Application of long-stemmed holly glycosides in promoting hair growth and preventing hair loss

Abstract

This invention belongs to the fields of biomedicine and cosmetics, and discloses the application of holly glycoside in promoting hair growth and preventing hair loss. Holly glycoside promotes the transition of hair follicles from the telogen phase to the anagen phase (i.e., shortening the telogen phase and prolonging the anagen phase), and inhibits cyclophosphamide-induced apoptosis of hair follicle cells and DHT-induced miniaturization of hair follicles. In androgenetic alopecia, telogen effluvium, and chemical alopecia models, its hair growth-promoting effect is comparable to that of the positive control drug minoxidil. It has broad application prospects in pharmaceutical formulations, cosmetics, skincare products, and beauty products.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and cosmetics, and relates to the application of long-stemmed holly glycoside in promoting hair growth and preventing hair loss. Background Technology

[0002] As the largest organ in the human body, the skin constitutes the body's first line of defense, effectively resisting damage from external physical, chemical, and biological factors. Hair is an important appendage of the skin, participating in various physiological functions such as barrier protection, sensory transmission, and immune defense. However, with the accelerated pace of life and increased social pressure, hair loss is becoming increasingly common and showing a trend towards affecting younger people. Hair loss not only accelerates scalp aging and affects skin immune function, but also causes personal image problems and can even lead to emotional trauma or depression. According to data from the "2024 China Hair Health Management Industry In-Depth Research Report" and the "Chinese Scalp Health White Paper," the number of people with hair loss in my country has exceeded 200 million and continues to grow at a rate of 15%-18% annually, meaning that on average, one in six people suffers from hair loss.

[0003] Despite the large number of people suffering from hair loss, current treatment options remain relatively limited. Among the various types of hair loss, androgenetic alopecia (AGA) is the most common. Currently, only two main medications are approved globally—topical minoxidil and oral finasteride. Minoxidil, as a first-line topical medication for AGA, has long suffered from limited clinical adherence, primarily due to the following four unavoidable drawbacks: First, the incidence of local irritation is approximately 7%, manifesting as itching, erythema, and dandruff-like desquamation. The propylene glycol-containing solvent may induce contact dermatitis, leading to premature discontinuation of treatment in about 4% of patients. Second, transdermal absorption can cause systemic adverse reactions such as palpitations, dizziness, and weight gain. Third, transient excessive hair loss often occurs during the initial treatment phase (2-8 weeks), accompanied by uncontrollable hirsutism on the face or forearms in 3-5% of female patients, significantly impacting aesthetics and psychological acceptance. Fourth, efficacy depends on continuous use; new hair growth rapidly falls out 3-4 months after discontinuation, requiring lifelong maintenance treatment twice daily. Discontinuation of treatment results in a return to baseline, lacking radical cure and involving a lengthy treatment course, further weakening patient adherence. However, long-term oral administration of finasteride may cause side effects such as erectile dysfunction and decreased libido, and poses a teratogenic risk to male fetuses; therefore, it is contraindicated in women of childbearing age. Furthermore, while autologous hair transplantation can achieve hair regrowth, it has limitations such as surgical risks and low post-operative hair follicle survival rates. In conclusion, there is an urgent need to develop new, safe, and effective drugs for treating hair loss.

[0004] Pedunculoside is a characteristic triterpenoid saponin compound of Ilex genus plants, possessing a wide range of pharmacological activities, including antitumor, anti-inflammatory, cardiovascular protective, and Alzheimer's disease treatment effects. However, to date, no publicly available research or literature has been published on this ingredient in the fields of promoting hair growth and treating hair loss. Summary of the Invention

[0005] This invention addresses common problems in the clinical and cosmetic fields, such as a lack of active ingredients for promoting hair growth, insufficient safety profiles, and poor patient compliance, and proposes the following solutions:

[0006] 1. The present invention aims to provide a small molecule compound with a well-defined structure, natural origin and high safety—pedunculoside—as a novel hair follicle growth stimulant to overcome the defects of existing therapies (such as minoxidil) such as local irritation, systemic adverse reactions and lifelong drug dependence;

[0007] 2. Ilex chinensis glycoside promotes the transition of hair follicles from the telogen phase to the anagen phase (i.e., shortens the telogen phase and prolongs the anagen phase), and inhibits cyclophosphamide-induced apoptosis of hair follicle cells and DHT-induced miniaturization of hair follicles. In androgenetic alopecia, telogen effluvium and chemical alopecia models, its hair growth-promoting effect is comparable to that of the positive control drug minoxidil.

[0008] This invention addresses the existing problems of hair loss and the scarcity of effective treatments for hair loss, proposing a novel compound called long-stemmed holly glycoside to promote hair growth. Firstly, long-stemmed holly glycoside is a triterpenoid saponin compound (… Figure 1 As shown). In one specific embodiment of the invention, experimental evidence systematically verified its effectiveness and safety: CCK-8 assay showed that long-stemmed holminoside had no toxic effect on human dermal papilla cells (HDPC). Figure 2 This confirmed good biocompatibility; in in vitro mouse whisker hair follicle culture, 20 μM long-stem holmin significantly promoted hair shaft growth, superior to the solvent control group and the minoxidil group. Figure 3 , Figure 4 In an in vivo hair removal-induced synchronized hair regeneration model, 1 mM and 2 mM hollyoside accelerated the transition of hair from the telogen to the anagen phase, with effects comparable to the minoxidil group. Figures 5-7 After 7 days of treatment, the body weight of mice in the minoxidil group increased significantly, which is consistent with the reported side effects of minoxidil in the literature, while the body weight of mice in the holly glycoside treatment group did not change significantly. Figure 8 This highlights its safety advantages; in the cyclophosphamide-induced alopecia (CIA) model, ilexoside alleviated hair follicle apoptosis and hair thinning. Figures 9-11Meanwhile, long-stemmed holly glycosides significantly alleviated DHT-induced HDPC viability damage and improved cell viability ( Figure 12 This indicates its potential in treating androgenetic alopecia. In summary, ilexoside, as a novel hair follicle growth stimulant, combines high efficacy and safety, and is expected to overcome the shortcomings of existing therapies (such as minoxidil) such as local irritation, systemic exposure, and lifelong dependence.

[0009] Theoretical basis for the innovation of this invention:

[0010] 1. Targeting the fundamental regulation of the hair follicle growth cycle. The innovative theoretical starting point lies in breaking through the limitations of existing therapies that mostly focus on local stimulation or single pathway inhibition, proposing that long-stemmed holmside can act as a "hair follicle growth phase initiator and maintainer". Its core basis is that it can directly promote the hair follicle to re-enter the active growth phase from the quiescent phase (Telogen).

[0011] 2. Multi-target mechanism hypothesis: Based on the diversity of hair loss types, a multi-target action theory of ilex glycoside is innovatively constructed: A. Anti-apoptotic protection: For chemical alopecia caused by chemotherapy, the theoretical basis is that it can inhibit hair follicle cell apoptosis and maintain the integrity of hair follicle structure. B. Antagonism of androgen toxicity: For androgenetic alopecia, the theoretical basis is that it can alleviate the damage of dihydrotestosterone (DHT) to dermal papilla cells, thereby preventing hair follicle miniaturization—a key pathological link in the action of androgens on hair follicles.

[0012] The technical challenges / difficulties that this invention needs to overcome are as follows: Challenge 1: Identifying candidate compounds from a "knowledge blind spot." The biggest difficulty lies in the fact that the hair growth-promoting activity of ilexoside is completely unknown. Research must start from scratch; without any prior clues, the primary technical challenge is how to screen and confirm it as the target compound from thousands of natural compounds. Challenge 2: Constructing a pharmacodynamic evaluation system that transcends multiple barriers. To fully confirm its universal efficacy of "promoting hair growth and preventing hair loss," a complex evaluation system must be established, encompassing microscopic to macroscopic levels and in vitro to in vivo findings.

[0013] This invention relates to the fields of medicine and cosmetics, specifically disclosing the use of the compound *Ilex longiflora* glycoside in promoting hair growth and preventing hair loss. This compound can be used to treat various types of hair loss, including chemotherapy-induced alopecia, androgenetic alopecia, alopecia areata, neurogenic alopecia, seborrheic alopecia, and cicatricial alopecia, and is characterized by its natural origin and high safety. In practical applications, *Ilex longiflora* glycoside can be added at an effective concentration to topical preparations such as basic cosmetic creams for hair growth and hair loss prevention care.

[0014] This invention proposes the application of long-stemmed holly glycoside in promoting hair growth, preventing hair loss, abnormal hair loss, thinning hair, hair aging, fine and soft hair, and hair growth stagnation.

[0015] This invention proposes the application of long-stemmed holly glycoside in the preparation of drugs, cosmetics, personal care products, beauty products, food, and health products that promote hair growth, prevent hair loss, treat abnormal hair loss, thinning hair, aging hair, fine and soft hair, and hair growth stagnation.

[0016] The present invention also proposes a regulator / promoter / activator, which includes, as described above, ide glycosides, etc.

[0017] In one specific embodiment, the regulator / promoter / activator further includes one or more of the following: α-estradiol, stilbene glycoside, cyclosporine, anthraquinone, pilocarpine, carboplatin chloride, dutasteride, cimetidine, caffeine, vitamin E, dipotassium glycyrrhizate, copper peptide (GHK-Cu), acetyl tetrapeptide-3, Kisspeptin-234 (peptide 234), adenosine, etc.

[0018] The present invention also proposes a cosmetic / care product / beauty product / food / health product, wherein the cosmetic / care product / beauty product / food / health product contains one or more of the above-mentioned holly glycosides, regulators / promoters / activators, etc.

[0019] In one specific embodiment, the cosmetics / care products / beauty products / food / health products also include ginger, ginseng, ganoderma lucidum, rhubarb, he shou wu, Sichuan pepper, pinellia, psoralea corylifolia, black sesame, gastrodia elata, arborvitae leaf, peppermint, artemisia argyi, saposhnikovia divaricata, vitex trifolia, wolfberry, safflower, angelica sinensis, salvia miltiorrhiza, citronella cirrhosa, pumpkin seeds, saw palmetto, astragalus membranaceus, privet fruit, abelmoschus manihot, hawthorn, polyporus umbellatus, sandalwood, ginkgo biloba, chuanxiong rhizome, sanguisorba officinalis, black bean, licorice, starch, maltodextrin, gelatin, vegetable oil, and dietary fiber. Water, ethanol, isopropanol, propylene glycol, butylene glycol, glycerin, 1,3-propanediol, caprylic / capric triglyceride, mineral oil, petrolatum, silicone oil (such as cyclopentamethoxysiloxane, polydimethylsiloxane), jojoba oil, almond oil, olive oil, castor oil, shea butter, squalane, hyaluronic acid, ceramide, panthenol, sodium pyrrolidone carboxylate, sodium lactate, salicylic acid, zinc pyrithione (ZPT), ketoconazole, clomiphene citrate, selenium disulfide, microneedles, needle rollers, etc.

[0020] The present invention also proposes a reagent / kit containing one or more of the following: long-stemmed holly glycosides, regulators / promoters / activators, cosmetics / care products / beauty products / foods / health products, etc., as described above.

[0021] The present invention also proposes a drug / drug composition comprising one or more of the following: *Ilex longiflorum glycoside* as described above, regulators / promoters / activators as described above, cosmetics / care products / beauty products / foods / health products as described above, reagents / kits as described above.

[0022] Furthermore, the drug / drug composition further includes a pharmaceutically acceptable carrier.

[0023] Specifically, the drug / drug composition includes liquid dosage forms, gaseous dosage forms, solid dosage forms, and semi-solid dosage forms.

[0024] Specifically, the drug / drug composition is administered orally, by injection, nasal administration, transdermal administration, or mucosal administration.

[0025] Preferably, the pharmaceutically acceptable carrier refers to a carrier that, when properly administered to animals or humans, does not produce adverse, allergic, or other adverse reactions. Pharmaceutically acceptable carriers include, but are not limited to: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0026] Specifically, the drug / drug composition may also contain physiologically compatible excipients, including buffers, diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc.

[0027] Specifically, the drug / drug composition can be formulated into injections, sterile powders for injection, tablets, pills, capsules, lozenges, liniments, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalers, or suppositories, etc. All of the above dosage forms of the drug / drug composition can be prepared according to conventional methods in the pharmaceutical field. In the above pharmaceutical formulation, the dosage form of the drug / drug composition is an injection.

[0028] Specifically, the drug / drug composition can be introduced into the body, such as into muscles, intradermal tissues, subcutaneous tissues, veins, or mucous membranes, via injection, spray, nasal drops, eye drops, penetration, absorption, or physical or chemical mediated methods; or it can be introduced into the body after being mixed with or encapsulated by other substances. Preferably, it is administered by injection. The drug / drug composition can also be used in combination with other treatment methods, including surgery, radiotherapy, chemotherapy, and targeted therapy.

[0029] The dosage level of the drug / drug composition of the present invention can be adjusted according to the amount of composition required to achieve the desired diagnostic or therapeutic outcome. The administration regimen can also be a single injection or multiple injections, or adjustments thereof. The selected dosage level and regimen are rationally adjusted based on various factors including the activity and stability (i.e., half-life) of the cellular drug / drug composition, the formulation, the route of administration, combination with other drugs or treatments, the disease or condition to be detected and / or treated, and the health status and prior medical history of the subject to be treated.

[0030] The therapeutically effective dose of the drug / pharmaceutical composition of the present invention can initially be estimated in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs, and / or primates. Animal models can also be used to determine suitable concentration ranges and routes of administration. These can then be used to determine the useful dose and route of administration in humans. Generally, the determination and adjustment of the effective amount or dose, and the assessment of when and how to make such adjustments, are known to those skilled in the art.

[0031] For further guidance on formulations, dosages, administration regimens, and measurable treatment outcomes, see Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology.

[0032] Specifically, the drug / drug composition may be used alone and / or in combination with one or more of the following drugs: minoxidil, finasteride, anthraquinol, pilocarpine, latanoprost, bimatoprost, spironolactone, flutamide, ketoconazole, fluconazole, glucocorticoids (such as hydrocortisone, triamcinolone), immunosuppressants (such as tacrolimus, pimecrolimus), biologics (such as JAK inhibitors tofacitinib, baricitinib), vasodilators (such as nitroglycerin), anti-inflammatory drugs (such as aspirin), and antioxidants (such as vitamin C, coenzyme Q10).

[0033] The present invention also proposes a method for promoting hair growth, the method comprising administering to a subject in need an effective amount of one or more of the following: *Ilex longiflora glycoside* as described above, a regulator / promoter / activator as described above, a cosmetic / care product / beauty product / food / health product as described above, a reagent / kit as described above, a drug / drug composition as described above.

[0034] Specifically, the preferred target is a mammal, including but not limited to humans, primates, livestock (such as sheep, cattle, horses, donkeys, and pigs), pets (such as dogs and cats), laboratory test animals (such as mice, rabbits, rats, guinea pigs, and hamsters), or captured wild animals (such as foxes and deer). Preferably, the target is a primate. Most preferably, the target is a human.

[0035] This invention also proposes a method for preventing / inhibiting / alleviating / reducing / treating diseases, the method comprising administering to a subject in need an effective amount of one or more of the following: *Ilex longiflora glycoside* as described above, *modifiers / promoters / activators* as described above, *cosmetics / care products / beauty products / foods / health products* as described above, *reagents / kits* as described above, *medicines / pharmaceutical compositions* as described above. The method may also be in vitro or non-therapeutic.

[0036] Specifically, the preferred target is a mammal, including but not limited to humans, primates, livestock (such as sheep, cattle, horses, donkeys, and pigs), pets (such as dogs and cats), laboratory test animals (such as mice, rabbits, rats, guinea pigs, and hamsters), or captured wild animals (such as foxes and deer). Preferably, the target is a primate. Most preferably, the target is a human.

[0037] Specifically, the target may be a patient with a disease or an individual who wishes to prevent the disease. One or more of the following can be administered to the target before, during, or after receiving disease treatment: *Ilex longiflora glycoside*, *modifiers / promoters / activators*, *cosmetics / care products / beauty products / foods / health products*, *reagents / kits*, *medicines / pharmaceutical compositions*, etc.

[0038] The present invention also proposes the application of the above-mentioned holly glycoside, the above-mentioned regulator / promoter / activator, the above-mentioned cosmetics / care products / beauty products / foods / health products, the above-mentioned reagents / kits, the above-mentioned drugs / drug compositions, or the above-mentioned methods in promoting hair growth, preventing hair loss, abnormal hair loss, thinning hair, fine hair, hair aging, and hair growth stagnation.

[0039] The present invention also proposes the application of the above-mentioned holly glycoside, the above-mentioned regulators / promoters / activators, the above-mentioned cosmetics / care products / beauty products / foods / health products, the above-mentioned reagents / kits, the above-mentioned drugs / drug compositions, or the above-mentioned methods in the preparation of drugs / cosmetics / care products / beauty products / foods / health products that promote hair growth, prevent hair loss, abnormal hair loss, thinning hair, fine hair, hair aging, and hair growth stagnation.

[0040] In this invention, the diseases include hair loss, abnormal hair loss, sparse hair, fine and soft hair, hair aging, and hair growth stagnation.

[0041] In one specific embodiment of the present invention, the hair loss includes various types of hair loss such as chemotherapy-induced hair loss, androgenetic alopecia, alopecia areata, neurogenic alopecia, seborrheic alopecia, and scarring alopecia.

[0042] This invention proposes a novel use for the natural product *Ilex longiflorus* glycoside, which can be used to promote hair growth in pharmaceutical formulations, cosmetics, skincare products, and beauty products. The compound can be used to address hair regrowth stagnation in patients with hair loss, stimulating hair growth, preventing hair follicle cell apoptosis, and its efficacy is comparable to the FDA-approved drug minoxidil. Evaluations have shown that the ingredient is safe, effective, and has few toxic side effects, exhibiting broad application prospects in pharmaceutical formulations, cosmetics, skincare products, and beauty products. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 This is a schematic diagram of the molecular structure of the compound long-stemmed holly glycoside.

[0045] Figure 2 The effect of the compound long-stemmed holly glycoside on the viability of human dermal papilla cells (HDPC);

[0046] Figure 3 A schematic diagram showing the effect of the compound long-stemmed holly glycoside on promoting the growth of mouse whisker hair follicles in vitro.

[0047] Figure 4 A schematic diagram showing the statistical results of the compound long-stem holly glycoside promoting the growth of whisker hair follicles in cultured mice in vitro.

[0048] Figure 5 A schematic diagram showing the effect of the compound long-stemmed holly glycoside on promoting hair growth in hair-removing mice;

[0049] Figure 6 The statistical results of relative grayscale of mouse dorsal skin after treatment with the compound long-stem holly glycoside for 7 days;

[0050] Figure 7 The statistical results of relative grayscale of mouse dorsal skin after 11 days of treatment with the compound longistyloside.

[0051] Figure 8 A statistical analysis of mouse body weight after 7 days of treatment with the compound long-stem holly glycoside.

[0052] Figure 9 A schematic diagram showing the results of treating chemotherapy-induced alopecia with the compound long-stem holly glycoside.

[0053] Figure 10 Representative results of mouse hair in each group after treatment with the compound ileoside for 22 days;

[0054] Figure 11 A schematic diagram showing the hair weight of mice in each group after 22 days of treatment with the compound holly glycoside.

[0055] Figure 12 A schematic diagram illustrating the effect of the compound long-stemmed holly glycoside in alleviating DHT-induced damage to the viability of human dermal papilla cells. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0057] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0058] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0059] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0060] In this embodiment of the invention, the experimental materials used were as follows: 7-week-old male C57BL / 6 mice were obtained from the SPF Experimental Animal Center of East China Normal University. Human dermal papilla cells (Catalog No.: QuiCell-Y1755-M500) and cell culture medium (QuiCell-Y1755-M500) were purchased from Kuisai Biotechnology; commercially available minoxidil was purchased from Mandy Pharmaceuticals (National Approval Number H20010714); Williams' E medium was purchased from Gibco (Catalog No.: 12551032); CCK8 reagent (Catalog No.: C0005) and holly glycoside (Catalog No.: 230119) were purchased from Shanghai Taoshu Biotechnology Co., Ltd.; cyclophosphamide was purchased from Baxteroncology GmbH, specification: 0.2g; DHT was purchased from KKL Med Inc. (Catalog No.: KP1222).

[0061] Example 1:

[0062] like Figure 1 The image shows the molecular structure of the compound long-stemmed holly glycoside.

[0063] like Figure 2 As shown, this invention treated human dermal papilla cells (HDPCs) with four concentrations of 10 μM, 20 μM, 40 μM, and 80 μM for 24 hours to evaluate their cytotoxicity. The specific experimental method is as follows: HDPCs were treated with 5 × 10... 3 Cells were seeded at a density of 100 μL / well in 96-well plates and cultured adherently for 12 hours. The culture medium was then replaced with a special medium containing a gradient concentration of lecithin and incubated for another 24 hours. After discarding the old medium, culture medium containing CCK-8 reagent was added and the plates were incubated at 37 °C in the dark for 1.5 hours. Finally, the absorbance was measured at 450 nm using a microplate reader.

[0064] like Figure 3 As shown, this invention evaluated the promoting effect of ilexobin at concentrations of 10 μM and 20 μM on hair follicle growth using an in vitro cultured mouse hair follicle model. The experimental groups included a control group (containing only culture medium), a minoxidil group (50 μM), and ilexobin groups (two concentrations: 10 μM and 20 μM). Hair shaft length changes were recorded on days 1 and 8 of culture for each group. The specific experimental method is as follows: Hair follicles from 8-week-old mice were placed in hair follicle culture medium to establish an in vitro mouse hair follicle model. According to the grouping, corresponding drugs were added: the control group contained only culture medium, the minoxidil group received 50 μM minoxidil, and the ilexobin groups received 10 μM or 20 μM ilexobin. After drug treatment, the hair shaft length was observed and recorded on days 1 and 8 using an inverted phase-contrast microscope.

[0065] like Figure 4 The figure shows the statistical results of the effect of the compound leucocelein on cultured mouse whisker hair follicles in vitro. The horizontal axis represents the four treatment groups: Group 1: control group, Group 2: minoxidil group, and Groups 3 and 4: 10 μM and 20 μM leucocelein treatment groups, respectively. Each group had 11-12 hair follicles. The vertical axis represents the relative elongation length of the hair shaft. P<0.05, P<0.01, P<0.001, P<0.0001, # P<0.05.

[0066] like Figure 5 The results show that the compound *Ilex chinensis* glycoside promoted hair growth in hair-regenerating mice. Specifically, 8-week-old male mice were dehaired using a hair-regenerating cream to establish a synchronized hair regeneration model. *Ilex chinensis* glycoside was diluted at 1 mM and 2 mM in a cosmetic base cream, and 0.1 g was applied to the backs of the mice daily. Observations and photographic records were taken. On days 7 and 11, the *Ilex chinensis* glycoside group significantly accelerated the transition of hair from the resting phase to the anagen phase on the backs of the mice, with better results than the control group and the minoxidil group.

[0067] like Figure 6 and Figure 7 As shown, this is the product after treatment with the compound longistinoside. Figure 5 The relative grayscale statistics of the back skin of hair-removed mice were obtained by statistically analyzing the grayscale values ​​of the back skin of the mice using ImageJ software. The results shown are the relative grayscale statistics of the back skin on days 7 and 11. Group 1: Control group ( Figure 5 The statistical results of the grayscale of the back skin of mice in the blank group corresponding to day 7 or 11), the second group: minoxidil group ( Figure 5 The statistical results of the gray scale of the back skin of mice in the minoxidil group corresponding to day 7 or 11), the third and fourth groups were 1mM ( Figure 5 The statistical results of grayscale of the dorsal skin of mice treated with 1mM long-stemmed holmide on day 7 or 11) and 2mM long-stemmed holmide ( Figure 5 The grayscale results of the back skin of mice in the 2mM long-stem holmine treatment group on day 7 or 11 are shown in the figure. Each group contains 7-8 mice, and the vertical axis is the relative grayscale value of the hairless mice. P<0.05, P<0.001.

[0068] like Figure 8The figure shows the results of holly glycoside treatment for chemotherapy-induced alopecia. On day 7, the body weight of mice in each group was measured. Group 1: control group, Group 2: minoxidil group, Groups 3 and 4: 1 mM and 2 mM holly glycoside groups, respectively. Each group contained 7-8 mice. The vertical axis represents the change in body weight of mice in each group. P<0.001.

[0069] like Figure 9 The diagram shows the results of treating chemotherapy-induced alopecia with the compound holly glycoside. The specific experimental method is as follows: Eight-week-old male mice were treated with a hair removal cream. The experimental groups included a control group, a model group, a minoxidil group (2% minoxidil, trade name Mandi), and a holly glycoside group (two concentrations: 1 mM and 2 mM). For the first 9 days, the control and model groups were treated with the blank cream, the minoxidil group with minoxidil, and the holly glycoside groups with the corresponding concentration of holly glycoside. The cream was applied daily at a dose of 0.1 g to the back of the mice, and observations and photographs were taken. On day 9, except for the control group, the other four groups of mice were injected with 125 mg / kg cyclophosphamide to induce chemotherapy-induced alopecia (CIA). On days 10 and 12, compared with the model group, the holly glycoside group significantly promoted the transition of hair on the back of the mice from the telogen phase to the anagen phase, indicating that holly glycoside can significantly accelerate hair growth. On day 16, all mice except the control group exhibited hair loss. By day 22, the regeneration rate of hair on the backs of mice in the minoxidil group and the holly glycoside treatment group was significantly faster than that in the model group, with hair essentially covering the entire back skin. These results indicate that holly glycoside can effectively inhibit hair follicle cell apoptosis, thereby preventing hair loss. Furthermore, the hair growth-promoting effect of the high-concentration holly glycoside treatment group was comparable to that of minoxidil.

[0070] like Figure 10 and Figure 11 As shown, this invention presents image recordings and statistical results of mouse hair in each group after 22 days of treatment with holly glycosides. The specific experimental method is as follows: On day 22, mice in each group underwent hair removal treatment, followed by weighing and photographing of the hair. The experimental groups included: control group (group 1), model group (group 2), minoxidil group (group 3), 1 mM holly glycoside treatment group (group 4), and 2 mM holly glycoside treatment group (group 5), with 5-6 mice in each group. In the statistical graph, the vertical axis represents the change in hair weight. P < 0.05 indicates a statistically significant difference.

[0071] like Figure 12 As shown, this invention demonstrates the effect of ilex longiflorum glycoside in alleviating DHT-induced damage to the activity of human dermal papilla cells (HDPCs). The specific experimental method is as follows: First, HDPCs were subjected to a concentration of 5 × 10⁻⁶... 3Cells were seeded at a density of 100 μL / well in 96-well plates and cultured adherently for 12 hours. Except for the control group, all other groups were treated with medium containing 50 μM DHT for 24 hours to induce lesions. The old medium was then discarded and replaced with complete medium containing gradient concentrations of ilexobin (10 μM and 20 μM), and treated for another 24 hours. Finally, CCK-8 reagent was added, and the reaction was carried out at 37 °C in the dark for 1.5 hours. The absorbance was measured at 450 nm using a microplate reader to evaluate the therapeutic potential of ilexobin for androgenetic alopecia.

[0072] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0073] As used in this invention, the terms "comprising" and "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0074] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0075] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of this invention are included in this invention and are protected by the appended claims.

[0076] References:

[0077] [1] Z. Shadi, Compliance to Topical Minoxidil and Reasons for Discontinuation among Patients with Androgenetic Alopecia, Dermatology andTherapy, 13 (2023) 1157-1169.

[0078] [2] L. Li, J. Sun, F. Chen, L. Xiong, L. She, T. Hao, Y. Zeng, L. Li,W. Wang, X. Zhao, G. Liang, Pedunculoside alleviates cognitive deficits andneuronal cell apoptosis by activating the AMPK signaling cascade, ChineseMedicine, 19 (2024) 163.

Claims

1. Application of long-stemmed holly glycoside in the preparation of drugs that promote hair growth and prevent hair loss.

2. Use of a modulator for the manufacture of a medicament for promoting hair growth and preventing hair loss, characterized in that, The regulator is holly glycoside.

3. Use of an agent in the manufacture of a medicament for promoting hair growth and preventing hair loss, characterized in that, The reagent is holly glycoside.

4. Use according to any one of claims 1 to 3, wherein the compound is ###0002### The hair loss includes chemotherapy-induced hair loss and / or androgenetic alopecia.

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