples 1 to 3 had a very strong cyclooxygenase (COX-2) activity inhibitory action.

Example 9
-Endothelin-1 (ET-1) mRNA expression increase inhibitory effect test-
Using each extract of Production Examples 1 to 3 as a sample, the endothelin-1 mRNA expression inhibitory action was tested as follows.
Normal human newborn infant keratinocyte (NHEK) is precultured in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermis keratinocytes (NHEK) in an 80 cm ² flask at 37 ° C. and 5% CO _2. Cells were collected by trypsinization.
Next, seeded by 40 × ¹⁰ 4 cells / 2mL / dish in 35mm petri dish (FALCON Corp.) using EpiLife-KG2, 37 ° C., and cultured overnight at 5% CO _2. After 24 hours, the culture solution was discarded, 1 mL of HEPES buffer was added, UV-B irradiation (50 mJ / cm ² ) was performed, and then 2 mL of each sample solution dissolved in EpiLife-KG2 to the required concentration was added to each dish. The cells were cultured at 37 ° C. and 5% CO ₂ for 24 hours. After culturing, the culture solution is discarded, total RNA is extracted with ISOGEN (Wako; Cat. No. 311-02501), the amount of each RNA is measured with a spectrophotometer, and the total RNA is adjusted to 200 ng / μL. Adjusted.
Using this total RNA as a template, the expression levels of endothelin-1 (ET-1) and the internal standard GAPDH mRNA were measured. The detection was performed by real-time RT-PCR reaction using Takara SYBR ExScript RT-PCR Kit (Perfect Real Time) using a real-time PCR apparatus (Smart Cycler ^(R) , manufactured by Cepheid).
The expression level of ET-1 is a total RNA preparation prepared from cells cultured in “no UV irradiation, no sample solution added”, “UV irradiation, no sample solution added” and “UV irradiation, no sample solution added”, respectively. Based on the above, the correction value is obtained with the value of GAPDH, and when the correction value of “no UV irradiation, no sample solution added” is 100, “UV irradiation, no sample solution added” and “UV irradiation, sample The correction value for “solution addition” was calculated.
And from the obtained result, the ET-1 mRNA expression increase suppression rate was computed by following numerical formula 9. Table 10 shows the inhibition rate of ET-1 mRNA expression increase at sample concentrations of 1 μg / mL and 0.1 μg / mL.
<Formula 9>
Endothelin-1 (ET-1) mRNA expression increase suppression rate (%)
= {(A−B) − (A−C)} / (A−B) × 100
In Formula 9, A is a correction value when no ultraviolet light is irradiated and the sample solution is not added, B is a correction value when the ultraviolet light is irradiated and the sample solution is not added, and C is a correction value when the ultraviolet light is irradiated and the sample solution is added. Represent.

表１０の結果から、製造例１〜３のブラックジンジャーの抽出物が、エンドセリン−１（ＥＴ−１）ｍＲＮＡ発現上昇抑制作用を有することが確認できた。

From the results in Table 10, it was confirmed that the black ginger extracts of Production Examples 1 to 3 had an endothelin-1 (ET-1) mRNA expression increase inhibitory action.

(Example 10)
-SCF mRNA expression increase inhibitory effect test-
Using the extracts of Production Examples 1 to 3 as samples, the SCF mRNA expression inhibitory action was tested as follows.
Normal human neonatal foreskin keratinocytes (NHEK) in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermis keratinocytes in an 80 cm ² flask at 37 ° C. under 5% CO _2. Cells were collected by trypsinization.
Next, seeded by 40 × ¹⁰ 4 cells / 2mL / dish in 35mm petri dish (FALCON Corp.) using EpiLife-KG2, 37 ° C., and cultured overnight at 5% CO _2. After 24 hours, the culture solution was discarded, 1 mL of HEPES buffer was added, UV-B irradiation (50 mJ / cm ² ) was performed, and then 2 mL of each sample solution dissolved in EpiLife-KG2 to the required concentration was added to each dish. The cells were cultured at 37 ° C. and 5% CO ₂ for 24 hours. After culturing, the culture solution is discarded, total RNA is extracted with ISOGEN (Wako; Cat. No. 311-02501), the amount of each RNA is measured with a spectrophotometer, and the total RNA is adjusted to 200 ng / μL. Adjusted.
Using this total RNA as a template, the expression level of SCF (Stem cell factor) and GAPDH mRNA which is an internal standard was measured. The detection was performed by a real-time RT-PCR reaction using a real-time PCR apparatus (Smart Cycler ^(R) , manufactured by Cepheid).
The expression level of SCF is based on a total RNA preparation prepared from cells cultured in “no UV irradiation, no sample solution added”, “UV irradiation, no sample solution added”, and “UV irradiation, no sample solution added”, respectively. Then, the correction value is obtained from the value of GAPDH, and “UV irradiation, no sample solution added” and “UV irradiation, sample solution added” when the correction value of “UV non-irradiation, no sample solution added” is set to 100. ”Was calculated.
And from the obtained result, the SCF mRNA expression increase suppression rate was calculated by the following formula 10. Table 11 shows the inhibition rate of SCF mRNA expression increase at a sample concentration of 1 μg / mL.
<Formula 10>
SCF mRNA expression increase suppression rate (%)
= {(A−B) − (A−C)} / (A−B) × 100
In Equation 10, A is a correction value when no UV irradiation is performed and no sample solution is added, B is a correction value when UV irradiation is performed and no sample solution is added, and C is a correction value when UV irradiation is performed and the sample solution is added. Represent.

表１１の結果から、製造例１〜３のブラックジンジャーの抽出物が、ＳＣＦｍＲＮＡ発現上昇抑制作用を有することが確認できた。

From the results of Table 11, it was confirmed that the black ginger extracts of Production Examples 1 to 3 had an SCF mRNA expression increase inhibitory action.

(Example 11)
-Matrix metalloproteinase-2 (MMP-2) activity inhibition test-
Using the extracts of Production Examples 1 to 3 as samples, matrix metalloproteinase-2 (MMP-2) activity inhibitory action was tested as follows.
(1) Preparation of MMP-2 Human MMP-2 cDNA was prepared by using 5 ′ PCR primer: 5′-ggcggatccatggcgccgtcgcccccatc-3 ′ and 3 ′ PCR primer: 3′-gccgtcgactacaatgtcctgttgtcagat-5 ′. PCR reaction was performed using pSG-GelA containing a 3 kb cDNA fragment as a template. The resulting 1.3 kb PCR fragment encoding 30 Ala to 474 Val was digested with Bam HI / Sal I and cloned into the Bam HI / Sal I site of the pTH-72 expression vector (pTH-MMP2-PC).
Expression of human MMP-2 is described in Itoh, M. et al. et al. J .; Biolchem. 119: 667-673 (1996), purification and unwinding (sometimes referred to as “refolding”), Yoshifumi Nishimura, Shigeo Ohno, supervised by: Protein Experiment Protocol, Cell Engineering Separate Volume Experiment Protocol Series 2 Structural Analysis, 1997 It went according to. CDNA is also described in Collier, I. et al. E. et al. J .; Biol. Chem. , 263 (
14), 6579-6687 (1998).
Specifically, an expression vector (pTH-MMP-2) containing MMP-2 cDNA was transfected into E. coli BL21 (DE3) strain, and expression was induced with IPTG. The expressed protein was affinity-purified using Ni-NTA resin (QUIIA Inc., USA), refolded, and reacted with 4-aminophenylmercuric acetate at 37 ° C. for 60 minutes to shift to the active form. Later, EDTA was added. Using this as an enzyme sample, a fluorescent peptide substrate (MOCAc / DNP peptide) cleavage activity reaction was performed, and high performance liquid chromatography (column: Wakosil 5C18, eluent: 30 mass% acetonitrile + 0.1 mass% THF, flow rate 1. (0 mL / min, detection: excitation wavelength: 325 nm, fluorescence wavelength: 410 nm), the peak area of the product was measured and used as an indicator of enzyme activity.

(2) Measurement of MMP-2 inhibitory activity of plant extract The test sample was dissolved in distilled water to 8.0 mg / mL, and then distilled water to 4.0 mg / mL and 2.0 mg / mL. Filtration was performed to dilute and remove the suspension. MMP-2 inhibitory activity was measured by 40 μL of active MMP-2, 20 μL of each sample solution, 20 μL of assay buffer [Tris-HCl buffer (pH 7.5) 500 mmol / L, sodium chloride 1.5 mol / L, calcium chloride 100 mmol / L, zinc sulfate 500 μmol / L, sodium azide 30 mmol / L, 0.05 mass% Brij 35] was preincubated at 37 ° C. for 15 minutes, and then MOCAc / DNP peptide was 120 μL (4.16 μmol / L). After adding and reacting at 37 ° C. for 2 hours, 10 μL (200 mmol / L) of EDTA was added. The peak area by the high performance liquid chromatography analysis was measured about the product in a reaction liquid. MMP-2 activity inhibition rate (%) at a sample concentration of 200 μg / mL and 400 μg / mL was determined with the product of the reaction solution added with distilled water instead of the sample solution as 100%. The results are shown in Table 12.

表１２の結果から、製造例１〜３のブラックジンジャーの抽出物が、ＭＭＰ−２活性阻害作用を有することが確認できた。

From the results of Table 12, it was confirmed that the black ginger extract of Production Examples 1 to 3 had an MMP-2 activity inhibitory action.

(Example 12)
-Matrix metalloprotease-9 (MMP-9) mRNA expression increase inhibitory test-
Using each extract of Production Examples 1 to 3 as a sample, MMP-9 mRNA expression inhibitory action was tested as follows.
Normal human neonatal foreskin keratinocytes (NHEK) in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermis keratinocytes in an 80 cm ² flask at 37 ° C. under 5% CO _2. Cells were collected by trypsinization.
Epilife-KG2 was used to seed 40 × 10 ⁴ cells / 2 mL / dish in 35 mm dishes (manufactured by FALCON) and cultured overnight at 37 ° C. under 5% CO ₂ . After 24 hours, the culture solution was discarded, 1 mL of HEPES buffer was added, and UV-B irradiation (50 mJ / cm ² ) was performed. Thereafter, 2 mL of each sample solution dissolved to the required concentration with EpiLife-KG2 was added to each petri dish and cultured at 37 ° C. under 5% CO ₂ for 24 hours. After culturing, the culture solution is discarded, total RNA is extracted with ISOGEN (Wako; Cat. No. 311-02501), the amount of each RNA is measured with a spectrophotometer, and the total RNA is adjusted to 200 ng / μL. did.
Using this total RNA as a template, the expression levels of MMP-9 and GAPDH mRNA as an internal standard were measured. Detection is performed using a real-time PCR apparatus (Smart Cycler ^(R) , manufactured by Cepheid), TaKaRa SYBR ^(R) PrimeScript ^™ RT-PCR Kit (Perfect Real Time) (code No. RR063A). It went by. As the MMP-9 primer, F: acgcacgacgtctttccagta and R: caactcactccgggaactca were used. The expression level of MMP-9 was corrected by GAPDH and calculated.
From the obtained results, the MMP-9 mRNA expression increase suppression rate was calculated according to the following formula 11. Table 13 shows the inhibition rate of MMP-9 mRNA expression increase at a sample concentration of 1 μg / mL.
<Formula 11>
MMP-9 mRNA expression increase suppression rate (%)
= {(A−B) − (A−C)} / (A−B) × 100
In Formula 11, A is a correction value when no ultraviolet light is irradiated and the sample solution is not added, B is a correction value when the ultraviolet light is irradiated and the sample solution is not added, and C is a correction value when the ultraviolet light is irradiated and the sample solution is added. Represent.

表１３の結果から、製造例１〜３のブラックジンジャーの抽出物が、ＭＭＰ−９ｍＲＮＡ発現上昇抑制作用を有することが確認できた。

From the results in Table 13, it was confirmed that the black ginger extracts of Production Examples 1 to 3 had an inhibitory effect on the increase in MMP-9 mRNA expression.

(Example 13)
-Skin keratinocyte proliferation promoting effect test-
Using each extract of Production Examples 1 to 3 as a sample, the epidermal keratinocyte proliferation promoting action was tested by the following test method.
Normal human neonatal foreskin keratinocytes (NHEK) were cultured using normal human epidermal keratinocyte long-term culture growth medium (EpiLife-KG2), and then cells were collected by trypsin treatment. The collected cells were diluted with EpiLife-KG2 to a concentration of 2.0 × 10 ⁴ cells / mL, seeded in a collagen-coated 96-well microplate at 100 μL per well, and cultured overnight. After completion of the culture, 100 μL of each sample solution dissolved in EpiLife-KG2 was added to each well and cultured for 3 days. The epidermal keratinocyte proliferation promoting action was measured using the MTT assay. After completion of the culture, the medium was removed, and MTT [3- (4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium Bromide] dissolved in PBS (−) at a final concentration of 0.4 mg / mL. Was added to each well in an amount of 100 μL. After culturing for 2 hours, blue formazan produced in the cells was extracted with 100 μL of 2-propanol. After extraction, the absorbance at a wavelength of 570 nm was measured. At the same time, the absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was used as the amount of blue formazan produced. In addition, a blank test was performed and corrected in the same manner.
And from the obtained measurement result, the epidermal keratinocyte proliferation promotion rate was computed by the following numerical formula 12. Table 14 shows the acceleration rate of epidermal keratinocyte proliferation at sample concentrations of 3.125 μg / mL and 12.5 μg / mL.
<Formula 12>
Epidermal keratinocyte proliferation promotion rate (%) = (St / Ct) × 100
In Formula 12, St represents the absorbance in the cells to which the sample solution was added, and Ct represents the absorbance in the cells to which the sample solution was not added.

表１４の結果から、製造例１〜３のブラックジンジャーの抽出物が、表皮角化細胞増殖促進作用を有することが確認できた。

From the results of Table 14, it was confirmed that the extracts of black ginger of Production Examples 1 to 3 have an action to promote epidermal keratinocyte proliferation.

(Example 14)
-Androgen receptor antagonism test-
Using the extracts of Production Examples 1 to 3 as samples, androgen receptor antagonistic activity was tested by the following test method.
First, after culturing SC-3 cells cloned from mouse spontaneous breast cancer (Shionogi cancer, SC115) using 2% DCC-FBS and ^10-8 mol / L testosterone-containing MEM (MEM / 2). Cells were collected by trypsinization. The collected cells were diluted with activated carbon-treated FBS-containing MEM (MEM / 2) to a concentration of 1.0 × 10 ⁵ cells / mL, seeded at 100 μL per well in a 96-well microplate, and cultured overnight. After completion of the culture, the medium was removed, and 100 μL of a sample solution dissolved in 0.5% by mass of BSA-containing Ham F12 + MEM (HMB) containing 10 ⁻⁹ mol / L dihydrotestosterone (DHT) was added and cultured for 48 hours. Thereafter, 100 μL of MTT dissolved in MEM / 2 containing FBS containing activated carbon at a final concentration of 0.4 g / mL was added to each hole. After culturing for 2 hours, blue formazan produced in the cells was extracted with 200 μL of 2-propanol. After extraction, the absorbance at a wavelength of 570 nm was measured. At the same time, the absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was used as the amount of blue formazan produced. As a blank test, a cell cultured with HMB alone was used as a positive control, and a cell cultured with HMB containing only 10 ⁻⁹ mol / L DHT was used as a positive control.
And from these results, the androgen receptor antagonistic rate was calculated by the following formula 13. Table 15 shows the androgen receptor antagonism at sample concentrations of 50 μg / mL, 25 μg / mL, and 12.5 μg / mL.
<Formula 13>
Androgen receptor antagonist rate (%)
= [1- (C−D) / (A−B)] × 100
However, in said Numerical formula 13, A represents the light absorbency in 570 nm-650 nm by DHT addition and sample solution non-addition. B represents the absorbance at 570 nm to 650 nm with no DHT added and no sample solution added. C represents the absorbance at 570 nm to 650 nm when DHT is added and the sample solution is added. D represents the absorbance at 570 nm to 650 nm when DHT is not added and the sample solution is added.

表１５の結果から、製造例１〜３のブラックジンジャーの抽出物が、アンドロゲンレセプター拮抗作用を有することが確認できた。

From the results of Table 15, it was confirmed that the black ginger extracts of Production Examples 1 to 3 had androgen receptor antagonistic action.

(Example 15)
-Papilla cell growth promoting effect test-
Each extract of Production Examples 1 to 3 was used as a sample, and the dermal papilla cell growth promoting effect was tested by the following test method.
After culturing using a hair papilla cell growth medium (TOYOBO, TPGM-250) containing normal human hair papilla cells (TOYOBO, CA60205), the cells were collected by trypsin treatment. The collected cells were diluted with Dulbecco's MEM (DMEM) containing 10% fetal bovine serum (FBS) to a concentration of 2.0 × 10 ⁴ cells / mL, and then 100 μL per well in a collagen-coated 96-well microplate. Seeding and culturing for 3 days. After the culture, the medium was removed, 200 μL of each sample solution dissolved in serum-free Dulbecco MEM (DMEM) was added to each well, and further cultured for 4 days. The dermal papilla cell proliferation promoting effect was measured using MTT assay. Specifically, after completion of the culture, the medium was removed and MTT [3- (4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl- dissolved in serum-free DMEM at a final concentration of 0.4 mg / mL. 2H-tetrazolium Bromide] was added to each well in an amount of 100 μL. After culturing for 2 hours, blue formazan produced in the cells was extracted with 100 μL of 2-propanol. After extraction, the absorbance at a wavelength of 570 nm was measured. At the same time, the absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was used as the amount of blue formazan produced. In addition, a blank test was performed and corrected in the same manner.
And from the obtained measurement result, the dermal papilla cell proliferation promotion rate was calculated from the following formula 14. Table 16 shows the proliferation rate of dermal papilla cells at a sample concentration of 1.5625 μg / mL.
<Formula 14>
Growth rate of dermal papilla cells (%) = A / B × 100
In Formula 14, A represents the absorbance when the sample solution was added, and B represents the absorbance when no sample solution was added.

表１６の結果から、製造例１〜３のブラックジンジャーの抽出物が、毛乳頭細胞増殖促進作用を有することが確認できた。

From the results shown in Table 16, it was confirmed that the black ginger extracts of Production Examples 1 to 3 had a dermal papilla cell proliferation promoting effect.

(Example 16)
-Cyclic AMP phosphodiesterase activity inhibition test-
Using each extract of Production Examples 1 to 3 as a sample, the cyclic AMP phosphodiesterase activity inhibitory action was tested as follows.
First, 0.2 mL of a 2.5 mg / mL bovine serum albumin (BSA) solution was added to 0.2 mL of 50 mmol / L Tris-HCl buffer (pH 7.5) containing 5 mmol / L magnesium chloride, and 0.1 mg / L 0.1 mL of a phosphodiesterase solution of mL and 0.05 mL of each sample solution were added, and pre-reacted at 37 ° C. for 5 minutes. To this was added 0.05 mL of a 0.5 mg / mL cAMP solution and reacted at 37 ° C. for 60 minutes. The reaction was stopped by boiling on a boiling water bath for 3 minutes. This was centrifuged (2260 × g, 10 minutes, 4 ° C.), and the supernatant was used as a sample reaction solution for HPLC analysis under the following conditions. A blank test was performed and corrected in the same manner.
[HPLC conditions]
Column: Wakosil _C18- ODS 5 μm
Mobile phase: 1 mmol / L TBAP in 25 mmol / L KH ₂ PO ₄ : CH ₃ CN = 90: 10
・ Flow rate: 1.0 mL / min
・ Detection: UV, 260 nm

Next, the peak area of the cyclic AMP standard product (A), the peak area of the supernatant of the cyclic AMP standard product and the phosphodiesterase reaction (B1), and the peak area of the cyclic AMP standard product, the test sample and the supernatant of the phosphodiesterase reaction Based on Equation 18 below, (B2) was used to determine the degradation rate (C) of the standard product and the degradation rate (D) of the test sample.
<Formula 18>
Decomposition rate (%) = (A−B) / A × 100
However, B in the formula represents either B1 or B2.

Thereafter, the phosphodiesterase activity inhibition rate was calculated based on the following formula 19 according to the degradation rate obtained from the formula 18.
<Formula 19>
Phosphodiesterase activity inhibition rate (%) = (C−D) / C × 100

  Next, the sample concentration of the sample solution is decreased stepwise to measure the cyclic AMP phosphodiesterase activity inhibition rate, and the sample concentration (μg / mL) that inhibits the cyclic AMP phosphodiesterase activity by 50% is interpolated. Asked. The results are shown in Table 17.

表１７の結果から、製造例１〜３のブラックジンジャーの抽出物が、高いサイクリックＡＭＰホスホジエステラーゼ活性阻害作用を有することが確認された。

From the results in Table 17, it was confirmed that the extracts of black ginger of Production Examples 1 to 3 have a high cyclic AMP phosphodiesterase activity inhibitory action.

(Example 17)
-Lipolysis promotion test using rat epididymal fat cells-
Using each extract of Production Examples 1 to 3 as a sample, the lipolysis promoting action using rat epididymal fat cells was tested as follows.
(1) Preparation of adipocytes Seven Wistar male rats (7 weeks old) were subjected to ether anesthesia and exsanguinated by decapitation under anesthesia. The adipose tissue on the accessory testicle was cut out, and the tissue was cut finely with scissors in physiological saline kept at 37 ° C. Place the tissue piece into a small Erlenmeyer flask and add 10 mL of Buffer A (119 mmol / L sodium chloride, 4.7 mmol / L potassium chloride, 2.6 mmol / L calcium chloride, 1.2 mmol / L phosphorus). 10 mg collagenase dissolved in potassium dihydrogenate, 1.2 mmol / L magnesium sulfate, 32.3 mmol / L HEPES (pH 7.4), 20 mg / mL bovine serum albumin (BSA), 2 mmol / L glucose) And reacted for 1 hour at 37 ° C. with stirring (100 rpm / min). After the reaction, it was filtered with gauze to remove undigested tissue. The filtrate was collected in a spitz tube with a lid and centrifuged (180 × g, 20 seconds), and the lower layer was removed with a Pasteur pipette. 10 mL of buffer A was added thereto, mixed, and then centrifuged again. This operation was repeated 4 times to sufficiently remove collagenase. Finally, about 10 mL of buffer A was added to obtain an adipocyte solution.

(2) Lipolysis promotion test 90 μL per well of the adipocyte solution prepared above was seeded in a 96-well microplate, and 10 μL of each sample solution dissolved in a soluble solvent was added to each well. Incubated for 5 hours. After completion of the culture, 5 μL was collected from each well, and the free fatty acid was measured with NEFA-C Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.).
And from the obtained measurement result, the lipolysis promotion rate was computed by the following numerical formula 16. Table 18 shows the rate of acceleration of lipolysis at a sample concentration of 400 μg / mL.
<Formula 16>
Lipolysis promotion rate (%) = (A / B) × 100
In Formula 16, A represents the amount of free fatty acid when the sample was added, and B represents the amount of free fatty acid when the sample solution was not added (control).

表１８の結果から、製造例１〜３のブラックジンジャーの抽出物が、ラット副睾丸脂肪細胞を用いた脂肪分解促進作用を有することが確認できた。

From the results of Table 18, it was confirmed that the black ginger extract of Production Examples 1 to 3 had a lipolysis promoting action using rat accessory testicular fat cells.

(Formulation Example 1)
-Emulsion-
An emulsion was prepared from the following composition by a conventional method.
・ Water extract of black ginger of Production Example 1 ... 0.10g
・ Jojoba oil: 4.00 g
・ 1,3-Butylene glycol ・ ・ ・ 3.00g
・ Polyoxyethylene cetyl ether (20E.O.) ... 2.50 g
・ Olive oil ... 2.00g
・ Squalane ... 2.00g
・ Cetanol ... 2.00g
・ Glyceryl monostearate ... 2.00g
・ Oleic acid polyoxyethylene sorbitan (20E.O.) ... 2.00g
・ Methyl paraoxybenzoate 0.15 g
・ Twilight extract ... 0.10g
・ Dipotassium glycyrrhizinate ... 0.10g
・ Ginkgo biloba extract ... 0.10g
・ Conchiolin ... 0.10g
・ Oat extract ... 0.10g
・ Chicken extract ... 0.10g
・ Fragrance ... 0.05g
・ Purified water: remainder (total 100.00 g)

(Formulation Example 2)
-Lotion-
A lotion was produced from the following composition by a conventional method.
-50% by weight ethanol extract of black ginger of Production Example 2 ... 0.10g
・ Glycerin ... 3.00g
・ 1,3-Butylene glycol ・ ・ ・ 3.00g
・ Oleic acid polyoxyethylene sorbitan (20E.O.) ... 2.00g
・ Methyl paraoxybenzoate 0.15 g
・ Citric acid ... 0.10g
・ Sodium citrate: 0.10 g
・ Oil-soluble licorice extract ... 0.10g
・ Seaweed extract ... 0.10g
・ Cudin extract ... 0.10g
・ Xylobiose Mixture ... 0.05g
・ Fragrance ... 0.05g
・ Purified water: remainder (total: 100.00 g)

(Formulation Example 3)
-Cream-
A cream was produced from the following composition by a conventional method.
・ Black ginger ethanol extract of Production Example 3 ... 0.10g
・ Squalane ... 10.00g
・ 1,3-butylene glycol ... 6.00g
・ Liquid paraffin ・ ・ ・ 5.00g
・ Salah beeswax 4.00 g
・ Cetanol ... 3.00g
・ Glyceryl monostearate ... 3.00g
・ Lanoline ... 2.00g
・ Oleic acid polyoxyethylene sorbitan (20E.O.) ... 1.50 g
・ Methyl paraoxybenzoate ... 1.50g
・ Stearic acid: 1.00 g
・ Yeast extract ... 0.10g
・ Perilla extract ... 0.10g
-Linden extract ... 0.10g
・ Jiuyu extract ... 0.10g
・ Fragrance ... 0.10g
・ Purified water: remainder (total: 100.00 g)

(Formulation Example 4)
−Pack−
A pack was produced from the following composition by a conventional method.
-Water extract of black ginger of Production Example 1 ... 0.20 g
・ Polyvinyl alcohol: 15.00g
・ Ethanol ... 10.00g
・ Propylene glycol: 7.00 g
・ Polyethylene glycol ... 3.00g
・ Sage extract ... 0.10g
・ Toki extract ... 0.10g
・ Carrot extract ... 0.10g
・ Ethyl paraoxybenzoate 0.05g
・ Fragrance ... 0.05g
・ Purified water: remainder (total: 100.00 g)

(Formulation Example 5)
-Hair tonic-
A hair tonic having a hair growth action having the following composition was produced by a conventional method.
・ Pyridoxine hydrochloride ... 0.1g
・ Resorcin ・ ・ ・ 0.01g
・ D-pantothenyl alcohol ... 0.1g
・ Dipotassium glycyrrhizinate ... 0.1g
・ L-Menthol 0.05g
・ 1,3-Butylene glycol ・ ・ ・ 4.0g
・ Carrot extract ... 0.5g
・ Ethanol ... 25.0g
-50% ethanol extract of black ginger of Production Example 2 0.2g
-Fragrance: appropriate amount-Purified water: remainder (the total amount is 100.0 g)

(Formulation Example 6)
-Shampoo-
A shampoo (cream shampoo) having a hair-growing action having the following composition was produced by a conventional method.
・ Polyoxyethylene alkyl ether sodium sulfate 30.0 g
・ Polyoxyethylene alkyl ether ammonium sulfate ... 20.0g
・ Palm oil fatty acid amidopropyl betaine ... 6.0g
・ Palm oil fatty acid modiethanolamide: 4.0 g
・ Ethylene glycol distearate ... 2.0g
・ Preservative (methyl paraoxybenzoate) ... 0.15g
・ Water extract of black ginger of Production Example 1 0.2g
・ Muguroji extract ... 0.2g
・ Cotton extract ... 0.5g
・ Oat extract ... 0.3g
・ Rosemary extract 0.5g
・ Perfume 0.01g
・ 1,3-Butylene glycol ・ ・ ・ 3.0g
・ Purified water: remainder (the total amount is 100.0 g)

(Formulation Example 7)
-Rinse-
A rinse having a hair-growth effect having the following composition was produced by a conventional method.
・ Stearyltrimethylammonium chloride 1.5g
・ Polyoxyethylene cetyl ether ... 1.0g
・ Cetyl alcohol: 2.0g
・ Octyldodecanol ・ ・ ・ 1.0g
・ Cationized cellulose: 0.5g
・ Propylene glycol ... 5.0g
・ Water extract of black ginger of Production Example 1 0.2g
・ Muguroji extract ... 0.2g
・ Cotton extract ... 0.5g
・ Oat extract ... 0.3g
・ Rosemary extract 0.5g
・ Fragrance ... 3.0g
・ Purified water: remainder (the total amount is 100.0 g)

(Formulation Example 8)
-Rinse-
A rinse having a hair-growth effect having the following composition was produced by a conventional method.
・ Stearyltrimethylammonium chloride 1.5g
・ Polyoxyethylene cetyl ether ... 1.0g
・ Cetyl alcohol: 2.0g
・ Octyldodecanol ・ ・ ・ 1.0g
・ Cationized cellulose: 0.5g
・ Propylene glycol ... 5.0g
・ Black ginger ethanol extract of Production Example 3 0.2g
・ Muguroji extract ... 0.2g
・ Cotton extract ... 0.5g
・ Oat extract ... 0.3g
・ Rosemary extract 0.5g
・ Fragrance ... 3.0g
・ Purified water: remainder (the total amount is 100.0 g)

(Formulation Example 9)
-Tablet dietary supplements-
The following mixture was tableted to produce a tablet-shaped dietary supplement.
-Black ginger water extract of Production Example 1 30g
・ Powdered sugar (sucrose) 178g
・ Sorbit ・ ・ ・ 10g
・ Glycerin fatty acid ester: 12g

(Formulation Example 10)
-Granular dietary supplement-
The following mixture was formed into granules to produce a dietary supplement.
-50% ethanol extract of black ginger of Production Example 2 20g
・ Beat oligosaccharide ... 1000g
・ Vitamin C ... 167g
・ Stevia extract ... 10g

(Formulation Example 11)
-Granular dietary supplement-
The following mixture was formed into granules to produce a dietary supplement.
-Ethanol extract of black ginger of Production Example 3 ... 20g
・ Beat oligosaccharide ... 1000g
・ Vitamin C ... 167g
・ Stevia extract ... 10g

A cosmetic comprising at least one of the antioxidant, anti-aging agent, anti-inflammatory agent, hair restorer, anti-obesity agent, and whitening agent of the present invention has an excellent antioxidant effect, anti-aging effect, anti-inflammatory effect, It has at least one of hair growth, anti-obesity, and whitening effects, and is effective in preventing oxidation in the body, preventing hair growth, whitening, preventing and improving skin wrinkles and skin elasticity, preventing rough skin, and preventing rough skin. For example, it is widely used in ointments, creams, emulsions, lotions, packs, jellies, lip balms, lipsticks, bath preparations, astringents, hair tonics, hair creams, hair liquids, shampoos, pomades, rinses and the like.
In addition, the food and drink for cosmetics to which at least one of the antioxidant, anti-aging agent, anti-inflammatory agent, hair-restoring agent, anti-obesity agent, and whitening agent of the present invention is added has an excellent anti-oxidation effect even when taken orally. It has at least one of an anti-aging action, an anti-inflammatory action, a hair growth action, an anti-obesity action, and a whitening action, and is excellent in safety.

Claims (5)

An obesity-relieving agent comprising an extract of black ginger ( Kaempferia parviflora ).   The obesity-eliminating agent according to claim 1, which has at least one of a cyclic AMP-phosphodiesterase activity inhibitory action and a lipolysis promoting action using rat epididymal fat cells. A lipolysis promoter comprising an extract of black ginger ( Kaempferia parviflora ). A cyclic AMP-phosphodiesterase activity inhibitor characterized by containing an extract of black ginger ( Kaempferia parviflora ). An agent for promoting lipolysis of rat epididymal fat cells, comprising an extract of black ginger ( Kaempferia parviflora ).