Skin cosmetic, hair cosmetic and food or drink

Abstract

To discover an ingredient having anti-obesity action, a cyclic AMP phosphodiesterase activity inhibitory action, a dipeptidyl peptidase IV activity inhibitory action, anti-aging action, hair-growing action, antiandrogen action, skin-whitening action or anti-inflammatory action from compounds derived from a natural product and to provide an anti-obesity agent, a cyclic AMP phosphodiesterase activity inhibitor, a dipeptidyl peptidase IV activity inhibitor, an anti-aging agent, a hair-growing agent, an antiandrogen agent, a skin whitening agent or an anti-inflammatory agent, which uses the ingredient as an active ingredient.SOLUTION: Dihydroferulic acid is used as an active ingredient of an anti-obesity agent, a cyclic AMP phosphodiesterase activity inhibitor, a dipeptidyl peptidase IV activity inhibitor, an anti-aging agent, a hair-growing agent, an anti-androgen agent, a skin-whitening agent and an anti-inflammatory agent. Further, dihydroferulic acid is blended in a skin cosmetic, a hair cosmetic and a food or drink.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to anti-obesity agents, cyclic AMP phosphodiesterase activity inhibitors, dipeptidyl peptidase IV activity inhibitors, anti-aging agents, hair growth agents, anti-androgenic agents, skin whitening agents, anti-inflammatory agents, skin cosmetics, hair cosmetics, and foods and beverages. [Background technology]

[0002] In recent years, lifestyle habits such as overeating and lack of exercise have led to an increase in body fat and obesity. This increase in obesity is not only seen in humans, but also in pets and livestock. Obesity can lead to lifestyle-related diseases such as hyperlipidemia and arteriosclerosis, so it is not only a problem in terms of beauty but also a major health issue.

[0003] Cyclic AMP (cAMP) is known to be involved in lipolysis in vivo. cAMP activates lipase present in the body, and the activated lipase breaks down fat into fatty acids and glycerol. However, activation of cAMP phosphodiesterase induces the breakdown of cAMP, inhibiting lipase activation. Therefore, it is thought that inhibiting the activity of cAMP phosphodiesterase increases intracellular cAMP and promotes fat breakdown.

[0004] Furthermore, platelet aggregation, which causes inflammatory reactions, is related to the concentration of cyclic AMP (cAMP) in platelets. It is known that when cAMP is decomposed by cAMP phosphodiesterase and the cAMP concentration decreases, platelets become more susceptible to aggregation. Therefore, it is believed that platelet aggregation can be prevented by inhibiting the action of cAMP phosphodiesterase to prevent a decrease in cAMP concentration, thereby making it possible to prevent, treat, or ameliorate allergic diseases, inflammatory diseases, etc. Tubeimoside I (see Patent Document 1) and the like are known to have cAMP phosphodiesterase activity inhibitory effects.

[0005] Obesity causes abnormalities in lipid metabolism, which, as mentioned above, can lead to lifestyle-related diseases such as dyslipidemia. Dyslipidemia is a condition characterized by an abnormal increase in one or more serum lipids, i.e., cholesterol, triglycerides, phospholipids, and free fatty acids, resulting in various disorders. Cholesterol is a vital component of animal biomembranes and a precursor to steroid hormones. However, excessive blood cholesterol can accumulate within blood vessels, leading to atherosclerosis and potentially fatal diseases such as ischemic heart disease and cerebral infarction. Free fatty acids are released into the bloodstream from adipocytes and used as an energy source, but excess free fatty acids are absorbed by the liver and resynthesized into triglycerides. Excess free fatty acids in the blood can lead to a chronic state of triglyceride excess, resulting in persistent dyslipidemia and increased risk of atherosclerosis. Furthermore, excessive free fatty acids in the blood can increase insulin resistance and contribute to the progression of diabetes.

[0006] Therefore, if excessively high blood lipids (e.g., blood cholesterol, blood free fatty acids, etc.) caused by obesity can be reduced, it is believed that the symptoms of arteriosclerosis and diabetes, which progress with obesity, can be prevented, alleviated, or improved. Extracts of roasted yellow quince leaves (Patent Document 2) are known to have the effect of reducing blood cholesterol and blood free fatty acids.

[0007] Dipeptidyl peptidase IV (hereinafter also referred to as "DPPIV") is a serine protease that recognizes the second N-terminal proline or alanine and cleaves it at its C-terminus. DPPIV is expressed on the cell surface of epithelial and endothelial cells in tissues such as the kidney, liver, intestine, and placenta, as well as on T cells, and is thought to be involved in various physiological phenomena through its enzymatic activity.

[0008] Substrates of DPPIV include hormones called incretins. Incretins are a collective term for hormones secreted from the intestinal tract in response to nutrient stimulation and promote insulin secretion from pancreatic β cells in a blood glucose-dependent manner. Known examples include GLP-1 and GIP. These incretins not only promote blood glucose-dependent insulin secretion, but also suppress glucagon secretion from pancreatic α cells, lower blood pressure, inhibit gastric emptying, and even suppress food intake by acting on the hypothalamus (see Non-Patent Document 1). However, since incretins are degraded by DPPIV, the half-life of GLP-1 in vivo is known to be approximately 1.5 minutes. Therefore, inhibiting the enzymatic activity of DPPIV could extend the half-life of incretins in vivo, which is expected to be useful in treating type 2 diabetes, obesity, hypertension, insulin resistance, and other conditions through the aforementioned incretin actions.

[0009] DPPIV is also identical to CD26, a T cell activation marker, and is known to regulate the activity of many immunoregulatory peptides as substrates. Therefore, regulating DPPIV activity may be useful in controlling immune responses, including autoimmune diseases such as rheumatoid arthritis and transplant rejection. Furthermore, DPPIV is known to be involved in the metabolism of several neuropeptides and growth hormone; cancer invasion, metastasis, and angiogenesis; and HIV infection of lymphocytes. Therefore, inhibiting DPPIV activity may be useful in treating pain, neurodegenerative diseases, neuropsychiatric disorders, and other neurological disorders (e.g., sciatica, Alzheimer's disease, depression, etc.); growth hormone deficiency and diseases for which growth hormone is used; cancers (e.g., T-cell lymphoma, acute lymphoblastic leukemia, thyroid cancer, basal cell carcinoma, breast cancer, etc.); and HIV infection (AIDS).

[0010] A basement membrane exists at the boundary between the epidermis and dermis that make up the skin. The basement membrane not only connects the epidermis and dermis, but also plays an important role in maintaining skin function (see Non-Patent Document 2). The basement membrane's main skeleton has a mesh structure made of type IV collagen. Various glycoproteins, primarily composed of laminin 5, exist at the boundary between the basement membrane and the epidermis and connect the two, and laminin 5 is produced by epidermal keratinocytes present in the epidermis. In young skin, the interaction between the epidermis and dermis is maintained at homeostasis through the function of the basement membrane, ensuring moisture retention, flexibility, elasticity, etc., and maintaining the skin's appearance of firmness, luster, and freshness.

[0011] However, when exposed to certain external factors, such as ultraviolet radiation, extremely dry air, or excessive skin cleansing, or due to aging, laminin 5, a major component of the basement membrane, decomposes and alters, destroying the basement membrane structure (see Non-Patent Document 3). As a result, the skin's moisturizing function and elasticity decline, and the keratin begins to peel abnormally, causing the skin to lose firmness and luster and to exhibit aging symptoms such as roughness and wrinkles. Thus, changes associated with skin aging, such as wrinkles, dullness, loss of texture, and loss of elasticity, are thought to be related to a decrease in basement membrane components and structural changes in the basement membrane. Therefore, promoting the production of laminin 5 may be able to prevent or improve skin aging symptoms.

[0012] Laminins consist of various combinations of α, β, and γ chains, and currently 15 types (laminin 1 to laminin 15) are known. Among these, laminin 5 (α3β3γ2) is abundantly present in the basement membranes of epithelial tissues, such as the skin, digestive tract, kidneys, and lungs. A genetic disease (lethal congenital epidermolysis bullosa, or Herlitz junctional epidermolysis bullosa) caused by congenital abnormalities in the genes encoding each of the laminin 5 chains is known to cause fatal symptoms, including complete epidermal peeling. Furthermore, laminin 5 is known to strongly adhere cells (high cell adhesion activity) and strongly promote cell motility (high cell motility activity) compared to other extracellular matrix molecules.

[0013] As described above, laminin-5 is known to promote cell migration in damaged skin and promote wound healing due to its high cell motility activity (see Patent Document 3). In other words, promoting the production of laminin-5 is important for promoting the healing of skin injuries that destroy the basement membrane structure.

[0014] In recent years, the involvement of matrix metalloproteinases (MMPs) has been suggested as a factor in inducing changes associated with skin aging. Among these MMPs, matrix metalloproteinase-2 (MMP-2), a gelatinase enzyme, is known to degrade type IV collagen and laminin 5, major components of basement membranes. It has been shown that MMP-2 expression and activity are significantly increased by UV irradiation, causing UV-induced reduction of basement membrane components and structural changes in the basement membrane, which are major factors in the formation of wrinkles and sagging skin (see Non-Patent Document 4). Furthermore, MMP-2 degrades type IV collagen and other components of the basement membrane present beneath vascular endothelial cells. The degraded vascular endothelial cells migrate into the interstitium, proliferate there, form lumens, and establish new blood vessels. These newly formed blood vessels then reach tumor cells, supplying them with nutrients and oxygen, leading to tumor growth (see Non-Patent Document 5).

[0015] Therefore, it is believed that inhibiting the activity of MMP-2 can suppress the decrease in basement membrane components and structural changes in the basement membrane, improve skin function, and also suppress angiogenesis and tumor cell proliferation. For example, extracts from black gram (see Patent Document 4) are known to have MMP-2 inhibitory activity.

[0016] Among MMPs, matrix metalloproteinase-1 (MMP-1), an enzyme belonging to the collagenase group, is known to degrade collagen, the main component of the aforementioned dermal extracellular matrix. MMP-1 expression is significantly increased by UV irradiation, contributing to the reduction and degeneration of collagen, which is thought to be a major factor in the formation of wrinkles and loss of elasticity in the skin. Furthermore, increased MMP-1 activity leads to the destruction of the extracellular matrix. Extracellular matrix destruction is known to be associated with various diseases, including cancer invasion and metastasis, rheumatoid arthritis, osteoarthritis, periodontal disease, and age-related macular degeneration.

[0017] Therefore, it is believed that inhibiting the activity of MMP-1 can prevent, treat, or improve symptoms of skin aging, as well as treat or prevent diseases associated with the destruction of the extracellular matrix. Corosolic acid (see Patent Document 5), for example, is known to have an inhibitory effect on MMP-1 activity.

[0018] The epidermis and dermis of the skin are composed of epidermal cells, fibroblasts, and the extracellular matrix that is located outside these cells and supports the skin structure, such as collagen, elastin, and hyaluronic acid. In young skin, fibroblast proliferation is active, and the interaction between skin tissues such as fibroblasts and collagen maintains homeostasis, ensuring moisture retention, flexibility, elasticity, etc., and maintaining the skin in a firm, glossy, and moist state in appearance.

[0019] However, under the influence of certain external factors, such as ultraviolet radiation, extremely dry air, and excessive skin cleansing, or due to aging, the production of elastin and hyaluronic acid, the major components of the extracellular matrix, decreases and undergoes degradation and alteration. As a result, the skin's moisturizing function and elasticity decline, and abnormal peeling of the keratin occurs, causing the skin to lose firmness and luster, leading to signs of aging such as rough skin and wrinkles. Thus, changes associated with skin aging, such as wrinkles, dullness, loss of texture, and loss of elasticity, are associated with the reduction and denaturation of matrix components such as elastin and hyaluronic acid. Therefore, promoting the production of elastin or hyaluronic acid is important for preventing, treating, or improving skin aging.

[0020] Among these extracellular matrix components, hyaluronic acid is a type of mucopolysaccharide, and has the function of retaining cells by filling the intercellular spaces, and also has many other functions, such as retaining moisture in the intercellular spaces, imparting lubricity and flexibility to tissues, and resisting external forces such as mechanical damage.If the production of hyaluronic acid can be promoted, it is thought that skin aging symptoms such as rough skin, wrinkles, dullness, loss of texture, loss of elasticity, and loss of moisturizing function can be prevented, treated, or improved.

[0021] Hyaluronic acid is also present in connective tissues such as cartilage, synovial fluid, umbilical cord, vitreous body, and other tissues, in addition to skin tissue. Hyaluronic acid in synovial fluid coats the surface of articular cartilage, contributing to the smooth functioning of joints through its lubricating and cartilage-covering / protecting functions. Meanwhile, it is known that the concentration of hyaluronic acid in synovial fluid decreases in cases of arthritis such as rheumatoid arthritis. Therefore, promoting hyaluronic acid production is believed to be effective in preventing or treating arthritis such as rheumatoid arthritis, osteoarthritis, septic arthritis, gouty arthritis, traumatic arthritis, and osteoarthritis. Furthermore, granulation tissue is formed during the healing process of wounds or burns, and it is known that hyaluronic acid levels increase significantly in these tissues. Therefore, promoting hyaluronic acid production is believed to promote wound or burn healing. Extracts from Camphor Tree (see Patent Document 6) and other substances are known to have the effect of promoting hyaluronic acid production.

[0022] Among the extracellular matrix components, elastin is a fiber that provides elasticity to skin tissue. Promoting elastin production may reduce wrinkles and sagging, and may prevent, treat, or improve skin aging symptoms such as loss of firmness and elasticity.

[0023] In addition to skin tissue, elastin is widely expressed in tissues in the body that require elasticity, such as the lungs and blood vessels. It is known that a decrease in normal elastin from these tissues with aging leads to a decrease in elasticity in the lungs, blood vessels, etc., which can cause pulmonary diseases such as emphysema, hypertension, and vascular diseases such as aneurysms. Therefore, if elastin production can be promoted, it is thought that a decrease in elasticity in the lungs, blood vessels, etc. will be less likely to occur, and pulmonary diseases such as emphysema, hypertension, and vascular diseases such as aneurysms will be prevented or treated. For example, extracts from plants belonging to the genus Hippophae in the Elaeagnaceae family are known to have the effect of promoting elastin production (see Patent Document 7).

[0024] The aforementioned extracellular matrix components, such as collagen, are produced by fibroblasts. In young skin, fibroblast proliferation is active, and the interaction between fibroblasts, collagen, and other skin tissues maintains homeostasis, ensuring moisture retention, flexibility, elasticity, and other properties, maintaining the skin's appearance of firmness, luster, and freshness. However, under the influence of certain external factors, such as ultraviolet rays, extremely dry air, and excessive skin cleansing, or with aging, fibroblast proliferation slows, reducing the skin's moisturizing function and elasticity. As a result, the skin loses firmness and luster and exhibits aging symptoms such as roughness and wrinkles. Therefore, promoting fibroblast proliferation is considered to be extremely important for preventing, treating, or improving skin aging.

[0025] Fibroblasts also play an important role in the healing process of wounds such as trauma and burns, as well as in the healing process of skin diseases (e.g., skin ulcers such as pressure sores, burn ulcers, and diabetic ulcers). Therefore, it is believed that wounds and skin diseases can be treated by promoting the proliferation of fibroblasts. Furthermore, in the field of regenerative medicine, a known treatment for patients with wounds that are difficult to heal themselves (such as severe burns) involves culturing and growing skin cells from a fragment of the patient's own skin and transplanting them into the patient. The use of a cell proliferation promoter is expected to shorten the skin cell culture period. Extracts from Camphor Tree (see Patent Document 6 mentioned above) and the like are known to have the effect of promoting fibroblast proliferation.

[0026] The epidermis functions to mitigate external stimuli and control the loss of body components such as water, and is composed of a four-layer structure, starting with the basal layer at the bottom, followed by the spinous layer, granular layer, and stratum corneum. Most of the cells in each layer are keratinocytes that differentiate from the basal layer. Keratinocytes that divide and proliferate in the basal layer differentiate as they pass through the spinous and granular layers to become corneocytes, which then form the stratum corneum, made up of keratin protein fibers with strong cross-links, and are eventually shed from the stratum corneum as dirt.

[0027] The stratum corneum is the outermost layer of the skin and serves as a physical barrier against external stimuli. To maintain this barrier function, the skin undergoes a cycle of epidermal metabolism (keratinization), typically repeated every four weeks, from the time keratinocytes are produced in the basal layer until they become dandruff and are shed. However, the metabolic function of the stratum corneum also declines with age, resulting in skin problems such as fine wrinkles, dullness, pigmentation, and rough skin. Therefore, promoting keratinocyte proliferation and restoring the skin's metabolic function is thought to improve skin aging symptoms such as fine wrinkles, dullness, and pigmentation. Conventionally, extracts of Aster crustacea (see Patent Document 8) and the like are known to have the effect of promoting epidermal keratinocyte proliferation.

[0028] In skin cells, aquaporins, known as water channels, are expressed on the cell membrane and are known to take up small molecules such as water from the intercellular space into the cells. Thirteen types of aquaporins (AQP0-AQP12) are known to exist in humans. Epidermal cells primarily contain AQP3, which is thought to take up not only water but also small molecules such as glycerol and urea, which are involved in moisture retention.

[0029] However, AQP3 decreases with age, and it has been suggested that this is one of the reasons for the decline in water retention function. Therefore, it is thought that promoting AQP3 expression may be able to control water retention ability and barrier function due to aging (see Non-Patent Document 6). For example, extracts from star fruit leaves (see Patent Document 9) are known to have the effect of promoting AQP3 expression.

[0030] Carbohydrates are extremely important as an energy source for humans and other living organisms. However, they are known to undergo glycation reactions with proteins. Glycation is a series of reactions that begins with a non-enzymatic reaction between the carbonyl group of a carbohydrate and an amino group in a protein, leading to the formation of Schiff bases, Amadori compounds, and finally to the formation of advanced glycation end products (hereinafter sometimes referred to as "AGEs"). Glycation non-enzymatically modifies proteins with sugars, which can cause denaturation of the protein and cross-linking between proteins, resulting in a decrease in protein function.

[0031] Glycation not only causes direct damage by modifying and structurally altering extracellular matrix proteins such as collagen, but also induces cellular responses through recognition by receptors that use glycated proteins as ligands. The impact of glycation is particularly severe for diabetic patients with high blood glucose levels. Protein glycation is known to be a contributing factor in diabetic complications such as diabetic neuropathy, diabetic retinopathy, and diabetic nephropathy. Glycation in vascular walls is also known to contribute to the progression of arteriosclerosis through endothelial cell damage and the accumulation of denatured proteins. Furthermore, extracellular matrix components, including collagen, account for more than half of the dry weight of tissues such as bone and skin. Therefore, for example, glycation of collagen, which becomes abnormally cross-linked, can lead to osteoporosis and osteoarthritis in bone and cartilage tissues, and to a decrease in skin elasticity and dullness due to yellowing. Furthermore, abnormally cross-linked collagen and the like is less susceptible to degradation by collagenase and the like, which leads to problems such as the induction of collagenase and the like, resulting in the degradation of normal collagen as well.

[0032] Therefore, if glycation reactions could be inhibited in some way, for example, by inhibiting the formation of AGEs, it would be expected to be useful in preventing or treating the aforementioned diseases, namely, diabetic complications, arteriosclerosis, osteoporosis, osteoarthritis, etc. Furthermore, it is expected to be effective in preventing or improving loss of skin elasticity, dullness, etc.

[0033] Many steroid hormones exert their effects by binding to receptors in the molecular form secreted from the organs that produce them, but in the case of male hormones collectively known as androgens, for example, testosterone enters the cells of the target organ and is reduced to 5α-dihydrotestosterone (5α-DHT) by testosterone 5α-reductase before binding to receptors and exerting its androgen effects.

[0034] Androgens are important hormones, but excessive action of androgens can induce various undesirable symptoms, such as male pattern baldness, hirsutism, seborrheic dermatitis, acne (e.g., acne), benign prostatic hyperplasia, prostatic tumors, and precocious puberty in boys. To alleviate these symptoms, methods for suppressing the action of excess androgens have been known, specifically, methods for suppressing the production of active 5α-DHT by inhibiting the action of testosterone 5α-reductase, which reduces testosterone to active 5α-DHT. To date, extracts from Eastern perilla (see Patent Document 10) and the like have been known to have testosterone 5α-reductase inhibitory activity.

[0035] Hair grows and falls out repeatedly according to a cyclical hair cycle (hair cycle) consisting of an anagen phase, a catagen phase, and a telogen phase. The stage of this hair cycle, during which new hair follicles are formed from the telogen phase to the anagen phase, is considered to be the most important for hair growth, and dermal papilla cells are thought to play an important role in the proliferation and differentiation of hair follicle epithelial cells during this stage. Dermal papilla cells are located inside hair follicle epithelial cells, which are composed of outer root sheath cells and matrix cells near the hair root, in the basement membrane-enclosed shaft portion of the hair root. They act on hair follicle epithelial cells to promote their proliferation, and thus play an important role in the proliferation and differentiation of hair follicle epithelial cells and hair formation (see Non-Patent Document 7).

[0036] Thus, dermal papilla cells play an important role in the proliferation and differentiation of hair follicle epithelial cells and hair formation, and it is believed that promoting the proliferation of dermal papilla cells can prevent or improve alopecia. To date, substances known to have the effect of promoting the proliferation of dermal papilla cells include, for example, wild thyme extract (see Patent Document 11).

[0037] In the skin, melanin plays a role in protecting the body from ultraviolet rays, but excessive production or uneven accumulation can cause skin darkening and age spots. Melanin is generally thought to be formed through the conversion of tyrosine to dopa, dopa to dopaquinone, and then via intermediates such as 5,6-dihydroxyindophenol by the action of the enzyme tyrosinase, which is biosynthesized in pigment cells. Therefore, inhibiting melanin production is considered to be a way to prevent, treat, or improve skin darkening (cutaneous pigmentation), age spots, freckles, etc.

[0038] Conventionally, skin whitening agents containing chemically synthesized products such as hydroquinone as an active ingredient have been used to prevent, treat, or improve skin pigmentation, age spots, freckles, and the like. However, chemically synthesized products such as hydroquinone may cause side effects such as skin irritation and allergies. Therefore, the development of skin whitening agents containing highly safe natural ingredients as active ingredients is desired, and extracts from plants of the genus Saussurea (see Patent Document 12) are known to have melanin production inhibitory effects.

[0039] The causes and mechanisms of inflammatory diseases, such as contact dermatitis (rash), psoriasis, pemphigus vulgaris, atopic dermatitis, and other inflammatory skin diseases accompanied by rough skin, as well as rheumatoid arthritis, osteoarthritis, asthma, etc., are diverse and are primarily due to increased hyaluronidase activity, histamine release, and cyclooxygenase-2 (COX-2) activity.

[0040] Hyaluronidase is a hydrolase that hydrolyzes hyaluronic acid. Hyaluronate, which maintains its affinity for body tissues, is decomposed by ultraviolet light, oxygen, etc. in aqueous systems, and its moisture-retaining effect decreases as its molecular weight decreases. Hyaluronic acid also exists in the body as intercellular tissue and is involved in vascular permeability. Furthermore, hyaluronidase is present in mast cells, but is released by degranulation caused by their activation and acts as an inflammatory chemical mediator. Therefore, inhibiting hyaluronidase activity is expected to enhance moisture retention and prevent or reduce inflammation. Extracts from plants of the genus Osbeckia (see Patent Document 13), for example, are known to have hyaluronidase activity inhibitory effects.

[0041] Histamine release is a phenomenon in which histamine in mast cells is released outside the cells, and the released histamine causes an inflammatory response. Therefore, attempts have been made to prevent or treat allergic diseases and inflammatory diseases using substances that inhibit or suppress histamine release. However, it is difficult to directly evaluate histamine release, and histamine release can be evaluated using the release of hexosaminidase, which has been confirmed to be released simultaneously with histamine release, as an indicator. Therefore, by inhibiting the release of hexosaminidase, histamine release can also be inhibited, which is thought to be effective in preventing, treating, or improving inflammatory diseases, etc.

[0042] Histamine also mediates intercellular communication as a local neurotransmitter, promoting gastric acid secretion in the digestive tract and functioning as a neurotransmitter in the central nervous system, contributing to maintaining wakefulness. Excessive histamine release can cause ulcers due to excess stomach acid in the digestive tract and contribute to sleep disorders in the central nervous system. As mentioned above, inhibiting the release of hexosaminidase can also inhibit the release of histamine, which is thought to prevent, treat, or improve gastric ulcers, sleep disorders, and other conditions caused by excess stomach acid. For example, extracts from wisteria tea (see Patent Document 14) are known to have the effect of inhibiting hexosaminidase release.

[0043] Inflammation is a complex reaction that manifests with symptoms such as redness, edema, fever, pain, and functional impairment. Microscopically, inflammation consists of common reactions, such as vascular responses resulting in plasma leakage, leukocyte infiltration, and tissue destruction by inflammatory cells. It can also cause systemic reactions, including fever and hyperalgesia, which involve the central nervous system. Prostaglandins play an important role in each of these inflammatory reactions, and it has been shown that the production of prostaglandins during inflammation is primarily mediated by cyclooxygenase-2, an inducible cyclooxygenase. For this reason, many cyclooxygenase inhibitors, such as aspirin, are used to prevent and prevent inflammatory reactions (see Non-Patent Document 8).

[0044] It is known that dihydroferulic acid can be detected when certain types of lactic acid bacteria are cultured in a medium containing ferulic acid or ethyl ferulate (see Non-Patent Document 9 and Patent Document 15). [Prior art documents] [Patent documents]

[0045] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-056855 [Patent Document 2] Japanese Patent Application Publication No. 7-274832 [Patent Document 3] Japanese Patent Application Laid-Open No. 2006-063033 [Patent Document 4] Japanese Patent Application Laid-Open No. 2007-217352 [Patent Document 5] Japanese Patent Application Laid-Open No. 2006-265232 [Patent Document 6] Japanese Patent Application Laid-Open No. 2003-146837 [Patent Document 7] Japanese Patent Application Laid-Open No. 2005-022993 [Patent Document 8] Japanese Patent Application Laid-Open No. 2006-056854 [Patent Document 9] Japanese Patent Application Laid-Open No. 2009-191039 [Patent Document 10] Japanese Patent Application Laid-Open No. 2010-184915 [Patent Document 11] Japanese Patent Application Laid-Open No. 2006-219407 [Patent Document 12] Japanese Patent Application Laid-Open No. 2002-201122 [Patent Document 13] Japanese Patent Application Laid-Open No. 2003-055242 [Patent Document 14] Japanese Patent Application Laid-Open No. 2003-012532 [Patent Document 15] Japanese Patent Application Laid-Open No. 2014-003929 [Non-patent literature]

[0046] [Non-Patent Document 1] Japanese Journal of Pharmacology, 2005, Vol. 125, pp. 379-384 [Non-patent document 2] J. Cell. Biol., 1992, Vol. 199, p. 695-703 [Non-patent document 3] J. Invest. Dermatol.,1979,Vol.73,p.59-66 [Non-patent document 4] Nature, 1996, Vol. 379, p. 335-339 [Non-patent document 5] "Angiogenesis and Matrix Metalloproteinases," Proceedings of the 120th Japanese Medical Association Symposium, Fundamentals and Clinical Practice of Angiogenesis, December 13, 2001, pp. 43-49 [Non-patent document 6] "Fragrance Journal", 2006, Vol. 34, pp. 19-23 [Non-Patent Document 7] Trends Genet.,1992,Vol.8,p.55-61 [Non-patent document 8] "Pharmacology Atlas", Takehiko Fukuhara, Bunkodo, 1995, p.184 [Non-Patent Document 9] J. Sci. Food Agric.,2012,Vol.92,pp.2291-2296 Summary of the Invention [Problem to be solved by the invention]

[0047] The present invention aims to discover compounds derived from natural products that have anti-obesity effects, cyclic AMP phosphodiesterase activity inhibitory effects, dipeptidyl peptidase IV activity inhibitory effects, anti-aging effects, hair growth effects, anti-androgenic effects, whitening effects, or anti-inflammatory effects, and to provide anti-obesity agents, cyclic AMP phosphodiesterase activity inhibitors, dipeptidyl peptidase IV activity inhibitors, anti-aging agents, hair growth agents, anti-androgenic agents, whitening agents, and anti-inflammatory agents that contain these compounds as active ingredients, as well as skin cosmetics, hair cosmetics, and foods and beverages that incorporate these compounds. [Means for solving the problem]

[0048] To achieve the above object, the anti-obesity agent, cyclic AMP phosphodiesterase inhibitor, dipeptidyl peptidase IV inhibitor, anti-aging agent, hair growth agent, anti-androgenic agent, whitening agent, and anti-inflammatory agent of the present invention are characterized by containing dihydroferulic acid as an active ingredient.Furthermore, the skin cosmetic, hair cosmetic, and food and beverage of the present invention are characterized by containing dihydroferulic acid. [Effects of the Invention]

[0049] According to the present invention, by using dihydroferulic acid as an active ingredient, it is possible to provide an anti-obesity agent, a cyclic AMP phosphodiesterase inhibitor, a dipeptidyl peptidase IV inhibitor, an anti-aging agent, a hair growth agent, an anti-androgenic agent, a skin-whitening agent, and an anti-inflammatory agent, which have excellent effects. Furthermore, by incorporating dihydroferulic acid, it is possible to provide skin cosmetics, hair cosmetics, and foods and beverages, which have excellent effects as described above. DETAILED DESCRIPTION OF THE INVENTION

[0050] Hereinafter, an embodiment of the present invention will be described. The anti-obesity agent, cyclic AMP phosphodiesterase inhibitor, dipeptidyl peptidase IV inhibitor, anti-aging agent, hair growth agent, anti-androgenic agent, whitening agent, and anti-inflammatory agent of this embodiment contain dihydroferulic acid as an active ingredient. Furthermore, the skin cosmetic, hair cosmetic, and food and beverage of this embodiment contain dihydroferulic acid.

[0051] Dihydroferulic acid is a cinnamic acid derivative with the chemical structure shown below.

[0052] [ka]

[0053] Dihydroferulic acid can also be produced by fermenting ferulic acid or a ferulic acid derivative such as ethyl ferulate, or a composition containing the same (e.g., crushed or extracted plant material) with a microorganism having phenolic acid reductase to convert the ferulic acid to dihydroferulic acid, followed by extraction, isolation, and purification of the resulting fermented product. Examples of compositions containing ferulic acid include crushed or extracted plant material such as coffee, wheat, corn, tomato, yerba mate, mugwort, and burdock. Furthermore, because ferulic acid is a component of lignin in woody plants, lignin or a composition containing lignin may be used as the composition containing ferulic acid. On the other hand, examples of microorganisms having phenolic acid reductase include lactic acid bacteria such as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus crispatus, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus delbrueckii, Lactobacillus buchneri, Lactobacillus kefiranofaciens, Lactobacillus gallinarum, and Enterococcus faecalis.

[0054] The method for extracting, isolating, and purifying dihydroferulic acid from the above-mentioned plants or fermented products is not particularly limited, and can be carried out according to conventional methods. For example, the extraction process can be carried out by drying the above-mentioned plants or fermented products as the extraction raw material, and then subjecting them to extraction with an extraction solvent either directly or after pulverization using a crusher. Drying can be carried out in the sun or using a commonly used dryer. Furthermore, the raw material can be used after pretreatment such as degreasing with a nonpolar solvent such as hexane. Pretreatment such as degreasing allows for efficient extraction with a polar solvent.

[0055] As the extraction solvent, it is preferable to use a polar solvent, such as water or a hydrophilic organic solvent, which is preferably used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent.

[0056] Water that can be used as an extraction solvent includes pure water, tap water, well water, mineral water, hot spring water, spring water, fresh water, etc., as well as water that has undergone various treatments. Treatments that can be applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, etc. Therefore, water that can be used as an extraction solvent in this embodiment also includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate-buffered physiological saline, etc.

[0057] Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic 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; and polyhydric alcohols having 2 to 5 carbon atoms, such as 1,3-butylene glycol, propylene glycol, and glycerin.

[0058] When a mixture of two or more polar solvents is used as the extraction solvent, the mixing ratio can be adjusted appropriately. For example, when a mixture of water and a lower aliphatic alcohol is used as the extraction solvent, the mixing ratio of water to lower aliphatic alcohol is preferably 9:1 to 1:9 (volume ratio), and more preferably 7:3 to 2:8 (volume ratio). When a mixture of water and a lower aliphatic ketone is used, the mixing ratio of water to lower aliphatic ketone is preferably 9:1 to 2:8 (volume ratio), and when a mixture of water and a polyhydric alcohol is used, the mixing ratio of water to polyhydric alcohol is preferably 5:5 to 1:9 (volume ratio).

[0059] The extraction process is not particularly limited as long as it can dissolve the soluble components contained in the extraction raw material into the extraction solvent, and can be carried out according to conventional methods. For example, the extraction raw material is immersed in an extraction solvent in an amount (mass ratio) 5 to 15 times the amount of the extraction raw material, and the soluble components are extracted at room temperature or under reflux heating, followed by filtration to remove the extraction residue, to obtain an extract. The solvent is distilled off from the obtained extract to obtain a paste-like concentrate, which is then further dried to obtain a dried product.

[0060] The method for isolating and purifying dihydroferulic acid from the extract, concentrate, or dried extract obtained as described above is not particularly limited and can be performed by conventional methods. For example, the extract may be dissolved in a developing solvent and subjected to column chromatography using a porous material such as silica gel or alumina, or a porous resin such as a styrene-divinylbenzene copolymer or polymethacrylate, to recover a fraction containing dihydroferulic acid. In this case, the developing solvent may be appropriately selected depending on the stationary phase used. For example, when the extract is separated by normal-phase chromatography using silica gel as the stationary phase, the developing solvent may be chloroform:methanol=95:5. Furthermore, the fraction containing dihydroferulic acid obtained by column chromatography may be purified using any organic compound purification method, such as reverse-phase silica gel chromatography using ODS, recrystallization, liquid-liquid countercurrent extraction, or column chromatography using an ion-exchange resin.

[0061] [Anti-obesity agent, cyclic AMP phosphodiesterase inhibitor, dipeptidyl peptidase IV inhibitor, anti-aging agent, hair growth agent, anti-androgenic agent, skin whitening agent, anti-inflammatory agent] The dihydroferulic acid obtained as described above has excellent anti-obesity effects, cyclic AMP (cAMP) phosphodiesterase activity inhibitory effects, dipeptidyl peptidase IV (DPPIV) activity inhibitory effects, anti-aging effects, hair growth effects, anti-androgenic effects, whitening effects, and anti-inflammatory effects, and can therefore be used as an active ingredient in anti-obesity agents, cAMP phosphodiesterase activity inhibitors, DPPIV activity inhibitors, anti-aging agents, hair growth agents, anti-androgenic agents, whitening agents, and anti-inflammatory agents. The anti-obesity agents, cAMP phosphodiesterase activity inhibitors, DPPIV activity inhibitors, anti-aging agents, hair growth agents, anti-androgenic agents, whitening agents, and anti-inflammatory agents of this embodiment can be used in a wide range of applications, such as pharmaceuticals, quasi-drugs, and cosmetics.

[0062] In addition, a composition containing dihydroferulic acid may be used instead of isolated dihydroferulic acid as the active ingredient of the anti-obesity agent, cAMP phosphodiesterase inhibitor, DPPIV inhibitor, anti-aging agent, hair growth agent, anti-androgen agent, skin-whitening agent, and anti-inflammatory agent according to this embodiment. Here, the "composition containing dihydroferulic acid" in this embodiment includes an extract obtained using a plant containing dihydroferulic acid as an extraction raw material, a fermented product containing dihydroferulic acid, and an extract obtained using the fermented product as an extraction raw material. Furthermore, the "extract" includes an extract obtained by extraction treatment, a diluted or concentrated solution of the extract, or a dried product obtained by drying the extract.

[0063] When using a composition containing dihydroferulic acid as the active ingredient of the anti-obesity agent, cAMP phosphodiesterase inhibitor, DPPIV inhibitor, anti-aging agent, hair growth agent, anti-androgen agent, whitening agent, and anti-inflammatory agent according to the present embodiment, it is preferable to use dihydroferulic acid that has been purified to increase its purity.By using dihydroferulic acid that has increased its purity as the active ingredient, it is possible to obtain anti-obesity agents, cAMP phosphodiesterase inhibitors, DPPIV inhibitors, anti-aging agents, hair growth agents, anti-androgen agents, whitening agents, and anti-inflammatory agents that have even more excellent effects.

[0064] The anti-obesity effect of dihydroferulic acid is exerted based on one or more actions selected from the group consisting of cAMP phosphodiesterase activity inhibitory action, blood cholesterol-lowering action, blood free fatty acid-lowering action, and DPPIV activity inhibitory action, but the anti-obesity effect of dihydroferulic acid is not limited to the anti-obesity effect exerted based on the above actions.

[0065] Furthermore, dihydroferulic acid may be used as an active ingredient in a blood lipid-reducing agent, a blood cholesterol-reducing agent, or a blood free fatty acid-reducing agent, taking advantage of its blood lipid-reducing effect (particularly the blood cholesterol-reducing effect and the blood free fatty acid-reducing effect).

[0066] The anti-aging effect of dihydroferulic acid is exerted by one or more actions selected from the group consisting of promoting laminin 5 production, inhibiting matrix metalloproteinase-2 (MMP-2) activity, promoting hyaluronic acid production, promoting epidermal keratinocyte proliferation, promoting elastin production, inhibiting matrix metalloproteinase-1 (MMP-1) activity, promoting fibroblast proliferation, promoting aquaporin 3 (AQP3) mRNA expression, and inhibiting the formation of advanced glycation end products (AGEs). However, the anti-aging effect of dihydroferulic acid is not limited to the anti-aging effects exerted by the above actions.

[0067] Furthermore, dihydroferulic acid may be used as the active ingredient of a laminin 5 production promoter, MMP-2 activity inhibitor, hyaluronic acid production promoter, epidermal keratinocyte proliferation promoter, elastin production promoter, MMP-1 activity inhibitor, fibroblast proliferation promoter, AQP3 mRNA expression promoter, or AGE formation inhibitor, taking advantage of its laminin 5 production promoting action, MMP-2 activity inhibitor, hyaluronic acid production promoter, epidermal keratinocyte proliferation promoter, elastin production promoter, MMP-1 activity inhibitor, fibroblast proliferation promoter, AQP3 mRNA expression promoter, or AGE formation inhibitor, respectively.

[0068] The hair-growth effect of dihydroferulic acid is exerted, for example, based on its testosterone 5α-reductase activity inhibitory effect and / or its hair papilla cell proliferation-promoting effect. However, the hair-growth effect of dihydroferulic acid is not limited to the hair-growth effect exerted based on the above-mentioned effects. Furthermore, dihydroferulic acid may be used as the active ingredient of a testosterone 5α-reductase activity inhibitor or a hair papilla cell proliferation promoter, respectively, by utilizing its testosterone 5α-reductase activity inhibitory effect or hair papilla cell proliferation-promoting effect.

[0069] The antiandrogenic effect of dihydroferulic acid is exerted, for example, through its inhibitory effect on testosterone 5α-reductase activity, but is not limited to the antiandrogenic effect exerted through the above-mentioned action.

[0070] The whitening effect of dihydroferulic acid is exerted, for example, based on its melanin production inhibitory effect. However, the whitening effect of dihydroferulic acid is not limited to the whitening effect exerted based on the above-mentioned effect. Furthermore, dihydroferulic acid may be used as an active ingredient of a melanin production inhibitor, taking advantage of its melanin production inhibitory effect.

[0071] The anti-inflammatory effect of dihydroferulic acid is exerted, for example, by one or more actions selected from the group consisting of hyaluronidase activity inhibitory action, hexosaminidase release inhibitory action, and cyclooxygenase-2 (COX-2) activity inhibitory action. However, the anti-inflammatory effect of dihydroferulic acid is not limited to the anti-inflammatory action exerted by the above actions. Furthermore, dihydroferulic acid may be used as the active ingredient of a hyaluronidase activity inhibitor, a hexosaminidase release inhibitor, or a COX-2 activity inhibitor, respectively, by utilizing its hyaluronidase activity inhibitory action, hexosaminidase release inhibitory action, or COX-2 activity inhibitory action.

[0072] The anti-obesity agent, cAMP phosphodiesterase activity inhibitor, DPPIV activity inhibitor, anti-aging agent, hair growth agent, anti-androgenic agent, whitening agent, or anti-inflammatory agent of this embodiment may consist solely of dihydroferulic acid or a composition containing dihydroferulic acid, or may be a formulation of dihydroferulic acid or a composition containing dihydroferulic acid.

[0073] The anti-obesity agent, cAMP phosphodiesterase inhibitor, DPPIV inhibitor, anti-aging agent, hair growth agent, anti-androgen agent, whitening agent, and anti-inflammatory agent of the present embodiment can be formulated into any dosage form, such as powder, granules, tablets, or liquid, using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin, or any other auxiliary agent, according to a conventional method. In this case, examples of auxiliary agents that can be used include excipients, binders, disintegrants, lubricants, stabilizers, and flavorings / flavoring agents. The anti-obesity agent, cAMP phosphodiesterase inhibitor, DPPIV inhibitor, anti-aging agent, hair growth agent, anti-androgen agent, whitening agent, and anti-inflammatory agent can be incorporated into other compositions (e.g., skin cosmetics, hair cosmetics, etc.) and used in the form of ointments, topical liquids, patches, etc.

[0074] When the anti-obesity agent, cAMP phosphodiesterase activity inhibitor, DPPIV activity inhibitor, anti-aging agent, hair growth agent, anti-androgenic agent, whitening agent, or anti-inflammatory agent of this embodiment is formulated, the content of dihydroferulic acid or a composition containing dihydroferulic acid is not particularly limited and can be set appropriately depending on the purpose.

[0075] In addition, the anti-obesity agent, cAMP phosphodiesterase inhibitor, DPPIV inhibitor, anti-aging agent, hair growth agent, anti-androgenic agent, whitening agent, or anti-inflammatory agent of this embodiment can be used as an active ingredient by blending other natural extracts having anti-obesity activity, cAMP phosphodiesterase activity inhibitory activity, DPPIV activity inhibitory activity, anti-aging activity, hair growth activity, anti-androgenic activity, whitening activity, or anti-inflammatory activity together with dihydroferulic acid or a composition containing dihydroferulic acid, as necessary.

[0076] The method of administering the anti-obesity agent, cAMP phosphodiesterase inhibitor, DPPIV inhibitor, anti-aging agent, hair growth agent, anti-androgen agent, whitening agent, or anti-inflammatory agent of this embodiment to a patient includes transdermal administration, oral administration, etc., and a method suitable for the prevention or treatment, etc., may be appropriately selected depending on the type of disease. Furthermore, the dosage of the anti-obesity agent, cAMP phosphodiesterase inhibitor, DPPIV inhibitor, anti-aging agent, hair growth agent, anti-androgen agent, whitening agent, or anti-inflammatory agent of this embodiment may be appropriately increased or decreased depending on the type and severity of the disease, individual patient differences, administration method, administration period, etc.

[0077] The anti-obesity agent of this embodiment can promote cAMP production and promote fat breakdown in adipocytes through one or more actions selected from the group consisting of cAMP phosphodiesterase activity inhibitory action, blood cholesterol-lowering action, blood free fatty acid-lowering action, and DPPIV activity inhibitory action possessed by the active ingredient dihydroferulic acid, and can also reduce blood lipid levels and extend the half-life of incretin in vivo. As a result, the anti-obesity agent of this embodiment can prevent or ameliorate various diseases associated with obesity, such as arteriosclerosis, diabetes, and metabolic syndrome. However, in addition to its use as a cAMP phosphodiesterase activity inhibitor or DPPIV activity inhibitor described below, the anti-obesity agent of this embodiment can also be used for any purpose in which exerting a blood cholesterol-lowering effect or a blood free fatty acid-lowering effect is significant.

[0078] For example, the anti-obesity agent of this embodiment or the aforementioned blood lipid-reducing agent, blood cholesterol-reducing agent, or blood free fatty acid-reducing agent can improve dyslipidemia not caused by obesity (e.g., symptoms of high blood cholesterol or high blood free fatty acids), and can prevent or improve various diseases not associated with obesity, such as arteriosclerosis, diabetes, and metabolic syndrome.

[0079] The cAMP phosphodiesterase activity inhibitor of this embodiment promotes cAMP production through the cAMP phosphodiesterase activity inhibitory effect of the active ingredient dihydroferulic acid, thereby suppressing platelet aggregation and thereby preventing, treating, or ameliorating allergic diseases, various inflammatory diseases, etc. Furthermore, the cAMP phosphodiesterase activity inhibitor of this embodiment promotes fat breakdown in adipocytes through the cAMP phosphodiesterase activity inhibitory effect of dihydroferulic acid, thereby preventing or ameliorating obesity and various lifestyle-related diseases associated therewith, such as arteriosclerosis, diabetes, and metabolic syndrome. However, the cAMP phosphodiesterase activity inhibitor of this embodiment can be used in all applications in which exerting cAMP phosphodiesterase activity inhibitory effect is significant, in addition to these applications.

[0080] The DPPIV activity inhibitor of this embodiment can prevent or treat type 2 diabetes, obesity, hypertension, insulin resistance, and the like through the DPPIV activity inhibitory activity of dihydroferulic acid, as well as autoimmune diseases such as rheumatoid arthritis and transplant rejection. However, the DPPIV activity inhibitor of this embodiment can also be used for any other applications in which exerting DPPIV activity inhibitory activity is meaningful. For example, the DPPIV activity inhibitor of this embodiment can prevent or treat diseases such as pain, neurodegenerative diseases, and neuropsychiatric disorders (e.g., sciatica, Alzheimer's disease, depression, etc.); growth hormone deficiency and diseases treated with growth hormone; cancer (e.g., T-cell lymphoma, acute lymphoblastic leukemia, thyroid cancer, basal cell carcinoma, breast cancer, etc.); and HIV infection (AIDS), through the DPPIV activity inhibitory activity of dihydroferulic acid.

[0081] The anti-aging agent of this embodiment can prevent, treat, or improve skin aging symptoms such as wrinkle formation, loss of elasticity, and loss of moisturizing function through one or more actions selected from the group consisting of laminin 5 production promotion, MMP-2 activity inhibition, hyaluronic acid production promotion, epidermal keratinocyte proliferation promotion, elastin production promotion, MMP-1 activity inhibition, fibroblast proliferation promotion, AQP3 mRNA expression promotion, and AGE formation inhibition, possessed by the active ingredient dihydroferulic acid. However, in addition to these uses, the anti-aging agent of this embodiment can also be used for any purpose in which it is significant to exert the effects of laminin 5 production promotion, MMP-2 activity inhibition, hyaluronic acid production promotion, epidermal keratinocyte proliferation promotion, elastin production promotion, MMP-1 activity inhibition, fibroblast proliferation promotion, AQP3 mRNA expression promotion, or AGE formation inhibition.

[0082] For example, the anti-aging agent of this embodiment or the laminin 5 production promoter described above can promote the production of laminin 5 by utilizing the action of dihydroferulic acid. This can induce the reconstruction of the basement membrane structure and treat or improve skin wounds. Furthermore, the anti-aging agent of this embodiment or the laminin 5 production promoter described above can be used as an agent for preventing or treating diseases (such as epidermolysis bullosa) caused by a deficiency (defect) of laminin 5.

[0083] In addition to the uses described above, the anti-aging agent of this embodiment or the aforementioned MMP-2 activity inhibitor or MMP-1 activity inhibitor can be used for the treatment and prevention of diseases in which destruction of the extracellular matrix is ​​known to be associated with the pathology (e.g., cancer invasion and metastasis, rheumatoid arthritis, knee osteoarthritis, periodontal disease, age-related macular degeneration, etc.) through the MMP-2 activity inhibitory activity or MMP-1 activity inhibitory activity of dihydroferulic acid. Furthermore, the anti-aging agent or MMP-2 activity inhibitor of this embodiment can suppress angiogenesis and tumor cell proliferation through its MMP-2 activity inhibitory activity.

[0084] In addition to the uses described above, the anti-aging agent of this embodiment or the hyaluronic acid production promoter described above can be used, due to its hyaluronic acid production-promoting activity, for the prevention, treatment, or amelioration of arthritis such as rheumatoid arthritis, osteoarthritis, septic arthritis, gouty arthritis, traumatic arthritis, and osteoarthritis; for the promotion of wound or burn healing; etc. Furthermore, the anti-aging agent of this embodiment or the epidermal keratinocyte proliferation promoter described above can be used, due to the epidermal keratinocyte proliferation-promoting activity of dihydroferulic acid, to restore skin metabolism and prevent, treat, or ameliorate fine wrinkles, dullness, pigmentation, etc.; regenerative medicine; etc.

[0085] In addition to the uses described above, the anti-aging agent of this embodiment or the elastin production promoter described above can be used, due to its elastin production-promoting activity, to prevent, treat, or improve pulmonary diseases such as emphysema; vascular diseases such as hypertension and aneurysms; etc. Furthermore, the anti-aging agent of this embodiment or the fibroblast proliferation promoter described above can be used, due to the fibroblast proliferation-promoting activity of dihydroferulic acid, to promote the healing of wounds such as trauma and burns; treat or improve skin diseases (e.g., skin ulcers such as bedsores, burn ulcers, and diabetic ulcers); regenerative medicine; etc.

[0086] In addition to the uses described above, the anti-aging agent of this embodiment or the AQP3 mRNA expression promoter described above can improve moisture retention ability and barrier function caused by aging through the AQP3 mRNA expression-promoting effect of dihydroferulic acid.

[0087] In addition to the uses described above, the anti-aging agent of this embodiment or the aforementioned AGE formation inhibitor can, through the AGE formation inhibitory effect of dihydroferulic acid, prevent or treat diabetic complications such as diabetic neuropathy, diabetic retinopathy, and diabetic nephropathy; arteriosclerosis caused by protein glycation; osteoporosis and osteoarthritis caused by protein glycation; etc. Furthermore, the anti-aging agent of this embodiment or the aforementioned AGE formation inhibitor can, through its AGE formation inhibitory effect, prevent or suppress hair damage caused by protein glycation, thereby preventing or suppressing coarse hair, restoring hair elasticity and suppleness, and imparting firmness and strength to hair.

[0088] The hair growth agent of the present embodiment can prevent, treat, or improve alopecia such as androgenetic alopecia, alopecia areata, and trichotillomania through the testosterone 5α-reductase activity inhibitory effect and / or dermal papilla cell proliferation promoting effect of the active ingredient dihydroferulic acid, and is particularly suitable for the prevention, treatment, or improvement of androgenetic alopecia. However, in addition to these uses, the hair growth agent of the present invention can also be used for all uses in which it is significant to exert the testosterone 5α-reductase activity inhibitory effect or the dermal papilla cell proliferation promoting effect.

[0089] For example, the hair growth agent of this embodiment or the aforementioned hair papilla cell proliferation promoter can activate hair papilla cells and promote the proliferation and differentiation of hair follicle epithelial cells and hair formation through the hair papilla cell proliferation-promoting activity of dihydroferulic acid, and can also prevent the transition from the anagen phase to the catagen and telogen phase in the hair cycle, thereby extending the anagen phase. This makes it possible to prevent or improve alopecia. Furthermore, the hair growth agent of this embodiment or the aforementioned hair papilla cell proliferation promoter can also be used in applications in the field of regenerative medicine, such as hair regeneration using hair papilla cells, through the dermal papilla cell proliferation-promoting activity of dihydroferulic acid.

[0090] The antiandrogenic agent of this embodiment or the above-described testosterone 5α-reductase inhibitor can prevent, treat, or improve androgenic diseases, such as male pattern baldness, hirsutism, seborrhea, acne (e.g., acne), benign prostatic hyperplasia, prostatic tumors, and precocious male puberty, through the testosterone 5α-reductase inhibitory activity of the active ingredient, dihydroferulic acid. However, the antiandrogenic agent of the present invention can also be used for all other applications in which it is significant to exert a testosterone 5α-reductase inhibitory activity.

[0091] The whitening agent of the present embodiment can prevent or improve pigmentation such as skin darkening, age spots, and freckles through the melanin production inhibitory effect of the active ingredient dihydroferulic acid. However, the whitening agent of the present embodiment can also be used for all other applications in which exerting an inhibitory effect on melanin production is significant.

[0092] The anti-inflammatory agent of this embodiment can prevent, treat, or improve various inflammatory skin diseases associated with rough skin, such as contact dermatitis (rash), psoriasis, pemphigus vulgaris, atopic dermatitis, and other skin conditions, through one or more actions selected from the group consisting of hyaluronidase activity inhibitory action, hexosaminidase release inhibitory action, and COX-2 activity inhibitory action possessed by the active ingredient dihydroferulic acid. However, in addition to these uses, the anti-inflammatory agent of this embodiment can also be used for any use in which exerting hyaluronidase activity inhibitory action, hexosaminidase release inhibitory action, or COX-2 activity inhibitory action is meaningful.

[0093] For example, the anti-inflammatory agent of this embodiment or the aforementioned hyaluronidase activity inhibitor, hexosaminidase release inhibitor, or COX-2 activity inhibitor can prevent, treat, or ameliorate rheumatoid arthritis, osteoarthritis, asthma, etc. through the hyaluronidase activity inhibitory effect, hexosaminidase release inhibitory effect, or COX-2 activity inhibitory effect of dihydroferulic acid. Furthermore, the anti-inflammatory agent of this embodiment or the aforementioned hexosaminidase release inhibitor can prevent, treat, or ameliorate gastric ulcers, sleep disorders, etc. caused by gastric hyperacidity through the hexosaminidase release inhibitory effect of dihydroferulic acid.

[0094] Furthermore, the anti-obesity agent, cAMP phosphodiesterase inhibitor, DPPIV inhibitor, anti-aging agent, hair growth agent, anti-androgenic agent, whitening agent, or anti-inflammatory agent of the present embodiment has excellent anti-obesity activity, cAMP phosphodiesterase inhibitory activity, DPPIV inhibitory activity, anti-aging activity, hair growth agent, anti-androgenic agent, whitening agent, or anti-inflammatory activity, and is therefore suitable for incorporation into, for example, external skin preparations. In this case, dihydroferulic acid or a composition containing dihydroferulic acid may be incorporated as is, or an anti-obesity agent, cAMP phosphodiesterase inhibitor, DPPIV inhibitor, anti-aging agent, hair growth agent, anti-androgenic agent, whitening agent, or anti-inflammatory agent formulated from a composition containing dihydroferulic acid or dihydroferulic acid may be incorporated.

[0095] Here, the topical skin preparation is not limited to any particular category, and includes a wide range of products such as skin cosmetics, which will be described later, as well as quasi-drugs and pharmaceuticals that are used transdermally.

[0096] Furthermore, the anti-obesity agent, cAMP phosphodiesterase activity inhibitor, DPPIV activity inhibitor, anti-aging agent, hair growth agent, anti-androgenic agent, whitening agent, or anti-inflammatory agent of the present embodiment has excellent anti-obesity action, cAMP phosphodiesterase activity inhibitory action, DPPIV activity inhibitory action, anti-aging action, hair growth action, anti-androgenic action, whitening action, or anti-inflammatory action, and can therefore be suitably used as a reagent for research on the mechanisms of these actions.

[0097] [Skin cosmetics, hair cosmetics] Dihydroferulic acid has excellent anti-aging, hair-restoring, anti-androgenic, whitening, anti-inflammatory, cAMP phosphodiesterase inhibitory, DPPIV inhibitory, and anti-obesity effects, making it suitable for incorporation into skin or hair cosmetics. In this case, dihydroferulic acid or a composition containing dihydroferulic acid may be incorporated as is, or an anti-aging agent, hair-restoring agent, anti-androgenic agent, whitening agent, anti-inflammatory agent, cAMP phosphodiesterase inhibitor, DPPIV inhibitor, or anti-obesity agent formulated from a composition containing dihydroferulic acid or dihydroferulic acid may be incorporated.

[0098] By incorporating dihydroferulic acid or the above-mentioned anti-aging agents, hair growth agents, anti-androgen agents, whitening agents, anti-inflammatory agents, cAMP phosphodiesterase activity inhibitors, DPPIV activity inhibitors, or anti-obesity agents, skin cosmetics or hair cosmetics can have anti-aging effects, laminin 5 production promotion effects, MMP-2 activity inhibition effects, hyaluronic acid production promotion effects, epidermal keratinocyte proliferation promotion effects, elastin production promotion effects, MMP-1 activity inhibition effects, fibroblast proliferation promotion effects, and AQP3 mRNA expression promotion effects. The compounds can have the following effects: hair growth promoting effect, AGE formation inhibitory effect, hair growth promoting effect, testosterone 5α-reductase activity inhibitory effect, hair follicle papilla cell proliferation promoting effect, anti-androgenic effect, skin whitening effect, melanin production inhibitory effect, anti-inflammatory effect, hyaluronidase activity inhibitory effect, hexosaminidase release inhibitory effect, COX-2 activity inhibitory effect, cAMP phosphodiesterase activity inhibitory effect, DPPIV activity inhibitory effect, anti-obesity effect, blood cholesterol reducing effect, or blood free fatty acid reducing effect.

[0099] All of these effects are favorable when imparted to skin or hair cosmetics. Among these, the following effects are particularly favorable when imparted to skin cosmetics: anti-aging effect, laminin 5 production promotion effect, MMP-2 activity inhibition effect, hyaluronic acid production promotion effect, epidermal keratinocyte proliferation promotion effect, elastin production promotion effect, MMP-1 activity inhibition effect, fibroblast proliferation promotion effect, AQP3 mRNA expression promotion effect, AGE formation inhibition effect, whitening effect, melanin production inhibition effect, anti-inflammatory effect, hyaluronidase activity inhibition effect, hexosaminidase release inhibition effect, COX-2 activity inhibition effect, cAMP phosphodiesterase activity inhibition effect, testosterone 5α-reductase activity inhibition effect, and anti-androgenic effect, because these effects are more easily exerted.

[0100] Furthermore, among the above-mentioned effects, hair growth effect, testosterone 5α-reductase activity inhibitory effect, hair papilla cell proliferation promoting effect, anti-androgenic effect, anti-inflammatory effect, hyaluronidase activity inhibitory effect, hexosaminidase release inhibitory effect, COX-2 activity inhibitory effect, cAMP phosphodiesterase activity inhibitory effect, laminin 5 production promoting effect, hyaluronic acid production promoting effect, elastin production promoting effect, fibroblast proliferation promoting effect, or AGE formation inhibitory effect are particularly preferred when imparted to hair cosmetics, as these effects are more likely to be exhibited.

[0101] The type of skin cosmetic or hair cosmetic that can be formulated with dihydroferulic acid or the above-mentioned anti-aging agent, hair growth agent, anti-androgenic agent, whitening agent, anti-inflammatory agent, cAMP phosphodiesterase activity inhibitor, DPPIV activity inhibitor, or anti-obesity agent is not particularly limited. Examples of skin cosmetic products include ointments, creams, emulsions, lotions, packs, and foundations, and examples of hair cosmetic products include hair tonics, hair creams, hair liquids, shampoos, pomades, and rinses.

[0102] When dihydroferulic acid or the above-mentioned anti-aging agent, hair growth agent, anti-androgenic agent, whitening agent, anti-inflammatory agent, cAMP phosphodiesterase inhibitor, DPPIV inhibitor, or anti-obesity agent is incorporated into a skin cosmetic or hair cosmetic, the amount incorporated can be adjusted appropriately depending on the type of skin cosmetic or hair cosmetic. A suitable incorporation rate is about 0.0001 to 10% by mass, and a particularly suitable incorporation rate is about 0.001 to 1% by mass calculated as a standard extract.

[0103] The skin cosmetic or hair cosmetic of this embodiment has the following properties: anti-aging effect, laminin 5 production promoting effect, MMP-2 activity inhibitory effect, hyaluronic acid production promoting effect, epidermal keratinocyte proliferation promoting effect, elastin production promoting effect, MMP-1 activity inhibitory effect, fibroblast proliferation promoting effect, AQP3 mRNA expression promoting effect, AGE formation suppression effect, hair growth effect, testosterone 5α-reductase activity inhibitory effect, dermal papilla cell proliferation promoting effect, anti-androgenic effect, whitening effect, melanin production suppression effect, anti-inflammatory effect, hyaluronidase activity inhibitory effect, hexosaminidase inhibitory effect, and the like, which are possessed by dihydroferulic acid. As long as the enzyme release inhibitory effect, COX-2 activity inhibitory effect, cAMP phosphodiesterase activity inhibitory effect, DPPIV activity inhibitory effect, anti-obesity effect, blood cholesterol lowering effect, or blood free fatty acid lowering effect are not interfered with, the main ingredients, auxiliary ingredients, or other ingredients typically used in the manufacture of skin or hair cosmetics, such as astringents, germicides / antibacterial agents, whitening agents, UV absorbers, moisturizers, cell activators, anti-inflammatory / antiallergic agents, antioxidants / active oxygen scavengers, oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances, etc., can be used in combination. Such combinations can result in more general-purpose products, and the synergistic effect with the other active ingredients used in combination can sometimes produce superior effects beyond those normally expected.

[0104] The skin cosmetic of this embodiment has the following properties: anti-aging effect, laminin 5 production promoting effect, MMP-2 activity inhibitory effect, hyaluronic acid production promoting effect, epidermal keratinocyte proliferation promoting effect, elastin production promoting effect, MMP-1 activity inhibitory effect, fibroblast proliferation promoting effect, AQP3 mRNA expression promoting effect, AGE formation suppression effect, whitening effect, melanin production suppression effect, anti-inflammatory effect, hyaluronidase activity inhibitory effect, hexosaminidase release suppression effect, COX-2 activity inhibitory effect, cAMP phosphodiesterase activity inhibitory effect, hair growth effect, testosterone 5α-reductase activity inhibitory effect, dermal papilla cell proliferation promoting effect, anti-androgenic effect, DPPIV activity inhibitory effect, anti-obesity effect, blood cholesterol reducing effect, and blood migration suppression effect, all of which are possessed by dihydroferulic acid. and the like. Through one or more actions selected from the group consisting of the action of reducing free fatty acids, the compound can prevent, treat, or improve symptoms of skin aging such as the formation of wrinkles, loss of elasticity, and loss of moisturizing function; promote the healing of wounds or burns; prevent, treat, or improve rough skin, dry skin, and dry skin diseases (e.g., atopic dermatitis, psoriasis, ichthyosis, etc.); prevent, treat, or improve pigmentation such as skin darkening, age spots, and freckles; prevent, treat, or improve contact dermatitis (rash), psoriasis, pemphigus vulgaris, and other various inflammatory skin diseases associated with rough skin; prevent, treat, or improve seborrhea, acne (pimples, etc.); and prevent, treat, or improve obesity and lifestyle-related diseases associated with it such as arteriosclerosis, diabetes, and metabolic syndrome.

[0105] Furthermore, the hair cosmetic composition of the present embodiment has the following effects: hair growth effect, testosterone 5α-reductase activity inhibitory effect, hair papilla cell proliferation promoting effect, anti-androgenic effect, anti-inflammatory effect, hyaluronidase activity inhibitory effect, hexosaminidase release inhibitory effect, COX-2 activity inhibitory effect, cAMP phosphodiesterase activity inhibitory effect, anti-aging effect, laminin 5 production promoting effect, MMP-2 activity inhibitory effect, hyaluronic acid production promoting effect, epidermal keratinocyte proliferation promoting effect, elastin production promoting effect, MMP-1 activity inhibitory effect, fibroblast proliferation promoting effect, AQP3 mRNA expression promoting effect, AGE formation suppression effect, whitening effect, melanin Through one or more actions selected from the group consisting of inhibitory action on collagen production, inhibitory action on DPPIV activity, anti-obesity action, blood cholesterol-lowering action, and blood free fatty acid-lowering action, it is possible to prevent, treat, or improve alopecia such as male pattern baldness, alopecia areata, and trichotillomania; treat rough skin, dry skin, and dry skin diseases (e.g., atopic dermatitis, psoriasis, ichthyosis, etc.); prevent, treat, or improve contact dermatitis (rash), psoriasis, pemphigus vulgaris, and other various inflammatory skin diseases associated with rough skin; prevent or suppress coarse hair, restore hair elasticity and suppleness, and give hair firmness and strength; etc.

[0106] [Food and beverages] Dihydroferulic acid has excellent anti-obesity effects, cAMP phosphodiesterase activity inhibitory effects, DPPIV activity inhibitory effects, anti-aging effects, hair growth effects, anti-androgenic effects, whitening effects, and anti-inflammatory effects, and is therefore suitable for incorporation into foods and beverages. In this case, dihydroferulic acid may be incorporated as is, or anti-obesity agents, cAMP phosphodiesterase activity inhibitors, DPPIV activity inhibitors, anti-aging agents, hair growth agents, anti-androgenic agents, whitening agents, and anti-inflammatory agents formulated from dihydroferulic acid may be incorporated.

[0107] Here, food and drink refers to those that are unlikely to be harmful to human health and are taken orally or by administration through the digestive tract in normal social life, and are not limited to administrative classifications such as food, medicine, quasi-drug, etc. Therefore, in this embodiment, "food and drink" broadly includes general foods, health foods (functional food and drink), health functional foods (foods for specified health uses, foods with nutrient functions), quasi-drugs, medicines, etc. that are taken orally.

[0108] By incorporating dihydroferulic acid or the above-mentioned anti-obesity agents, cAMP phosphodiesterase inhibitors, DPPIV inhibitors, anti-aging agents, hair growth agents, anti-androgen agents, anti-inflammatory agents, or skin whitening agents into foods and beverages, it is possible to achieve anti-obesity effects, blood cholesterol reducing effects, blood free fatty acid reducing effects, cAMP phosphodiesterase activity inhibitory effects, DPPIV activity inhibitory effects, anti-aging effects, laminin 5 production promoting effects, MMP-2 activity inhibitory effects, hyaluronic acid production promoting effects, and epidermal The following effects can be imparted to foods and beverages: keratinocyte proliferation promotion, elastin production promotion, MMP-1 activity inhibition, fibroblast proliferation promotion, AQP3 mRNA expression promotion, AGE formation inhibition, hair growth promotion, testosterone 5α-reductase activity inhibition, hair papilla cell proliferation promotion, anti-androgenic effect, anti-inflammatory effect, hyaluronidase activity inhibition, hexosaminidase release inhibition, COX-2 activity inhibition, skin whitening effect, or melanin production inhibition effect. These effects are preferably imparted to foods and beverages because they are easily exerted when imparted to foods and beverages.

[0109] When dihydroferulic acid or an anti-obesity agent, cAMP phosphodiesterase inhibitor, DPPIV inhibitor, anti-aging agent, hair growth agent, anti-androgen agent, whitening agent, or anti-inflammatory agent formulated from dihydroferulic acid is incorporated into a food or beverage, the amount of active ingredient can be appropriately changed taking into account the intended use, symptoms, gender, etc., but taking into account the general intake of the food or beverage to be added, it is preferable to adjust the amount of extract to about 1 to 1000 mg per day for an adult. Note that when the food or beverage to be added is in the form of granules, tablets, or capsules, the amount of dihydroferulic acid or an anti-obesity agent, cAMP phosphodiesterase inhibitor, DPPIV inhibitor, anti-aging agent, hair growth agent, anti-androgen agent, whitening agent, or anti-inflammatory agent added is usually 0.1 to 100% by mass, preferably 5 to 100% by mass, of the food or beverage to be added.

[0110] The food and drink of this embodiment may be any food and drink containing dihydroferulic acid that does not interfere with its activity, or may be a nutritional supplement containing dihydroferulic acid as a main ingredient.

[0111] When producing the food and beverage of this embodiment, any auxiliary agent can be added, such as sugars such as dextrin and starch; proteins such as gelatin, soy protein and corn protein; amino acids such as alanine, glutamine and isoleucine; polysaccharides such as cellulose and gum arabic; and fats and oils such as soybean oil and medium-chain fatty acid triglycerides, to form the food and beverage into any shape.

[0112] There are no particular limitations on the foods and beverages that can contain dihydroferulic acid, but specific examples include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and lactic acid drinks (including concentrated concentrates and powders for adjusting these beverages); frozen desserts such as ice cream, ice sorbet, and shaved ice; noodles such as soba, udon, harusame, gyoza wrappers, shumai wrappers, Chinese noodles, and instant noodles; sweets such as candy, chewing gum, candy, chewing gum, chocolate, tablet sweets, snacks, biscuits, jellies, jams, creams, and baked goods; These include processed seafood and livestock foods such as maboko, ham, and sausage; dairy products such as processed milk and fermented milk; oils and fats such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings, as well as oil-based processed foods; seasonings such as sauces and dressings; soups, stews, salads, side dishes, and pickles; and various other forms of health and nutritional supplements; tablets, capsules, and drinks.When dihydroferulic acid is added to these foods and beverages, it can also be used in conjunction with commonly used auxiliary ingredients and additives.

[0113] The anti-obesity agents, cAMP phosphodiesterase activity inhibitors, DPPIV activity inhibitors, anti-aging agents, hair growth agents, anti-androgen agents, whitening agents, anti-inflammatory agents, skin cosmetics, hair cosmetics, and foods and beverages of this embodiment are preferably applied to humans, but can also be applied to animals other than humans (e.g., mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) as long as their respective effects are achieved. [Example]

[0114] The present invention will be specifically explained below by showing test examples, but the present invention is not limited to the following examples. In these test examples, dihydroferulic acid (Tokyo Chemical Industry Co., Ltd., Sample 1) was used as the test sample.

[0115] [Test Example 1] Cyclic AMP phosphodiesterase activity inhibitory effect test Dihydroferulic acid (Sample 1) was tested for its inhibitory effect on cyclic AMP phosphodiesterase activity as follows.

[0116] To 0.2 mL of 50 mmol / L Tris-HCl buffer (pH 7.5) containing 5 mmol / L magnesium chloride, 0.1 mL of 2.5 mg / mL bovine serum albumin solution, 0.1 mL of 0.1 mg / mL cyclic AMP phosphodiesterase solution, and 0.05 mL of test sample solution (Sample 1, sample concentrations are shown in Table 1 below) were added and incubated at 37°C for 5 minutes. Then, 0.05 mL of 0.5 mg / mL cyclic AMP solution was added and incubated at 37°C for 60 minutes. After completion of the reaction, the mixture was boiled in a boiling water bath for 3 minutes, centrifuged (2260 × g, 10 minutes, 4°C), and the reaction substrate cyclic AMP in the supernatant was analyzed using high-performance liquid chromatography (HPLC) condition 3 described below. A control was also performed using the same procedure with the solvent alone, without the sample.

[0117] <High-performance liquid chromatography condition 3> Product name: Chromatocorder 12 (manufactured by SYSTEM INSTRUMENTS) Stationary phase: Wakosil C 18 -ODS 5μm (Wako Pure Chemical Industries, Ltd.) Column length: 250 mm Mobile phase: 1mmol / L TBAP in 25mmol / L KH2PO4:CH3CN=90:10 Mobile phase flow rate: 1.0mL / min Detection: 260nm

[0118] Next, the peak area (A) of the cyclic AMP standard, the peak area (B1) of the supernatant of the reaction solution of the cyclic AMP standard and cyclic AMP phosphodiesterase without the addition of a sample, and the peak area (B2) of the supernatant of the reaction solution of the cyclic AMP standard and cyclic AMP phosphodiesterase with the addition of a test sample were determined. From the obtained results, the decomposition rates of the cyclic AMP standard without the addition of a sample (C) and with the addition of a test sample (D) were calculated using the following formula:

[0119] Decomposition rate of standard without sample (C,%) = (1-B1 / A) x 100 Decomposition rate of standard product when test sample is added (D,%) = (1-B2 / A) x 100

[0120] Thereafter, based on the decomposition rates (C, D) calculated by the above formula, the cyclic AMP phosphodiesterase activity inhibition rate (%) was calculated by the following formula. cAMP phosphodiesterase activity inhibition rate (%) = (1-D / C) x 100 The results are shown in Table 1.

[0121] [Table 1]

[0122] As shown in Table 1, it was confirmed that dihydroferulic acid (sample 1) has an excellent inhibitory effect on cyclic AMP phosphodiesterase activity.

[0123] [Test Example 2] Blood lipid lowering effect test Dihydroferulic acid (sample 1) was tested for its blood lipid-lowering effect as follows.

[0124] Four-week-old TSOD mice (purchased from Shimizu Experimental Materials) were thoroughly quarantined and acclimatized, and then observed for general condition and weighed. Animals in good health were selected for use at 5 weeks of age. The animals were housed in plastic cages at a temperature of 22±3°C, humidity of 55±15%, ventilation with a constant all-fresh system, and lighting for 12 hours per day (6:00 AM to 6:00 PM). They were fed a solid laboratory animal feed, Lab MR Stock (Nippon Nosan Kogyo Co., Ltd.), ad libitum, and provided with tap water via an automatic watering system.

[0125] Five-week-old mice were divided into a dihydroferulic acid 50 mg / kg / day group and a control group (7 mice per group, total 14 mice). They were administered dihydroferulic acid intragastrically once daily using a gastrostomy tube and maintained for 7 weeks. They were fasted for 16 hours starting the day before the end of the study, and blood was collected under isoflurane anesthesia at the end of the study. Plasma was prepared from the collected blood by centrifugation (3,000 rpm, 10 minutes), and the amounts of total cholesterol and free fatty acids were measured. Total cholesterol (T-CHO) was measured using a DRI-CHEM3030 (Fujifilm Corporation), and free fatty acids (NEFA) were measured using a NEFA-C Test Wako (Wako Pure Chemical Industries, Ltd.). The results are shown in Table 2.

[0126] [Table 2]

[0127] As shown in Table 2, it was confirmed that dihydroferulic acid (sample 1) has excellent effects of reducing blood cholesterol and blood free fatty acids.

[0128] [Test Example 3] DPPIV activity inhibitory effect test Dihydroferulic acid (Sample 1) was tested for its dipeptidyl peptidase IV (DPPIV) inhibitory activity as follows.

[0129] In a 96-well plate, 25 μL of a test sample (Sample 1, see Table 3 below for final concentration) prepared in 25 mM Tris-HCl buffer (pH 8.0) was mixed with 25 μL of a 0.4 μg / mL DPPIV (rhCD26, R&D Systems) solution prepared in the same buffer, and the mixture was preincubated at 37°C for 5 minutes. Then, 50 μL of 0.5 mM Gly-Pro-p-NA·Tos (Peptide Institute) prepared in the same buffer was added, and the mixture was incubated at 37°C for 90 minutes. After the reaction, the absorbance at 415 nm was measured. The DPPIV inhibition rate (%) was calculated from the results using the following formula:

[0130] DPPIV inhibition rate (%) = {1-(CD) / (AB)} × 100 The terms in the formula represent the following: A: Absorbance at 415 nm with no sample added and enzyme added B: Absorbance at 415 nm without adding sample or enzyme C: Absorbance at 415 nm after adding test sample and enzyme D: Absorbance at 415 nm with test sample and without enzyme added The results are shown in Table 3.

[0131] [Table 3]

[0132] As shown in Table 3, dihydroferulic acid (sample 1) exhibited excellent DPPIV inhibitory activity.

[0133] [Test Example 4] Laminin 5 production promoting effect test Dihydroferulic acid (sample 1) was tested for its laminin 5 production promoting effect as follows.

[0134] Human normal neonatal epidermal keratinocytes (NHEK) were cultured at 80cm 2 The cells were pre-cultured in a flask using human normal epidermal keratinocyte medium (KGM) at 37°C, 5% CO2, and 95% air, and then harvested by trypsinization. The harvested cells were collected at a concentration of 1.0 × 10 5 The cells were diluted with KGM medium (KGM-BPE) to a cell density of 100 cells / mL, and then seeded in a 24-well plate at 500 μL per well. The cells were cultured at 37°C in an atmosphere of 5% CO2 and 95% air for two days.

[0135] After incubation, the medium was removed, and 500 μL of the test sample (Sample 1, see Table 4 below for sample concentrations) dissolved in KGM-BPE medium was added to each well and incubated for 48 hours at 37°C in 5% CO2 and 95% air. As a control, cells were incubated in the same manner using KGM-BPE medium without the sample. After incubation, 100 μL of the supernatant was transferred to an ELISA plate and allowed to adsorb to the plate for 2 hours at 37°C. The amount of adsorbed laminin-5 was measured by ELISA using a monoclonal anti-human laminin-5 antibody (mouse IgG, Chemicon). The laminin-5 production promotion rate (%) was calculated from the results using the following formula:

[0136] Laminin 5 production promotion rate (%) = A / B x 100 The terms in the formula represent the following: A: Absorbance at a wavelength of 405 nm when the test sample is added B: Absorbance at 405 nm without sample addition The results are shown in Table 4.

[0137] [Table 4]

[0138] As shown in Table 4, it was confirmed that dihydroferulic acid (sample 1) has an excellent effect of promoting laminin 5 production.

[0139] [Test Example 5] MMP-2 activity inhibitory effect test MMP-2 was prepared according to the method described in Japanese Patent Application Laid-Open No. 2007-217352. Specifically, MMP-2 protein was mass-expressed in an Escherichia coli gene expression system as a recombinant proMMP protein with six histidine residues at the C-terminus, purified using Ni-NTA resin, refolded, and then converted to an active form. This enzyme preparation was diluted appropriately to prepare an enzyme solution. Using the obtained enzyme solution, the inhibitory effect of dihydroferulic acid (sample 1) on MMP-2 activity was tested as follows.

[0140] A 96-well plate for measuring fluorescence intensity was used to mix 20 μL of test sample (Sample 1; see Table 5 below for sample concentrations), 40 μL of enzyme solution, and 20 μL of buffer solution (0.05 mol / L Tris, 150 mmol / L NaCl, 10 mmol / L CaCl2, 50 μmol / L ZnSO4, 0.02% NaN3, 0.05% Brij35 (pH 7.5)) and incubate at 37°C for 15 minutes. Then, 120 μL of a 4.16 μmol / L fluorescent substrate peptide (MOCAc / DNP peptide) was added. The fluorescence intensity was measured immediately at an excitation wavelength of 340 nm and an emission wavelength of 420 nm. This was recorded as the fluorescence intensity immediately after substrate addition. After the measurement, the reaction was allowed to continue for 120 minutes at 37°C. During this time, the fluorescence intensity at an excitation wavelength of 340 nm and an emission wavelength of 420 nm was measured every 15 minutes. This was recorded as the fluorescence intensity after substrate addition. A blank experiment was also performed in the same manner to correct for the fluorescence intensity. From the obtained results, the MMP-2 activity inhibition rate (%) was calculated using the following formula.

[0141] MMP-2 activity inhibition rate (%) = {1-(CD) / (AB)} × 100 The terms in the formula represent the following: A: Fluorescence intensity at 420 nm 120 minutes after adding substrate without adding sample B: Fluorescence intensity at 420 nm immediately after adding substrate without adding sample C: Fluorescence intensity at 420 nm 120 minutes after addition of the substrate in the test sample D: Fluorescence intensity at 420 nm immediately after adding the substrate when the test sample was added The results are shown in Table 5.

[0142] [Table 5]

[0143] As shown in Table 5, it was confirmed that dihydroferulic acid (sample 1) has an excellent inhibitory effect on MMP-2 activity.

[0144] [Test Example 6] Dermal hyaluronic acid production promotion test Human normal skin fibroblasts (NB1RGB) were cultured in α-MEM medium containing 10% FBS, and then the cells were harvested by trypsinization. The harvested cells were collected at a concentration of 1.6 × 10 5 The cells were diluted with α-MEM medium containing 0.5% FBS to a cell density of 100 cells / mL, and then seeded in a 96-well plate at 100 μL per well and cultured overnight.

[0145] After the incubation, the medium was removed, and 100 μL of the test sample (Sample 1, see Table 6 below for sample concentration) dissolved in 0.5% FBS-containing α-MEM medium was added to each well and incubated for 3 days. As a control, 0.5% FBS-containing α-MEM medium without sample was used for incubation in the same manner. After incubation, the amount of hyaluronic acid in the medium of each well was measured by the sandwich method using hyaluronic acid binding protein (HABP). From the results obtained, the hyaluronic acid production promotion rate (%) was calculated using the following formula.

[0146] Dermal hyaluronic acid production promotion rate (%) = A / B x 100 The terms in the formula represent the following: A: Amount of hyaluronic acid when test sample was added B: Amount of hyaluronic acid when no test sample is added The results are shown in Table 6.

[0147] [Table 6]

[0148] As shown in Table 6, it was confirmed that dihydroferulic acid (sample 1) has an excellent effect of promoting dermal hyaluronic acid production.

[0149] [Test Example 7] Epidermal keratinocyte proliferation promoting activity test Dihydroferulic acid (Sample 1) was tested for its effect of promoting epidermal keratinocyte proliferation as follows.

[0150] Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM), and then the cells were harvested by trypsinization. The harvested cells were collected at a concentration of 3.0 × 10 4 After diluting the cells with KGM medium to a cell density of 100 cells / mL, 100 μL of the cells were seeded per well of a collagen-coated 96-well plate and cultured overnight. After the culture was completed, 100 μL of the test sample (Sample 1, see Table 7 below for sample concentration) dissolved in KGM medium was added to each well and cultured for 3 days. As a control, KGM medium without the sample was used and cultured in the same manner.

[0151] The epidermal keratinocyte proliferation-promoting activity was measured using the MTT assay. Specifically, after 3 days of culture, the medium was removed, and 100 μL of MTT dissolved in PBS(-) buffer at a final concentration of 0.4 mg / mL was added to each well. After 2 hours of culture, the blue formazan produced within 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 to determine the amount of blue formazan produced. From the results obtained, the epidermal keratinocyte proliferation promotion rate (%) was calculated using the following formula:

[0152] Epidermal keratinocyte proliferation promotion rate (%)=St / Ct×100 The terms in the formula represent the following: St: Amount of blue formazan produced in cells to which the test sample was added Ct: Amount of blue formazan produced in cells without sample added The results are shown in Table 7.

[0153] [Table 7]

[0154] As shown in Table 7, it was confirmed that dihydroferulic acid (sample 1) has an excellent effect of promoting the proliferation of epidermal keratinocytes.

[0155] [Test Example 8] Elastin production promoting effect test Dihydroferulic acid (Sample 1) was tested for its elastin production-promoting effect as follows.

[0156] Human normal fibroblasts (NB1RGB) were cultured in Dulbecco's MEM medium containing 10% FBS, and then the cells were harvested by trypsinization. The harvested cells were collected at a concentration of 2.2 × 10 5 After diluting the cells with the above medium to a cell density of 100 cells / mL, the cells were seeded in a 96-well microplate at 100 μL per well and cultured overnight.

[0157] After incubation, the medium was removed, and 150 μL of the test sample (Sample 1, see Table 8 below for sample concentration) dissolved in 0.25% FBS-containing Dulbecco's MEM medium was added to each well and incubated for two days. As a control, cells were incubated in the same manner using 0.25% FBS-containing Dulbecco's MEM medium without the sample. After incubation, the supernatant was collected, and the amount of elastin released into the culture supernatant was measured by ELISA. The elastin production promotion rate (%) was calculated from the measurement results using the following formula:

[0158] Elastin production promotion rate (%) = A / B x 100 The terms in the formula represent the following: A: Amount of elastin when test sample was added B: Amount of elastin without sample The results are shown in Table 8.

[0159] [Table 8]

[0160] As shown in Table 8, dihydroferulic acid (sample 1) exhibited an excellent effect of promoting elastin production.

[0161] [Test Example 9] MMP-1 activity inhibitory effect test Dihydroferulic acid (Sample 1) was tested for its inhibitory effect on MMP-1 activity as follows.

[0162] In a capped test tube, 50 μL of the test sample (Sample 1, see Table 9 below for sample concentration) dissolved in 0.1 mol / L Tris-HCl buffer (pH 7.1) containing 20 mmol / L calcium chloride, 50 μL of MMP-1 solution (Sigma, COLLAGENASE Type IV from Clostridium histolyticum), and 400 μL of Pz peptide solution (BACHEM Feinchemikalien AG, Pz-Pro-Leu-Gly-Pro-D-Arg-OH) were mixed and reacted at 37°C for 30 minutes, after which 1 mL of 25 mmol / L citric acid solution was added to stop the reaction.

[0163] After that, 5 mL of ethyl acetate was added and the mixture was vigorously shaken. The mixture was centrifuged (1600 × g, 10 minutes), and the absorbance of the ethyl acetate layer was measured at a wavelength of 320 nm. A blank test was also performed in the same manner, and correction was performed. From the obtained results, the MMP-1 activity inhibition rate (%) was calculated using the following formula.

[0164] MMP-1 activity inhibition rate (%) = {1-(CD) / (AB)} x 100 The terms in the formula represent the following: A: Absorbance at 320 nm with no sample added and enzyme added B: Absorbance at 320 nm without adding sample or enzyme C: Absorbance at 320 nm after adding sample and enzyme D: Absorbance at 320 nm with sample and without enzyme added The results are shown in Table 9.

[0165] [Table 9]

[0166] As shown in Table 9, dihydroferulic acid (sample 1) was found to have an excellent inhibitory effect on MMP-1 activity.

[0167] [Test Example 10] Skin fibroblast proliferation promoting activity test Dihydroferulic acid (Sample 1) was tested for its skin fibroblast proliferation promoting effect as follows.

[0168] Normal human dermal fibroblasts (NB1RGB) were cultured in α-MEM medium containing 10% FBS, and then the cells were harvested by trypsinization. The harvested cells were collected at a concentration of 7.0 × 10 4 After diluting the cells with α-MEM medium containing 5% FBS to a cell density of 100 cells / mL, 100 μL of the cells were seeded per well of a 96-well plate and cultured overnight. After the culture was completed, the medium was removed, and 100 μL of the test sample (Sample 1, see Table 10 below for sample concentration) dissolved in α-MEM medium containing 5% FBS was added to each well and cultured for 3 days. As a control, α-MEM medium containing 5% FBS without the sample was used and cultured in the same manner.

[0169] The fibroblast proliferation-promoting activity was measured using the MTT assay. After 3 days of culture, the medium was removed, and 100 μL of MTT dissolved in PBS(-) buffer at a final concentration of 5 mg / mL was added to each well. After 2 hours of culture, the blue formazan produced within the cells was extracted with 100 μL of 2-propanol. After extraction, the absorbance at 570 nm was measured. At the same time, the absorbance at 650 nm was measured as turbidity, and the difference between the two was used to determine the amount of blue formazan produced. The fibroblast proliferation promotion rate (%) was calculated from the results using the following formula:

[0170] Fibroblast proliferation promotion rate (%) = St / Ct x 100 The terms in the formula represent the following: St: Amount of blue formazan produced when test sample is added Ct: Amount of blue formazan produced without adding sample The results are shown in Table 10.

[0171] [Table 10]

[0172] As shown in Table 10, it was confirmed that dihydroferulic acid (sample 1) has an excellent fibroblast proliferation promoting effect.

[0173] [Test Example 11] Aquaporin 3 (AQP3) mRNA expression promoting effect test Dihydroferulic acid (sample 1) was tested for its AQP3 mRNA expression promoting effect as follows.

[0174] Human normal neonatal epidermal keratinocytes (NHEK) were cultured at 80cm 2 The cells were pre-cultured in a flask using human normal epidermal keratinocyte medium (KGM) at 37°C, 5% CO2, and 95% air, and then harvested by trypsinization. The harvested cells were collected at a concentration of 20 × 10 4 After diluting with KGM medium to a cell density of 40 × 10 cells / mL, 2 mL of the medium was seeded into 35 mm dishes (Falcon). 4 The cells were cultured at 37°C in an atmosphere of 5% CO2 and 95% air for 24 hours.

[0175] After incubation, the medium was removed, and 2 mL of the test sample (Sample 1, see Table 11 below for sample concentration) dissolved in KGM medium was added to each dish. The cultures were incubated for 24 hours at 37°C in 5% CO2 and 95% air. As a control, KGM medium without sample was used for incubation in the same manner. After incubation, the medium was removed, and total RNA was extracted using ISOGEN (Nippon Gene Co., Ltd., Cat. No. 311-02501). The amount of RNA was measured using a spectrophotometer, and total RNA was adjusted to 200 ng / μL.

[0176] Using this total RNA as a template, the mRNA expression levels of AQP3 and the internal standard GAPDH were measured. Detection was performed by real-time 2-step RT-PCR using a real-time PCR device, Smart Cycler (Cepheid), with the TaKaRa SYBR Prime Script RT-PCR kit (Perfect Real Time) (Takara Bio, code No. RR063A). The expression level of AQP3 mRNA was calculated based on the total RNA samples prepared from cells cultured with and without the test sample, and corrected for the GAPDH level. The AQP3 mRNA expression promotion rate (%) was calculated from the obtained values ​​using the following formula:

[0177] AQP3 mRNA expression promotion rate (%) = A / B × 100 The terms in the formula represent the following: A: Correction value when test sample is added B: Corrected value when no sample is added The results are shown in Table 11.

[0178] [Table 11]

[0179] As shown in Table 11, it was confirmed that dihydroferulic acid (sample 1) has an excellent effect of promoting AQP3 mRNA expression.

[0180] [Test Example 12] AGE formation inhibitory effect test Dihydroferulic acid (sample 1) was tested for its inhibitory effect on the formation of advanced glycation end products (AGEs) as follows.

[0181] A 100 μL mixture of 0.2M D(-)-ribose prepared in PBS(-) buffer and a test sample (sample 1; see Table 14 for sample concentration) was added to a 96-well type I collagen-coated plate (Asahi Glass Co., Ltd.), and then allowed to stand at 37°C for two weeks to allow AGEs to form. As a negative control, PBS(-) buffer alone, and as a positive control, a 0.2M D(-)-ribose solution prepared in PBS(-) buffer were also allowed to stand in the same manner. After two weeks, the amount of AGEs was measured by ELISA using an anti-AGE antibody (Transgenic Co., Ltd.) to evaluate the inhibitory effect on AGE formation. From the results obtained, the AGE formation inhibition rate (%) was calculated using the following formula:

[0182] AGEs formation inhibition rate (%)={(BC) / (BA)}×100 The terms in the formula represent the following: A: Absorbance at 405 nm for negative control B: Absorbance at 405 nm for positive control C: Absorbance at a wavelength of 405 nm when test sample is added The results are shown in Table 12.

[0183] [Table 12]

[0184] As shown in Table 12, dihydroferulic acid (sample 1) exhibited excellent AGE formation inhibitory activity.

[0185] [Test Example 13] Testosterone 5α-reductase inhibitory activity test Dihydroferulic acid (Sample 1) was tested for its testosterone 5α-reductase inhibitory activity as follows.

[0186] In a capped V-bottom test tube, 20 μL of a 4.2 mg / mL testosterone (Wako Pure Chemical Industries, Ltd.) solution prepared in propylene glycol was mixed with 825 μL of 5 mmol / L Tris-HCl (pH 7.13) buffer solution containing 1 mg / mL NADPH.

[0187] Furthermore, 80 μL of a test sample (Sample 1, see Table 12 below for sample concentration) solution prepared with 50% ethanol was added to 75 μL of S-9 (rat liver homogenate, Oriental Yeast Co., Ltd.), mixed, and incubated at 37°C for 30 minutes. The reaction was then stopped by adding 1 mL of methylene chloride. The mixture was centrifuged (1600 × g, 10 minutes), and the methylene chloride layer was separated and subjected to gas chromatography analysis under the following conditions to quantify the concentrations of 3α-androstanediol, 5α-dihydrotestosterone (5α-DHT), and testosterone. As a control, the same volume (80 μL) of sample solvent was used instead of the test sample solution, and the mixture was subjected to gas chromatography analysis in the same manner.

[0188] <Gas chromatography conditions> Equipment used: Shimadzu GC-2010 (Shimadzu Corporation) Column: DB-1701 (inner diameter: 0.53 mm, length: 30 m, film thickness: 1.0 μm, manufactured by J&W Scientific) Column temperature: 240℃ Inlet temperature: 300℃ Detector: FID Sample injection volume: 1 μL Split ratio: 1:2 Carrier gas: Nitrogen gas Carrier gas flow rate: 3 mL / min

[0189] The concentrations of 3α-androstanediol, 5α-DHT and testosterone were quantified by the following method. Standards of 3α-androstanediol, 5α-DHT, and testosterone were dissolved in methylene chloride, and the solution was subjected to gas chromatography analysis. The correlation between the peak area and the concentration of each compound was previously determined from the concentration (μg / mL) and peak area of ​​each compound. The concentrations of 3α-androstanediol, 5α-DHT, and testosterone per peak area after the reaction of testosterone with S-9 were then calculated using the previously determined correlation according to the following formula (1):

[0190] A = B × C / D (1) The terms in the formula represent the following: A: 3α-androstanediol, 5α-DHT, or testosterone concentration B: Peak area of ​​3α-androstanediol, 5α-DHT or testosterone C: Concentration of the standard D: Peak area of ​​standard

[0191] Using the compound concentrations calculated based on formula (1), the conversion rate (the ratio of the concentrations of 3α-androstanediol and 5α-DHT produced by reduction of testosterone by testosterone 5α-reductase to the initial concentration of testosterone) was calculated based on the following formula (2).

[0192] Conversion rate = (E + F) / (E + F + G) (2) The terms in the formula represent the following: E: 3α-androstanediol concentration (μg / mL) F: 5α-DHT concentration (μg / mL) G: Testosterone concentration (μg / mL)

[0193] Using the conversion rate calculated based on formula (2), the testosterone 5α-reductase inhibition rate (%) was calculated based on the following formula (3). Testosterone 5α-reductase inhibition rate (%) = (1 − H / I) × 100 (3) The terms in the formula represent the following: H: Conversion rate when test sample is added I: Conversion rate without sample addition The results are shown in Table 13.

[0194] [Table 13]

[0195] As shown in Table 13, it was confirmed that dihydroferulic acid (sample 1) has excellent testosterone 5α-reductase inhibitory activity.

[0196] [Test Example 14] Test of dermal papilla cell proliferation promoting activity Dihydroferulic acid (Sample 1) was tested for its ability to promote proliferation of hair papilla cells as follows.

[0197] Normal human hair dermal papilla cells (HFDPC, derived from the male scalp) were cultured in a dermal papilla cell growth medium (PCGM, manufactured by Toyobo Co., Ltd.) containing 1% FCS and growth additives, and then the cells were harvested by trypsinization. The harvested cells were diluted to 1.0 × 10 in DMEM medium containing 10% FBS. 4 After dilution to a cell density of 1000 cells / mL, 200 μL of the solution was seeded into each well of a collagen-coated 96-well plate and cultured for 3 days. As a control, serum-free DMEM medium without the sample was used for culture in the same manner.

[0198] The medium was then removed, and 200 μL of a test sample (Sample 1, see Table 16 for sample concentration) dissolved in serum-free DMEM medium was added to each well, followed by further incubation for 4 days. After incubation, the dermal papilla cell proliferation-promoting activity was measured using an MTT assay. Specifically, the medium was removed, and 200 μL of 0.4 mg / mL MTT prepared in serum-free DMEM medium was added. After incubation for another 2 hours, the blue formazan produced within the cells was extracted with 100 μL of 2-propanol. The absorbance of this extract was measured at 570 nm, where the absorption maximum of blue formazan is located. At the same time, the absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was used to determine the amount of blue formazan produced. From the measurement results, the dermal papilla cell proliferation promotion rate (%) was calculated using the following formula:

[0199] Hair papilla cell proliferation promotion rate (%) = A / B x 100 The terms in the formula represent the following: A: Amount of blue formazan produced when test sample is added B: Amount of blue formazan produced without adding sample The results are shown in Table 14.

[0200] [Table 14]

[0201] As shown in Table 14, dihydroferulic acid (Sample 1) was found to have an excellent effect of promoting the proliferation of dermal papilla cells.

[0202] [Test Example 15] Test of melanin production inhibitory effect on B16 melanoma cells Dihydroferulic acid (Sample 1) was tested for its melanin production inhibitory effect on B16 melanoma cells as follows.

[0203] B16 melanoma cells were cultured in Dulbecco's MEM medium containing 10% FBS, and then harvested by trypsinization. The harvested cells were collected at a concentration of 24.0 × 10 4The cells were diluted with Dulbecco's MEM medium containing 10% FBS and 1 mmol / L theophylline to a cell density of 100 cells / mL, and then seeded in a 48-well plate at 300 μL per well and cultured for 6 hours.

[0204] After incubation, 300 μL of the test sample (Sample 1, see Table 13 below for sample concentrations) dissolved in Dulbecco's MEM medium containing 10% FBS and 1 mmol / L theophylline was added to each well and incubated for 4 days. As a control, cells were incubated in Dulbecco's MEM medium containing 10% FBS and 1 mmol / L theophylline without the sample. After incubation, the medium was removed, 200 μL of 2 mol / L NaOH solution was added, and the cells were disrupted using an ultrasonic disrupter. The absorbance at 475 nm was measured. The amount of melanin was calculated from the measured absorbance based on a calibration curve prepared using synthetic melanin (manufactured by SIGMA).

[0205] To measure cell viability, the cells were cultured as described above, then the medium was removed and washed with 400 μL of PBS buffer. 200 μL of neutral red dissolved in 10% FBS-containing Dulbecco's MEM (FBS) at a final concentration of 0.05 mg / mL was added to each well and cultured for 2.5 hours. After incubation, the neutral red solution was removed, and 200 μL of an ethanol-acetic acid solution (ethanol:acetic acid:water = 50:1:49) was added to each well to extract the pigment. After extraction, the absorbance at 540 nm was measured. From the results obtained, the melanin production inhibition rate (%), corrected for cell viability, was calculated using the following formula:

[0206] Melanin production inhibition rate (%) = {1-(B / D) / (A / C)} x 100 The terms in the formula represent the following: A: Amount of melanin without sample addition B: Amount of melanin after addition of test sample C: Absorbance at 540 nm without sample addition D: Absorbance at 540 nm when test sample is added The results are shown in Table 15.

[0207] [Table 15]

[0208] As shown in Table 15, dihydroferulic acid (sample 1) was found to have an excellent melanin production inhibitory effect.

[0209] [Test Example 16] Hyaluronidase activity inhibitory effect test To 0.2 mL of the test sample (Sample 1, see Table 14 below for sample concentration) dissolved in 0.1 mol / L acetate buffer (pH 3.5), 0.1 mL of hyaluronidase solution (Type IV-S (from bovine testes), 400 NF units / mL, SIGMA) was added and the mixture was incubated at 37°C for 20 minutes. 0.2 mL of 2.5 mmol / L calcium chloride was added as an activator, and the mixture was incubated at 37°C for 20 minutes. 0.5 mL of 0.8 mg / mL sodium hyaluronate solution (from rooster comb) was added, and the mixture was incubated at 37°C for 40 minutes. 0.2 mL of 0.4 mol / L sodium hydroxide was added to stop the reaction. After cooling, 0.2 mL of boric acid solution was added to each reaction solution, and the mixture was boiled for 3 minutes. After cooling on ice, 6 mL of p-DABA reagent was added, and the mixture was incubated at 37°C for 20 minutes. The absorbance at a wavelength of 585 nm was then measured.

[0210] As a blank, the same procedure and absorbance measurement were performed without adding the enzyme solution. Furthermore, as a control, the same measurement was performed with distilled water added without adding the sample solution. From the obtained results, the hyaluronidase activity inhibition rate (%) was calculated using the following formula. Hyaluronidase activity inhibition rate (%) = {1-(St-Sb) / (Ct-Cb)} x 100 The terms in the formula represent the following: St: absorbance of the test sample solution at a wavelength of 585 nm Sb: absorbance of the test sample solution blank at a wavelength of 585 nm Ct: absorbance of the control solution at a wavelength of 585 nm Cb: Absorbance of control solution blank at 585 nm The results are shown in Table 16.

[0211] [Table 16]

[0212] As shown in Table 16, it was confirmed that dihydroferulic acid (Sample 1) has excellent hyaluronidase activity inhibitory effect.

[0213] [Test Example 17] Hexosaminidase release inhibitory effect test Dihydroferulic acid (sample 1) was tested for its inhibitory effect on hexosaminidase release as follows.

[0214] Rat basophilic leukemia cells (RBL-2H3) were cultured in S-MEM medium containing 15% FBS, and then harvested by trypsinization. The harvested cells were collected at a concentration of 4.0 × 10 5 The cells were diluted with 15% FBS-containing S-MEM medium to a cell density of 0.50 cells / mL, and DNP-specific IgE was added to a final concentration of 0.5 μL / mL. 100 μL of the cells were then seeded into a 96-well plate at a volume of 100 μL per well and cultured overnight.

[0215] After incubation, the medium was removed and the wells were washed twice with 100 μL of Silagahanian buffer. Next, 10 μL of the test sample (Sample 1, see Table 15 below for sample concentration) dissolved in the same buffer and 30 μL of the same buffer were added to each well and allowed to stand at 37°C for 10 minutes. As a control, the same procedure was performed using 40 μL of Silagahanian buffer without sample. Next, 10 μL of 100 ng / mL DNP-BSA solution was added and allowed to stand at 37°C for 15 minutes to release hexosaminidase.

[0216] The 96-well plate was then placed on ice to stop the release. 10 μL of cell supernatant from each well was transferred to a new 96-well plate, and 10 μL of 1 mmol / L p-nitrophenyl-N-acetyl-β-D-glucosaminide (p-NAG) solution was added to each well. The reaction was allowed to proceed at 37°C for 1 hour.

[0217] After the reaction was completed, 250 μL of 0.1 mol / L Na2CO3 / NaHCO3 was added to each well, and the absorbance at 415 nm and 650 nm was measured. The absorbance at 650 nm was subtracted from the absorbance at 415 nm to obtain a correction value. A blank was also prepared by measuring the absorbance at 415 nm and 650 nm of a mixture of 10 μL of cell supernatant and 250 μL of 0.1 mol / L Na2CO3 / NaHCO3, and calculating the correction value. The hexosaminidase release inhibition rate (%) was calculated from the measurement results using the following formula:

[0218] Hexosaminidase release inhibition rate (%) = {1-(BC) / A} x 100 The terms in the formula represent the following: A: Corrected value without sample addition B: Corrected value with test sample added C: Corrected value with test sample and without p-NAG The results are shown in Table 17.

[0219] [Table 17]

[0220] As shown in Table 17, it was confirmed that dihydroferulic acid (sample 1) has an excellent inhibitory effect on hexosaminidase release.

[0221] [Test Example 18] COX-2 activity inhibitory effect test in mouse macrophages (PGE2 production inhibitory effect test) Mouse macrophage cells (RAW264.7) were cultured in Dulbecco's MEM medium containing 10% FBS, and then the cells were collected using a cell scraper. The collected cells were collected at a concentration of 2.0 × 105 After diluting with Dulbecco's MEM medium containing 10% FBS to a concentration of 100 cells / mL, the cells were seeded in a 96-well plate at 100 μL per well and cultured for 18 hours.

[0222] After incubation, the medium was replaced with 500 μmol / L aspirin-containing medium to inactivate pre-existing COX-1 and low levels of COX-2. The cells were then washed three times with PBS(-) buffer. 100 μL of the test sample (Sample 1, see Table 16 below) dissolved in 10% FBS-containing Dulbecco's MEM medium containing 0.5% DMSO was added to each well. 100 μL of lipopolysaccharide (LPS, E. coli 0111; B4, DIFCO) dissolved in 10% FBS-containing Dulbecco's MEM medium at a final concentration of 1 μg / mL was then added and incubated for 16 hours. A control was cultured in 10% FBS-containing Dulbecco's MEM medium containing 0.5% DMSO without the sample. After the incubation, the amount of prostaglandin E2 in the culture supernatant of each well was quantified using a PGE2EIA Kit (Cayman Chemical Co.). From the results obtained, the COX-2 activity inhibition rate (%, PGE2 production inhibition rate) was calculated using the following formula.

[0223] Macrophage COX-2 activity inhibition rate (%) = {1-(AC) / (BC)} x 100 The terms in the formula represent the following: A: Amount of prostaglandin E2 after addition of test sample and stimulation with LPS B: Amount of prostaglandin E2 when stimulated with LPS without adding sample C: Amount of prostaglandin E2 without sample addition and without LPS stimulation The results are shown in Table 18.

[0224] [Table 18]

[0225] As shown in Table 18, it was confirmed that dihydroferulic acid (Sample 1) has an excellent inhibitory effect on COX-2 activity in macrophages.

[0226] [Formulation example 1] An emulsion having the following composition was prepared by a conventional method. Dihydroferulic acid 0.01g Jojoba oil 4.00g 1,3-butylene glycol 3.00g Arbutin 3.00g Polyoxyethylene cetyl ether (20E.O.) 2.50g 2.00g olive oil Squalane 2.00g Cetyl alcohol 2.00g Glyceryl monostearate 2.00g Polyoxyethylene sorbitan oleate (20E.O.) 2.00g Methyl parahydroxybenzoate 0.15g Stearyl glycyrrhizinate 0.10g Phellodendron bark extract 0.10g Dipotassium glycyrrhizinate 0.10g Ginkgo biloba extract 0.10g Conchiolin 0.10g Phellodendron bark extract 0.10g Chamomile extract 0.10g Fragrance 0.05g Purified water Rest (total amount is 100g)

[0227] [Formulation example 2] A cream having the following composition was prepared by a conventional method. Dihydroferulic acid 0.05g Sophora root extract 0.1g Scutellaria root extract 0.1g Liquid paraffin 5.0g White beeswax 4.0g Squalane 10.0g Cetanol 3.0g Lanolin 2.0g Stearic acid 1.0g Polyoxyethylene sorbitan oleate (20E.O.) 1.5g Glyceryl monostearate 3.0g Oil-soluble licorice extract 0.1g 1,3-butylene glycol 6.0g Methyl parahydroxybenzoate 1.5g Fragrance 0.1g Purified water Rest (total amount is 100g)

[0228] [Formulation example 3] A cosmetic essence having the following composition was prepared by a conventional method. Dihydroferulic acid 0.01g Chamomile extract 0.1g Carrot extract 0.1g Xanthan gum 0.3g Hydroxyethyl cellulose 0.1g Carboxyvinyl polymer 0.1g 1,3-butylene glycol 4.0g Dipotassium glycyrrhizinate 0.1g Glycerin 2.0g Potassium hydroxide 0.25g Fragrance 0.01g Preservative (methyl parahydroxybenzoate) 0.15g Ethanol 2.0g Purified water Rest (total amount is 100g)

[0229] [Formulation example 4] A hair tonic having the following composition was prepared by a conventional method. Dihydroferulic acid 0.4g Tocopherol acetate (appropriate amount) Cephalatin 0.002g Isopropylmethylphenol 0.1g Sodium hyaluronate 0.15g Glycerin 15.0g Ethanol 15.0g Fragrance (appropriate amount) Chelating agent (sodium edetate) appropriate amount Preservative (hinokitiol) appropriate amount Solubilizer (polyoxyethylene cetyl ether) appropriate amount Purified water Rest (total amount is 100g)

[0230] [Formulation example 5] A shampoo having the following composition was prepared by a conventional method. Dihydroferulic acid 0.5g Marjoram extract 1.0g Plum fruit extract 0.2g Sodium coconut oil fatty acid methyl taurate 10.0g Coconut oil fatty acid amidopropyl betaine 10.0g Sodium polyoxyethylene alkyl ether sulfate 20.0g Coconut oil fatty acid diethanolamide 4.0g Propylene glycol 2.0g Fragrance (appropriate amount) Purified water Rest (total amount is 100g)

[0231] [Formulation example 6] Tablets having the following composition were prepared by a conventional method. Dihydroferulic acid 5.0mg Dolomite (contains 20% calcium and 10% magnesium) 83.4mg Casein phosphopeptide 16.7mg Vitamin C 33.4mg Maltitol 136.8mg Collagen 12.7mg Sucrose fatty acid ester 12.0mg

[0232] [Formulation example 7] An oral liquid preparation having the following composition was prepared by a conventional method. <Composition in 1 ampoule (100 mL)> Dihydroferulic acid 0.3% by mass Sorbitol 12.0% by mass Sodium benzoate 0.1% by mass Fragrance 1.0% by mass Calcium sulfate 0.5% by mass Purified water remainder (100% by mass) [Industrial Applicability]

[0233] The anti-obesity agents, cAMP phosphodiesterase activity inhibitors, DPPIV activity inhibitors, anti-aging agents, hair growth agents, anti-androgen agents, whitening agents, and anti-inflammatory agents of the present invention can significantly contribute to the prevention, treatment, or improvement of skin aging symptoms; promotion of wound or burn healing; prevention, treatment, or improvement of dry skin diseases; prevention, treatment, or improvement of alopecia, particularly male pattern baldness; prevention or improvement of pigmentation such as skin darkening, age spots, and freckles; prevention, treatment, or improvement of type 2 diabetes, obesity, hypertension, insulin resistance, etc.; prevention or improvement of various inflammatory skin diseases; and prevention, treatment, or improvement of obesity.

Claims

1. An anti-obesity agent characterized by containing dihydroferulic acid as an active ingredient.

2. The anti-obesity agent according to claim 1, characterized in that the dihydroferulic acid has one or more effects selected from the group consisting of an inhibitory effect on cAMP phosphodiesterase activity, a blood cholesterol-lowering effect, a blood free fatty acid-lowering effect, and a dipeptidyl peptidase IV activity inhibitory effect.

3. A cyclic AMP phosphodiesterase activity inhibitor characterized by containing dihydroferulic acid as an active ingredient.

4. A dipeptidyl peptidase IV activity inhibitor characterized by having dihydroferulic acid as an active ingredient.

5. An anti-aging agent characterized by containing dihydroferulic acid as an active ingredient.

6. The anti-aging agent according to claim 5, characterized in that the dihydroferulic acid has one or more effects selected from the group consisting of laminin 5 production promoting effect, matrix metalloproteinase-2 activity inhibiting effect, hyaluronic acid production promoting effect, epidermal keratinocyte proliferation promoting effect, elastin production promoting effect, matrix metalloproteinase-1 activity inhibiting effect, fibroblast proliferation promoting effect, aquaporin 3 mRNA expression promoting effect, and advanced glycation end product formation inhibiting effect.

7. A hair growth agent characterized by containing dihydroferulic acid as an active ingredient.

8. The hair growth agent according to claim 7, wherein the dihydroferulic acid has one or more effects selected from the group consisting of an inhibitory effect on testosterone 5α-reductase activity and / or an effect of promoting the proliferation of hair papilla cells.

9. An anti-androgenic agent characterized by containing dihydroferulic acid as an active ingredient.

10. A skin whitening agent characterized by containing dihydroferulic acid as an active ingredient.

11. 11. The skin whitening agent according to claim 10, wherein the dihydroferulic acid has an inhibitory effect on melanin production.

12. An anti-inflammatory agent characterized by containing dihydroferulic acid as an active ingredient.

13. The anti-inflammatory agent according to claim 12, characterized in that the dihydroferulic acid has one or more effects selected from the group consisting of hyaluronidase activity inhibitory effect, hexosaminidase release inhibitory effect, and cyclooxygenase-2 activity inhibitory effect.

14. A skin cosmetic characterized by containing dihydroferulic acid.

15. A hair cosmetic characterized by containing dihydroferulic acid.

16. A food or drink containing dihydroferulic acid.

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