Hair growth tonic

Abstract

The present invention provides a hair growth agent that contains a rice bran-derived component mixture and that is characterized in that the rice bran-derived component mixture is a rice oil containing 20 mass% or more of (1) a water-soluble rice bran component, (2) an ester between a plant sterol and a fatty acid and / or an ester between a triterpene alcohol and a fatty acid, (3) a rice bran oil unsaponifiable concentrate, or (4) γ-oryzanol.

Description

Technical Field

[0001] The present invention relates to a hair growth agent.

Background Art

[0002] Many people are troubled by thinning hair or hair loss on the head due to causes such as aging, genetic factors, and social stress, and various attempts have been made to provide excellent hair growth agents, hair growth stimulants, and hair loss inhibitors. As factors involved in hair growth, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-1), etc. have been reported. Minoxidil has the function of promoting the production of VEGF and IGF-1, and pharmaceuticals containing this as an active ingredient are sold, but side effects such as skin diseases have been reported in women, and the dosage is restricted. As therapeutic agents for male pattern baldness, there are dutasteride and finasteride that inhibit 5α-reductase, but they have serious side effects such as sexual dysfunction and liver dysfunction, and the use by pregnant women is particularly taboo. In recent years, due to such circumstances, there has been an increasing interest in alternatives derived from natural products with less risk. For example, it has been shown that phytic acid and inositol derived from natural products have the effect of promoting hair growth (Patent Documents 1 and 2). In addition, it has been shown that an extract of yacon has an anti-hair loss effect by inhibiting 5α-reductase (Patent Document 3).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] The objective of this invention is to provide a novel hair growth agent derived from natural products. [Means for solving the problem]

[0005] To solve the above problems, the present invention includes the following inventions. [1] A hair growth product containing a mixture of rice bran-derived ingredients. [2] The hair growth agent according to [1], wherein the rice bran-derived component mixture is selected from (1) to (4) below: (1)Water-soluble rice bran ingredients (2) Esters of plant sterols and fatty acids and / or esters of triterpene alcohols and fatty acids (3) Rice bran oil unsaponifiable concentrate (4) Rice oil containing 20% ​​by mass or more of γ-oryzanol [3] The hair growth agent according to [2], wherein (1) contains phytic acid and inositol. [4] The hair growth agent according to [3], wherein (1) contains 15% by mass or more and 45% by mass or less of phytic acid and 5% by mass or more and 20% by mass or less of inositol. [5] A hair growth agent containing purified inositol and purified phytic acid. [6] The hair growth agent according to [5], wherein the ratio of inositol and phytic acid is 1:2 or more and 1:9 or less by mass. [7] A pharmaceutical product characterized by containing the hair growth agent described in [1] above. [8] Cosmetics characterized by containing the hair growth agent described in [1] above. [9] Food and beverages characterized by containing the hair growth agent described in [1] above.

[10] A hair papilla cell proliferation promoter containing a mixture of rice bran-derived components.

[11] A vascular endothelial growth factor production promoter containing a mixture of rice bran-derived components.

[12] An insulin-like growth factor-1 production promoter containing a mixture of rice bran-derived components.

[13] A type 2 5α-reductase production inhibitor containing a mixture of rice bran-derived components. [Effects of the Invention]

[0006] The present invention provides a novel hair growth agent derived from natural products. This hair growth agent can be used as a pharmaceutical, cosmetic, or food / beverage. [Brief explanation of the drawing]

[0007] [Figure 1] Figure 1 shows the relative gene expression levels of the VEGF gene in human primary dermal papilla cells after RSE, RTN, or RICEO treatment compared to a negative control. [Figure 2] Figure 2 shows the relative gene expression levels of the IGF-1 gene in human primary dermal papilla cells after RSE and RICEO treatment compared to a negative control. [Figure 3] Figure 3 shows the relative gene expression levels of the 5α-R2 gene in human prostate cancer cells after GX-N or RTN treatment compared to negative controls. [Figure 4] Figure 4 shows the cell proliferation rate of human primary dermal papilla cells after RICEO treatment compared to a negative control. [Figure 5] Figure 5 shows the relative gene expression levels of the VEGF gene in human primary dermal papilla cells after treatment with PA, IN, or IN / PA mix compared to a negative control. [Modes for carrying out the invention]

[0008] [Hair growth tonic] The present invention provides a hair growth agent containing a mixture of rice bran-derived components as an active ingredient. The mixture of rice bran-derived components in the hair growth agent of the present invention is not particularly limited as long as it contains two or more components obtained from rice bran as a raw material. Examples of the mixture of rice bran-derived components include water-soluble rice bran components, esters of plant sterols and fatty acids and / or esters of triterpene alcohols and fatty acids, rice bran oil unsaponifiable concentrate, rice oil containing 20% ​​by mass or more of γ-oryzanol, and combinations thereof.

[0009] The water-soluble rice bran component is not particularly limited as long as it is a mixture of water-soluble components obtained from rice bran. The water-soluble rice bran component includes phytic acid, inositol, minerals (such as potassium, magnesium, calcium, sodium, iron, zinc, copper, selenium, etc.), dietary fiber, protein, water-soluble vitamins (such as vitamin B group, etc.). The water-soluble rice bran component preferably contains phytic acid and inositol, more preferably contains more phytic acid than inositol, further preferably contains 15% by mass or more and 45% by mass or less of phytic acid and 5% by mass or more and 20% by mass or less of inositol, and particularly preferably contains 20% by mass or more and 40% by mass or less of phytic acid and 7% by mass or more and 15% by mass or less of inositol.

[0010] The water-soluble rice bran component may be dissolved in an aqueous solution or may have a form such as a powder by drying.

[0011] The water-soluble rice bran component may be a water-soluble rice bran component extracted from rice bran using a known method or a commercially available water-soluble rice bran component. The method for obtaining the water-soluble rice bran component from rice bran is not particularly limited. For example, there are methods of extracting with one or more acids such as organic acids, acetic acid, oxalic acid, tartaric acid, phosphoric acid, lactic acid, butyric acid, citric acid, succinic acid, etc. contained in rice bran, and the method described in JP-A-2002-20307. Examples of commercially available water-soluble rice bran components include RICEO-EX (trade name) manufactured by Tsukino Food Industry Co., Ltd. RICEO-EX contains about 30% by mass of phytic acid and about 10% by mass of inositol. Although the composition of the water-soluble rice bran component is not limited, as an example, it contains 30.0 g of carbohydrates, 5.3 g of potassium, 2.7 g of magnesium, 121 mg of calcium, 30.9 mg of sodium, 19.1 mg of iron, 13.2 mg of zinc, 0.4 mg of copper, 7.2 mg of vitamin B1, 0.6 mg of vitamin B2, 10.2 mg of vitamin B6, 11.2 g of dietary fiber, 8.2 g of protein, 27.8 g of phytic acid, and 11.0 g of inositol per 100 g.

[0012] The ester of a plant sterol and a fatty acid and / or the ester of a triterpene alcohol and a fatty acid is not particularly limited as long as it is a mixture containing a sterol ester obtained from rice bran, and may contain an ester of a plant sterol and a fatty acid, may contain an ester of a triterpene alcohol and a fatty acid, or may contain a mixture thereof.

[0013] A sterol refers to a compound (steroid) having a cyclopentanoperhydrophenanthrene skeleton (sterane skeleton) and having a hydroxyl group at the 3-position. Sterols are usually widely distributed in the animal and plant kingdoms in the form of free, ester-type, glycosides, etc., and are also important components of biological membranes. The ester of a plant sterol and a fatty acid and / or the ester of a triterpene alcohol and a fatty acid contained in the hair growth agent of the present invention is a sterol in the ester form with a fatty acid.

[0014] Sterols may be unsaturated compounds having 27 to 30 carbon atoms and having one double bond at the 5 / 6 position, 7 / 8 position, 8 / 9 position or other positions, or may be saturated compounds obtained by hydrogenation. Examples of plant sterols that are abundant in plants among sterols include β-sitosterol, stigmasterol, campesterol, etc. Triterpene alcohols are sterols having 30 carbon atoms and are components abundantly contained in rice bran or rice oil.

[0015] The ester in the ester of a plant sterol and a fatty acid and the ester of a triterpene alcohol and a fatty acid is of the formula (A): R’CO-OH (A) [In the formula, R’CO has 14 to 22 carbon atoms and is a linear fatty acid acyl group having any of 0 to 3 double bonds.] It may be derived from a carboxylic acid represented by the formula (A). Examples of the carboxylic acid represented by the formula (A) include myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, behenic acid, etc.

[0016] The esters of plant sterols and fatty acids and / or triterpene alcohols and fatty acids may be sterol esters purified from rice bran using known methods, or commercially available sterol esters. The method for obtaining sterol esters from rice bran is not particularly limited and includes, for example, a method using by-products such as soup stock and deodorized scum generated in the process of producing rice oil from rice bran as raw materials, and extracting and purifying them with a solvent such as acetone or hexene; the method described in the publicly available literature (Japanese Patent Publication No. 2014-47311, Taniguchi et al., Journal of the Japan Society for Food Science and Technology, 2012, Vol. 59, No. 7, pp. 301-318); and a method of esterifying at least one of the plant sterols and triterpene alcohols obtained from rice bran using an enzyme that esterifies nonspecifically or specifically. Examples of commercially available sterol esters include Rice Sterol Ester (trade name) manufactured by Tsukuno Foods Industry Co., Ltd.

[0017] The rice bran oil unsaponifiable matter concentrate is not particularly limited as long as it is a concentrate of unsaponifiable matter obtained from rice bran, and may also be a concentrate of unsaponifiable matter obtained by neutralizing and removing fatty acids from the rice oil deodorized distillate, which is a by-product of the deodorization process in the process of refining rice oil from rice bran. The rice bran oil unsaponifiable matter concentrate contains vitamin E derivatives (tocopherols, tocotrienols, etc.), sterols (plant sterols, triterpene alcohols, their fatty acid esters, etc.), squalene, etc.

[0018] Examples of plant sterols and triterpene alcohols included in sterols are those similar to those exemplified as sterols in the esters of plant sterols and fatty acids and / or triterpene alcohols and fatty acids in the present invention.

[0019] The total content of fatty acid esters of plant sterols and / or fatty acid esters of triterpene alcohols in the rice bran oil unsaponifiable concentrate may be 3% by mass or more and 70% by mass or less, or 5% by mass or more and 60% by mass or less. The content of plant sterols in the rice bran oil unsaponifiable concentrate may be 5% by mass or more and 20% by mass or less. The content of tocopherols in the rice bran oil unsaponifiable concentrate may be 1% by mass or more and 5% by mass or less. The content of tocotrienols in the rice bran oil unsaponifiable concentrate may be 1% by mass or more and 5% by mass or less. The content of squalene in the rice bran oil unsaponifiable concentrate may be 1% by mass or more and 10% by mass or less.

[0020] The rice bran oil unsaponifiable concentrate may be a concentrate of unsaponifiable matter obtained in the process of extracting and refining oil from rice bran using a known method, or it may be a commercially available rice bran oil unsaponifiable concentrate. The method for obtaining the rice bran oil unsaponifiable concentrate from rice bran is not particularly limited, and examples include a method in which rice bran oil is produced using a known manufacturing method, and then the deodorized scum obtained in the deodorization process is used to deacidify the large amount of fatty acids contained therein with the minimum necessary amount of alkali to produce a lipid-soluble rice bran oil unsaponifiable concentrate, or the method described in Japanese Patent Application Publication No. 2005-255746. Examples of commercially available rice bran oil unsaponifiable concentrates include Ricetrienol (trade name) manufactured by Tsukuno Foods Industry Co., Ltd. While not limiting the composition of rice bran oil unsaponifiable concentrates, one example is one with an acid value of 0.8 mg KOH / g, containing 5.8 g of squalene, 10.1 g of plant sterols, 5.2 g of triterpene alcohols, 3.7 g of tocopherol, 3.0 g of tocotrienol, and 10.0 g of fatty acid esters of plant sterols and triterpene alcohols per 100 g.

[0021] Rice bran oil containing 20% ​​by mass or more of γ-oryzanol is not particularly limited as long as it contains 20% by mass or more of γ-oryzanol in the rice bran oil containing γ-oryzanol. The γ-oryzanol content of the γ-oryzanol-containing rice bran oil is preferably 25% by mass or more, more preferably 30% by mass or more, and the upper limit of the γ-oryzanol content of the γ-oryzanol-containing rice bran oil is not particularly limited and may be, for example, 60% by mass. The γ-oryzanol-containing rice bran oil may also be γ-oryzanol-containing rice germ oil.

[0022] γ-oryzanol-containing rice oil may be rice oil to which γ-oryzanol has not been added externally. The method for producing such γ-oryzanol-containing rice oil is not particularly limited, but for example, it can be produced by the method described in International Publication No. 2012 / 063794. Specifically, the method may include the steps of (A) obtaining an oil treatment product having an oil layer and an alkaline oil residue layer containing a γ-oryzanol salt, and (B) adding acid to the oil treatment product obtained in (A) to transfer γ-oryzanol from the alkaline oil residue layer to the oil layer, and recovering the oil with an increased γ-oryzanol content. Using this method, rice oil containing about 60% by mass of γ-oryzanol can be produced.

[0023] The gamma-oryzanol-containing rice oil may be a commercially available gamma-oryzanol-containing rice oil. Examples of commercially available gamma-oryzanol-containing rice oils include Rice Germ Oil GX-N (product name) manufactured by Tsukuno Foods Industry Co., Ltd. Rice Germ Oil GX-N contains approximately 30% by mass of gamma-oryzanol.

[0024] The γ-oryzanol content in rice bran oil can be measured by known methods. For example, it can be done by the following method, but is not limited to this method. Weigh 2 to 5 g of oil and dilute it with n-hexane to 100 mL. Take 2 mL of this solution and dilute it with n-hexane to 100 mL, then measure the absorbance at 315 nm. If the weight of the oil is X (g) and the absorbance is A, the γ-oryzanol content G (mass%) can be calculated using the following formula (B). G = (A × 5000) / (X × 359) (B)

[0025] Gamma-oryzanol-containing rice oil may also contain other components commonly found in rice oil, such as ferulic acid, sterols, waxes, glucoside ceramides, tocopherols (vitamin E), and tocotrienols, in addition to gamma-oryzanol.

[0026] The γ-oryzanol-containing rice oil may be used as an edible oil. The edible oil is not particularly limited as long as it contains γ-oryzanol-containing rice oil, and may also be a blended oil mixed with γ-oryzanol-containing rice oil and other edible oils. Examples of edible oils other than γ-oryzanol-containing rice oil include palm oil, rapeseed oil, soybean oil, sesame oil, corn oil, cottonseed oil, coconut oil, olive oil, palm oil, safflower oil, sunflower oil, almond oil, cashew oil, hazelnut oil, macadamia nut oil, mongongo oil, pecan oil, pine nut oil, walnut oil, camellia oil, tea oil, beef tallow, lard, chicken fat, horse oil, and fish oil. The blended oil may be a mixture of γ-oryzanol-containing rice oil and two or more other edible oils.

[0027] The present invention provides a hair growth agent containing purified inositol and purified phytic acid. The inositol and phytic acid in the hair growth agent of the present invention may be any form containing purified inositol and purified phytic acid, respectively.

[0028] "Purified inositol" refers to inositol obtained from a mixture containing inositol through a purification process to increase the purity of the inositol. "Purified inositol" may be in powder form. "Purified phytic acid" refers to phytic acid obtained from a mixture containing phytic acid through a purification process to increase the purity of the phytic acid. "Purified phytic acid" may be in liquid form or in powder form.

[0029] The purified inositol and purified phytic acid may be extracted and purified by known methods using the same biological species as raw materials, or they may be extracted and purified by known methods using different biological species as raw materials, or they may be artificially synthesized inositol and / or phytic acid that has been purified.

[0030] The organisms that serve as raw materials for refined inositol are not particularly limited as long as they contain inositol in their bodies. Examples include rice bran and other grain bran, soybeans and other legumes, fruits, nuts and other plants, and meat and fish.

[0031] The organisms that serve as raw materials for refined phytic acid are not particularly limited as long as they contain phytic acid in their bodies, and examples include grains such as rice bran and legumes such as soybeans.

[0032] The ratio of inositol and phytic acid in the hair growth agent of the present invention is not particularly limited, but may be, for example, 1:2 or more and 1:9 or less by mass ratio, and preferably 1:2.5 or more and 1:5 or less by mass ratio.

[0033] The rice bran-derived component mixture contained in the hair growth agent of the present invention, and the mixture of purified inositol and purified phytic acid, have hair growth, hair regeneration, hair nourishment, and hair loss suppression effects, and are therefore suitably used as active ingredients in hair growth agents, hair regeneration agents, hair nourishment agents, and hair loss suppressants. The hair growth agent of the present invention can be rephrased as a hair regeneration agent, a hair nourishment agent, and a hair loss suppressant.

[0034] The rice bran-derived component mixture contained in the hair growth agent of the present invention is a component also used in food, and therefore has high safety for the body, allowing for continuous and long-term use or ingestion. Because the rice bran-derived component mixture contained in the hair growth agent of the present invention contains multiple rice bran-derived components, it is believed that the additive or synergistic effects of these components contribute to excellent hair growth. The same applies to the mixture of purified inositol and purified phytic acid.

[0035] The hair growth agent of the present invention is characterized by having a hair growth effect. The hair growth effect of the hair growth agent of the present invention can be confirmed, for example, by treating dermal papilla cells with the hair growth agent of the present invention and measuring the cell proliferation rate of the dermal papilla cells, measuring the protein expression level of vascular endothelial growth factor (VEGF) or the VEGF gene expression level, measuring the protein expression level of insulin-like growth factor (IGF-1) or the IGF-1 gene expression level, and by treating prostate cancer cells with the hair growth agent of the present invention and measuring the protein expression level of type 2 5α-reductase (5α-R2) or the 5α-R2 gene expression level of the prostate cancer cells.

[0036] The cell proliferation rate of dermal papilla cells can be measured, for example, using a commercially available cell proliferation measurement kit in the treated cell culture medium. The protein expression levels of VEGF, IGF-1, or 5α-R2 can be measured, for example, by extracting proteins from treated dermal papilla cells or prostate cancer cells and using known methods such as SDS-PAGE, Western blotting, ELISA, or immunoprecipitation. The gene expression levels of VEGF, IGF-1, or 5α-R2 can be measured, for example, by extracting nucleic acids from treated dermal papilla cells or prostate cancer cells and using known methods such as RT-PCR, quantitative PCR, or microarrays.

[0037] The content of the rice bran-derived component mixture and the mixture of purified inositol and purified phytic acid in the hair growth agent of the present invention is not particularly limited as long as it is within a range in which a hair growth effect is achieved. For example, the total content of the rice bran-derived component mixture and the purified inositol and purified phytic acid may be 0.01 (wt / v)% to 80 (wt / v)%, 0.05 (wt / v)% to 50 (wt / v)%, or 0.1 (wt / v)% to 30 (wt / v)% relative to the entire hair growth agent.

[0038] The target animals for administration of the hair growth agent of the present invention are not particularly limited, and include, for example, humans, non-human mammals (livestock animals such as cattle, horses, pigs, sheep, goats, llamas, alpacas, camels, rabbits, minks, foxes, chinchillas, geese, ducks, and pet animals such as dogs and cats).

[0039] The frequency of use or intake of the hair growth agent of the present invention is not particularly limited, and examples include once or more times a day, or within the range of 1 to 5 times a day.

[0040] The period of use or intake of the hair growth agent of the present invention is not particularly limited and may be, for example, 4 weeks or more, 8 weeks or more, or 12 weeks or more.

[0041] The dosage form of the hair growth agent of the present invention is not particularly limited and may be, for example, a liquid preparation such as a low viscosity liquid or lotion, a suspension, an emulsion, a gel, a paste, a cream, a foam, a sheet, an ointment, a powder, an aerosol, an inhalant, an eye drop, a nasal spray, a suppository, or a patch for external use; or an oral preparation such as a tablet, granules, a capsule, or an oral solution; or an injectable preparation administered directly into the muscle or blood vessel.

[0042] [Pharmaceuticals, cosmetics, or food and beverages] The present invention provides pharmaceuticals, cosmetics, or food and beverages. The pharmaceuticals, cosmetics, or food and beverages of the present invention may contain the hair growth agent of the present invention. The form of the pharmaceuticals, cosmetics, or food and beverages of the present invention can be used in the same form as the dosage form of the hair growth agent described above.

[0043] If the present invention relates to food or beverages, the food or beverages of the present invention may be, for example, health foods, functional foods, foods for specified health uses, supplements, foods for sick people, etc. The form of food and beverages is not particularly limited and may be processed forms such as natural liquid food, semi-digested nutritional food, elemental nutritional food, or drinkable beverages; beverages such as tea drinks, soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and lactic acid drinks; noodles such as soba, udon, Chinese noodles, and instant noodles; sweets and breads such as candy, candy, gum, chocolate, snacks, biscuits, jelly, jam, cream, baked goods, and bread; processed seafood and livestock products such as kamaboko, ham, and sausage; dairy products such as processed milk and fermented milk; oils and fats and processed oils such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings; seasonings such as sauces and dips; retort pouch foods such as curry, stew, donburi, porridge, and rice gruel; and frozen desserts such as ice cream, sherbet, and shaved ice.

[0044] The pharmaceuticals, cosmetics, or food products of the present invention may contain, in addition to the hair growth agent of the present invention, other pharmaceutically or physiologically acceptable ingredients. The pharmaceutically or physiologically acceptable ingredients are not particularly limited and may include water, oils and fats, waxes, hydrocarbons, fatty acids, higher alcohols, esters, plant extracts, vitamins, water-soluble polymers, surfactants, alcohols, polyhydric alcohols, and the like.

[0045] The pharmaceuticals, cosmetics, or food and beverages of the present invention may, as necessary, contain known additives used in the compositions of pharmaceuticals, quasi-drugs, cosmetics, food and beverages, such as antioxidants, thickeners, preservatives, pH adjusters, stabilizers, irritation reducers, antiseptics, colorants, fragrances, etc. These additives may be used individually or in combination of two or more.

[0046] The pharmaceutical, cosmetic, or food product of the present invention may further contain other active ingredients used to promote hair growth or hair regeneration. These other active ingredients may be used alone or in combination of two or more. Examples of other active ingredients include minoxidil and finasteride.

[0047] The present invention includes a hair papilla cell proliferation promoter, a vascular endothelial growth factor production promoter, an insulin-like growth factor-1 production promoter, and a type 2 5α-reductase production inhibitor, all of which contain a mixture of rice bran-derived components. The present invention also includes a hair papilla cell proliferation promoter, a vascular endothelial growth factor production promoter, an insulin-like growth factor-1 production promoter, and all of which contain a mixture of purified inositol and purified phytic acid.

[0048] The present invention is characterized by administering an effective amount of the hair growth agent of the present invention to an animal that requires hair growth. This includes hair growth promotion methods. The animals are not particularly limited, but may include, for example, humans, non-human mammals, etc. Non-human mammals are not particularly limited, but may include cows, horses, pigs, sheep, goats, llamas, alpacas, camels, rabbits, minks, foxes, chinchillas, geese, ducks, dogs, cats, etc. [Examples]

[0049] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0050] [Example 1: Confirmation of hair-related gene expression levels] [Test materials and methods] 1. Rice bran-derived component mixture, control sample, reagent, and solvent 1.1 Rice bran-derived ingredient mixture The following four types of rice bran-derived component mixtures were used. • RICEO-EX (product name, manufactured by Tsukuno Foods Industry Co., Ltd.) • Rice sterol ester (product name, manufactured by Tsukuno Foods Co., Ltd.) • Rice Trienol (product name, manufactured by Tsukuno Foods Co., Ltd.) • Rice germ oil GX-N (product name, manufactured by Tsukuno Foods Co., Ltd.)

[0051] 1.2 Control Samples The following two types were used as control samples. • Phytic acid (product name, manufactured by Tsukuno Foods Co., Ltd.) • Inositol (product name, manufactured by Tsukuno Foods Co., Ltd.)

[0052] 1.3 Reagents The following reagents were used: • D-MEM (High Glucose) (Manufacturer: Fujifilm Wako Pure Chemical Corporation) • Fetal bovine serum (Manufacturer: Biosera) • 0.25 w / v% Trypsin-1 mmol / 1 EDTA·4Na Solution with Phenol Red (Trypsin / EDTA Solution) (Manufacturer: Fujifilm Wako Pure Chemical Industries, Ltd.) • Penicillin / Streptomycin solution (x100) (Manufacturer: Fujifilm Wako Pure Chemical Corporation) • Dulbecco PBS(-) "Nissui" (Manufacturer: Nissui Pharmaceutical Co., Ltd.) • Dimethyl sulfoxide (DMSO) (Manufacturer: Fujifilm Wako Pure Chemical Corporation) • Isogen II (Manufacturer: Fujifilm Wako Pure Chemical Corporation (Nippon Gene Co., Ltd.)) • 2-Propanol (Manufacturer: Fujifilm Wako Pure Chemical Corporation) • Ethanol (99.5%) (Manufacturer: Fujifilm Wako Pure Chemical Industries, Ltd.) • Distilled Water, Deionized, Sterile (RNase-free distilled water) (Manufacturer: Nippon Gene Co., Ltd.) • PrimeScript RT Reagent kit (Manufacturer: Takara Bio Inc.) ·PowerUp SYBR Green Master Mix (manufacturer: applied biosystems)

[0053] 2. Preparation of reagents 2.1 Culture medium 500 mL of D-MEM (high glucose) was mixed with 5 mL of penicillin / streptomycin solution and 55 mL of fetal bovine serum (FBS) to prepare 10% FBS culture medium. Additionally, the 10% FBS culture medium was diluted 10-fold with a mixed solution of D-MEM and penicillin / streptomycin to prepare 1% FBS culture medium.

[0054] 2.2 Dalbecco PBS (-) 9.8 g of Dulbecco's PBS(-) "Nissui" powder, weighed using an electronic balance, was dissolved in 1000 mL of distilled water, and then autoclaved to obtain Dulbecco's PBS(-) for cell culture.

[0055] 2.3 75% ethanol solution 30 mL of ethanol (99.5%) and 10 mL of RNase-free distilled water were mixed to obtain 40 mL of a 75% ethanol solution.

[0056] 3. Preparation of culture media containing each sample 3.1 Preparation of RICEO-EX-containing medium Using a microelectronic balance (Sartorius), 50 mg of RICEO-EX (RICEO) was weighed into a 15 mL plastic tube, and 10 mL of 1% FBS culture medium was added and dissolved using a disposable pipette. To remove impurities, the prepared solution was transferred to a 10 mL syringe fitted with a 0.45 μm filter and filtered to obtain a 0.5% RICEO-containing medium. 2 mL of the 0.5% RICEO-containing medium was mixed with 8 mL of 1% FBS culture medium to obtain a 0.1% RICEO-containing medium. Furthermore, 1 mL of the 0.1% RICEO-containing medium was mixed with 9 mL of 1% FBS culture medium to obtain a 0.01% RICEO-containing medium.

[0057] 3.2 Preparation of phytic acid-containing culture medium 4.5 mL of 50% phytic acid solution was dispensed into a 15 mL plastic tube using a disposable pipette, and 5.5 mL of 25% NaOH aqueous solution was added to obtain a 22.5% phytic acid neutralization solution (pH 6.13). 1.25 mL of distilled water was added to this solution to obtain a 20% phytic acid neutralization solution. 4.875 mL of 1% FBS culture medium was added to a 15 mL plastic tube using a disposable pipette, and 125 μL of the aforementioned 20% phytic acid neutralization solution was dissolved in it using a micropipette to obtain 0.5% phytic acid-containing media. These media were then diluted five-fold with 1% FBS culture medium to obtain a 0.1% phytic acid-containing media.

[0058] 3.3 Preparation of inositol-containing culture medium Using a microelectronic balance (Sartorius), 50 mg of inositol powder was weighed into a 15 mL plastic tube. 10 mL of 1% FBS culture medium was added and dissolved using a disposable pipette to obtain a 0.5% inositol-containing medium. The 0.5% inositol-containing medium was diluted fivefold with 1% FBS culture medium to prepare a 0.1% inositol-containing medium.

[0059] The concentrations were adjusted according to the intended use to prepare culture media containing RICEO, phytic acid, and inositol at the desired concentrations. 1% FBS culture medium was used as a negative control for the RICEO, phytic acid, and inositol-containing media.

[0060] 3.4 Preparation of culture medium containing rice sterol esters, rice trienols, and rice germ oil GX-N Using a microelectronic balance (Sartorius), 100 mg each of lysterol ester (RSE), lysterenol (RTN), and rice germ oil GX-N (GX-N) were weighed into 1.5 mL tubes, and 1 mL of DMSO was added to each using a micropipette. The solutions were heated to 50°C using a small heat block (AS ONE), and clear 10% solutions of RSE, RTN, and GX-N were obtained by inversion mixing. 450 μL of DMSO was added to a 1.5 mL tube, heated to 50°C, and 50 μL of the 10% solutions of RSE, RTN, and GX-N were added to obtain 1% solutions of RSE, RTN, and GX-N. The concentrations were adjusted according to the application to prepare RSE, RTN, and GX-N solutions of the desired concentration. 4.95 mL of 1% FBS culture medium was added to a 15 mL plastic tube using a disposable pipette. Then, 50 μL each of 10% and 1% solutions of RSE, RTN, and GX-N, or their serial dilutions, were added using a micropipette to obtain culture media containing RSE, RTN, and GX-N at concentrations of 0.1%, 0.01%, or 1 / 100th of their serial dilutions, respectively. As a negative control for the RSE, RTN, and GX-N-containing media, a medium prepared by adding 50 μL of DMSO to 4.95 mL of 1% FBS culture medium was used.

[0061] 4 Cell culture Human primary dermal papilla cells (HFDPC) and human lymph node carcinoma of the prostate (LNCaP) were used as test subjects. Under sterile conditions in a cabinet, the target cells stored in vials in liquid nitrogen were thawed and seeded onto a 10 cm culture dish (Biolamo). 10 mL of 10% FBS culture medium was used. All subsequent cell handling procedures were performed under ethanol disinfection and flame sterilization in the cabinet. The cells were incubated in a CO2 incubator (PHC) at 37°C in a 5% CO2 atmosphere for 3 days.

[0062] The supernatant of cultured cells was aspirated using a suction pump (Hi-Tec). 5 mL of autoclaved Dulbecco's PBS(-) was added to the dish using a 10 mL disposable pipette (Biolamo) to wash the cells. The solution was then removed using the suction pump. 0.5 mL of trypsin / EDTA solution was added to the dish using a micropipette (Thermo Scientific) and allowed to stand for 2 minutes. The cells were suspended by continuous aspiration and dispensing with the micropipette, and the reaction was stopped by adding 5 mL of 10% FBS culture medium using a disposable pipette.

[0063] The solution after stopping the reaction was transferred to a 15 mL plastic tube (Biolamo Co., Ltd.), and the cells were collected by centrifugation at 1200 rpm for 5 minutes at room temperature using a bucket-type centrifuge (Tommy Seiko Co., Ltd.). The solution was removed using a suction pump, and 10 mL of 10% FBS culture medium was added using a new disposable pipette to suspend the cells. The cell count was measured using a fully automated cell counter (Bio-Rad Co., Ltd.). Specifically, 30 μL each of trypan blue and cell suspension provided with the instrument were mixed, the mixed solution was added to two locations on the dedicated counting slide, and the added portion was inserted into the instrument for measurement. The dilution ratio was calculated from the obtained concentration, and 1.0 × 10⁻⁶ was calculated. 5 A cell suspension was prepared at a concentration of cells / mL.

[0064] 1.0 x 10 on a 24 Well Culture Plate (Biolamo Co.) 5 The target cells were seeded to achieve the desired cell / well ratio. They were then cultured overnight in a CO2 incubator at 37°C under a 5% CO2 atmosphere.

[0065] Human primary dermal papilla cells (HFDPCs) were cultured in a CO2 incubator at 37°C under a 5% CO2 atmosphere for 24 hours. Each well contained 1 mL of RICEO-containing medium (0.01%, 0.1%, 0.5%), RSE-containing medium (0.01%, 0.1%), RTN-containing medium (0.01%, 0.1%), or 1% FBS culture medium (negative control). Four wells were used for each group (n=4).

[0066] Cell culture was performed in the same manner, except that the culture media containing each rice bran-derived component mixture was changed to phytic acid-containing media (0.1%, 0.5%) and inositol-containing media (0.1%, 0.5%).

[0067] Human prostate cancer cells (LNCaP) were incubated in a CO2 incubator at 37°C under a 5% CO2 atmosphere for 24 hours. Each well contained 1 mL of either RTN-containing medium (0.01%, 0.1%), GX-N-containing medium (0.01%, 0.1%), or 0.05(v / v)% DMSO, 1% FBS culture medium (negative control). Four wells were used for each group (n=4).

[0068] 5. Gene Expression Analysis 5.1 RNA Extraction Using the cultured cells obtained in step 4, RNA extraction was performed according to the modified guanidine thioisocyanate-phenol chloroform method (Chomczynski P and Sacchi N (1987) Anal. Biochem., 162(1); 156-159) using the following procedure. The culture medium from the 24-well culture plate was removed by suction pump, and 1 mL of sterile Dulbecco's PBS(-) was added per well to wash the medium residue with water. The solution was then removed again using the suction pump.

[0069] 250 μL of ISOGEN II (Nippon Gene Co., Ltd.) was added to each well, and the cells were lysed by pipetting. The lysate was collected in a 1.5 mL tube. 100 μL of RNase-free distilled water (Nippon Gene Co., Ltd.) was added and mixed by vortex mixing. After standing at room temperature for 15 minutes, the mixture was centrifuged at 12,000 × g for 15 minutes at room temperature using a micro-high-speed centrifuge (Tommy Seiko Co., Ltd.). Approximately 250 μL of the supernatant was collected without aspirating the precipitate and transferred to a new 1.5 mL tube. 250 μL of 2-propanol (Fujifilm Wako Pure Chemical Industries, Ltd.) was added, mixed by inversion, and then stood at room temperature for 15 minutes.

[0070] Using a micro-high-speed centrifuge, the solution was centrifuged at 12,000 × g for 15 minutes at room temperature. The supernatant was removed by decanting, and the mouth of the 1.5 mL tube was held against a Kimwipe (Nippon Paper Crecia Co., Ltd.) for a few seconds to remove any dripping. 500 μL of 75% ethanol solution was added and mixed by inversion. Using a micro-high-speed centrifuge, the solution was centrifuged at 12,000 × g for 5 minutes at room temperature. The supernatant was removed by decanting, and the mouth of the tube was held against a Kimwipe for a few seconds to remove any dripping. Another 500 μL of 75% ethanol was added and mixed by inversion. Using a micro-high-speed centrifuge, the solution was centrifuged at 12,000 × g for 5 minutes at room temperature. The supernatant was removed by decanting, and the mouth of the tube was held against a Kimwipe for a few seconds to remove any remaining moisture. The solution was then collected at the bottom of the 1.5 mL tube by performing a gentle centrifuge again. The solution at the bottom was removed using a micropipette, and then the 1.5 mL tubes were placed upright on a Kimwipe with the opening facing downwards and air-dried for 15 minutes. 10 μL of RNase-free distilled water was added to each 1.5 mL tube to dissolve the RNA. The RNA concentration was calculated using an Epoch absorbance plate reader (BioTek).

[0071] 5.2 cDNA synthesis cDNA synthesis was performed using the extracted RNA as a template. The cDNA synthesis was carried out using the PrimeScript RT Reagent kit (Takara Bio). Specifically, an enzymatic method was performed that utilized the activity of a modified M-MLV reverse transcriptase (Roth MJ, Tanese N and Goff SP (1985) J. Biol. Chem., 260(16); 9326-9335) to convert the entire sequence, starting from the complementary sites of random hexamer primers and oligo-dT primers, into DNA. Each extracted RNA was diluted appropriately with RNase-free distilled water to a 50 μg / μL solution. Using a micropipette, the following volumes of the mixture were prepared in 1.5 mL tubes. · 5 × PrimeScript Buffer (for Real Time) 26μL · Oligo dT Primer 50μM 6.5μL · Random 6 mers 100μM 6.5μL • RNase-free distilled water 84.5 μL Finally, add the following on the ice. · PrimeScript RT Enzyme Mix I 6.5μL Total 130 μL for 26 reactions

[0072] Eight PCR tubes (Thermo Fisher Scientific) were prepared on a cooled rack, and 5 μL of the mixture was added to each. 5 μL of 50 μg / μL RNA solution was spotted onto the lid of each eight PCR tube containing the mixture. The lids were closed, and the solutions were mixed by inversion or tapping. The solutions were then collected at the bottom of the tubes using a swing-type plate centrifuge (Kubota Seisakusho). A reverse transcription reaction was carried out using a thermal cycler (Applied Biosystems) according to the following program, and then the reaction solution was diluted with 50 μL of distilled water. I) 37℃ for 15 minutes II) 85℃ 5 seconds III) 4℃ heat retention

[0073] 5.3 Real-time quantitative PCR (RT-qPCR) The expression levels of each target gene were evaluated using the cDNA library synthesized in 5.2 by real-time quantitative PCR (Higuchi R, Fockler C, Dollinger G and Watson R (1993) Biotechnology, 11: 1026-1030). The CyberGreen method (Kitahara T, Li HS and Balaban CD (2004) Hear Res., 196: 39-48) was performed using PowerUp SYBR Green Master Mix (Applied Biosystems). The primers were designed based on previous literature (Kim J, Kim M, Yun JG and Hwang J (2017), Pekmezci E, Turkoglu M (2017), and Nakamura T, Yamamura H (2018)) and purchased from Thermo Fisher Scientific.

[0074] [Table 1]

[0075] 10 μL each of forward and reverse primer solutions (100 μM concentration) for amplifying each target gene was used and diluted with 80 μL of distilled water to prepare a premix solution containing 100 μL of each primer at 10 μM concentration. The following solutions were mixed in 1.5 mL tubes to prepare the reaction solutions (one tube was prepared for each primer set). Power Up SYBR Green Master Mix (2×) 130μL Primer 10 μM premix solution 20.8 μL Sterile purified water 57.2μL Total 208 μL

[0076] Two μL of cDNA obtained by cDNA synthesis was added to each well of a PCR plate. Eight μL of reaction solution was added to each well of the PCR plate containing the cDNA using a micropipette, and the mixture was mixed by repeatedly aspirating and discharging. The plate was sealed with a dedicated seal. The reaction was carried out using the following program. The expression levels of each target gene were compared based on the difference in cycle numbers between the housekeeping gene and the target gene. T The calculations were performed according to the method (Livak KJ and Schmittgen TD (2001) Methods., 25(4); 402-408). Statistical analysis was performed using the MEPHAS statistical analysis program for medical and pharmaceutical data provided by the Genetic Information Experiment Center, Osaka University, and Dunnett's multiple testing was conducted.

[0077] [Table 2]

[0078] [result] Figure 1 shows the relative gene expression levels of vascular endothelial growth factor (VEGF) in human primary dermal papilla cells (HFDPCs) treated with each concentration of RSE, RTN, or RICEO, with the gene expression level in the negative control set to 1. A p-value of <0.05 was considered statistically significant. With RSE, a 0.1% treatment resulted in approximately a twofold increase in VEGF gene expression compared to the negative control. With RTN, 0.01% and 0.1% treatments resulted in approximately a threefold and sixfold increase in VEGF gene expression compared to the negative control, respectively. With RICEO, a 0.5% treatment resulted in approximately a 17-fold increase in VEGF gene expression compared to the negative control.

[0079] Similarly, when treated with 0.1% and 0.5% phytic acid or inositol, respectively, no significant change in VEGF gene expression was observed with 0.1% phytic acid, but an approximately twofold increase in VEGF gene expression compared to the negative control was observed with 0.5% phytic acid. With inositol, no significant change in VEGF gene expression was observed with 0.1% inositol, but an approximately 1.5-fold increase in VEGF gene expression compared to the negative control was observed with 0.5% inositol. In RSE and RTN, an increase in VEGF gene expression was observed at lower treatment concentrations than with phytic acid or inositol alone. Furthermore, although 0.5% RICEO actually contains approximately 0.15% phytic acid + approximately 0.05% inositol, treatment with 0.5% RICEO showed a significantly higher VEGF gene expression-promoting effect than treatment with 0.5% phytic acid or 0.5% inositol alone. This suggests that RSE, RTN, and RICEO exhibit superior hair growth effects compared to phytic acid or inositol alone, and in particular, RICEO demonstrates remarkable hair growth effects due to the synergistic effects of phytic acid, inositol, and other rice bran-derived components.

[0080] Figure 2 shows the relative gene expression levels of insulin-like growth factor (IGF-1) in HFDPC treated with RSE or RICEO at various concentrations, with the gene expression level in the negative control HFDPC set to 1. A statistically significant difference was considered to be p<0.05. With RSE, an increase of approximately 2-fold and 3-fold in IGF-1 gene expression was observed compared to the negative control with 0.01% and 0.1% treatments, respectively. With RICEO, an increase of approximately 4-fold and 6.5-fold in IGF-1 gene expression was observed compared to the negative control with 0.1% and 0.5% treatments, respectively.

[0081] Similarly, when treated with 0.1% and 0.5% phytic acid or inositol, respectively, the 0.1% phytic acid treatment increased IGF-1 gene expression compared to the negative control, but the difference was not statistically significant. The 0.5% treatment showed an approximately 6.5-fold increase in IGF-1 gene expression compared to the negative control. With inositol, there was no significant difference in the increase in IGF-1 gene expression with either the 0.1% or 0.5% treatments. RSE showed an increase in IGF-1 gene expression at lower treatment concentrations than treatment with phytic acid or inositol alone. Furthermore, treatment with 0.1% RICEO (equivalent to approximately 0.03% phytic acid + approximately 0.01% inositol) showed a significantly higher increase in IGF-1 gene expression than treatment with 0.1% phytic acid or 0.1% inositol alone. This suggests that RSE and RICEO exhibit superior hair growth effects compared to phytic acid and inositol, and in particular, RICEO demonstrates remarkable hair growth effects through synergistic effects with phytic acid, inositol, and other rice bran-derived components.

[0082] Figure 3 shows the relative gene expression levels of the type 2 5α-reductase (5α-R2) gene in human prostate cancer cells (LNCaP) treated with each concentration of GX-N or RTN, with the gene expression level in the negative control set to 1. A p<0.05 value was considered statistically significant. With GX-N, a decrease in 5α-R2 gene expression of approximately 0.85-fold and 0.75-fold was observed compared to the negative control with 0.01% and 0.1% treatments, respectively. With RTN, a decrease in 5α-R2 gene expression of approximately 0.7-fold was observed with 0.1% treatment compared to the negative control.

[0083] [Example 2: Confirmation of hair papilla cell proliferation rate] [Test Method] Except for using human primary dermal papilla cells (HFDPC), the procedure is the same as in Example 1, with a difference of 1.0 × 10⁻¹⁴ cells. 5 A cell suspension was prepared at a concentration of cells / mL. The resulting cell suspension was placed in a 96-well culture plate (Biolamo Co.) at a rate of 1.0 × 10⁶. 4 Cells were seeded to a cell / well ratio and cultured overnight in a CO2 incubator at 37°C under a 5% CO2 atmosphere. 0.1 mL each of 0.005%, 0.01%, 0.05%, and 0.1% RICEO-containing medium or 1% FBS culture medium (negative control) was added to each well, and the cells were cultured for 24 hours in a CO2 incubator at 37°C under a 5% CO2 atmosphere. Five wells were used for each group (n=5). After culture, the medium was removed, and 100 μL / well of culture medium containing 10% tetrazolium salt reagent (CCK-8) (0% FBS) was added, and the cells were cultured in a CO2 incubator for 2 hours. Absorbance at 450 nm was measured using a microplate reader (Tecan). The relative cell percentage was calculated with the number of cells in the negative control set to 100%. Statistical analysis was performed using MEPHAS, a statistical analysis program for medical and pharmaceutical data provided by the Genetic Information Experiment Center, Osaka University, and Dunnett's multiple testing was conducted.

[0084] The culture was carried out in the same manner, except that the RICEO-containing medium was replaced with phytic acid-containing medium (0.016%, 0.06%, 0.25%, 1%) or inositol-containing medium (0.016%, 0.06%, 0.25%, 1%).

[0085] [result] Figure 4 shows the cell proliferation rates when HFDPC was treated with RICEO at various concentrations. A statistically significant difference was considered to be p<0.05. A significant increase in cell number was observed when treated with 0.01%, 0.05%, and 0.1% RICEO, with particularly remarkable cell proliferation of approximately 150-160% observed with 0.1% RICEO treatment.

[0086] Similarly, when treated with 0.016%, 0.06%, 0.25%, and 1% phytic acid or inositol, respectively, phytic acid treatment showed approximately 120-130% cell proliferation at 0.016%, 0.06%, and 0.25%, while the 1% treatment reduced the cell count to approximately 70%. Inositol treatment showed a significant, gradual increase in cell count from approximately 120% to 150% in a concentration-dependent manner, progressing from 0.06% to 0.25% and then to 1%. Treatment with 0.1% RICEO (equivalent to approximately 0.03% phytic acid + approximately 0.01% inositol) showed a more significant cell proliferation effect than treatment with 0.06% phytic acid or 0.016% inositol alone. This suggests that RICEO exhibits remarkable hair growth effects through synergistic interactions with other rice bran-derived components, including phytic acid and inositol.

[0087] [Example 3: Verification of the effect of a mixture of inositol and phytic acid on VEGF gene expression levels] [Test materials] The following two types of purified inositol and purified phytic acid were used. • Phytic acid (product name, manufactured by Tsukuno Foods Co., Ltd.) • Inositol (product name, manufactured by Tsukuno Foods Co., Ltd.)

[0088] The effect of the inositol (IN) and phytic acid (PA) ratio (IN:PA = approximately 1:3) on VEGF gene expression was investigated. A 0.5% inositol-containing medium and a 0.5% phytic acid-containing medium were mixed in a 1:3 ratio to obtain a 0.5% IN / PA mixture medium. This medium was then diluted 5-fold or 10-fold with 1% FBS culture medium to obtain a 0.1% IN / PA mixture medium and a 0.05% IN / PA mixture medium. Table 3 shows the inositol and phytic acid content in each IN / PA mixture medium. [Table 3]

[0089] Human primary dermal papilla cells (HFDPCs) were cultured and VEGF gene expression levels were analyzed in the same manner as in Example 1, except that 1 mL of phytic acid-containing medium (0.1%, 0.5%), inositol-containing medium (0.1%, 0.5%), IN / PA mixture-containing medium (0.05%, 0.1%, 0.5%), or 1% FBS culture medium (negative control) was added per well.

[0090] [result] Figure 5 shows the relative gene expression levels of vascular endothelial growth factor (VEGF) in HFDPCs treated with various concentrations of phytic acid (PA), inositol (IN), or a mixture of inositol and phytic acid (IN / PA mix), with the gene expression level of human primary dermal papilla cells (HFDPCs) in the negative control set to 1. A p-value of <0.05 was considered statistically significant. When treated with 0.1% and 0.5% phytic acid or inositol, respectively, no significant change in VEGF gene expression was observed with 0.1% phytic acid, but an approximately twofold increase in VEGF gene expression was observed with 0.5% phytic acid compared to the negative control. With inositol, no significant change in VEGF gene expression was observed with 0.1% phytic acid, but an approximately 1.5-fold increase in VEGF gene expression was observed with 0.5% inositol compared to the negative control. In the case of a mixture of inositol and phytic acid, a VEGF gene expression level increased by approximately 2.5 times and 4 times, respectively, at treatment concentrations of 0.05% and 0.1%, lower than that observed with phytic acid or inositol alone. This confirms that using a mixture of inositol and phytic acid results in a significantly higher VEGF gene expression promotion effect at low concentrations compared to using phytic acid or inositol alone, due to the synergistic effect of inositol and phytic acid.

[0091] It should be noted that the present invention is not limited to the embodiments and examples described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included within the technical scope of the present invention. Furthermore, all academic and patent documents mentioned herein are incorporated herein by reference.

Claims

1. It contains purified inositol and purified phytic acid, and the ratio of inositol to phytic acid is 1:2 or more and 1:4 or less by mass. A hair growth product in which the total content of purified inositol and purified phytic acid is 0.01% (wt / v) or more and 80% (wt / v) or less of the total hair growth product.

2. The hair growth agent according to claim 1, wherein the ratio of inositol and phytic acid is 1:2.5 or more and 1:4 or less by mass.

3. A pharmaceutical product characterized by containing the hair growth agent described in claim 1.

4. A cosmetic product characterized by containing the hair growth agent described in claim 1.

5. A food or beverage characterized by containing the hair growth agent described in claim 1.

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