Composition for preventing hair loss and promoting hair growth comprising connarus conchocarpus extract, and methods of use and preparation thereof

Abstract

This invention relates to a composition for promoting hair growth containing extracts of *Achyranthes bidentata* as an active ingredient, and to a composition for preventing hair loss and promoting hair growth, which promotes hair growth and contains ingredients that inhibit male pattern baldness, inhibit alopecia areata, and induce new hair growth or hair regrowth. Using the extracts of *Achyranthes bidentata* of this invention can effectively prevent or treat hair loss and promote hair regrowth.

Description

Technical Field

[0001] This invention relates to a composition for promoting hair growth containing extract of *Cephalotaxus fortunei* as an active ingredient, and to a composition for preventing hair loss and promoting hair growth, which promotes hair growth and contains ingredients that inhibit male pattern baldness, inhibit alopecia areata, and induce new hair growth or hair growth.

[0002] This patent application claims priority to Korean Patent Application No. 10-2024-0092487, filed with the Korean Intellectual Property Office on July 12, 2024, the disclosure of which is incorporated herein by reference.

[0003] This patent application claims priority to Korean Patent Application No. 10-2025-0083786, filed with the Korean Intellectual Property Office on June 24, 2025, the disclosure of which is incorporated herein by reference. Prior Technology

[0004] In modern society, hair loss is a common cosmetic and psychological problem for both men and women, and the demand for its prevention and treatment is increasing. Hair loss is caused by a variety of factors, including genetic factors, hormonal imbalances, stress, medication, aging, and environmental pollution, either individually or in combination, and its pathophysiological mechanisms are quite complex.

[0005] The hair growth cycle repeatedly goes through the anagen (growth) phase, catagen (transitional) phase, and telogen (resting) phase. However, if this cycle is imbalanced or shortened, it can lead to miniaturization of hair follicles and reduction of hair, resulting in alopecia. Hair loss can be classified into congenital alopecia, acquired alopecia, and pathological alopecia based on its etiology. Among acquired alopecia, male pattern baldness and alopecia areata are known to have higher incidence rates and relatively clear pathogenesis.

[0006] Male pattern baldness occurs because dihydrotestosterone (DHT) binds to the androgen receptor (AR) in dermal papilla cells, inducing the expression of hair growth inhibitory factors such as DKK1, TGF-β, and IL-6. This inhibits the Wnt / β-catenin signaling pathway, leading to hair follicle cell apoptosis and a shortened anagen phase. Furthermore, the conversion of testosterone to DHT by 5α-reductase further promotes this mechanism.

[0007] Alopecia areata is an autoimmune disease caused by the collapse of immune privilege in hair follicles and overactivation of the JAK-STAT signaling pathway. Increased secretion of cytokines such as IFN-γ and IL-15 promotes the expression of inflammatory genes through phosphorylation of JAK1 / 2 / 3 and STAT1 / 3 / 5, leading to CD8+ T cell invasion of hair follicles and excessive secretion of granzyme B and perforin, ultimately destroying hair follicle cells. Furthermore, activation of the JAK-STAT signaling pathway inhibits the transition of hair follicle stem cells from the resting phase to the anagen phase, resulting in persistent hair loss.

[0008] To date, minoxidil, finasteride, steroids, immunosuppressants, and phototherapy have all been considered as treatments for hair loss, but their effectiveness is limited due to various reported side effects, such as increased body hair, skin irritation, decreased sexual function, depression, and Cushing's syndrome with long-term use.

[0009] Therefore, there is an urgent need to develop a safe and effective natural hair loss treatment material with few side effects that can comprehensively regulate the function of hair papilla cells and hair follicles.

[0010] F. Muell., a member of the family F. Muellaceae, is an evergreen shrub native to tropical regions such as New Guinea and Queensland. Its main components are flavonoids and phenolic compounds, known to possess antioxidant, anti-inflammatory, and antibacterial activities. Traditionally, it has been used to treat digestive and respiratory disorders, but scientific research on its efficacy in preventing hair loss and promoting hair regrowth is lacking. Summary of the Invention

[0011] The problem that the invention aims to solve In order to develop an effective treatment for hair loss, the inventors conducted in-depth research and found that the use of extracts from *Smilax china* can achieve excellent effects in preventing and treating hair loss and promoting hair regrowth by promoting the proliferation of dermal papilla cells, activating the Wnt / β-catenin signaling pathway, inhibiting the expression of JAK and AR, and inhibiting the activity of 5α-reductase. This led to the completion of this invention.

[0012] Therefore, the object of the present invention is to provide a pharmaceutical composition or agent comprising extract of *Cynanchum paniculatum* for the prevention or treatment of hair loss; or the use of preparing said composition or agent.

[0013] Another object of the present invention is to provide a method for preparing an extract of *Gnaphalium affine*.

[0014] Another object of the present invention is to provide a cosmetic composition for preventing hair loss or promoting hair growth, comprising extract of *Cynanchum paniculatum*.

[0015] Another object of the present invention is to provide a food composition for preventing hair loss or promoting hair growth, comprising extract of *Cynanchum paniculatum*.

[0016] Another object of the present invention is to provide a feed composition for preventing hair loss or promoting hair growth, comprising extract of *Cynanchum paniculatum*.

[0017] Other objects and advantages of the present invention will become more apparent from the following description of the invention, the scope of protection claimed, and the accompanying drawings.

[0018] Technical means to solve the problem In one aspect of the invention, the invention provides a pharmaceutical composition or agent comprising an extract of *Cynanchum paniculatum* for the prevention or treatment of hair loss; or the use of preparing said composition or agent.

[0019] In order to develop an effective treatment for hair loss, the inventors conducted in-depth research and found that the use of extracts from *Cephalotaxus fortunei* can achieve excellent effects in preventing and treating hair loss and promoting hair regrowth by promoting the proliferation of dermal papilla cells, activating the Wnt / β-catenin signaling pathway, inhibiting the expression of JAK and AR, and inhibiting the activity of 5α-reductase.

[0020] The term "Connaraceae" in this manual refers to an evergreen shrub or vine belonging to the Connaraceae family, native to tropical regions such as New Guinea and northeastern Australia (Queensland). Traditionally used in folk medicine to treat digestive and respiratory ailments, this plant has recently gained attention as a medicinal plant containing various physiologically active substances, including flavonoids and phenolic compounds.

[0021] The *Gnaphalium affine* can be the whole plant, its above-ground parts (leaves, stems, etc.), and its underground parts (roots, rhizomes, tubers, etc.). Furthermore, it can be processed into powder through processes such as washing, drying, and pulverizing, but is not limited to these methods. This powder can be used as a starting material for the solvent extraction process described below.

[0022] As used in this specification, the term "prevention" refers to the prevention or protective treatment of a disease or disease state. The term "treatment" as used in this specification refers to the reduction, suppression, alleviation, or eradication of a disease state.

[0023] The pharmaceutical compositions of the present invention can be formulated into unit dose forms by means of methods readily practiced by those skilled in the art, using pharmaceutically acceptable carriers and / or excipients, or by placing them in multi-dose containers. In this case, the dosage form can be a solution, suspension, or emulsion in an oily or aqueous medium, or it can be an extract, powder, suppository, granule, tablet, or capsule, and may also contain dispersants or stabilizers.

[0024] The pharmaceutical compositions or formulations of the present invention may contain pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are those commonly used in formulations, including, but not limited to, 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, and mineral oil. In addition to the above-mentioned components, the pharmaceutical compositions of the present invention may also contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0025] The pharmaceutical compositions or agents of the present invention can be administered orally or parenterally, and can be administered by, for example, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intrasternal injection, intratumoral injection, local administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0026] The appropriate dosage of the pharmaceutical composition or agent of the present invention depends on factors such as the preparation method, route of administration, patient's age, weight, sex, pathological condition, diet, administration time, route of administration, excretion rate, and reaction sensitivity. Generally, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dose of the anticancer composition is 0.0001-100 mg / kg.

[0027] In one embodiment of the present invention, the composition or agent is administered in combination with a drug for the prevention or treatment of hair loss.

[0028] In one specific embodiment of the present invention, the drug for preventing or treating hair loss may be, for example, minoxidil, finasteride, dutasteride, spironolactone, ketoconazole or a combination thereof, but is not limited thereto.

[0029] The pharmaceutical compositions or agents of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with existing therapeutic agents, or can be administered once or multiple times. Considering all the above factors, it is important to achieve the maximum effect with the minimum dosage and without side effects, which can be easily determined by those skilled in the art to which this invention pertains.

[0030] The extract of *Hemiberlesia lingua* used in this invention is commercially available or can be obtained through direct extraction. When the extract of *Hemiberlesia lingua* used in the compositions of this invention is obtained by treatment with an extraction solvent, various extraction solvents can be used, such as polar solvents or non-polar solvents.

[0031] Suitable polar solvents may include, but are not limited to: (i) water; (ii) low molecular weight alcohols and polyols (e.g., methanol, ethanol, n-propanol, isopropanol, n-butanol, 1-pentanol, 2-butoxyethanol, ethylene glycol, butylene glycol, glycerol, etc.); (iii) acetic acid; (iv) dimethylformamide (DMF); and (v) dimethyl sulfoxide (DMSO).

[0032] Suitable nonpolar solvents may include, but are not limited to: acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkanes, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, 1,2-dichloroethane, aniline, diethylamine, diethyl ether, carbon tetrachloride, and tetrahydrofuran (THF).

[0033] In this invention, the term "extract" has the meaning described above, referring to crude extract as commonly understood in the art, but broadly also including fractions obtained through further fractionation of the extract. That is, the *Achyranthes bidentata* extract includes not only extracts obtained using the aforementioned extraction solvents, but also extracts obtained through additional purification processes. For example, fractions obtained by passing the extract through an ultrafiltration membrane with a specific molecular weight cutoff, as well as fractions obtained through various additional purification methods such as chromatographic separation (separation based on size, charge, hydrophobicity, or affinity), can all be included in the *Achyranthes bidentata* extract of this invention.

[0034] In this invention, when extracting the extract of *Cephalotaxus fortunei*, hot water extraction, pressurized hot water extraction, cold soaking extraction, reflux extraction, room temperature extraction, or ultrasonic extraction can be used, but it is not limited to these methods.

[0035] To prepare the extract of an example of the present invention, the amount of solvent used for extraction can be appropriately selected according to the amount of *Achyranthes bidentata* used. Specifically, the weight or volume of the solvent can be 1 to 20 times, 1 to 18 times, 1 to 16 times, 1 to 14 times, 1 to 12 times, 1 to 10 times, 1 to 8 times, 1 to 6 times, 1 to 4 times, 1 to 2 times, 2 to 20 times, 4 to 20 times, 6 to 20 times, 8 to 20 times, 10 to 20 times, 12 to 20 times, 14 to 20 times, 16 to 20 times, 18 to 20 times, 5 to 15 times, or 10 to 15 times the weight or volume of *Achyranthes bidentata* used to prepare the extract, but is not limited thereto.

[0036] The extraction temperature of the extract in one embodiment of the present invention can be, for example, 10°C to 110°C, 20°C to 110°C, 30°C to 110°C, 40°C to 110°C, 50°C to 110°C, 60°C to 110°C, 70°C to 110°C, 80°C to 110°C, 90°C to 110°C, 100°C to 110°C, 10°C to 100°C, 10°C to 90°C, 10°C to 80°C, 10°C to 70°C, 10°C to 60°C, 10°C to 50°C, 10°C to 40°C, 10°C to 30°C, 10°C to 20°C, 20°C to 30°C, 60°C to 90°C, 70°C to 90°C, 75°C to 85°C, 90°C to 100°C, or 90°C to 105°C, but is not limited thereto.

[0037] The extraction time of the extract in one embodiment of the present invention can be, for example, 1 hour to 100 hours, 1 hour to 90 hours, 1 hour to 80 hours, 1 hour to 72 hours, 1 hour to 70 hours, 1 hour to 60 hours, 1 hour to 50 hours, 1 hour to 40 hours, 1 hour to 30 hours, 1 hour to 20 hours, 1 hour to 10 hours, 10 hours to 100 hours, 20 hours to 100 hours, 30 hours to 100 hours, 40 hours to 100 hours, 50 hours to 100 hours, 60 hours to 100 hours, 70 hours to 100 hours, 80 hours to 100 hours, 90 hours to 100 hours, 10 hours to 80 hours, 20 hours to 60 hours, 40 hours to 60 hours, 2 hours to 10 hours, 3 hours to 10 hours, 3 hours to 5 hours, 60 hours to 80 hours, or 65 hours to 75 hours, but is not limited thereto.

[0038] The extract of *Gnaphalium affine* used in this invention can be prepared into powder form through additional processes such as vacuum distillation, freeze drying, or spray drying.

[0039] In one embodiment of the present invention, the extract of *Hemiberlesia lataniae* is a solvent extract obtained by extraction with a solvent selected from the group consisting of diol solvents, water, alcohols, or combinations thereof.

[0040] The solvent is a means of effectively extracting active ingredients from the plant *Gnaphalium affine*. The types and physiological activities of the extracted ingredients can vary depending on the characteristics of each solvent.

[0041] For example, diol solvents can be butylene glycol, propylene glycol, ethylene glycol, dipropylene glycol, but are not limited to these. These solvents have excellent water solubility and skin penetration, and can provide extracts suitable for cosmetic and quasi-drug compositions.

[0042] Using water or purified water as a solvent, water-soluble polysaccharides, flavonoids, and phenolic compounds are mainly extracted through high-temperature hot water extraction or leaching.

[0043] For example, alcohol solvents can be low molecular weight alcohols, such as methanol, ethanol, n-propanol, isopropanol, n-butanol, n-propanol, n-butanol, 1-pentanol, 2-butoxyethanol, etc., but are not limited to these. They can effectively extract not only polar compounds, but also some non-polar physiologically active substances. These solvents can be used alone, in the form of aqueous solutions with a concentration of 20-80% (v / v), or in the form of complex solvent components.

[0044] In one embodiment of the present invention, the extract is as follows:

[0045] (a) The diol solvent is a diol solvent with a concentration of 20% (v / v) to 80% (v / v); or

[0046] (b) The alcohols are alcohols with a concentration of 20% (v / v) to 80% (v / v).

[0047] In one embodiment of the present invention, the extract of *Achyranthes bidentata* is a hot water extract of *Achyranthes bidentata* residue extracted with a diol solvent at a concentration of 20% (v / v) to 80% (v / v).

[0048] In one specific embodiment of the present invention, the preparation methods of *Achyranthes bidentata* extract vary depending on the type and concentration of the solvent used. In particular, the concentration of the extraction solvent affects the dissolution efficiency, stability, and physiological activity of the active ingredients; therefore, setting it within a suitable concentration range is crucial.

[0049] Specifically, the diol solvent extract is obtained by using diol solvents such as butanediol and propylene glycol diluted in purified water to a concentration of 20% (v / v) to 80% (v / v) as the extraction solvent, and then immersing or stirring the *Cephalotaxus fortunei* plant body in the solution for extraction.

[0050] Furthermore, the preparation method of the water extract involves a first extraction with a diol solvent (20-80% v / v), followed by a second extraction with hot water (purified water at approximately 100°C) on the remaining solid residue. This hot water extraction method can additionally obtain water-soluble components (e.g., polysaccharides, water-soluble phenols, flavonoids, etc.) that were not dissolved in the first solvent extraction, which is beneficial for developing compositions with complex physiological activities.

[0051] On the other hand, alcohol extracts are obtained by diluting low molecular weight alcohols such as methanol, ethanol, propanol, and butanol in purified water to a concentration of 20% (v / v) to 80% (v / v), and then soaking or stirring the *Cynanchum paniculatum* in this solution for extraction. This type of extract can extract not only polar components but also some semi-polar components. Ethanol is a preferred solvent due to its high safety profile, especially suitable for food and pharmaceutical applications.

[0052] Thus, the extract of *Achyranthes bidentata* of the present invention can be prepared by stepwise extraction using single or combined solvents such as diol solvents, water, and alcohols. By setting the concentration of the extraction solvent in the range of 20% (v / v) to 80% (v / v), the effective components suitable for the purpose can be stably ensured.

[0053] The concentration of the diol solvent or alcohol may be 20% (v / v) to 80% (v / v), 20% (v / v) to 75% (v / v), 20% (v / v) to 70% (v / v), 20% (v / v) to 60% (v / v), 20% (v / v) to 55% (v / v), 20% (v / v) to 50% (v / v), 20% (v / v) to 50% (v / v), 20% (v / v) to 50% (v / v). ) to 45%(v / v), 20%(v / v) to 40%(v / v), 20%(v / v) to 35%(v / v), 20%(v / v) to 30%(v / v), 2 0%(v / v) to 25%(v / v), 25%(v / v) to 80%(v / v), 30%(v / v) to 80%(v / v), 35%(v / v) to 80%( v / v), 40%(v / v) to 80%(v / v), 45%(v / v) to 80%(v / v), 50%(v / v) to 80%(v / v), 55%(v / v ) to 80%(v / v), 60%(v / v) to 80%(v / v), 65%(v / v) to 80%(v / v), 70%(v / v) to 80%(v / v), 7 5% (v / v) to 80% (v / v), 30% (v / v) to 70% (v / v), 40% (v / v) to 60% (v / v), 25% (v / v) to 35% (v / v), 40% (v / v) to 60% (v / v), 45% (v / v) to 55% (v / v), or 65% (v / v) to 75% (v / v), but not limited thereto.

[0054] In one embodiment of the present invention, the extract has the properties of inhibiting the expression of 5α-reductase, inhibiting the expression of androgen receptor, inhibiting the activity of Janus kinase (JAK), activating the Wnt / β-catenin signaling pathway, or a combination thereof.

[0055] The extract of *Achyranthes bidentata* from this invention exerts its preventive or therapeutic effects on hair loss by acting on various molecular biological mechanisms associated with hair loss, such as inhibiting 5α-reductase, inhibiting androgen receptors, inhibiting the JAK signaling pathway, and activating the Wnt / β-catenin signaling pathway. Each of these mechanisms is a scientifically established target in the pathophysiology of hair loss, and various literature and clinical studies have demonstrated that their individual or combined effects can improve hair loss.

[0056] First, 5α-reductase is an enzyme that converts testosterone, a male hormone, into dihydrotestosterone (DHT), a more potent androgen. DHT binds to androgen receptors in dermal papilla cells, inducing the expression of hair loss-related genes, leading to follicle atrophy and a shortened growth phase. Therefore, finasteride and dutasteride, as 5α-reductase inhibitors, have been approved by the FDA for use as treatments for male pattern baldness, and long-term use has been reported to increase hair quantity and improve hair thickness. Inhibiting this enzyme, such as the extract of this invention, can scientifically induce improvements in hair loss.

[0057] Furthermore, the androgen receptor (AR) is a major target protein that binds to DHT and induces the transcription of hair loss genes; its elevated expression is known to accelerate the progression of male pattern baldness. Some studies have reported that AR inhibition affects the restoration of Wnt / β-catenin signaling and the activation of hair follicle stem cells, while RNA-based candidates for indirect AR regulation are under active preclinical investigation. Therefore, compositions that inhibit AR expression, such as the extract of this invention, can effectively alleviate the progression of hair loss.

[0058] The Janus kinase (JAK) signaling pathway is a key pathway in the pathogenesis of autoimmune alopecia, especially alopecia areata. Inflammatory cytokines IFN-γ and IL-15 induce phosphorylation of JAK1 / 2 / 3 and STAT1 / 3 / 5, leading to CD8+ T cell invasion and destruction of hair follicle cells. Therefore, drugs such as tofacitinib and ruxolitinib, as JAK inhibitors, have shown clinical efficacy in promoting hair regeneration in patients with alopecia areata, while baricitinib, another JAK inhibitor, was approved by the FDA in 2022 for use as a treatment for alopecia areata. Therefore, the extract of this invention, which inhibits JAK activity, can be effectively applied to inflammatory alopecia such as alopecia areata.

[0059] Finally, the Wnt / β-catenin signaling pathway is essential for inducing hair follicle stem cell survival and entry into the anagen phase. Inhibiting this pathway inhibits hair regeneration and induces telogen effluvium. Studies have shown that mouse models lacking the β-catenin gene induce hair loss, while inducing Wnt3a or β-catenin expression promotes hair follicle regeneration and hair growth. In fact, minoxidil has been reported to exert its hair growth effect by indirectly activating the Wnt / β-catenin signaling pathway. The extract of this invention can induce hair follicle cell survival and restore the growth cycle by exhibiting activity that increases the expression of Wnt3a and β-catenin.

[0060] In this way, the extract of *Gnaphalium affine* of the present invention can contribute to the treatment of hair loss through individual mechanisms, and when multiple mechanisms work in combination, it can block the root cause of hair loss or promote hair growth in multiple ways, thereby providing a superior therapeutic effect. This scientific and clinical background indicates that the composition of the present invention is a technology sufficient to effectively prevent or treat hair loss.

[0061] In one embodiment of the present invention, the hair loss is selected from the group consisting of male pattern baldness, female pattern baldness, inflammatory hair loss, alopecia areata, telogen effluvium, traumatic hair loss, trichotillomaniatic hair loss, pressure-induced hair loss, anagen effluvium, pityriasis alopecia, syphilitic hair loss, seborrheic alopecia, symptomatic hair loss, non-scarring hair loss, scarring hair loss, congenital hair loss, and combinations thereof.

[0062] In particular, the extract of *Gnaphalium affine* from this invention can effectively inhibit the progression of hormone-induced alopecia such as androgenetic alopecia by suppressing the expression of 5α-reductase and androgen receptors, and also shows significant effects on autoimmune alopecia such as alopecia areata by inhibiting the JAK pathway. Furthermore, activation of the Wnt / β-catenin signaling pathway can help restore telogen effluvium, seborrheic alopecia, or inflammatory alopecia by inducing hair follicle cell regeneration and growth phase.

[0063] Therefore, the extracts of the present invention can be widely used for hair loss caused by various etiologies and can serve as a natural source of therapeutic material that exhibits excellent efficacy, especially for hair loss types involving hormone, autoimmune and inflammatory mechanisms.

[0064] In one aspect of the invention, a method for preparing an extract of *Gnaphalium affine* is provided, comprising the step of extracting the extract by adding a solvent selected from the group consisting of diols, water, alcohols, or combinations thereof to *Gnaphalium affine*.

[0065] The preparation methods of the extract of *Achyranthes bidentata* of the present invention have commonalities in obtaining the above-mentioned extract of *Achyranthes bidentata*. Therefore, they have commonalities in terms of the pretreatment or state of *Achyranthes bidentata*, the type, amount or concentration of solvent, extraction time or temperature and other extraction conditions, extraction methods, etc. In order to avoid excessive repetition in the specification, the description of the common parts is omitted.

[0066] In one embodiment of the present invention, the concentration of the diol solvent is 20% (v / v) to 80% (v / v); or the concentration of the alcohol is 20% (v / v) to 80% (v / v).

[0067] In one embodiment of the present invention, the preparation method includes the following steps: after extraction with a diol solvent at a concentration of 20% (v / v) to 80% (v / v), the remaining residue is subjected to hot water extraction.

[0068] In one aspect of the invention, *Achyranthes bidentata* extract can be prepared via a two-step continuous extraction process. In particular, further hot water extraction of the plant residue remaining after the first extraction step using a diol solvent ensures a more comprehensive preservation of the active ingredients contained in *Achyranthes bidentata*.

[0069] More specifically, a 20% (v / v) to 80% (v / v) diol solvent (e.g., butanediol, propylene glycol, etc.) is first diluted in purified water, and the dried powder of *Cynanchum paniculatum* is then impregnated or stirred for extraction. Extraction can be performed at a temperature of 25°C to 60°C for 10 to 100 hours, followed by filtration to separate the diol solvent extract.

[0070] The solid residue remaining after filtration may contain water-soluble bioactive substances (e.g., polysaccharides, phenols, water-soluble flavonoids, etc.) that were not recovered during the first extraction. Therefore, a complex extract containing a wider variety of components can be obtained by performing a second hot water extraction on this residue.

[0071] Add 10-20 times the weight of purified water to the above residue, and extract by heating and stirring or soaking at about 100°C for 2-5 hours or longer, thus performing a second extraction. Afterwards, obtain the final hot water extract through filtration and vacuum concentration. This hot water extract can be combined with the diol solvent extract from the first extraction, fractionated separately, or used as a complement in a composition.

[0072] As described above, compared with single solvent extraction, the extraction method of the present invention has the advantages of obtaining polar and water-soluble active ingredients in the plant body of *Achyranthes bidentata* in a more quantitative and effective manner. Furthermore, since the range of physiologically active ingredients is expanded through two-step extraction, the effect of the final composition in inhibiting hair loss and promoting hair growth can also be improved.

[0073] In one embodiment of the present invention, the preparation method further includes a vacuum concentration step performed after extraction.

[0074] In the preparation method described above, by adding a vacuum concentration step after the extraction process, the solvent can be removed and the concentration of the active ingredient can be increased. Vacuum concentration is typically carried out using a rotary evaporator or a vacuum concentrator, evaporating the solvent at low pressure and a temperature of 40°C to 60°C, thereby concentrating the extract. The advantage of this process is that it maximizes concentration efficiency while minimizing the decomposition of heat-sensitive active ingredients, ultimately yielding a high-concentration extract suitable for preparing the composition.

[0075] In one aspect of the invention, a cosmetic composition for preventing hair loss or promoting hair growth is provided, comprising an extract of *Cynanchum paniculatum*.

[0076] The cosmetic compositions of the present invention for preventing hair loss or promoting hair growth can be formulated into any dosage form commonly used in the art, such as solutions, suspensions, emulsions, pastes, gels, creams, lotions, loose powders, soaps, facial cleansers containing surfactants, makeup remover oils, pressed powders, liquid foundations, foundation sticks, and sprays, but are not limited thereto. More specifically, they can be formulated into toners, nourishing lotions, lotions, nourishing creams, massage creams, serums, eye creams, makeup remover balms, facial cleansers, makeup remover waters, sheet masks, sprays, or loose powders.

[0077] In addition to the active ingredient, the cosmetic composition of the present invention may also contain a cosmetically acceptable carrier. The term "cosmetically acceptable carrier" refers to an additive known to be included in a cosmetic formulation that does not significantly reduce the main efficacy of the active ingredient or cause side effects to the human body, and can improve the application of the active ingredient to the skin, enhance user convenience and preference.

[0078] The carrier content relative to the total weight of the composition of the present invention can be from about 1% by weight to about 99.99% by weight, preferably from about 50% by weight to about 99% by weight of the total weight of the composition. However, the carrier content can be appropriately adjusted according to the dosage form of the cosmetic, the specific application site, and the preferred application amount, but there are no particular limitations.

[0079] When the dosage form of the present invention is a paste, cream, emulsion or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicon dioxide, talc or zinc oxide can be used as carrier components.

[0080] When the dosage form of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder can be used as carrier components. In particular, in the case of a spray, it may also contain propellants such as chlorofluorocarbons, propane / butane or dimethyl ether.

[0081] When the dosage form of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, including water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitol.

[0082] When the dosage form of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester and polyoxyethylene dehydrated sorbitan ester, and carrier components such as microcrystalline cellulose, aluminum hydroxide, bentonite, agar or tragacanth gum can be used.

[0083] When the dosage form of the present invention is a facial cleanser containing surfactants, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinate monoesters, hydroxyethyl sulfonates, imidazoline derivatives, methyl taurate, sarcosinates, fatty acid amide ether sulfates, alkyl amide betaine, fatty alcohols, fatty acid glycerides, fatty acid diethanolamide, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters can be used as carrier components.

[0084] In addition to the active ingredient and the carrier ingredient, the cosmetic composition of the present invention also includes ingredients commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances.

[0085] In one embodiment of the present invention, the dosage form of the composition is hair conditioner, hair cream, hair lotion, shampoo, hair conditioner, hair spray, hairspray, hair gel, hair oil, hair powder, hair gel, hair mask, hair paste, eyebrow growth agent, eyelash growth agent, eyelash nutrient agent, or ointment.

[0086] The cosmetic composition of the present invention is intended for administration to individuals who require prevention or improvement of hair loss. Such individuals may include all animals, including humans, that have experienced or may experience the aforementioned hair loss. These animals may include, but are not limited to: non-human primates, such as chimpanzees, other apes, or monkeys; livestock, such as cattle, horses, sheep, goats, and pigs; domesticated animals, such as rabbits, dogs, or cats; laboratory animals, such as rodents, such as rats, mice, or guinea pigs; and non-mammals, such as birds or fish.

[0087] In one aspect of the invention, a food composition for preventing hair loss or promoting hair growth is provided, comprising an extract of *Syngonium arvense*.

[0088] The food may be a functional food, a health food, or a food additive, and in this case, there are no special restrictions on the type of food. Examples of the food include: dairy products including functional beverages, meat, sausages, bread, biscuits, rice cakes, chocolate, candy, snacks, pastries, pizza, ramen, other noodles, chewing gum, ice cream, various soups, beverages, alcoholic beverages, vitamin complexes, dairy products, and all conventionally defined functional foods and health food compositions, such as tablets, capsules, pills, or liquids.

[0089] The health food, health food and food additive composition containing the extract of the fruit of the present invention can be directly added to food, or used in combination with other food or food ingredients, and can be used appropriately according to conventional methods.

[0090] In this invention, the amount of the composition added to the health functional food, health food, or food additive can be from 0.1 parts by weight to 90 parts by weight of the total weight of the food. However, in cases of long-term intake for maintaining or regulating health, the above-mentioned amount can be below the above range.

[0091] In this invention, besides containing the aforementioned *Achyranthes bidentata* extract, there are no particular restrictions on other added ingredients. Various flavoring agents and natural carbohydrates can be added as additives, similar to conventional beverages. Examples of the aforementioned natural carbohydrates include: conventional sugars, such as monosaccharides like glucose and fructose; disaccharides like maltose and sucrose; polysaccharides like dextrin and cyclodextrin; and sugar alcohols like xylitol, sorbitol, and erythritol. In addition to the aforementioned flavoring agents, natural flavoring agents such as sematriene and stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can also be advantageously used. In every 100g of the health food, health food, or food additive composition of this invention, the proportion of the aforementioned natural carbohydrates is typically from about 1g to 20g, preferably from about 5g to 12g.

[0092] In this invention, the food composition containing *Gnaphalium affine* extract may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. Furthermore, the health functional foods and health food compositions of this invention may contain fruit pulp used in the production of natural fruit juices, fruit juice beverages, and vegetable beverages.

[0093] In one embodiment of the present invention, the composition can be formulated into any dosage form selected from powder, granules, pills, tablets, capsules, candy, syrup and beverage.

[0094] In one aspect of the invention, a feed composition for preventing hair loss or promoting hair growth is provided, comprising extract of *Cynanchum paniculatum*.

[0095] The feed composition may contain feed or feed additives for feeding animals.

[0096] When the extract of *Cynanchum paniculatum* of the present invention is included in a feed composition or feed additive composition, the composition may be a high-concentration concentrate of 20% to 90%, or it may be prepared in powder or granule form. The feed additive may also contain one or more of the following: organic acids, such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, etc.; phosphates, such as sodium phosphate, potassium phosphate, acid pyrophosphate, etc.; natural antioxidants, such as polyphenols, catechins, α-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, etc.

[0097] In this invention, when the extract of *Gnaphalium affine* is used as feed, the composition can be formulated into a conventional feed form and can simultaneously contain conventional feed ingredients. The feed additives and feed may also contain: grains, such as crushed or ground wheat, oats, barley, corn, and rice; plant-based protein feeds, such as feeds primarily composed of rapeseed, soybeans, and sunflower; animal-based protein feeds, such as blood meal, meat meal, bone meal, and fish meal; dry components composed of sugars and dairy products, such as various types of milk powder and whey powder; and may also contain nutritional supplements, digestive and absorption promoters, growth promoters, etc.

[0098] In this invention, when the extract of *Gnaphalium affine* is used as the feed additive, it can be administered to animals alone or in combination with other feed additives in an edible carrier. Furthermore, the feed additive can be conveniently administered to animals as a top dressing, directly mixed into animal feed, or administered in an oral dosage form separate from the feed. When the feed additive is administered separately from animal feed, it can be combined with pharmaceutically acceptable edible carriers known in the art to formulate immediate-release or sustained-release dosage forms. Such edible carriers can be solid or liquid, such as corn starch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil, and propylene glycol. When using a solid carrier, the feed additive can be a tablet, capsule, powder, lozenge, sugar tablet, or a non-dispersible top dressing. When using a liquid carrier, the feed additive can be a gelatin soft capsule, syrup, suspension, emulsion, or solution. The feed can contain any protein-containing organic cereal powder commonly used to meet the dietary needs of animals. Such protein-containing cereal powders are typically composed of corn, soybean meal, or a corn / soybean meal mixture. Furthermore, feed compositions may contain, for example, preservatives, stabilizers, wetting agents or emulsifiers, and solubilizers. In addition, feed additive compositions can be added to animal feed by soaking, spraying, or mixing.

[0099] The feed or feed additive of the present invention is intended for individuals who need to prevent or improve hair loss. These individuals may include all animals other than humans that have experienced or may experience the aforementioned hair loss. These animals may include, but are not limited to: non-human primates, such as chimpanzees, other apes or monkeys; livestock, such as cattle, horses, sheep, goats, and pigs; domesticated animals, such as rabbits, dogs, or cats; laboratory animals, such as rodents, such as rats, mice, or guinea pigs; and non-mammals, such as birds or fish.

[0100] Compared with the efficacy of previous technologies The features and advantages of this invention are summarized as follows:

[0101] (a) The present invention provides a pharmaceutical composition or agent comprising extract of *Cynanchum paniculatum* for the prevention or treatment of hair loss; and the use of preparing said composition or agent.

[0102] (b) This invention provides a method for preparing an extract of *Gnaphalium affine*.

[0103] (c) The present invention provides a cosmetic composition for preventing hair loss or promoting hair growth, comprising extract of *Cynanchum paniculatum*.

[0104] (d) The present invention provides a food composition for preventing hair loss or promoting hair growth, comprising extract of *Cynanchum paniculatum*.

[0105] (e) The present invention provides a feed composition for preventing hair loss or promoting hair growth, comprising extract of *Cynanchum paniculatum*.

[0106] (f) The extract of the fruit of the present invention, *Cephalotaxus fortunei*, can effectively prevent or treat hair loss and promote hair growth. Simple Explanation of the Diagram

[0107] Figure 1 shows the results of the analysis of the inhibitory effect of the extract of *Cephalotaxus fortunei* on Janus kinase (JAK) 1.

[0108] Figure 2 shows the results of the analysis of the inhibitory effect of the extract of *Cephalotaxus fortunei* on 5α-reductase.

[0109] Figure 3 shows the results of the analysis of the inhibitory effect of the extract of *Cephalotaxus fortunei* on androgen receptor production.

[0110] Figure 4 shows the results of the analysis of the inhibitory effect of the extract of *Cephalotaxus fortunei* on the activation of the Wnt / β-catenin signaling pathway. Implementation

[0111] The present invention will now be described in more detail through embodiments. These embodiments are only used to illustrate the invention more specifically, and it will be apparent to those skilled in the art that the scope of the invention is not limited to these embodiments.

[0112] Example Example 1: Preparation of extract from *Gnaphalium affine* 1-1. Preparation of Butylene Glycol Extract from *Cephalotaxus fortunei* Butylene glycol (BG) was mixed with purified water to prepare 30% (v / v), 50% (v / v), and 70% (v / v) aqueous solutions. These solutions were then added to 200g of dried *Cephalotaxus fortunei* (also known as *Cephalotaxus fortunei*) at a concentration 10 times its weight, and extracted at 25°C for 3 days. Each extract was concentrated using a vacuum condenser to obtain more than 10g of *Cephalotaxus fortunei* extract. These were named CC-BG30, CC-BG50, and CC-BG70, respectively.

[0113]

[0114] 1-2. Preparation of hot water extract of *Gnaphalium affine* After initial extraction with 30% (v / v), 50% (v / v), and 70% (v / v) aqueous solutions of butylene glycol, 15 times the volume of purified water was added to the remaining residue, and extraction was carried out at 100°C for 4 hours. Each extract was concentrated using a vacuum concentrator to obtain more than 10g of each extract. These were named CC-W30, CC-W50, and CC-W70, respectively.

[0115]

[0116] 1-3. Preparation of Ethanol Extract of *Gnaphalium affine* Ethanol was mixed with purified water to prepare 30% (v / v), 50% (v / v), and 70% (v / v) aqueous solutions. These solutions were then added to 200g of dried *Achyranthes bidentata* (also known as *Achyranthes bidentata* var. *pinnata*) at a concentration 10 times its weight, and extracted at 80°C for 4 hours. Each extract was concentrated using a vacuum condenser to obtain more than 10g of *Achyranthes bidentata* var. *pinnata* extract, which were named CC-EE30, CC-EE50, and CC-EE70, respectively.

[0117]

[0118] The preparation methods of the above extracts are summarized in Table 1.

[0119] Table 1 Sample number name Preparation method 1 CC-BG30 Extracted with 30% (v / v) butylene glycol at 25°C for 3 days. 2 CC-BG50 Extracted with 50% (v / v) butanediol at 25°C for 3 days. 3 CC-BG70 Extracted with 70% (v / v) butanediol at 25°C for 3 days. 4 CC-W30 Add 15 times the amount of purified water to the CC-BG30 extraction residue and extract at 100℃ for 4 hours. 5 CC-W50 Add 15 times the volume of purified water to the residue extracted by CC-BG50 and extract at 100°C for 4 hours. 6 CC-W70 Add 15 times the amount of purified water to the residue extracted by CC-BG70 and extract at 100℃ for 4 hours. 7 CC-EE30 Extracted with 30% (v / v) ethanol at 80°C for 4 hours. 8 CC-EE50 Extracted with 50% (v / v) ethanol at 80°C for 4 hours. 9 CC-EE70 Extracted with 70% (v / v) ethanol at 80°C for 4 hours.

[0120]

[0121] Example 2: Evaluation of the inhibitory effect on Janus kinase (JAK)1 To evaluate the inhibitory effect of the samples prepared in Example 1 on JAK1 (Janus kinase 1), a major biomarker of alopecia areata, experiments were conducted using HEK293T cells with high transfection efficiency. JAK1 overexpression conditions were established by transfecting FLAG-tagged JAK1 (FALG-JAK1) into HEK293T cells, and 500 μL of each experimental sample was processed according to the specified concentration.

[0122] After sample processing, cells were collected with 1 mL of phosphate-buffered saline (PBS) and transferred to a microcentrifuge tube (e-tube). The tube was then centrifuged at 12,000 rpm for 3 minutes to remove the supernatant, leaving only the pellet. Cells were then lysed using a lysis buffer containing the following components: 50 mM Tris-HCl (pH 7.4), 120 mM NaCl, 25 mM β-glycerol phosphate (pH 7.5), 20 mM sodium fluoride (NaF), 2% Nonidet P-40, and protease inhibitors.

[0123] After lysis, the sample was sonicated and then centrifuged again at 12,000 rpm for 3 minutes at 4°C to obtain the supernatant. The supernatant was separated by 10-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting analysis was performed using antibodies against each of p-JAK1, JAK1, p-STAT3, STAT3, and β-actin.

[0124] The results are shown in Figure 1.

[0125] As shown in Figure 1, compared with the control group (Normal), the band intensities of both FLAG and JAK1 proteins in the FLAG-JAK1 transfection group were increased. Comparison of the JAK1 inhibitory effects of various extracts showed that the 50% ethanol extract (sample 8, CC-EE30) exhibited the best inhibitory activity, while the 30% and 70% ethanol extracts (sample 7, CC-EE30; sample 9, CC-EE70) also showed a significant reduction in JAK1 phosphorylation. Furthermore, the 70% butylene glycol extract (sample 3, CC-BG70) and the hot water extract (sample 6, CC-W70) also showed some degree of inhibitory effect.

[0126] These results indicate that the ethanol extract of *Gnaphalium affine* is a naturally derived material with significant inhibitory activity against JAK1, the main target of alopecia areata.

[0127]

[0128] Example 3: Evaluation of inhibitory ability against 5α-reductase 5α-reductase is one of the main targets of male pattern baldness. It is an enzyme that converts testosterone, a male hormone, into dihydrotestosterone (DHT). When the expression of this enzyme increases, the production of DHT also increases. DHT is known to induce androgen receptor (AR) activity more strongly than testosterone, thereby promoting hair follicle atrophy and hair loss.

[0129] The inhibitory effect of the extract of *Spatholobus suberectus* prepared in Example 1 on 5α-reductase was evaluated. Ten-week-old male Sprague-Dawley rats were used in the experiment. The entire liver of the rat was removed, and the enzyme was extracted by homogenization using a lysis buffer containing the following components: 7.5 nM K₂HPO₄, 3.25 nM KH₂PO₄, 1 mM dithiothreitol (DTT), 32 mM sucrose, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and additional protease inhibitors.

[0130] For 4 μL of extracted enzyme, 34 mM nicotinamide adenine dinucleotide phosphate (NADPH) and 0.4 mM testosterone were added to a reaction system containing the target sample at a concentration of 20 μM. McIlvaine buffer (pH 5.0) was immediately added. The reaction was terminated by heating at 80 °C for 5 minutes. Changes in NADPH oxidation were assessed by measuring absorbance changes at 340 nm, thereby evaluating changes in enzyme activity.

[0131] The results are shown in Figure 2.

[0132] As shown in Figure 2, neither butylene glycol (BG) extract nor hot water extract showed any inhibitory effect; in fact, they tended to increase enzyme activity. The ethanol extracts (CC-EE30, sample 7; CC-EE50, sample 8; CC-EE70, sample 9) all exhibited excellent inhibitory activity. In particular, the 30% ethanol extract (CC-EE30, sample 7) showed higher inhibitory activity than finasteride, the positive control group. The 50% and 70% ethanol extracts (CC-EE50, sample 8; CC-EE70, sample 9) also showed strong inhibitory effects.

[0133] These results indicate that the ethanol extract of *Gnaphalium affine* effectively inhibits 5α-reductase activity, especially the 30% and 50% ethanol extracts (CC-EE30, sample 7; CC-EE50, sample 8), which show excellent inhibitory effects on the main pathogenic mechanism of male pattern baldness. Furthermore, since the ethanol extract also uniformly exerts the previously confirmed JAK1 inhibitory effect, it is determined to have dual efficacy and can be applied simultaneously to alopecia areata. In contrast, the butylene glycol extract and hot water extract only show limited effects on specific targets.

[0134]

[0135] Example 4: Evaluation of the inhibitory effect on androgen receptor (AR) generation [estimate] One of the main pathological mechanisms of male pattern baldness is the elevated concentration of dihydrotestosterone (DHT) in the blood, which promotes the expression of androgen receptors (AR), ultimately leading to hair follicle atrophy and hair loss. Therefore, the inhibitory ability of the extract of *Gnaphalium affine* prepared in Example 1 on androgen receptor production was evaluated.

[0136] In this experiment, HEK293T cells with high transfection efficiency were used. To establish an androgen receptor overexpression model, the Myc-DDK-SRD5A2 plasmid was transfected. Cells were then treated with testosterone to establish androgen receptor overexpression conditions, and 500 μL of each experimental sample was processed.

[0137] Cells were washed with 1 mL of phosphate-buffered saline and transferred to microcentrifuge tubes (e-tubes). The tubes were then centrifuged at 12,000 rpm for 3 minutes to remove the supernatant, leaving only the pellet. Cells were then lysed using a lysis buffer consisting of: 50 mM Tris-HCl (pH 7.4), 120 mM NaCl, 25 mM β-glycerol phosphate (pH 7.5), 20 mM sodium fluoride (NaF), 2% Nonidet P-40, and protease inhibitors.

[0138] After lysis, the samples were sonicated and then centrifuged again at 12,000 rpm for 3 minutes at 4°C to obtain the supernatant. The obtained samples were then subjected to SDS-PAGE (10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to separate proteins, and Western blotting analysis was performed using antibodies against Myc, androgen receptor (AR), and β-actin.

[0139] The results are shown in Figure 3.

[0140] As shown in Figure 3, among the nine samples, the 30% butylene glycol extract (CC-BG30, sample 1), the hot water extract (CC-W50, sample 5), and the 50% ethanol extract (CC-EE50, sample 8) exhibited excellent inhibitory activity against androgen receptor production, even stronger than the inhibitory activity of dutasteride, which served as the positive control. Furthermore, CC-BG50 (sample 2), CC-W70 (sample 6), and CC-EE30 (sample 7) also showed some degree of inhibitory activity.

[0141] These results indicate that extracts from *Gnaphalium affine* have physiological activity that effectively inhibits DHT-induced androgen receptor expression. In particular, not only the ethanol extract, but also some BG and water extracts can act on specific targets.

[0142]

[0143] Example 5: Evaluation of the activity of the Wnt / β-catenin signaling pathway As male pattern baldness progresses, elevated dihydrotestosterone (DHT) levels promote androgen receptor (AR) expression, thereby inhibiting the Wnt / β-catenin signaling pathway and interfering with the induction of hair follicle anagen phase, leading to hair loss. Therefore, this study evaluated whether extracts from *Gnaphalium affine* could activate the Wnt / β-catenin signaling pathway.

[0144] The experiment was conducted using HEK293T cells with high transfection efficiency. After establishing the experimental conditions by transfecting HEK293T cells with the SRD5A1-GFP plasmid, 500 μL of the sample prepared in Example 1 was taken and processed according to the concentration.

[0145] Cells were washed with 1 mL of phosphate-buffered saline and transferred to a microcentrifuge tube (e-tube). The tube was then centrifuged at 12,000 rpm for 3 minutes to remove the supernatant, leaving only the pellet. Cells were then lysed using a lysis buffer containing the following components: 50 mM Tris-HCl (pH 7.4), 120 mM NaCl, 25 mM β-glycerol phosphate (pH 7.5), 20 mM sodium fluoride (NaF), 2% Nonidet P-40, and protease inhibitors.

[0146] After lysis, the proteins were sonicated and then centrifuged again at 12,000 rpm for 3 minutes at 4°C to obtain the supernatant. The obtained proteins were separated by 10-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting analysis was performed using antibodies against each of GFP, Wnt3a, β-catenin, and β-actin.

[0147] The results are shown in Figure 4. As shown in Figure 4, among the samples with increased expression of Wnt3a and β-catenin, the 50% ethanol extract (CC-EE50, sample 8) exhibited the best activation effect, while the water extracts (CC-W30, sample 4; CC-W70, sample 6) also showed excellent Wnt3a expression induction effects. Furthermore, the 50% and 70% butylene glycol extracts (CC-W50, sample 5; CC-W70, sample 6) also showed some degree of signaling pathway activation induction.

[0148] Based on the results of the above embodiments, when ethanol was used as a solvent to prepare the extract of *Achyranthes bidentata*, it exhibited various physiological activities, especially the 30% and 50% ethanol extracts (CC-EE30, sample 7; CC-EE50, sample 8), which showed excellent inhibitory effects on the main biomarkers related to hair loss. Furthermore, in the evaluation of Wnt3a and β-catenin expression, not only the 50% ethanol extract (CC-EE50, sample 8) but also the hot water extract (CC-W30, sample 4; CC-W70, sample 6) showed excellent expression induction effects, thus confirming that the hot water extract also has a hair regrowth promoting effect.

[0149] Therefore, these results indicate that extracts of *Gnaphalium affine*, especially 30% or 50% ethanol extracts (CC-EE30, sample 7; CC-EE50; sample 8), and hot water extracts prepared from the residue after extraction with 30% or 70% butanediol (CC-W30, sample 4; CC-W70, sample 6), activate the Wnt / β-catenin signaling pathway. On the other hand, Wnt signaling pathway activity is known to be closely related to the proliferation of dermal papilla cells. The Wnt / β-catenin signaling pathway regulates the survival and proliferation of dermal papilla cells and plays an important role in the hair growth cycle. When the Wnt signaling pathway is activated, β-catenin is not degraded in the cytoplasm but enters the nucleus, thereby inducing the expression of genes that promote cell proliferation and differentiation. Therefore, it is shown that the extract of *Cephalotaxus fortunei* of the present invention can increase the proliferation of dermal papilla cells and promote hair growth by activating the Wnt / β-catenin signaling pathway, thereby effectively promoting hair follicle growth and inducing hair regrowth.

Claims

1. An use of extracts from *Gnaphalium affine* in the preparation of a pharmaceutical preparation for the treatment of hair loss.

2. As in the purpose of request item 1, wherein, The extract of *Gnaphalium affine* is a solvent extract obtained by extraction with a solvent selected from the group consisting of butylene glycol, water, ethanol, or combinations thereof.

3. As in the purpose of request item 2, wherein, The extract is as follows: (a) the butanediol solvent is a butanediol solvent with a concentration of 20% (v / v) to 80% (v / v); or (b) the ethanol is an ethanol with a concentration of 20% (v / v) to 80% (v / v).

4. As in the purpose of request item 1, wherein, The extract of *Hemiberleinii Sinensis* is a hot water extract of *Hemiberleinii Sinensis* residue extracted with butylene glycol solvent at a concentration of 20% (v / v) to 80% (v / v).

5. As in the purpose of request item 1, wherein, The extract has the properties of inhibiting the expression of 5α-reductase, inhibiting the expression of androgen receptor, inhibiting the activity of Janus kinase (JAK), activating the Wnt / β-catenin signaling pathway, or a combination thereof.

6. As in the purpose of request item 1, wherein, The hair loss is selected from the group consisting of male pattern baldness, inflammatory baldness, alopecia areata, telogen effluvium, traumatic baldness, trichotillomania, pressure baldness, anagen effluvium, pityriasis alopecia, syphilitic baldness, seborrheic baldness, symptomatic baldness, non-scarring baldness, scarring baldness, congenital baldness, and combinations thereof.

7. A method for preparing an extract of *Gnaphalium affine*, wherein, This includes the step of extraction by adding a solvent selected from the group consisting of butylene glycol, water, ethanol, or combinations thereof to the extract of *Cynanchum paniculatum*.

8. The method for preparing the extract of *Gnaphalium affine* as claimed in claim 7, wherein, The concentration of the butanediol solvent is from 20% (v / v) to 80% (v / v); or the concentration of the ethanol is from 20% (v / v) to 80% (v / v).

9. The method for preparing the extract of *Cephalotaxus fortunei* as claimed in claim 7, wherein, The preparation method includes the following steps: after extraction with butanediol solvent at a concentration of 20% (v / v) to 80% (v / v), the remaining residue is extracted with hot water.

10. A method for preparing the extract of *Gnaphalium affine* as claimed in any of claims 7 to 9, wherein, The preparation method also includes a vacuum concentration step performed after extraction.

11. A cosmetic composition for preventing hair loss or promoting hair regrowth, wherein, It contains extracts of *Gnaphalium affine*.

12. The cosmetic composition for preventing hair loss or promoting hair growth as claimed in claim 11, wherein, The dosage form of the composition is hair conditioner, hair cream, hair lotion, shampoo, hair conditioner, hair spray, hairspray, hair gel, hair oil, hair powder, hair gel, hair mask, hair paste, eyebrow growth agent, eyelash growth agent, eyelash nutrient, or ointment.

13. A food composition for preventing hair loss or promoting hair growth, wherein, It contains extracts of *Gnaphalium affine*.

14. A feed composition for preventing hair loss or promoting hair growth, wherein, It contains extracts of *Gnaphalium affine*.

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