A composition for improving, preventing or treating hair loss, comprising catechin 7-O-β-D-apiofuranoside as an active ingredient

Abstract

The present invention relates to a composition for improving, preventing or treating alopecia, which contains a catechin 7-O-β-D-apiofuranoside compound as an active ingredient. The compound of the present invention inhibits the conversion of testosterone in human hair follicle dermal papilla cells into dihydrotestosterone (DHT) through the action of 5α-reductase, and inhibits the apoptosis of dermal papilla cells caused by oxidative stress, and can be used as an active substance for treating, preventing or improving alopecia.

Description

Technical Field

[0001] The present invention relates to a composition for improving, preventing or treating alopecia, which contains catechin 7-O-β-D-apiofuranoside as an active ingredient. Background Art

[0002] Although alopecia is not a pathological problem compared with other pathological diseases, it has a great impact on alopecia patients socially and psychologically because it affects appearance. In the past, alopecia was considered an aging phenomenon, but in recent years, it has been recognized that many factors can cause alopecia, such as genetic factors, stress, nutritional imbalance, changes in social activities, diseases, childbirth, changes in eating habits, irregular life, and excessive use of chemical hair cosmetics. The types of alopecia are generally divided into male pattern alopecia, female pattern alopecia, alopecia areata, postpartum alopecia, and seborrheic dermatitis alopecia, among which the proportion of male pattern alopecia is the highest.

[0003] Male pattern alopecia is caused by genetic factors, and the earlier alopecia appears, the more severe it tends to be. On the other hand, in addition to genetic factors, the male hormone testosterone is converted into dihydrotestosterone (DHT) by the action of 5α-reductase, and DHT causes atrophy of dermal papilla cells, inhibits cell division, and leads to hair thinning or alopecia.

[0004] Alopecia caused by stress mainly presents as alopecia areata. Alopecia areata is a disease in which some or all body hairs fall out. It is common to have several coin-sized bald patches on the scalp, which may be caused by psychological stress and can develop into permanent alopecia with the loss of scalp hair, or body hairs such as beards and eyebrows.

[0005] In addition, alopecia caused by stress may appear in the form of telogen effluvium. Telogen effluvium refers to alopecia that occurs when hairs in the normal growth cycle enter the telogen phase. Hairs grow and fall out in a cycle of 3 - 6 years. Hairs stay in the anagen phase for 3 - 6 years, then enter a 3 - 4-week catagen phase, and fall out after a 3 - 5-month telogen phase. The hairs entering the telogen phase usually account for about 10% (about 10,000 strands) of the total hair volume, and alopecia caused by premature entry of hairs into the telogen phase is called telogen effluvium.

[0006] IGF-1 promotes the proliferation of epithelial cells in culture and prolongs the length of hair follicle tissue, and is an important growth factor for promoting hair growth and regulating hair growth (Itami et al., 1995; Hibino & Nishiyama, 2004).

[0007] Studies have shown that the IGF-1 growth factor increases its expression under the action of androgens, participates in the effect of testosterone on hair, and plays an important role in the mechanism leading to androgenetic alopecia. It also helps prevent aging, improve dementia and depression, prevent lifestyle diseases such as diabetes or hypertension, enhance immunity and increase bone density, prevent skin aging, and is used to treat hair loss because it can induce hair to enter the growth phase from the resting phase.

[0008] Studies have shown that TGF-β1 can induce the growth of the catagen phase. When TGF-β1 is injected into the back skin of mice, the number of keratinocytes can be reduced through hair follicle proliferation. When compared with the control group, the number of TUNEL cells increases in mice treated with TGF-β1 administration. The double visualization of TGF-β type II receptor (TGF-βII) and TUNEL reactivity shows that the apoptotic nuclei of catagen-phase hair follicles are colocalized with TGFRII, enabling TGF-βII agonists and antagonists to be used as effective treatment tools for human hair growth diseases based on advancing or delaying catagen-phase hair loss and hirsutism.

[0009] (Foitzik K, Lindner G, Mueller-Roever S, Maurer M, Botchkareva N, Botchkarev V, Handjiski B, Metz M, Hibino T, Soma T, Dotto GP, Paus R (2000). In vivo control of murine hair follicle regression (catagen) by TGF-β1. The FASEB Journal, 14(5): 752 - 812).

[0010] Shin et al. (2013) pointed out that androgenetic alopecia is closely related to the secretion of TGF-β1 (Shin H, Yoo HG, Inui S, Itami S, Kim IG, Cho AR, Lee DH, Park WS, Kwon O, Cho KH, Won CH (2013). Mediation of reactive oxygen species on androgen-induced growth factor-β1 conversion in human dermal papilla cells. BMB Reports, 46(9): 460 - 464.).

[0011] In mouse bladder papillary cells (DP-6) transfected with androgen receptor, ROS production increased after androgen treatment, and TGF-β1 secretion in DP-6 also increased after androgen treatment. Conversely, androgen-induced TGF-β1 was significantly inhibited by N-acetylcysteine, indicating that androgen-induced TGF-β1 is mediated by ROS in follicular DPCs and can thus be used in antioxidant therapy for androgenetic alopecia treatment.

[0012] Regarding the effects of growth factors on hair growth, many experiments have currently demonstrated that growth factors can participate in hair growth by acting on specific sites in the hair follicle. These growth factors include epidermal growth factor (EGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF), etc. (Tsuboi R (1997). Growth factors and hair growth. Kor J Invest Dermatil, 4(2): 103-108). In 1994, the research group of Hervert found that mice lacking the fibroblast growth factor (FGF)-5 gene had an abnormally long anagen phase, resulting in relatively long hair, indicating that FGF-7, IGF-1, and HGF are paracrine growth factors that promote hair growth (Hervert JM, Rosenquist T, Gotz J, Martin GR (1994). The hair growth cycle regulator FGF-5: targeted and spontaneous mutations demonstrated. Cell, 78: 1017-1025).

[0013] Jiang et al. (1995) found that among the endocrine factors affecting hair growth and shedding, TGF-β1 affects hair growth in the anagen stage, leading to premature entry of normal hair into the catagen stage and ultimately resulting in hair loss, while growth factors such as IGF-1, TGF-β2, EGF, and FGF promote hair growth and prevent hair cell degeneration, thus preventing hair loss (Jiang H, Yamamoto S, Kato R (1995). Induction of anagen in telogen mouse skin by topical application of the potent immunosuppressant FK506. J. Invest. Dermatol, 104: 523-525).

[0014] The research by Na Hyun-wook et al. (2006) showed that hair growth is closely related to blood circulation. Hormones such as insulin and growth factors like IGF-1 and EGF can promote hair growth and affect hair follicle growth. (Na Hyun-wook, Lee Chang-hyeon, Kwon Jin, Lee Moon-won, Jeong Han-sol, Lee Gwang-gyu (2006). Journal of Dong-Eui Physiology and Pathology, 20(2), 428-435).

[0015] Minoxidil, a vasodilator developed in the early 1970s for the treatment of hypertension, is used as a hair growth promoter due to the discovery of its side effect of causing hirsutism. Although the mechanism of hair growth is not yet clear, it has been confirmed that it promotes hair growth by supplying nutrients through vasodilation. However, minoxidil has no effect on the front part of the scalp that has already become bald, and it is effective only when applied to the scalp twice a day continuously for 6-12 months. The side effects of minoxidil include edema, tachycardia, local exfoliation, dermatitis, soreness at the administration site, erythema, skin peeling, itching, dryness, skin exfoliation, contact dermatitis, etc. In addition, it may also induce hirsutism. Minoxidil may also further exacerbate hair loss by affecting the hair cycle, resulting in excessive shedding during the telogen phase, and it will quickly return to the pre-treatment state after stopping the drug.

[0016] The patent documents and reference documents mentioned in this article are incorporated herein by reference and can be independently and clearly identified in this article.

[0017] Prior art documents

[0018] Patent documents

[0019] Korean Patent No. 10-2016002

[0020] Korean Patent No. 10-1659516

[0021] Korean Patent Publication No. 10-2013-0123626 Summary of the invention

[0022] Technical problems to be solved

[0023] The present inventors have been dedicated to developing a safer natural anti - hair loss agent with no side effects. Through research, the present inventors have found that the single compound catechin 7 - O - β - D - apiofuranoside isolated from the supercritical extraction residue of Ulmus plants can inhibit the conversion of testosterone to dihydrotestosterone (DHT) in human hair follicle dermal papilla cells, and can effectively inhibit apoptosis of human hair follicle dermal papilla cells due to oxidative stress. After confirming that the said compound can be used as a therapeutic agent for alopecia, the present invention has been completed.

[0024] An object of the present invention is to provide a composition for improving, preventing or treating hair loss.

[0025] Other objects and technical features of the present invention will be specifically embodied in the following description, claims and drawings.

[0026] Technical solutions for solving the problems

[0027] According to one aspect of the present invention, the present invention provides a composition for preventing, improving or treating alopecia, comprising catechin 7 - O - β - D - apiofuranoside compound as an active ingredient.

[0028] The active ingredient of the composition of the present invention, catechin 7 - O - β - D - apiofuranoside, is a catechin glycoside compound represented by the following Chemical Formula 1.

[0029] Chemical Formula 1

[0030]

[0031] The active ingredient of the composition of the present invention, catechin 7 - O - β - D - apiofuranoside, can be isolated from natural substances or can be chemically synthesized. It is obvious to those skilled in the art that chemical synthesis and natural extraction have the same effect.

[0032] The solvent extract of the supercritical extraction residue of natural Ulmus plants contains a large amount of the catechin 7 - O - β - D - apiofuranoside compound of the present invention, and can be obtained from this extract by conventional single - compound separation methods.

[0033] In the present invention, the supercritical extract residue of Ulmus plants refers to the residue remaining after extracting Ulmus plants by the supercritical extraction method.

[0034] The Ulmus plants used for preparing the supercritical extraction residue of the present invention include plants of the family Ulmaceae in the order Rosales, such as Ulmus davidiana, Ulmus davidiana var. japonica, Ulmus davidiana var. japonica f. suberosa, Ulmus parvifolia, Ulmus pumila, Ulmus laciniata, Ulmus macrocarpa, etc. The most representative ones, Ulmus davidiana and Ulmus davidiana var. japonica, can be used.

[0035] The whole Ulmus plants can be used for the supercritical extraction of the present invention; or a part of the Ulmus plants selected from the group consisting of stems, roots, leaves, flowers, fruits, and seeds can be used. Preferably, the stems, branches, or roots of the Ulmus plants can be used. In the present invention, all or part of the Ulmus plants can be dried and / or ground before supercritical extraction.

[0036] In the present invention, supercritical extraction refers to a method of extracting spices, pigments, oils, or functional substances contained in natural substances without denaturation by using a fluid in a supercritical state.

[0037] A fluid in a supercritical state refers to a fluid that exceeds the established high-temperature and high-pressure limits of the supercritical point and cannot distinguish between the gas and liquid states. The supercritical fluid can be supercritical carbon dioxide, etc. The supercritical carbon dioxide is carbon dioxide in a state exceeding the critical temperature (31°C) and critical pressure (7.5 MPa), and is a fluid with both gas and liquid properties.

[0038] Non-limiting examples of the supercritical extraction method of the present invention include the variable pressure method or the temperature change method. In the variable pressure method, the mixture of the supercritical fluid and the solute is depressurized and expanded at a temperature such as the extraction temperature, so that the dissolution ability of the supercritical fluid is reduced to separate the solute; in the temperature change method, the dissolution ability is reduced by raising the temperature of the supercritical fluid, thereby separating the supercritical fluid and the solute.

[0039] In the present invention, the supercritical fluid can be used together with a co-solvent, and the co-solvent includes polar solvents commonly used in the art. Preferably, a co-solvent selected from the group consisting of alcohols, water, ethylene glycol, polyethylene glycol, propylene carbonate, formic acid, acetic acid, acetonitrile, chlorodifluoromethane, or a mixture thereof is used.

[0040] In the present invention, the container for supercritical extraction is not particularly limited as long as it can control the temperature and pressure and bring the extraction raw material into contact with the supercritical fluid.

[0041] In the present invention, the pressure range for supercritical extraction can be within 100 - 600, the temperature range for supercritical extraction can be 10 - 100 °C, and the flow rates of the supercritical fluid and the cosolvent for supercritical extraction can be 10 - 100 g / min. Those skilled in the art can appropriately adjust and use the pressure, temperature, fluid, and flow rate of the cosolvent for supercritical extraction within the above ranges.

[0042] For the supercritical extraction residue of elm plants obtained by the above supercritical extraction method, an extract can be obtained by solvent extraction. Preferably, the solvent extraction is achieved through a process of bringing the supercritical extraction residue of elm into contact with an extraction solvent. Before solvent extraction, the supercritical extraction residue of elm can be dried and / or ground. In the present invention, the extraction solvent preferably used is an alcohol, and the alcohol can be an anhydrous or hydrated lower alcohol containing 1 - 4 carbon atoms, such as methanol, ethanol, propanol, butanol, n - propanol, isopropanol, and n - butanol.

[0043] In the present invention, a fraction can be obtained by further fractionating the solvent extract of the supercritical extraction residue of elm.

[0044] Similar to extraction, the fractionation is achieved by bringing a fractionation solvent into contact with the extract. The solvents used in the fractionation include (a) water, (b) alcohol, (c) a mixed solvent of the lower alcohol and water, (d) acetone, (e) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3 - butanediol, (i) hexane, and (j) ether. Preferably, the fraction obtained using ethyl acetate can be used.

[0045] Catechin 7 - O - β - D - apiofuranoside can be separated from the fraction using a single - compound separation method well - known in the art such as chromatography. The single - compound separation and purification methods applicable to the present invention include Thin Layer Chromatography (TLC) or High Performance Liquid Chromatography (HPLC), etc.

[0046] In this article, the term "as an active ingredient" means that the content of the catechin 7 - O - β - D - apiofuranoside compound of the present invention is sufficient to achieve the effect of preventing, improving, or treating alopecia.

[0047] As demonstrated by the specific embodiments of the present invention, the active compound catechin 7-O-β-D-apiofuranoside of the present invention can inhibit the conversion of testosterone to dihydrotestosterone (DHT) by 5-α-reductase in human hair follicle dermal papilla cells (HFDPC). Therefore, the compounds of the present invention can play a role in preventing, treating, and improving male pattern hair loss.

[0048] As demonstrated by another embodiment of the present invention, the active compound catechin 7-O-β-D-apiofuranoside of the present invention can inhibit apoptosis of human hair follicle dermal papilla cells (HFDPC) due to oxidative stress. Therefore, the compounds of the present invention can be used to improve, prevent, or treat alopecia, especially alopecia areata.

[0049] In the present invention, the term "hair loss" refers to the situation where there is no hair in the area where normal hair should be due to abnormal hair loss, and the term "alopecia" refers to the state caused by this hair loss.

[0050] In the present invention, "male pattern alopecia" is also known as androgenetic alopecia, which refers to alopecia caused by genetic factors, male hormones, or aging.

[0051] More specifically, in the present invention, it refers to alopecia caused by the conversion of testosterone, a male hormone, to dihydrotestosterone (DHT) by 5α-reductase.

[0052] In the present invention, "alopecia areata" refers to alopecia caused by psychological or physiological stress, including alopecia areata and telogen effluvium areata.

[0053] In the present invention, "alopecia areata" includes not only alopecia areata in humans but also alopecia areata in animals other than humans.

[0054] The composition of the present invention can be in the form of a pharmaceutical composition for preventing or treating alopecia, and the pharmaceutical composition can include a pharmaceutically acceptable carrier.

[0055] In the present invention, the term "prevention" refers to inhibiting the symptoms caused by hair loss or the symptoms caused by hair loss complications, and the term "treatment" refers to alleviating or eliminating the symptoms caused by the already occurring hair loss symptoms or hair loss complications.

[0056] The pharmaceutically acceptable carriers in the compositions of the present invention are carriers commonly used in formulations, and non-limiting examples include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, etc. In addition to the above components, the pharmaceutical compositions of the present invention may also include lubricants, wetting agents, sweeteners, fragrances, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are detailed in Remington's Pharmaceutical Sciences, 19th Edition, 1995.

[0057] The pharmaceutical compositions of the present invention can be administered orally or parenterally. When administered parenterally, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc.

[0058] The appropriate dosage of the pharmaceutical compositions of the present invention varies depending on factors such as the formulation method, administration route, patient's age, weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. Generally, a skilled doctor can easily determine the effective dosage required for treatment and prevention and prescribe it.

[0059] The daily dosage of the pharmaceutical compositions of the present invention is 0.001 - 10,000 mg / kg.

[0060] The pharmaceutical compositions of the present invention are prepared by using methods easily implemented by those skilled in the art and formulated into unit dosage forms or filled into large-capacity containers using pharmaceutically acceptable carriers and / or excipients. In this case, the dosage form can be a solution, suspension, or emulsion in an oil or water medium, or can be an extract, powder, granule, tablet, or capsule, and may also include dispersants or stabilizers.

[0061] According to another aspect of the present invention, there is provided a functional cosmetic composition for preventing or improving alopecia, which contains catechin 7-O-β-D-apiofuranoside as an active ingredient.

[0062] In the functional cosmetic composition of the present invention, alopecia may include male pattern alopecia or alopecia areata. The meanings and contents of the terms "alopecia", "male pattern alopecia", and "alopecia areata" are the same as those of the compositions for improving, preventing, or treating alopecia in other aspects of the present invention.

[0063] The content related to the active compound "catechin 7-O-β-D-apiofuranoside" contained in the functional cosmetic composition of the present invention is the same as that of the composition for improving, preventing or treating alopecia in other aspects of the present invention.

[0064] As used herein, the term "improve" means alleviating or relieving the symptoms caused by hair loss or the symptoms caused by hair loss complications.

[0065] The functional cosmetic composition according to the present invention can be prepared into any conventional dosage forms in the art, and non-limiting examples include solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleaners, oils, powder foundations, emulsion foundations, wax foundations and aerosols. More specifically, it can be prepared into skin softeners, nourishing lotions, nourishing creams, massage creams, serums, eye creams, cleansing creams, cleansing foams, cleansing waters, face masks, sprays or loose powders.

[0066] When the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, astragalus, cellulose derivatives, polyethylene glycols, silicones, bentonites, silica, talc or zinc oxide can be used as carrier components.

[0067] When the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder can be used as carrier components, and particularly when it is a spray, propellants such as chlorofluorocarbons, propane / butane or dimethyl ether can be additionally included.

[0068] When the present invention is a solution or emulsion, solvents, solubilizers or emulsifiers can be used as carrier components, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, glycerol fatty acid esters, polyethylene glycol or sorbitan.

[0069] When the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan esters and polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or astragalus can be used as carrier components.

[0070] When the present invention is a surfactant-containing cleaner, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic acid monoesters, hydroxyethanesulfonates, imidazoline derivatives, methyl taurine, sarcosinates, fatty acid amide ether sulfates, alkylamide betaines, fatty alcohols, fatty acid glycerol esters, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters can be used as carrier components.

[0071] In addition to the active ingredients and carrier ingredients, the functional cosmetic composition of the present invention further includes ingredients commonly used in cosmetic compositions, such as conventional auxiliaries like antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances.

[0072] According to another aspect of the present invention, there is provided a method for treating or improving alopecia, which includes the step of administering a catechin 7-O-β-D-apiofuranoside compound to a subject in need of treating alopecia.

[0073] In the method for treating or improving alopecia provided in one embodiment, the catechin 7-O-β-D-apiofuranoside compound has the activity of inhibiting the conversion of testosterone to dihydrotestosterone (DHT).

[0074] In the method for treating or improving alopecia provided in another embodiment, the alopecia is caused by dihydrotestosterone (DHT).

[0075] In the method for treating or improving alopecia provided in another embodiment, the catechin 7-O-β-D-apiofuranoside compound has the activity of inhibiting apoptosis of dermal papilla cells due to oxidative stress.

[0076] In the method for treating or improving alopecia provided in another embodiment, the alopecia is male pattern alopecia or alopecia areata.

[0077] Advantages of the Invention

[0078] The catechin 7-O-β-D-apiofuranoside compound of the present invention can inhibit the conversion of testosterone to dihydrotestosterone (DHT) in human dermal papilla cells of hair follicles, and inhibit apoptosis of human dermal papilla cells due to oxidative stress. Thus, it can be used as an active substance for treating, preventing, or improving alopecia. Brief Description of the Drawings

[0079] Figure 1a is a process flow chart summarizing the separation process of the compounds of the present invention.

[0080] Figure 1b is the chromatographic result for confirming catechin 7-O-β-D-apiofuranoside in the ethanol extract of the supercritical extraction residue of elm twigs.

[0081] Figure 1c is the chromatographic result for confirming catechin 7-O-β-D-apiofuranoside in the fraction obtained by fractionating the ethanol extract of the supercritical extraction residue of elm twigs with ethyl acetate.

[0082] Figure 1dIt is the result of catechin 7-O-β-D-apiofuranoside obtained by separation using Prep-LC from the fraction obtained by ethyl acetate fractionation of the ethanol extract of elm twig supercritical extraction residue.

[0083] Figure 2a It is the TOF-MS analysis result of the catechin 7-O-β-D-apiofuranoside compound.

[0084] Figure 2b It is the 13C-NMR data of the catechin 7-O-β-D-apiofuranoside compound.

[0085] Figure 2c It is the 1H-NMR data of the catechin 7-O-β-D-apiofuranoside compound.

[0086] Figure 3 It is the measurement result of the effect of the catechin 7-O-β-D-apiofuranoside compound on the production of dihydrotestosterone (DHT) by human hair follicle dermal papilla cells. *P<0.001 VS blank group. #P<0.001 and P<0.05 VS testosterone group.

[0087] Figure 4 It is the experimental result that the catechin 7-O-β-D-apiofuranoside compound inhibits the expression of Bax protein, increases the expression of Bcl-2 protein, and inhibits the expression of PARP-1 protein in dermal papilla cells treated with hydrogen peroxide.

[0088] Figure 5a It is a graph of the effect of catechin 7-O-β-D-apiofuranoside in promoting the expression of IGF, which is helpful for hair growth, in a concentration-dependent manner in dermal papilla cells treated with H2O2. *P<0.001 VS normal group. **P<0.005 VS H2O2 control group.

[0089] Figure 5b It is a graph of the effect of catechin 7-O-β-D-apiofuranoside in inhibiting the expression of TGF-β1 in a concentration-dependent manner in dermal papilla cells treated with H2O2. P<0.001 VS normal group. **P<0.005 VS H2O2 control group. Detailed implementation manners

[0090] The following specific examples in this specification are intended to represent the preferred embodiments or examples of the present invention and do not limit the scope of the present invention. It is obvious to those skilled in the art that the variations and other uses of the present invention do not depart from the scope recorded in the claims.

[0091] Examples

[0092] I. Experimental Materials and Methods

[0093] (1) Isolation of catechin 7-O-β-D-apiofuranoside compound

[0094] This experiment used a supercritical fluid extraction device (SCFE-P400, Ilsin Autoclave, Daejeon, Korea) to perform supercritical extraction on elm (Ulmus) branches. The elm branches were purchased from the Seoul Pharmaceutical Market. After removing impurities and washing, they were air-dried and used as experimental materials. 100 kg of the dried samples were crushed to pass through a 200-mesh crushing screen. The crushed elm branches were placed in the extraction tank and maintained at a temperature of 50 °C. After the temperature stabilized, the elm branch samples were put in. The isobaric CO2 was adjusted and injected into the CO2 using a high-pressure pump pipeline (line) and adjusted using a control valve until the experimental pressure reached 400 bar. After reaching the set pressure, ethanol (alcohol: edible alcohol) was injected into the bottom of the extraction tank at a rate of 0.5 - 0.6 L per minute for a total of 160 minutes, so that the total injection volume reached 60 - 80 L. In order to remove the residual ethanol remaining in the samples, CO2 was introduced using a high-pressure pump at the set pressure and temperature for 60 to 120 minutes to complete the extraction. After performing supercritical extraction on the elm branches as described above, the obtained 100 kg of elm supercritical extraction residue samples were air-dried, extracted once with 60% ethanol at room temperature, and then filtered. The extract was concentrated under reduced pressure and freeze-dried to obtain a final weight of 4.81 kg. 1 kg of the ethanol extract of the elm supercritical extraction residue (“USCFR”) obtained was weighed and fractionated with ethyl acetate solvent using a separating funnel to obtain 185.2 g of ethyl acetate solvent fractionated product (“USCFREA”). A single compound was purified from the obtained ethyl acetate solvent fractionated product (“USCFREA”) using Prep-LC (Waters, USA). When using silica gel resin, the mobile phase solvent was separated using chloroform: methanol: water (70:30:4); when using C18 column resin, the mobile phase condition of MeOH: water (0 - 100%) was used for separation (see Figures 1A to 1D).

[0095] (2) Using TOF-MS to analyze the target compounds in the extract

[0096] The high-resolution time-of-flight mass spectrometer (TOF-MS) uses a high-vacuum and high-sensitivity detector using TMP, and it uses a direct probe to determine the molecular weight and molecular structure of the main component catechin 7-O-β-D-apiofuranoside in the ethanol extract of elm branch supercritical residue.

[0097] Table 1 shows the sample information and analysis conditions.

[0098] Table 1

[0099]

[0100]

[0101] Figure 2A shows the TOF-MS analysis results; Figure 2B shows the 13C-NMR data; Figure 2C shows the 1H-NMR data.

[0102] The structural analysis results of the catechin 7-O-β-D-apiofuranoside compound of the present invention are shown as follows.

[0103] [Chemical formula 1]

[0104]

[0105] High Resolution TOF MS m / z: 422.17511 [M]+; 1H-NMR (400 MHz, DMSO-d6 + D2O): 6.74 (H-2’, 1H, d, J = 2.0 Hz), 6.69 (H-5’, 1H, d, J = 8.4 Hz), 6.59 (H-6’, 1H, dd J = 2.0, 8.4 Hz), 6.09 (H-8, 1H, d, J = 2.4 Hz), 5.90 (H-6, 1H, J = 2.4 Hz), 5.33 (H-1”, 1H, d, J = 4.0 Hz), 4.55 (H-2, 1H, d, J = 7.2 Hz), 4.03 (H-2”, 1H, d, J = 4.0 Hz), 4.00 (H-4a”, 1H, d, J = 9.6 Hz), 3.89 (H-3, 1H, m), 3.68 (H-4b”, 1H, d, J = 9.6 Hz), 3.45 (H-5”, 2H, m), 2.65 (H-4a, 1H, dd, J = 4.8, 16.0 Hz), 2.40 (H-4b, 1H, dd, J = 8.0, 16.0 z); 13C-NMR (100 MHz, DMSO-d6 + D2O): δ 156.7 (C-7), 156.5 (C-5), 155.7 (C-9), 145.2 (C-4’), 145.1 (C-3’), 130.8 (C-1’), 118.8 (C-6’), 115.6 (C-5’), 114.7 (C-2’), 107.3 (C-1”), 102.1 (C-10), 96.0 (C-8), 95.3 (C-6), 81.4 (C-2), 78.9 (C-3”), 76.3 (C-2”), 74.3 (C-4”), 66.3 (C-3), 62.4 (C-5”), 27.8 (C-4).

[0106] (3) Cell culture

[0107] The human hair follicle dermal papilla cells (HFDPC) used in this example were purchased from PromoCell (Heidelberg, Germany) and used as cells to demonstrate hair growth and maintenance characteristics. The HFDPC cells were inoculated in a special medium for dermal papilla cells recommended by the supplier (PromoCell in Heidelberg, Germany or Cell biologics Inc in Illinois, USA) and passaged and cultured at 37 °C and 5% CO2 for 24 - 48 hours for experiments.

[0108] (4) Analyze the activity of inhibiting dihydrotestosterone production

[0109] Through experiments, observe whether the compound catechin 7 - O - β - D - apiofuranoside inhibits the conversion of testosterone to dihydrotestosterone in dermal papilla cells. Immunochemical quantitative analysis using an ELISA kit was used for the quantitative analysis of dihydrotestosterone (DHT). The DHT ELISA kit was purchased from Alpco (New Hampshire, USA). The experiment was carried out using this kit according to the instructions provided by the manufacturer, and the antigen amount (pg / μg) per unit volume (ml) of the cell lysate was converted into a concentration unit to obtain the result.

[0110] (5) Effect of inhibiting apoptosis in dermal papilla cells

[0111] Through experiments, observe whether the compound catechin 7 - O - β - D - apiofuranoside can inhibit apoptosis of dermal papilla cells due to oxidative stress. In this experiment, the inhibitory effect of oxidative stress on dermal papilla cells (HFDPC) induced by hydrogen peroxide (H2O2) was measured by the MTT assay. The dermal papilla cells were inoculated into a 96 - well plate at 5×10 6 cells / mL. After 12 hours, 600 μM of H2O2 and the sample were treated simultaneously for 4 hours. The dermal papilla cells in this state were treated with the MTT stock solution until the final concentration was 0.2 mg / ml. After standing at 37 °C for 4 hours, the purple - stained formazan was dissolved in DMSO, and the absorbance was measured at 570 nm. The cell viability was expressed as a percentage value relative to the control group.

[0112] (6) Western blot analysis

[0113] After treating dermal papilla cells (HFDPC) with H2O2 and the sample, cells were collected at each time point and lysed in cell lysis buffer (RIPA buffer). Only the supernatant was taken after centrifuging the sample (13,000 g, about 10 - 15 minutes). After measuring the protein concentration of the supernatant using a BCA protein assay kit (Bio-Rad, Hercules, CA, USA), it was heated at 95 °C for 10 minutes. The prepared sample was electrophoresed on an SDS-polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane (PVDF, Bio-Rad, Hercules, CA, USA), blocked with 5% BSA, and reacted with the specified specific antibody. Then, after reacting with a secondary antibody conjugated with HRP (Santacruz, Hercules, CA, USA), it was detected using an ECL system (Bio-Rad, Hercules, CA, USA). The primary antibodies for Bax-1, Bcl-2, PARP-1, and β-actin were from Santacruz antibodies (Hercules, CA, USA).

[0114] (7) ELISA analysis

[0115] ELISA kits were purchased to perform immunochemical quantitative analysis related to hair growth promotion and hair loss inhibition. The TGF-β and IGF-1 kits were purchased from R&D systems (Hercules, CA, USA). Each kit was used for the experiment according to the supplier's manual, and the results were obtained by converting the antigen amount (pg or μg) per unit volume (ml) of the cell lysate into a concentration unit.

[0116] (8) Statistical analysis

[0117] All experiments were repeated three times, and the mean and standard deviation were obtained. In addition, using SPSS (Statistical Package for Social Science, ver. 18, IBM, Chicago, IL, USA), at the P < 0.05 level, a student's t-test was used to verify the significance of the mean values between the experimental group and the control group.

[0118] II. Experimental results

[0119] (1) Measurement results of the activity of catechin 7-O-β-D-apiofuranoside in inhibiting dihydrotestosterone (DHT)

[0120] After human dermal papilla cells (HFDPCs) were treated with 20 nM testosterone, the production of dihydrotestosterone (DHT) increased significantly statistically. Next, to confirm the ability of apigenin 7-O-β-D-apiofuranoside to inhibit DHT production, minoxidil, a well-known drug for preventing male pattern hair loss, was used as a positive control in a comparative experiment. In the positive control group treated with minoxidil (10 μM), it was confirmed that the production of DHT was significantly inhibited. When treated with apigenin 7-O-β-D-apiofuranoside (125 μg / ml, 150 μg / ml, 1100 μg / ml), although not as strong as the effect of minoxidil, it also showed a significant DHT inhibitory effect in a concentration-dependent manner statistically. Even compared with the negative control group, the production of DHT was significantly inhibited statistically ( Figure 3 ).

[0121] (2) Inhibitory effect on Bax protein expression

[0122] To induce apoptotic cytotoxicity in human dermal papilla cells (HFDPCs), hydrogen peroxide (H2O2) treatment was performed, and it was found that hydrogen peroxide (H2O2) showed 50% apoptosis at a concentration of 600 μM, so this concentration was used in the experiment. Bcl-2-associated X protein (Bax) is a representative apoptosis-inducing protein in apoptotic cytotoxicity. After treatment with H2O2, the expression of Bax molecules increased significantly. As a result of pretreatment with apigenin 7-O-β-D-apiofuranoside compounds at different concentrations, the expression of Bax decreased significantly in a concentration-dependent manner ( Figure 4 ). These results indicate that apigenin 7-O-β-D-apiofuranoside effectively inhibits apoptosis of human dermal papilla cells due to stress.

[0123] (3) Effect of increasing Bcl-2 protein expression

[0124] When inducing apoptotic cytotoxicity in human dermal papilla cells (HFDPCs), the method used was the same as that for evaluating the Bax expression inhibitory ability. Human dermal papilla cells were treated with 600 μM hydrogen peroxide (H2O2), and the induced apoptosis was evaluated. Bcl-2 is a representative protein molecule involved in inhibiting apoptosis. It was observed that when HFDPCs were treated with H2O2, the expression of Bcl-2 molecules decreased significantly. As a result of treatment with apigenin 7-O-β-D-apiofuranoside compounds at different concentrations, in the presence of H2O2, Bcl-2 expression increased significantly in a concentration-dependent manner ( Figure 4) These results indicate that catechin 7-O-β-D-apiofuranoside effectively inhibits stress-induced apoptosis of dermal papilla cells.

[0125] (4) Effect of inhibiting PARP-1 protein expression

[0126] PARP-1 uses intracellular NAD as a substrate to induce poly-ADP ribosylation of target proteins in the nucleus, thereby activating the signal transduction involved in downstream apoptosis. Thus, the activation of PARP-1 becomes an early indicator of apoptosis, especially as a representative marker related to apoptosis. Treatment with 600 μM hydrogen peroxide (H2O2) significantly increased the intracellular PARP-1 expression in HFDPC. These results demonstrate that apoptosis is occurring in HFDPC. On the other hand, the results of treatment with different concentrations of catechin 7-O-β-D-apiofuranoside compounds showed that in the presence of H2O2, the compound significantly inhibited the expression level of PARP-1 protein in a concentration-dependent manner ( Figure 4 ) These results indicate that the catechin 7-O-β-D-apiofuranoside compound effectively inhibits stress-induced apoptosis of dermal papilla cells.

[0127] (5) Effect of promoting IGF-1 expression and inhibiting TGF-β1 expression

[0128] In this experiment, it was confirmed that catechin 7-O-β-D-apiofuranoside compounds promoted the expression of IGF-1, which is beneficial for hair growth (Figure 5A), and inhibited TGF-β1, which hinders hair growth and accelerates hair loss (Figure 5B). The experimental results of (2) to (4) above indicate that catechin 7-O-β-D-apiofuranoside effectively inhibits stress-induced apoptosis of dermal papilla cells and is effective against alopecia areata.

[0129] The specific parts of the present invention have been described in detail above. Those of ordinary skill in the art should understand that these specific descriptions are only preferred embodiments, and the protection scope of the present invention is not limited thereto. The substantial scope of the present invention should be defined by the appended claims and their equivalents.

Claims

1. Use of the catechin 7-O-β-D-apiofuranoside compound in the preparation of a product for preventing or treating alopecia, wherein the catechin 7-O-β-D-apiofuranoside compound has the activity of inhibiting the conversion of testosterone into dihydrotestosterone.

2. The use according to claim 1, characterized in that the alopecia is caused by dihydrotestosterone.

3. The use according to claim 1, characterized in that the alopecia is male pattern alopecia or alopecia areata.

4. The use according to claim 1, characterized in that the product is a pharmaceutical composition.

5. Use of the catechin 7-O-β-D-apiofuranoside compound in the preparation of a functional cosmetic composition for preventing or improving alopecia, wherein the catechin 7-O-β-D-apiofuranoside compound has the activity of inhibiting the conversion of testosterone into dihydrotestosterone.

6. The use according to claim 5, characterized in that the alopecia is caused by dihydrotestosterone.

7. The use according to claim 5, characterized in that the alopecia is male pattern alopecia or alopecia areata.

Images