Application of Ilex kudingcha and ilex holly leaf extract in external preparations for preventing hair loss

Abstract

The present application relates to a kind of application of Ilex kudingcha leaf extract in anti-hair loss external preparation, Ilex kudingcha leaf extract is obtained by extracting Ilex kudingcha leaf of Ilex kudingcha leaf extract of Ilex kudingcha leaf extract of Ilex kudingcha leaf extract prepared in scalp lipid metabolism abnormality, oxidation stress damage and inflammation induced hair loss, all show outstanding effect of promoting hair growth and inhibiting hair loss;And Ilex kudingcha has high safety, its improvement effect for preventing hair loss is reflected in reducing the number of hair loss and increasing hair weight;Based on the above characteristics, Ilex kudingcha leaf extract can be added as functional additive to anti-hair loss external preparation.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, and in particular relates to the application of a bitter tea holly leaf extract in a topical preparation for preventing hair loss. Background Technology

[0002] Hair loss has become a global health challenge. With increasing mental stress and environmental pollution, the incidence of hair loss is rising rapidly and showing a trend towards affecting younger people, thus increasing the demand for hair loss prevention and hair regrowth products. Its pathogenesis is closely related to abnormal scalp lipid metabolism, oxidative stress damage, scalp microcirculation disorders, and hair follicle function decline.

[0003] Currently, the FDA has approved two main types of drugs for treating hair loss: topical minoxidil and oral finasteride. However, long-term use of minoxidil or finasteride has a series of toxic side effects, such as drug dependence, skin irritation, cardiovascular effects, and sexual dysfunction. Furthermore, compared to the complex causes of hair loss, these two drugs have a single mechanism of action and low transdermal efficiency, resulting in limited effectiveness in treating hair loss. In non-drug treatments, plant extracts, due to their natural and safe characteristics, are gradually becoming a research hotspot in the field of hair loss prevention and hair regrowth. For example, ginseng, he shou wu (Polygonum multiflorum), and arborvitae leaves have the effects of promoting scalp blood circulation, enhancing hair follicle nutrition, and inhibiting fungal growth, effectively improving the scalp environment and promoting hair growth. However, the application of plant extracts in current technologies still faces many problems. For example, the extraction process is complex, the active ingredients are unclear or have poor stability, and some products have poor absorption, making it difficult to penetrate deep into the hair follicles to exert their effects. In addition, the efficacy or mechanism of action of some hair loss prevention products on the market lacks scientific verification, or the products are expensive, making it difficult to meet the actual needs of consumers. Therefore, there is an urgent need to develop multi-mechanism synergistic, safe, and efficient alternative therapies to solve existing hair loss problems. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the application of Kuding tea holly leaf extract in topical preparations for preventing hair loss.

[0005] The technical solution adopted in this invention is: the application of bitter tea holly leaf extract in topical preparations for preventing hair loss.

[0006] Preferably, the extract of Ilex kudingcha leaves is Ilex kudingcha (from the genus Ilex of the family Aquifoliaceae). Ilex kudingcha Extracted from leaves of CJTseng.

[0007] Preferably, the extract of holly leaves from bitter tea contains kuding saponins as active substances.

[0008] Preferably, it is used to improve hair loss caused by abnormal lipid metabolism, or to alleviate hair loss caused by oxidative stress damage, or to improve hair loss caused by inflammatory response.

[0009] Preferably, the mass percentage of the extract of Ilex kudingcha leaves in the topical preparation is 0.05%–10%.

[0010] The preparation method of Kuding tea holly leaf extract involves extracting the active ingredients of Kuding tea holly leaves by decoction in water, removing solid substances and retaining the liquid obtained from the decoction to obtain Kuding tea holly leaf extract.

[0011] Preferably, the liquid obtained by decocting bitter tea holly leaves is concentrated to obtain bitter tea holly leaf extract;

[0012] Preferably, the saponin components are separated from the liquid obtained by decocting bitter tea leaves and holly leaves.

[0013] A method for preparing Kuding tea holly leaf extract.

[0014] A topical preparation for preventing hair loss, comprising an extract of bitter tea and holly leaves.

[0015] The advantages and positive effects of this invention are: the total saponin content in the leaves of Kuding tea holly is higher than that in the leaves of large-leaf holly and wolfberry, and the total saponin components of its specific composition have a significant effect on preventing hair loss; the same dose of Kuding tea holly leaf extract is more effective in preventing hair loss than Kuding tea, large-leaf holly, or wolfberry leaves.

[0016] Kuding tea holly can prevent abnormal scalp lipid metabolism, oxidative stress damage, and inflammation-induced hair loss. The examples also demonstrated that Kuding tea holly can significantly inhibit triglyceride, total cholesterol, or 5α-reductase levels in a mouse model of androgenetic alopecia; and inhibit excessively high ROS or MDA levels or abnormal expression of inflammatory factors TNF-α, IL-1β, IL-6, and INF-γ in mouse hair follicle stem cells induced by ultraviolet irradiation or hydrogen peroxide.

[0017] This invention utilizes bitter tea and holly in the field of hair loss prevention, effectively reducing hair follicle cell necrosis, thereby improving hair loss and promoting hair growth. Specifically, it can significantly reduce the number of hairs lost and promote the area of ​​hair growth.

[0018] This invention, referring to the "Cosmetic Safety Technical Specifications (2015 Edition)," investigates the safety of bitter tea holly. Local toxicity tests show the following results: Acute oral toxicity: LD50... 50 >5000 mg / kg, non-toxic; multiple skin irritation tests: no irritation; acute eye irritation: no irritation; skin allergic reaction: no sensitization; skin phototoxicity: no skin phototoxicity observed. Therefore, the Kuding tea holly exhibits high safety.

[0019] There is no existing research on the use of bitter tea and holly alone for preventing hair loss and promoting hair growth. This invention found that products with 0.05-10% bitter tea and holly added have significant effects in hair loss models. Among them, the effect of adding 2.5-10% is significantly better than that of commercially available 5% minoxidil tincture. Attached Figure Description

[0020] Figure 1 Comparison of the effects of active substances from bitter tea and holly leaves on the proliferation activity of hair follicle stem cells;

[0021] Figure 2 Mass spectrometry analysis of total saponin composition in holly leaf extract of bitter tea;

[0022] Figure 3 Determination of total saponin content in extracts of bitter tea holly, large-leaf holly, or wolfberry;

[0023] Figure 4 Comparison of the proliferative activity of total saponins from bitter tea holly, large-leaf holly, or wolfberry on hair follicle stem cell proliferation.

[0024] Figure 5 Mass spectrometry analysis of saponins in holly, large-leaf holly, or wolfberry;

[0025] Figure 6 Effects of extracts of bitter tea holly, large-leaf holly, or wolfberry on a mouse model of androgenetic alopecia.

[0026] Figure 7 Effects of Kuding tea and Ilex chinensis leaf extract on key lipid metabolism indicators in a mouse model of androgenetic alopecia; A: triglyceride level; B: total cholesterol level; C: 5α-reductase level;

[0027] Figure 8 Effects of Kuding tea and Ilex chinensis leaf extract on androgenetic alopecia mouse model; A: Effect on the amount of hair loss; B: Effect on hair weight;

[0028] Figure 9 Effects of bitter tea holly leaf extract on ROS levels in UV-induced mouse hair follicle stem cells.

[0029] Figure 10 Effects of Kuding tea holly leaf extract on the expression of inflammatory factors TNF-α, IL-1β, IL-6, and INF-γ in UV-induced mouse hair follicle stem cells;

[0030] Figure 11 Effects of bitter tea and holly leaf extract on ROS levels in hydrogen peroxide-induced mouse hair follicle stem cells;

[0031] Figure 12Effects of Kuding tea and Ilex chinensis leaf extract on the expression of inflammatory factors TNF-α, IL-1β, IL-6, and INF-γ in hydrogen peroxide-induced mouse hair follicle stem cells;

[0032] Figure 13 Effects of Kuding tea and holly leaf extract on a mouse model of alopecia following chemotherapy; A: Effect on the amount of hair loss; B: Effect on hair weight;

[0033] Figure 14 Effects of different concentrations of bitter tea and holly on a natural hair regrowth model; A: Effect on the amount of hair loss; B: Effect on hair weight;

[0034] Figure 15 Effects of different concentrations of bitter tea and holly on a shaved mouse model; where A represents the effect on the amount of hair loss and B represents the effect on hair weight.

[0035] Figure 16 Evaluation of the efficacy of bitter tea holly leaf extract on people suffering from hair loss and thinning hair (local hair map). Detailed Implementation

[0036] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0037] This invention relates to the application of a bitter tea holly leaf extract in a topical preparation for preventing hair loss. The bitter tea holly leaf extract is *Ilex kudingcha* (a type of holly) belonging to the genus *Ilex* of the family Aquifoliaceae. Ilex kudingcha Extracted from the leaves of *Ilex chinensis* (CJ Tseng). Experiments showed that the prepared *Ilex chinensis* leaf extract exhibited significant effects in promoting hair growth and inhibiting hair loss in cases of scalp lipid metabolism abnormalities, oxidative stress damage, and inflammation-induced hair loss.

[0038] Kuding tea has a long history in my country, and the representative varieties are large-leaf holly (…). Ilex latifolia Thunb.), bitter tea holly ( Ilex kudingcha CJ Tseng) and holly ( Ilex cornuta Lindl. et Paxt.). Kuding tea holly is a tree belonging to the genus *Ilex* of the family Aquifoliaceae, mainly produced in Hainan and Guangxi. Its dried leaves are primarily used medicinally and as food. They are cool in nature, bitter and slightly sweet in taste, and have the effects of dispelling wind and heat, clearing the head and eyes, relieving irritability and quenching thirst. The main component of Kuding tea holly is saponins, and it also contains flavonoids, polyphenols, polysaccharides, and volatile oils. Extracts obtained from the effective components of Kuding tea holly leaves can be used to prepare topical anti-hair loss preparations.

[0039] Kuding tea holly leaf extract is obtained by decoction of the leaves. The solution obtained after decoction can be further concentrated and dried, and the resulting Kuding tea holly leaf extract can be used as a raw material to prepare other products. In some embodiments of the present invention, the Kuding tea holly leaf extract contains effective components including total polysaccharides, total phenolic acids, total saponins, and total flavonoids. The total polysaccharides, total phenolic acids, total saponins, and total flavonoids in the aqueous extract of Kuding tea holly leaves can be separated using ethanol of different concentrations. The separated total polysaccharides, total phenolic acids, total saponins, and total flavonoids are then mixed and compounded to obtain a new type of Kuding tea holly leaf extract. In other embodiments of the present invention, Kuding tea holly leaf extract specifically refers to the saponin-like active substances extracted.

[0040] Boil holly leaves of Kuding tea in water, then add distilled water at a w / v ratio of 1:10-15 to extract the active ingredients. The extraction time is 0.5-3 hours. The filtered solution is concentrated and dried to obtain the holly leaf extract of Kuding tea. Alternatively, boil holly leaves of Kuding tea in water to extract the active ingredients. After concentrating the filtrate, add 70% ethanol and collect the precipitate as total polysaccharides. The supernatant is extracted and loaded onto an adsorption resin column. Eluenting with 30% ethanol yields total phenolic acids, 50% ethanol yields total saponins, and 80% ethanol yields total flavonoids. The total polysaccharides, total phenolic acids, total saponins, and total flavonoids are mixed and dried to obtain the holly leaf extract of Kuding tea. Alternatively, the total saponin components prepared by the aforementioned method can be used as the extract of Ilex kudingcha leaves; 70% ethanol is added to the aqueous extract of Ilex kudingcha leaves to remove total polysaccharides, the separated liquid portion is loaded onto an adsorption resin column, and the total phenolic acids are removed by elution with 30% ethanol. The eluted components with 50% ethanol are then collected, concentrated, and dried to obtain the total saponins of Ilex kudingcha leaves.

[0041] Experimental results showed that *Ilex kudingcha* leaf extract significantly inhibited the levels of triglycerides, total cholesterol, and 5α-reductase in a mouse model of androgenetic alopecia; it also inhibited excessively high levels of ROS or MDA or abnormal expression of inflammatory factors TNF-α, IL-1β, IL-6, and INF-γ in mouse hair follicle stem cells induced by ultraviolet irradiation or hydrogen peroxide. *Ilex kudingcha* leaf extract can be used to improve hair loss caused by various mechanisms, such as inhibiting hair loss caused by abnormally high sebum synthesis and secretion in the scalp or excessively high androgen levels, clearing hair loss caused by excessively high levels of reactive oxygen species (ROS) induced by physical damage or chemical stimulation, and inhibiting hair loss caused by abnormal expression of inflammatory factors induced by physical, chemical, or biological factors.

[0042] Research has revealed that total saponins in holly leaves from Kuding tea significantly outperform other types of active substances in promoting hair growth. Further analysis of the total saponin composition in the extract revealed four main categories: kudinoside G, kudinoside C, kudinoside A, and kudinoside D. The synergistic effect of these different types of saponins contributes to the outstanding anti-hair loss properties of the holly leaf extract. In particular, the holly leaf extract is superior to other Kuding tea varieties such as large-leaf holly or wolfberry leaves in preventing and treating hair loss. Using holly leaves in the field of hair loss prevention can effectively reduce hair follicle cell necrosis, thereby improving hair loss and promoting hair growth. Specifically, it can significantly reduce the number of hairs lost and promote the area of ​​hair growth. Furthermore, the holly leaf extract has a high safety profile.

[0043] Using holly leaf extract from bitter tea as a functional ingredient, various topical preparations for preventing hair loss can be formulated, such as shampoos, conditioners, and hair oils. The holly leaf extract contains bitter tea saponins, with a mass percentage ranging from 0.05% to 10%. Topical preparations may also include excipients, thickeners, emulsifiers, surfactants, preservatives, fragrances, flavorings, and pigments to improve product stability and user experience; other extracts or monomers may also be added.

[0044] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0045] Example 1: Preparation and characterization of Ilex kudingcha leaf extract

[0046] 1.1 Preparation of crude extract of bitter tea and holly leaf

[0047] The active ingredients of Kuding tea holly leaves can be extracted by crushing dried Kuding tea holly leaves and then boiling them in water.

[0048] Weigh 1000g of dried bitter tea holly leaves, pulverize them, add distilled water at a ratio of w / v = 1:15 and heat to boil for 2 hours to extract; filter while hot using a 100-mesh stainless steel sieve, and dry the concentrated product using a flash evaporator (JMF-320, Xi'an). The resulting solid substance is the crude extract of bitter tea holly leaves.

[0049] 1.2 Component Analysis of Ilex kudingcha Leaf Extract

[0050] To further analyze the key active components in the holly leaves of Kuding tea, different types of active components in the holly leaf extract of Kuding tea were separated and purified. Total polysaccharides, total phenolic acids, total saponins and total flavonoids were collected, and the functions of each type of active component were analyzed and verified.

[0051] 1000g of dried Kuding tea holly leaves were weighed, pulverized, and extracted by boiling with distilled water at a w / v ratio of 1:15 for 2 hours. The extract was then filtered while hot using a 100-mesh stainless steel sieve. The filtrate was concentrated to a suspension with a density of 1.1 (approximately 2.5 L) using a flash evaporator (JMF-320, Xi'an). Ethanol was added to the Kuding tea holly leaf extract suspension to adjust the ethanol content to 70%. After mixing, the solution was allowed to stand overnight. The precipitate was washed three times with anhydrous ethanol. The main component obtained was the total polysaccharide, totaling 35.4 g. The supernatant was further concentrated to prepare a solution with a solid content of approximately 1%. The solution was loaded onto an AB-8 macroporous adsorption resin column (diameter: 5.5 cm; column height: 35 cm; column volume: approximately 850 mL) at a flow rate of 2 mL / min. The column was equilibrated with 5% ethanol solution to elute water-soluble impurities. Gradient elution was then performed with ethanol solutions of different concentrations at a rate of 1 mL / min. The components obtained from the elution with 30% ethanol solution were mainly total phenolic acids, totaling 18.7 g; the components obtained from the elution with 50% ethanol solution were mainly total saponins, totaling 21.8 g; and the components obtained from the elution with 80% ethanol solution were mainly total flavonoids, totaling 28.4 g.

[0052] 1.3 Effects of active components from bitter tea and holly leaves on the proliferation activity of hair follicle stem cells

[0053] The proliferative activity of rat hair follicle stem cells (HFSCs) was tested using the isolated total polysaccharides, total phenolic acids, total saponins, and total flavonoids. Rat hair follicle stem cells (HFSCs) were purchased from Wuhan Pronosei Life Science Technology Co., Ltd. HFSCs were placed in a special culture medium and continuously cultured in a 37 ℃, 5% CO2 incubator, with the medium changed every other day. When the cells reached 70%–80% confluence, they were passaged. HFSCs in the logarithmic growth phase were harvested and cultured at a ratio of 1×10⁻⁶. 4100 μL of HFSCs (cells / well) were seeded into 96-well plates, with four groups: control group, bitter tea holly group, total polysaccharide group, total phenolic acid group, total saponin group, and total flavonoid group. The control group used basal medium, while the other groups used basal medium solutions containing 0.1% of the corresponding components. HFSCs were seeded into the control and experimental groups, respectively, and cultured for 24 h. 10 μL of CCK-8 reagent was added to each group, and the plates were returned to the incubator for another 1 h. Absorbance was measured at 450 nm. Each group was divided into three replicates to ensure accuracy. The increase in cell proliferation in each experimental group relative to the control group was compared, and the cell proliferation enhancement rate was calculated to measure the promoting effect of different experimental groups on cell proliferation.

[0054] Cell proliferation enhancement rate % = (experimental group absorbance value - control group absorbance value) / control group absorbance value × 100%.

[0055] Depend on Figure 1 It was found that the extract of Ilex kudingcha leaves significantly promoted the proliferation of hair follicle stem cells (HFSCs), with an average cell proliferation rate increase of 37.1% compared to the control group. Its main components (total polysaccharides, total phenolic acids, total saponins, and total flavonoids) all exhibited certain proliferative activities, with the total saponin group showing the most significant promoting effect (proliferation rate increased by 58.4%). This indicates that Ilex kudingcha has the effect of enhancing the activity of hair follicle stem cells, suggesting its potential application value in preventing hair loss, with total saponins being the core active component.

[0056] 1.4 Analysis and Performance Comparison of Total Saponins in Ilex kudingcha Leaf Extract

[0057] Further analysis was conducted on the key active components of total saponins in holly leaves of bitter tea, and the components were separated and identified by UPLC-Q-TOF / MS.

[0058] Chromatographic column: CORTECS T3 (2.1 mm × 150 mm, 1.6 μm); chromatographic gradient elution conditions: mobile phase A was acetonitrile, and mobile phase B was 0.1% formic acid aqueous solution; flow rate: 0.2 mL / min; gradient elution (0–5 min: 5% A, 95% B; 5–35 min: 25%, 75% B; 35–40 min: 40% A, 60% B; 40–45 min: 75% A, 25% B; 45–50 min: 95% A, 5% B). Results are as follows. Figure 2The key saponin components are shown in the figure. Table 1 illustrates the most representative saponin components identified through secondary mass spectrometry fragment analysis. The main saponin components are kudinoside G, kudinoside C, kudinoside A, and kudinoside D. Fractions were collected based on peak times, and the four saponin components were enriched and concentrated. The fraction from 36.5 min to 40.41 min was also collected and concentrated. The activity of the four kudinoside saponins in promoting hair follicle stem cell proliferation was determined according to the method described in section 1.3, and the results are shown in Table 1. It can be seen that all four saponin components at a concentration of 10 μg / mL have activity in promoting hair follicle stem cell proliferation, but their activity is lower than that of the fraction from 36.5 min to 40.41 min (total kudinoside saponins) at the same concentration. Comparison shows that a mixture of different types of kudinoside saponins (total kudinoside saponins) has a better effect.

[0059] Table 1. Total saponin composition and activity analysis in holly leaf extract of bitter tea.

[0060]

[0061] Example 2: Determination and performance analysis of total saponin content in bitter tea holly, large-leaf holly and wolfberry

[0062] 2.1 Comparison of total saponin content in bitter tea holly, large-leaf holly, and wolfberry

[0063] A comparison was made between representative Kuding tea leaves: Ilex kudingcha, Ilex macrocarpa, and Ilex chinensis. The leaves of Ilex kudingcha, Ilex macrocarpa, and Ilex chinensis were purchased from medicinal herb markets in Guangxi Zhuang Autonomous Region, Zhejiang Province, and Guizhou Province, respectively. The total saponin content of the three raw materials was tested.

[0064] The content of total saponins in three batches of Kuding tea (Ilex kudingcha, Ilex macrocarpa, and Ilex cornuta) was determined by ultraviolet-visible spectrophotometry, using ginsenoside Re (purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number A10036) as a reference standard and vanillin-glacial acetic acid method for color development. Specifically, 100 μL of the test sample or reference solution was accurately pipetted into a 10 mL stoppered test tube, the solvent was evaporated in a 60 °C water bath, 0.2 mL of 10% vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid were added, and the mixture was incubated in a 60 °C water bath for 15 min. After that, the tube was placed in an ice-water bath for 5 min, 5 mL of glacial acetic acid was added, and the mixture was stirred well. The absorbance was measured at 550 nm.

[0065] Preparation of reference solution: Accurately weigh 10 mg of ginsenoside Re reference standard, place it in a 10 mL volumetric flask, add methanol to make up to volume and shake well to obtain a 1 mg / mL ginsenoside Re reference solution.

[0066] Accurately pipette 0, 10, 20, 40, 60, 80, and 100 μL (1 mg / mL) of ginsenoside Re standard solution and measure the absorbance (A) using the method described above. Plot a standard curve with A concentration on the x-axis (X) and A value on the y-axis (Y). The obtained linear regression equation is y = 0.0204x + 0.0017, R0 2 =0.9995.

[0067] Preparation of the test solution: Weigh 100 g each of dried bitter tea holly leaves, large-leaf holly leaves, and wolfberry leaves, add 10 times the amount of water, soak for 30 minutes, heat to boiling and extract for 1 hour, filter, concentrate the filtrate to a relative density of 1.1-1.2, dry under reduced pressure, and pulverize to obtain each extract. Accurately weigh 10 mg of each extract powder, place it in a 10 mL volumetric flask, add methanol to make up to volume and shake well to obtain the test solution.

[0068] The total saponin content in the test samples was determined according to the above method, and the results are shown in Table 2. The highest total saponin content was found in *Ilex kudingcha*, with an average content of 7.43%; the total saponin content in *Ilex macrocarpa* was 5.18%; and the total saponin content in *Ilex cornuta* was 4.96%. Figure 3 As shown, the holly variety of Kuding tea has a higher content of total saponins, an active ingredient, compared to the other two types.

[0069] Table 2. Total saponin content of three extracts (Ilex kudingcha, Ilex macrocarpa, and Ilex cornuta) from different batches.

[0070]

[0071] 2.2 Comparison of the effects of total saponins from bitter tea holly, large-leaf holly, and wolfberry on the proliferative activity of hair follicle stem cells

[0072] Following the method for preparing total saponins in Example 1, total saponins from three species—Ilex kudingcha, Ilex macrocarpa, and Ilex cornuta—were separated, and the effects of the three total saponins on the proliferation activity of hair follicle stem cells were determined at a concentration of 0.1%.

[0073] When HFSCs reach a confluence of 70%–80%, passage them and collect HFSCs in the logarithmic growth phase at a ratio of 1 × 10⁻⁶. 4100 μL of reagent (per well) was inoculated into 96-well plates, with four groups: control group, Kuding tea group, Kuding tea holly group, large-leaf holly group, and holly group. The control group used basal medium, while the other groups used basal medium solutions containing 0.1% of the corresponding total saponins. The Kuding tea group consisted of one-third each of Kuding tea holly, large-leaf holly, and holly (0.033% of each). The plates were incubated for 24 h, then 10 μL of CCK-8 reagent was added, and the plates were returned to the incubator for another h. The absorbance was measured at 450 nm. Each group was tested in triplicate to ensure accuracy.

[0074] The cell proliferation enhancement rate was calculated according to the method in Example 1, and the results are as follows: Figure 4 As shown, the total saponins in the Kuding tea group, Kuding tea holly group, large-leaf holly group, and wolfberry group all had significant proliferative effects on HFSCs. Compared with the control group, the average cell proliferation rate of total saponins in Kuding tea increased by 41.1%, in large-leaf holly by 36.8%, in wolfberry by 27.9%, and in Kuding tea holly by 57.9%. This indicates that at the same level, the activity of total saponins in Kuding tea holly is significantly higher than that in large-leaf holly or wolfberry, or a combination of the three (Kuding tea), suggesting that the potential anti-hair loss effect of Kuding tea holly is the best among various Kuding teas.

[0075] 2.3 Analysis of saponin components in Ilex kudingcha, Ilex macrocarpa, or Ilex thunbergii

[0076] Furthermore, the key active components of total saponins from bitter tea holly, large-leaf holly, or wolfberry were identified and compared, and the components were separated and identified by UPLC-Q-TOF / MS.

[0077] Following the mass spectrometry analysis conditions shown in Example 1, parallel measurements were performed on three samples at 10 mg / mL, and the results are as follows: Figure 5 As shown, the three substances exhibit similar component categories, while their individual component contents also show significant differences. Based on a comparison of relative peak areas, the calculated contents of kudinoside G, kudinoside C, kudinoside A, and kudinoside D in Kuding tea holly are 1.57 times that in large-leaf holly and 2.54 times that in holly. This indicates that the content of active holly saponins in Kuding tea holly is significantly higher than that in large-leaf holly or holly.

[0078] Simultaneously, the fraction from 36.5 min to 40.41 min was collected and concentrated. The activity of four types of Kuding tea holly saponins in promoting hair follicle stem cell proliferation was determined according to the method described in Example 1, and the results are shown in Table 3. It can be seen that the saponins from Kuding tea holly, large-leaf holly, or wolfberry holly all exhibited activity in promoting hair follicle stem cell proliferation at a concentration of 10 μg / mL, but the activities varied considerably. The holly saponins from Kuding tea holly showed the best activity, significantly superior to those from large-leaf holly or wolfberry holly. This indicates that different total saponin extracts exhibit different abilities to promote hair follicle stem cell proliferation due to differences in the ratio of saponin components.

[0079] Table 3. Proliferative Activity of Ilex kudingcha saponins from Ilex kudingcha, Ilex macrocarpa, or Ilex cornuta

[0080]

[0081] The isolated total saponin active components were used as the extract of Ilex kudingcha leaves for subsequent experiments.

[0082] Example 3: Preparation of an extract containing bitter tea and holly leaf extract

[0083] The ingredients of the serum are shown in Table 4.

[0084] Table 4 Raw material composition and content

[0085]

[0086] In preparation, all raw materials except water in the aqueous phase are weighed into a beaker according to the above proportions, stirred evenly with a glass rod, and then distilled water is added. The beaker containing the aqueous phase is placed in a water bath and heated at 85°C with stirring for 20 to 30 minutes. After cooling to 45°C, the active ingredient is added and stirred evenly to obtain the different groups of essences. The product without added active ingredient is the blank matrix.

[0087] Following the above method, extracts of Kuding tea holly, large-leaf holly, and wolfberry were prepared with contents of 0.05%, 2.5%, and 10%, respectively.

[0088] Example 4: Effects of Kuding tea and Ilex chinensis leaf extract on androgenetic alopecia model mice

[0089] 4.1 Effects of different types of bitter tea extracts on mouse models

[0090] The experimental animals were male C57BL / 6J mice, 7 weeks old, weighing approximately 22 g, purchased from the Beijing Vital River Laboratory Animal Center. The C57BL / 6J mice were housed under normal conditions: temperature 25±2℃, humidity 50%–70%, alternating between 12-hour light and dark cycles, with free access to food and water, and regular bedding changes. Experiments began after the mice had adapted to their enclosure. Mice were anesthetized by intraperitoneal injection of 100 μL of 4% chloral hydrate. After anesthesia, all hair on the back was shaved using a shaver, and then depilated using commercially available depilatory wax (area 2 cm × 2.5 cm).

[0091] Thirty male C57BL / 6J mice were randomly divided into six groups: blank control group, model group, bitter tea group, bitter tea holly group, large-leaf holly group, and wolfberry group. The effective ingredient (active substance) in each test group was added to the essence at a concentration of 2.5%. The bitter tea group consisted of one-third each of bitter tea holly, large-leaf holly, and wolfberry, i.e., 0.833% added to each. Except for the blank control group, the other groups were treated daily with 200 μL of testosterone (5 mg / mL) for modeling. After complete absorption, 200 μL of the test substance was applied. The model group was treated with a blank matrix essence without active substances, while the other experimental groups were treated with essences containing the corresponding effective ingredients; this treatment lasted for 14 consecutive days.

[0092] On day 14, hair morphology was photographed using a scalposcope, and hair growth on the backs of the mice was recorded. A 1cm... 2 A tear-off test was conducted on the back of the test area using transparent tape. The number of hairs removed was counted to reflect the anti-hair loss effect of each group. The experimental results are as follows: Figure 6 As shown, compared with the blank group, the hair density in the modeling area of ​​the model group was significantly reduced, indicating that the modeling was successful. Compared with the model group, the Kuding tea holly group, the large-leaf holly group, and the holly group all showed some improvement in androgenetic alopecia. Among them, the hair loss prevention effect of Kuding tea holly was significantly better than the combination of the three (Kuding tea group), the large-leaf holly group, or the holly group.

[0093] Skin samples from the bald areas of mice were collected, and the levels of triglycerides and total cholesterol were measured using a kit. The expression level of 5α-reductase mRNA in each group was detected by qRT-PCR (specific primers required for qRT-PCR were designed using Primer 5.0 software and synthesized by Shanghai Sangon Biotech Co., Ltd., and their sequences are shown in Table 5). Results are as follows: Figure 7 As shown, the detection of key lipid metabolism indicators, total cholesterol and triglyceride levels, in the androgenic alopecia model revealed that the Kuding tea and wintergreen group significantly inhibited androgen-induced lipid metabolism disorders and reduced total cholesterol in the alopecia model. Figure 7 A) and triglycerides ( Figure 7 The level of B) inhibits 5α-reductase (B) Figure 7 C) activity, reducing the production of dihydrotestosterone, regulating lipid balance, and thus inhibiting the occurrence of androgen-dependent hair loss; and, comparative analysis showed that the inhibitory effect of the bitter tea holly group on various indicators was significantly better than that of the large-leaf holly and wolfberry treatment groups.

[0094] 4.2 Effects of different contents of bitter tea and holly leaf extract on model mice

[0095] Mice were fed and modeled according to the aforementioned steps. Thirty male C57BL / 6J mice were randomly divided into six groups: blank control group, model group, minoxidil group, low-dose (0.05%) holly extract group, medium-dose (2.5%) holly extract group, and high-dose (10%) holly extract group. Except for the blank control group, the other groups were treated daily with 200 μL of testosterone (5 mg / mL) evenly for modeling. After complete absorption, 200 μL of the test substance was applied. The minoxidil group used commercially available 5% minoxidil tincture, while the model group used a blank matrix without active ingredients as a control, for 14 consecutive days. After anesthetizing the mice, they were then subjected to treatment with a 1 cm... 2 A pull-out test was conducted on the back test area using transparent tape. The number of hairs removed was counted to reflect the anti-hair loss effect of each group. Hair that grew out in the shaved area was then shaved off with a razor, collected on weighing paper, and weighed using a 1 / 100,000 electronic balance.

[0096] The results are as follows Figure 8 As shown, the extract of Ilex kudingcha leaves can affect both the amount and weight of hair loss in mice with androgenetic alopecia. It is evident that the addition of 0.05%, 2.5%, and 10% doses of Ilex kudingcha leaves extract significantly inhibited hair loss in androgenetic alopecia mice and increased hair weight in the bald areas. Comparison shows that the treatment groups with 2.5% and 10% Ilex kudingcha leaves extract were more effective than those with 5% minoxidil.

[0097] Example 5: Effects of Kuding tea and Ilex chinensis leaf extract on a UV-induced mouse hair follicle stem cell model

[0098] The extract of Ilex kudingcha leaves can affect hair loss caused by oxidative stress or inflammation induced by physical factors. A rat hair follicle stem cell model induced by ultraviolet irradiation was constructed to evaluate the effect of Ilex kudingcha leaves.

[0099] When HFSCs reach a confluence of 70%–80%, they are passaged, and HFSCs in the logarithmic growth phase are selected and divided into (1×10⁻⁶) groups. 4100 μL of cells / well were seeded into 96-well plates, with a control group (basal medium) and a Kuding tea holly group (basal medium containing 0.1% Kuding tea holly). The cells were cultured for 24 h, and then the cell culture plates were placed under a UV crosslinker to construct a UV damage model at 750 mJ / cm². 2 Irradiation was performed for 30 min under the given conditions. After irradiation, the ROS levels in each group of cells were measured using a ROS detection kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.). The results are as follows: Figure 9 As shown.

[0100] Depend on Figure 9 It was found that, compared with the control group, after 24 h of intervention with HFSCs cells using Kuding tea holly leaf extract, the ROS level in the Kuding tea holly group was lower than that in the control group, significantly inhibiting ROS release with an average inhibition rate of 49.0%, thus reducing the occurrence of cellular oxidative stress damage. The difference was statistically significant (P < 0.05).

[0101] In addition, HFSCs in the same logarithmic growth phase were also selected, according to (1×10) 6 Cells were seeded in 6-well plates at 2 mL per well. A control group (basal medium) and a *Ilex kudingcha* (basal medium containing 0.1% *Ilex kudingcha* leaf extract) group were established to construct UV-induced injury models. Cells were collected, RNA was extracted, and the expression levels of inflammatory factors TNF-α, IL-1β, IL-6, and INF-γ mRNA in each group were detected by qRT-PCR. Specific primers required for qRT-PCR were designed using Primer 5.0 software and synthesized by Shanghai Sangon Biotech Co., Ltd. The primer sequences are shown in Table 5.

[0102] Table 5 qRT-PCR primer sequences

[0103]

[0104] The results are as follows Figure 10 As shown, Kuding tea holly has a greater effect on key inflammatory factors TNF-α, IL-1β, IL-6 and INF-γ in HFSCs cells; after treatment with Kuding tea holly leaf extract, cells can significantly inhibit the inflammatory response induced by ultraviolet light irradiation.

[0105] Example 6: Effects of Kuding tea and Ilex chinensis leaf extract on hydrogen peroxide-induced mouse hair follicle stem cell model

[0106] The extract of Ilex kudingcha leaves can affect hair loss caused by oxidative stress or inflammation induced by chemical factors. The effect of the extract was evaluated by constructing a hydrogen peroxide-induced rat hair follicle stem cell model.

[0107] When HFSCs reach a confluence of 70%–80%, they are passaged, and HFSCs in the logarithmic growth phase are selected and divided into (1×10⁻⁶) groups. 4 100 μL (per well) was seeded into a 96-well plate and then inoculated at a rate of (1 × 10⁻⁶) μL. 6 2 mL of the culture medium (cells / well) was seeded into 6-well plates, with a control group (basal culture medium) and a Kuding tea holly group (basal culture medium containing 0.1% Kuding tea holly leaf extract). The cells were cultured for 24 h, and then induced with 200 μmol / L hydrogen peroxide for 2 h to construct a hydrogen peroxide-induced mouse hair follicle stem cell model. ROS (96-well plate) or inflammatory factors (6-well plate) were measured in the cells according to the method in Example 5.

[0108] like Figure 11 As shown, compared with the control group, after 24 h of intervention with HFSCs cells using Kuding tea and wintergreen, the ROS level in the Kuding tea group was lower than that in the control group, significantly inhibiting ROS release and reducing the occurrence of cellular oxidative stress damage. The difference was statistically significant (P < 0.05). Figure 12 As shown, the extract of holly leaves from bitter tea can also significantly inhibit the inflammatory response in cells induced by hydrogen peroxide stimulation.

[0109] Example 7: Evaluation of the efficacy of Kuding tea and holly leaf extract on a chemotherapy-induced alopecia model mouse.

[0110] Thirty male C57BL / 6J mice were randomly divided into six groups: control group, model group, minoxidil group, low-dose (0.05%) holly extract group, medium-dose (2.5%) holly extract group, and high-dose (10%) holly extract group. Mice were intraperitoneally injected with 100 μL of 4% chloral hydrate. After anesthesia, all hair on the back of the mice was shaved, and commercially available depilatory wax (area 2 cm × 2.5 cm) was used for hair removal. Following hair removal, mice were treated with the corresponding test substance, 200 μL once daily for 14 consecutive days. On day 9 after hair removal, when hair entered growth phase VI, mice in all groups except the control group were intraperitoneally injected with 0.1 ml / 10 g B.W. 80 mg / kg cyclophosphamide injection to establish a chemotherapy-induced alopecia mouse model. The control group was injected with physiological saline. On day 6 after cyclophosphamide injection, hair loss reached its peak. The mice were anesthetized, and then a 1cm... 2 A pull-out test was conducted on the back test area using transparent tape. The number of hairs removed was counted to reflect the anti-hair loss effect of each group. Hair that grew out in the shaved area was then shaved off with a razor, collected on weighing paper, and weighed using a 1 / 100,000 electronic balance.

[0111] The results are as follows Figure 13 As shown, extracts containing different doses of Ilex kudingcha leaf extract significantly inhibited chemotherapy-induced hair loss and increased hair weight. In particular, the 2.5% and 10% Ilex kudingcha leaf extract treatments were more effective than minoxidil.

[0112] Example 8: Evaluation of the efficacy of bitter tea and holly leaf extract on a natural hair regrowth model

[0113] Twenty-five male C57BL / 6J mice were randomly divided into five groups: control group, minoxidil group, low-dose (0.05%) extract of *Ilex kudingcha*, medium-dose (2.5%) extract of *Ilex kudingcha*, and high-dose (10%) extract of *Ilex kudingcha*. Seven-week-old C57BL / 6 mice were in a quiescent phase of hair growth arrest, allowing for the construction of a natural hair growth model. Hair follicles were induced to synchronously enter the anagen phase, and these follicles were morphologically indistinguishable from spontaneously formed anagen follicles. Mice were treated with the test substance after waxing, once daily at 200 μL for 14 consecutive days. The mice were anesthetized and then subjected to a 1cm... 2 A pull-out test was conducted on the back test area using transparent tape. The number of hairs removed was counted to reflect the anti-hair loss effect of each group. Hair that grew out in the shaved area was then shaved off with a razor, collected on weighing paper, and weighed using a 1 / 100,000 electronic balance.

[0114] The results are as follows Figure 14 As shown, minoxidil solution and different concentrations of Kuding tea holly can increase the speed of hair growth, promote hair growth, increase hair weight, and reduce the amount of hair loss caused by tape removal. Furthermore, the group with a high amount of Kuding tea holly added showed better results than the minoxidil group.

[0115] Example 9: Evaluation of the therapeutic effect of bitter tea and holly leaf extract on a shaved mouse model

[0116] Experimental mice were divided into a control group, a minoxidil group, a low-dose (0.05%) holly extract group, a medium-dose (2.5%) holly extract group, and a high-dose (10%) holly extract group. 7-8 week old C57BL / 6 mice were in a telogen phase of hair regrowth, making them suitable for establishing a shaving model. All telogen hairs were shaved off using a shaver. After shaving, the mice were treated with the test substance, 200 μL once daily for 14 consecutive days. The mice were anesthetized and then subjected to a 1 cm... 2 A pull-out test was conducted on the back test area using transparent tape. The number of hairs removed was counted to reflect the anti-hair loss effect of each group. Hair that grew out in the shaved area was then shaved off with a razor, collected on weighing paper, and weighed using a 1 / 100,000 electronic balance.

[0117] The results are as follows Figure 15 As shown, the extract of Ilex kudingcha leaves significantly improved both the amount and weight of hair loss in shaved mouse models.

[0118] As verified by Examples 4-9, the extract of holly leaves from bitter tea exhibits significant therapeutic effects on different types of hair loss and has the function of preventing and treating hair loss.

[0119] Example 10: Local toxicological evaluation of holly leaf extract from bitter tea

[0120] Acute oral toxicity tests, multiple skin irritation tests, acute eye irritation tests, and skin allergy tests were conducted on the extract of *Ilex kudingcha* leaf according to the procedures outlined in the *Cosmetic Safety Technical Specifications* (2015 edition). All local toxicity tests showed no toxicity, as shown in Tables 6-9; the results indicate that *Ilex kudingcha* leaf extract has a high degree of safety.

[0121] Table 6 Results of Acute Oral Toxicity Tests

[0122]

[0123] Table 7 Results of multiple skin irritation tests on rabbits using Kuding tea and Ilex chinensis leaf extract.

[0124]

[0125] Table 8 Results of acute eye irritation test on rabbits by bitter tea and wintergreen.

[0126]

[0127] Table 9 Results of phototoxicity test of Kuding tea and Holly on guinea pig skin

[0128]

[0129] Example 11: Evaluation of the efficacy of bitter tea holly leaf extract on people with hair loss and thinning hair.

[0130] Subjects were recruited in accordance with Annex 8 of the Cosmetic Safety Technical Specifications (2015 Edition) Announcement (No. 17, 2021) to evaluate the efficacy of the 2.5% Kuding tea extract prepared in Example 3 of this invention. 1 mL of the anti-hair loss extract was sprayed onto the desired area 1-2 times daily, ensuring contact with the scalp; massage could be used to aid absorption. The desired area refers to the area of ​​hair loss or characteristic areas (such as thinning hair, receding hairline, or areas with reduced hair on the crown). The test product was used for 12 weeks. During the trial, subjects were required to record the usage time, evaluation of the effects during use, and any discomfort experienced.

[0131] Evaluation criteria for the therapeutic effects of bitter tea on humans:

[0132] (1) Ineffective: No improvement in hair loss, no new hair grows in the bald areas or no improvement in hair loss;

[0133] (2) Effective: Hair loss is improved, and some bald areas regrow fine hairs or partially improve the hair loss phenomenon;

[0134] (3) Significant effect: hair loss is significantly reduced, hair grows back in the bald areas or the hair loss phenomenon is significantly improved;

[0135] The results showed that among the 30 people who received clinical physiotherapy, 3 were ineffective, 4 were effective, and 23 were significantly effective; the significant effect rate was 76.6%, and the total effective rate was 90%.

[0136] To demonstrate the excellent therapeutic effect of this invention, typical cases are listed in Table 10;

[0137] Table 10

[0138]

[0139] The following is a partial microscopic image of hair. Figure 16 As shown, after using the Kuding tea and holly extract, hair density and the diameter of new hair growth were significantly improved. Combined with the subjects' self-reported results, Kuding tea and holly had a significant effect on hair loss and thinning hair.

[0140] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. The application of bitter tea holly leaf extract in the preparation of topical anti-hair loss formulations, characterized in that: Saponins were extracted from the leaves of Ilex kudingcha to obtain Ilex kudingcha leaf extract. The obtained extract can be used to prepare topical anti-hair loss preparations to relieve hair loss caused by oxidative stress or to improve hair loss caused by inflammatory response. The active ingredients of Kuding tea holly leaves were extracted by decoction. The filtrate was added with 70% ethanol to remove total polysaccharides. The separated liquid was loaded onto an adsorption resin column and eluted with 30% ethanol to remove total phenolic acids. The eluted fraction with 50% ethanol was then collected, which is the total saponins of Kuding tea holly leaves.

2. The application according to claim 1, characterized in that: The total saponins obtained from the leaves of Ilex kudingcha were concentrated and dried to obtain Ilex kudingcha leaf extract. The mass percentage of Ilex kudingcha leaf extract in topical preparations was 0.05%–10%.

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