5α-reductase inhibitor, composition for preventing and treating alopecia or promoting hair growth, method for inhibiting 5α-reductase in the scalp, and method for promoting hair growth

Abstract

A novel 5α-reductase inhibitor is provided. The 5α-reductase inhibitor contains fulvic acid as an active ingredient. Due to the 5α-reductase inhibitory effect of fulvic acid, it is expected to be used in the prevention and treatment of alopecia, as well as for promoting hair growth.

Description

[Technical Field]

[0001] The present invention relates to a novel 5α-reductase inhibitor and its application technology. [Background technology]

[0002] In men, male pattern baldness, which is related to male hormones, is well known. In addition, female androgen-related androgen-related hair loss is also known. It is thought that as women age, the level of female hormones decreases, and during menopause, the secretion of female hormones decreases, resulting in a relative excess of male hormones, making them more susceptible to the onset of this condition.

[0003] 5α-reductase is an enzyme that converts testosterone into the active form dihydrotestosterone. Testosterone, a male hormone, is converted to dihydrotestosterone (DHT) by 5α-reductase present in hair follicles, sebaceous glands, etc., and the resulting dihydrotestosterone inhibits the differentiation of hair matrix cells, promoting hair loss. Therefore, inhibiting the activity of 5α-reductase is expected to have beneficial effects such as alleviating alopecia.

[0004] Various 5α-reductase inhibitors have been reported to date, and their active ingredients include snow ganoderma extract (Patent Document 1), polynium oside (Patent Document 2), extract of a plant of the genus Closandra in the family Acanthaceae (Patent Document 3), extract of the Japanese broccoli (Patent Document 4), and aspalathin derived from non-fermented rooibos tea (Patent Document 5). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 566344 [Patent Document 2] Japanese Patent Application Laid-Open No. 2012-67014 [Patent Document 3] Patent No. 5632619 [Patent Document 4] Patent No. 5971833 [Patent Document 5] Japanese Patent Application Laid-Open No. 2009-249371 Summary of the Invention [Problem to be solved by the invention]

[0006] Some of the active ingredients in the above-mentioned conventional examples do not have sufficient 5α-reductase inhibitory activity, and further development has been carried out. Under these circumstances, an object of the present invention is to provide a novel 5α-reductase inhibitor and its application. [Means for solving the problem]

[0007] As a result of extensive research to solve the above problems, the present inventors discovered that fulvic acid has a testosterone 5α-reductase inhibitory effect, leading to the present invention.

[0008] That is, the present invention relates to the following inventions. <1> A 5α-reductase inhibitor containing purified fulvic acid as the active ingredient. <2> A composition containing purified fulvic acid for preventing and treating alopecia or promoting hair growth based on its 5α-reductase inhibitory effect. <3> A method for inhibiting 5α-reductase in the scalp by administering a composition containing purified fulvic acid to the scalp (excluding medical procedures for humans). <4> A method for promoting hair growth by administering a composition containing purified fulvic acid to the scalp (excluding medical procedures for humans). [Effects of the Invention]

[0009] According to the present invention, a 5α-reductase inhibitor capable of inhibiting the activity of 5α-reductase is provided by using fulvic acid as an active ingredient. Furthermore, by using fulvic acid as an active ingredient, it is possible to provide a composition that has excellent preventive and therapeutic effects for alopecia and hair growth promotion effects based on its 5α-reductase inhibitory effect. [Brief explanation of the drawings]

[0010] [Figure 1] 1 shows IR spectra of an example sample (fulvic acid A) and a commercially available fulvic acid. [Figure 2] 1 shows the UV spectra of an example sample (fulvic acid A) and a commercially available fulvic acid. [Figure 3] 1 is a H-NMR spectrum of an example sample (fulvic acid A). [Figure 4] 1 is a Vis-UV spectrum of an example sample (fulvic acid A). DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the examples below and can be practiced with any modifications within the scope of the gist of the present invention. In this specification, the symbol "to" is used as an expression including the numerical value or physical quantity before and after it.

[0012] <1.5α-reductase inhibitor> The 5α-reductase inhibitor of the present invention contains fulvic acid as an active ingredient and can inhibit the activity of 5α-reductase.

[0013] The term "fulvic acid" as used herein refers to an amorphous, high molecular weight organic acid that is contained in humic substances and does not precipitate with acid. In this specification, "humic substances" refers to organic components that are secondarily produced when organic matter such as plant leaves and stems is decomposed by a wide variety of microorganisms, and is a general term for organic matter that cannot be classified as sugars, proteins, lipids, etc. Fulvic acid is not a molecule with a single fixed chemical structure, but a polyvalent organic acid containing many carboxyl groups and phenolic hydroxyl groups within its molecule. Humic substances include humic acid as well as fulvic acid. According to the Japanese Humic Society, "the definition of humic substances is merely a tentative one," but humic substances from soil and sediments are generally defined based on their solubility in acids and bases: humic acid is generally soluble in basic aqueous solutions, while fulvic acid is generally soluble in both acidic and basic aqueous solutions. In the present invention, "fulvic acid" is defined as "amorphous high molecular weight organic acid that can be separated and purified from humic substances (including wood decomposition products) and is soluble in acidic and basic aqueous solutions."

[0014] The fulvic acid contained in the 5α-reductase inhibitor of the present invention is not particularly limited as long as it is acceptable for application to humans, particularly the human scalp, and can be, for example, any of fulvic acids derived from terrestrial soil, seabed soil, etc., naturally occurring fulvic acids such as those derived from water systems such as rivers, lakes, and marshes, and artificially produced fulvic acids such as fulvic acids derived from organic waste. One or more of these fulvic acids can be used.

[0015] As the fulvic acid according to the present invention, fulvic acid derived from wood decomposition products is preferably used. In the present invention, "fulvic acid derived from wood decomposition products" refers to fulvic acid separated and extracted from wood decomposition products obtained by using wood as a raw material and decomposing the wood with microorganisms, acid solution, etc. Although natural wood decomposition products can be used as the extraction source, wood decomposition products obtained artificially by decomposing wood with microorganisms, acid solution, etc. are usually used.

[0016] The raw wood material is preferably broad-leaved wood, but the raw wood material may contain materials other than broad-leaved wood (such as coniferous wood and non-wood lignin) within the scope of the present invention. Examples of fulvic acid derived from wood decomposition products include fulvic acid separated and purified from wood decomposition products obtained by decomposing sawtooth oak (oak tree) obtained by the method of the Examples described below using a specific white-rot fungus. Commercially available fulvic acid derived from wood decomposition products can also be used.

[0017] The fulvic acid of the present invention includes not only fulvic acid but also salts, esters, and derivatives of fulvic acid, as long as they have a 5α-reductase inhibitory effect. As the fulvic acid of the present invention, fulvic acid (particularly purified fulvic acid) and salts of fulvic acid can be suitably used.

[0018] In addition, as the fulvic acid of the present invention, as long as it has a 5α-reductase inhibitory effect, it is possible to use a substance containing humic acid, amino acids, vitamins, enzymes, minerals, and other trace elements, in addition to fulvic acid, but purified fulvic acid can also be used. In the present invention, "purified fulvic acid" refers to fulvic acid obtained by removing components other than fulvic acid from a mixture containing fulvic acid, followed by separation and purification. Any separation and purification process can be used as long as the desired fulvic acid is obtained, and can be carried out by passing a solution containing fulvic acid and other components through an ion exchange resin, for example. A method conforming to the IHSS method (operation and verification method specified by the International Humic Society) is particularly preferred. Specific examples of such separation and purification methods will be described in the Examples section.

[0019] Furthermore, as long as the fulvic acid of the present invention has a 5α-reductase inhibitory effect, various commercially available products can be used as is or after appropriate concentration or dilution. Examples of such commercially available products include "Lead Up," a humus precursor solution sold by T&G Co., Ltd., "Chelate Balance" and "Beauty Source Kirei-Ryu" sold by Miyamonte JAPAN Co., Ltd., "Concentrated Fulvic Acid Product" (fulvic acid plant activator "Green God") sold by Japan Fulvic Acid Research Institute Co., Ltd., and fulvic acid manufactured by Koyo Co., Ltd. Such commercially available products may contain impurities such as humic acid due to the purification process, but in such cases they can be separated and purified before use. In addition, fulvic acid (standard fulvic acid) derived from Danto Forest soil in Aichi Prefecture, distributed by the Japanese Society of Corrosive Materials, can also be used.

[0020] In the 5α-reductase inhibitor of the present invention, the content of fulvic acid may be appropriately determined within a range that provides a significant 5α-reductase inhibitory effect, for example, 0.0001 to 100% by mass in terms of the amount of fulvic acid.

[0021] The 5α-reductase inhibitory effect of fulvic acid as described above is expected to have various uses as a quasi-drug, particularly for male pattern baldness, female male pattern baldness, hair growth, prevention of thinning hair and hair loss, hair growth promotion, hair growth stimulation, and hair care.

[0022] The 5α-reductase inhibitors of the present invention can inhibit the activity of 5α-reductase and are therefore useful for preventing or treating various conditions caused by excessive testosterone 5α-reductase activity or excessive testosterone secretion, such as seborrhea, acne, and benign prostatic hyperplasia.

[0023] In addition to fulvic acid, the 5α-reductase inhibitor of the present invention can be appropriately blended with additives commonly used in the food, cosmetics, and pharmaceutical industries, such as excipients, moisture-proofing agents, preservatives, strengthening agents, thickeners, emulsifiers, antioxidants, sweeteners, acidulants, seasonings, coloring agents, fragrances, whitening agents, moisturizers, oily ingredients, UV absorbers, surfactants, alcohols, powder ingredients, coloring agents, aqueous ingredients, water, various skin nutrients, etc. Commercially available products can be suitably used as these additives.

[0024] <2. Composition for preventing and treating alopecia or promoting hair growth> The composition of the present invention for preventing or treating alopecia or promoting hair growth (hereinafter referred to as "the composition of the present invention") contains fulvic acid as an active ingredient and is characterized by its ability to prevent or treat alopecia and / or promote hair growth based on its 5α-reductase inhibitory activity. The fulvic acid used in the composition of the present invention is the same as that described in the 5α-reductase inhibitor of the present invention, so further description will be omitted.

[0025] The composition of the present invention has at least one of the effects of preventing alopecia, treating alopecia, and promoting hair growth, and particularly preferably has all of these effects. In this specification, "prevention of alopecia" includes the suppression and delay of alopecia.In addition, "treatment of alopecia" includes the improvement and remission of alopecia, and the suppression of the progression of alopecia.The alopecia that is the subject of the present invention is not limited as long as it is alopecia whose mechanism is the enzyme catalytic action of 5α-reductase, and typically includes androgenetic alopecia and female androgenetic alopecia.In addition, in this specification, "promoting hair growth" includes the improvement of hair growth rate and the promotion of hair growth.

[0026] The composition of the present invention is expected to be particularly effective in treating male pattern baldness, female male pattern baldness, promoting hair growth, preventing thinning hair and hair loss, promoting hair growth, promoting hair growth, and nourishing hair.

[0027] In the composition of the present invention, the amount of fulvic acid to be blended may be appropriately determined taking into consideration the purpose of use, gender, symptoms, etc., and is, for example, 0.0001 to 10 mass % in terms of the amount of fulvic acid.

[0028] The composition of the present invention can be prepared by mixing fulvic acid with various additives selected as needed. In addition to fulvic acid, additives commonly used in the food, cosmetic, and pharmaceutical industries, such as excipients, moisture-proofing agents, preservatives, strengthening agents, thickeners, emulsifiers, antioxidants, sweeteners, acidulants, seasonings, coloring agents, fragrances, whitening agents, moisturizers, oily ingredients, UV absorbers, surfactants, alcohols, powder ingredients, coloring agents, aqueous ingredients, water, and various skin nutrients, can be added as needed depending on the purpose. Commercially available additives can be used as these additives.

[0029] The composition of the present invention is preferably used in the form of an external preparation for the skin such as a solution, dispersion, emulsion, ointment, cream, gel, aerosol, or pack, and is particularly preferably used in the form of an external preparation for the scalp.

[0030] The composition of the present invention may be in any form, and is preferably used in the form of a composition for external application to the skin, particularly a composition for external application to the scalp, such as a solution, dispersion, emulsion, ointment, cream, gel, aerosol, or pack. Specific examples of the composition include, but are not limited to, hair tonic, hair cream, hair lotion, hair shampoo, hair rinse, hair conditioner, hair spray, hair aerosol, pomade, powder, and gel.

[0031] The composition of the present invention may contain other hair ingredients, provided that the ingredients do not impair the object of the present invention, such as ingredients that activate hair roots and hair follicle cells, ingredients that enhance blood flow to hair follicle cells, antibacterial ingredients, antidandruff agents, keratin softeners, refreshing agents, moisturizers, etc.

[0032] The composition of the present invention can be used in a method for inhibiting 5α-reductase in the scalp by administering it to the scalp. The composition of the present invention can also be used in a method for preventing and treating alopecia or a method for promoting hair growth by administering it to the scalp. Methods of transdermal administration include spraying, application, compresses, and the like, and are not particularly limited. The amount of the composition of the present invention used in this method can be appropriately determined taking into account the amount of fulvic acid contained, the purpose of use, gender, symptoms, etc.

[0033] The method for inhibiting 5α-reductase in the scalp by administering the composition of the present invention to the scalp and the method for promoting hair growth by administering the composition of the present invention to the scalp can be performed in addition to medical procedures for humans. Examples of such procedures that do not fall under medical procedures for humans include the application of the composition of the present invention to the human scalp at barber shops, beauty salons, hair care salons, etc. [Example]

[0034] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0035] In the examples, the following two types of fulvic acid were used to evaluate the inhibitory activity of 5α-reductase. Fulvic acid A: Fulvic acid derived from decomposed oak (manufacturing method described below) Fulvic Acid B: Fulvic Acid manufactured by Japan Fulvic Acid Research Institute Co., Ltd.

[0036] 1. Production and identification of fulvic acid A The method for producing fulvic acid A and the method for identifying it will be described below. The raw wood and white rot fungi used in the manufacturing method of Fulvic Acid A are as follows. (1) Raw material wood The raw wood used is sawtooth oak chips. These are taken from forest tree felling sites in Oita Prefecture, and do not contain wood derived from construction waste or industrial waste from demolition, etc. Furthermore, no chemical treatment is carried out after felling. (2)White rot fungi The test fungus used was a white rot fungus identified by the accession number NITE P-02428 (identification mark BMC-110012) at the Patent Microorganisms Deposit Center of the National Institute of Technology and Evaluation (hereinafter referred to as "white rot fungus (NITE P-02428)").

[0037] <Process (1)> Sawtooth oak wood (trunk) was crushed into chips of about 10 mm, and the resulting chips were further processed in a fiberizer (Nishiho Kiko Co., Ltd., product name: Love Machine) to defibrate them into powder (about 100 μm). The resulting powdered sawtooth oak, defatted rice bran (additive nutrients), and water were stirred and mixed in a weight ratio of 5:0.8:4, and the mixture (sawtooth oak wood flour medium) was then packed into 1.7 kg of mushroom bed bags (volume 3.6 L) and placed in a metal frame to shape. The moisture content at the time of packing was 57.9% by weight. The mixture was then sterilized in an autoclave (121°C, 60 minutes) according to standard methods, allowed to cool, and then inoculated with an inoculum (white rot fungus (NITE P-02428)) according to standard methods. The white rot fungus (NITE P-02428) used as the inoculum was pre-cultured in a 300 mL Erlenmeyer flask containing an appropriate amount of sawtooth oak wood flour medium. The inoculum was inoculated into the sawtooth oak wood flour medium in the mushroom bed bag by pressing the medium in the mushroom bed bag with a 15 mm diameter iron rod and puncturing two holes to form inoculation holes. The inoculum amount was 10 g per 1 kg of powdered sawtooth oak.

[0038] Using the wood decomposition product obtained in step (1), fulvic acid was separated and purified by the following procedure.

[0039] <Process (2)> 1 kg of the wood decomposition product (decomposed over 30 days) obtained in step (1) and 2 L of water were placed in a container and stirred with a mixer for a predetermined time (3 hours or more) to extract the components contained in the wood decomposition product into the water, thereby obtaining a liquid composition containing the wood decomposition product.

[0040] <Process (3)> The liquid composition containing the wood decomposition product obtained in step (2) was subjected to solid-liquid separation using a centrifuge, and the liquid obtained by decanting the liquid portion was dried under reduced pressure in a vacuum dryer to obtain a dry powder containing humic acid substances. The yield was about 5% (about 50 g produced from 1 kg of raw material).

[0041] <Process (4)> The dried powder obtained in step (3) was separated and purified by the following steps to obtain fulvic acid. This separation and purification method corresponds to the IHSS method (operation and verification method specified by the International Humic Society). First, the dried powder obtained in step (3) was placed in a 0.1 M NaOH aqueous solution and left for one day to form a precipitate, after which the precipitate and the aqueous solution were separated. Next, concentrated hydrochloric acid was added to the aqueous solution to adjust the pH to 1. A precipitate was formed again, so the precipitate and the aqueous solution were further separated. The resulting solution was passed through a column packed with approximately 150 mL of XRD resin, allowing the fulvic acid to be adsorbed onto the XRD resin. After the fulvic acid was adsorbed, the column was washed with 300 mL of 1 M hydrochloric acid, followed by 300 mL of 0.1 M hydrochloric acid, to remove impurities. Next, approximately 450 mL of 0.1 M NaOH aqueous solution was passed through the column to release the fulvic acid adsorbed on the XRD resin, and the released liquid containing fulvic acid was collected. The 0.1 M NaOH aqueous solution was passed through the column until the color of the liquid passing through the column became lighter.

[0042] The resulting free solution was passed through a column packed with approximately 300 mL of IRC resin to remove the NaOH solution. To recover the remaining fulvic acid, the column was washed with 300 mL of distilled water, and the washings were combined with the free fulvic acid solution. The resulting fulvic acid solution was dried under reduced pressure to remove water, yielding the desired dry powder (fulvic acid A). The yield was approximately 0.6% by weight (approximately 6 g produced from 1 kg).

[0043] The dried powder after separation and purification (hereinafter referred to as "Example sample") was analyzed by FT-IR, UV, 1 The product was analyzed by H-NMR and elemental analysis (C·H·N coder) and compared with commercially available fulvic acid (manufactured by Koyo Co., Ltd.). The equipment used is as follows: IR: JASCO Corporation, FT / IR-5000 UV: Nihon Bunko Co., Ltd., Ubest V-560 type NMR: Bruker BioSpin, S-NMR 600 Elemental analysis: PerkinElmer Japan, CHNS / O Analyzer 2400II

[0044] Figure 1 shows the IR spectra of the fulvic acid from the example and the commercially available product. -1 hydroxyl group (OH), 2980cm -1 methylene group (-CH2-) and 1700cm -1 A characteristic peak was observed at the wavelength of the carboxyl group (COOH). Similar peaks have also been confirmed in literature (Humus in the Environment - Their Characteristics and Research Methods, ISBN-10: 4782705778), and it is thought that fulvic acid has a structure with many hydroxyl and carboxyl groups, which are unique to fulvic acid. Figure 2 shows the UV spectra of the example and the commercial product. Both showed similar absorption curves, but the example had a stronger absorption band near 270 nm. This absorption band is thought to be derived from aromatic rings, and the example sample is thought to have a structure containing relatively more aromatic rings than the commercial product.

[0045] Figure 3 shows the sample of the example. 1 The results of H-NMR are shown below. A weak signal at 0.8 ppm was assigned to the terminal methyl group, signals at 1.1-1.3 ppm to the methyl group and methylene at the β-position, and signals at 3.5-4.0 ppm to the methoxyl group and substituted aliphatic hydrogen including alcoholic hydroxyl group. Figure 4 shows the results of the E4 / E6 ratio proposed by Kononova. The E4 / E6 ratio is the ratio of absorbance at 465 nm and 665 nm and is used to characterize fulvic acid. According to the literature (Humus Substances in the Environment - Their Characteristics and Research Methods, ISBN-10: 4782705778), the E4 / E6 ratio of fulvic acid is defined as varying between 6.0 and 8.5. The E4 / E6 ratio of the sample in this example was 7.8. Furthermore, elemental analysis revealed that the C, H, and N contents of each sample were the same as those of the commercial product (C: 29.16%, H: 4.71%, N: 4.99%) and the working example (C: 38.65%, H: 4.63%, N: 2.93%). Comparison with literature (Humus Substances in the Environment - Their Characteristics and Research Methods, ISBN-10: 4782705778) confirmed that the nitrogen content was high. The average molecular weight was also approximately 100,000. These results confirmed that the working example sample (dried powder after separation and purification) was fulvic acid.

[0046] Evaluation of 2.5α-reductase inhibitory activity The 5α-reductase inhibitory activity of fulvic acid A (fulvic acid obtained by the above method) and fulvic acid B (manufactured by Japan Fulvic Acid Research Institute Co., Ltd.) was evaluated using the following method. Note that fulvic acid B is a fulvic acid separated and purified from wood decomposition products (tree extracts) derived from broad-leaved trees. As the enzyme source (5α-reductase), rat liver homogenate (S-9) was used.

[0047] (1) Preparation of the measurement solution (Adjustment of standard solution) Approximately 1 mg of testosterone and dihydrotestosterone (DHT) were weighed out and dissolved in ethanol to make 1 mL, and the mixture was mixed in equal amounts to prepare a standard solution.

[0048] (Testosterone solution preparation) Approximately 4.2 mg of testosterone was precisely weighed and made up to 1 mL with propylene glycol to obtain a testosterone solution.

[0049] (Preparation of Tris-HCl buffer solution) 30.3 mg of 2-Amino-2-hydroxymethyl-1,3-propanediol was precisely weighed and dissolved in purified water, and then the pH was adjusted to 7.13 with 1 M HCl, and the solution was made up to 50 mL with purified water to obtain a Tris-HCl buffer solution.

[0050] (Preparation of NADPH solution) Approximately 10 mg of nicotinamide adenine dinucleotide phosphate (NADPH) was precisely weighed, dissolved in Tris-HCl buffer, and the solution was diluted to 10 mL to obtain an NADPH solution (prepared just before use).

[0051] (Preparation of test sample solution) 0.5 mg / mL of the fulvic acid to be evaluated (fulvic acid A or fulvic acid B) was precisely weighed and dissolved in purified water to obtain a test sample solution. The concentration of fulvic acid A is 0.05% by weight, and the concentration of fulvic acid B is 0.05% by weight.

[0052] (Preparation of rat liver homogenate (S-9) solution) Rat liver homogenate (S-9) frozen at −80° C. was thawed in ice to obtain a rat liver homogenate solution (S-9 solution). The concentration of S-9 was (S9 fraction: protein concentration 19.8 mg / mL S9 (55.4 mg / g liver)).

[0053] (2) 5α-reductase inhibition test Test Example 1: Testosterone + water reacted simultaneously (without S-9: testosterone standard) 20 μL of testosterone solution and 825 μL of NADPH solution were added to a 15 mL V-bottom test tube with a lid, mixed, and kept in a water bath at 37° C. 80 μL of the test sample solution (0.05 wt % fulvic acid A solution) was added to this, mixed again, and allowed to react in a water bath at 37° C. for 60 minutes. After the reaction was completed, exactly 1 mL of methylene chloride was added and the mixture was vigorously shaken to extract the substrate testosterone and its reaction products, thereby stopping the reaction. The mixture was then centrifuged at 1600 G for 10 minutes to separate the methylene chloride layer, and 300 μL of the resulting solution was removed, evaporated, dissolved in methanol, and then analyzed by gas chromatography-mass spectrometry (GC-MS). The GC-MS used was a Shimadzu GCMS QP2010 Ultra. The GC-MS measurement conditions are shown in Tables 1 and 2 below.

[0054] [Table 1]

[0055] [Table 2]

[0056] Test Example 2: Testosterone + water + S-9 reacted simultaneously 20 μL of testosterone solution and 825 μL of NADPH solution were added to a 15 mL V-bottom test tube with a lid, mixed, and kept in a water bath at 37°C. 80 μL of test sample solution (water) and 75 μL of S-9 solution were added to this, mixed again, and allowed to react in a water bath at 37°C for 60 minutes. After the reaction was completed, exactly 1 mL of methylene chloride was added and the mixture was vigorously shaken to extract the substrate testosterone and its reaction products, thereby stopping the reaction. Thereafter, the mixture was centrifuged at 1600 G for 10 minutes to separate the methylene chloride layer, and 300 μL of the layer was removed, vaporized, dissolved in methanol, and then measured by gas chromatography-mass spectrometry (GC-MS).

[0057] Test Example 3: Testosterone + Fulvic Acid A + S-9 reacted simultaneously 20 μL of testosterone solution and 825 μL of NADPH solution were added to a 15 mL V-bottom test tube with a lid, mixed, and kept in a water bath at 37°C. 80 μL of the test sample solution (0.05 wt% fulvic acid A solution) and 75 μL of S-9 solution were added to this, mixed again, and allowed to react in a water bath at 37°C for 60 minutes. After the reaction was completed, exactly 1 mL of methylene chloride was added and the mixture was vigorously shaken to extract the substrate testosterone and its reaction products, thereby stopping the reaction. Thereafter, the mixture was centrifuged at 1600 G for 10 minutes to separate the methylene chloride layer, and 300 μL of the layer was removed, vaporized, dissolved in methanol, and then measured by gas chromatography-mass spectrometry (GC-MS).

[0058] Test Example 4: Testosterone + Fulvic Acid B + S-9 reacted simultaneously 20 μL of testosterone solution and 825 μL of NADPH solution were added to a 15 mL V-bottom test tube with a lid, mixed, and kept in a water bath at 37°C. 80 μL of the test sample solution (0.05 wt% fulvic acid B solution) and 75 μL of S-9 solution were added to this, mixed again, and allowed to react in a water bath at 37°C for 60 minutes. After the reaction was completed, exactly 1 mL of methylene chloride was added and the mixture was vigorously shaken to extract the substrate testosterone and its reaction products, thereby stopping the reaction. Thereafter, the mixture was centrifuged at 1600 G for 10 minutes to separate the methylene chloride layer, and 300 μL of the layer was removed, vaporized, dissolved in methanol, and then measured by gas chromatography-mass spectrometry (GC-MS).

[0059] The 5α-reductase inhibitory activity (inhibition rate (%)) is calculated by the following formula, where the amount of testosterone remaining without being converted or decomposed into dihydrotestosterone or 3α-androstanediol by 5α-reductase (residual testosterone amount) is the ratio of the initial amount of testosterone (initial testosterone amount) (reference amount). Note that the peak area of ​​Test Example 1, which does not contain 5α-reductase (S-9), corresponds to the initial testosterone amount, and the peak areas of Test Examples 2 to 4 correspond to the residual testosterone amount. Inhibition rate (%) = (remaining testosterone amount) / (initial testosterone amount) x 100

[0060] The results are shown in Table 3.

[0061] [Table 3]

[0062] As shown in Table 3, the enzyme inhibition rate of testosterone by S-9 was measured using the amount of testosterone (334 μg) in Test Example 1 as the initial value as the reference value. In Test Example 2, when water was used as the test sample, S-9 enzyme activity was observed against testosterone, and most of the testosterone was converted or decomposed into dihydrotestosterone or 3-alpha-androstanediol, resulting in an 82.7% decrease in the initial testosterone amount. Furthermore, in Test Example 3, when fulvic acid B was added as the test sample, an inhibition rate of 66.3% was observed, demonstrating an inhibitory effect. On the other hand, in Test Example 4, when fulvic acid A was added as the test sample, the inhibition rate was 85.2%, demonstrating an even greater inhibitory effect. [Industrial Applicability]

[0063] The present invention provides a 5α-reductase inhibitor having excellent 5α-reductase inhibition properties and a composition for preventing and treating alopecia or promoting hair growth, and is therefore industrially promising.

Claims

[Claim 1] A 5α-reductase inhibitor containing purified fulvic acid as an active ingredient.

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