Glycycoumarin extract for preventing hair loss or promoting hair growth, composition, and use

Abstract

Disclosed are a glycycoumarin extract for preventing hair loss or promoting hair growth, a composition, and use. Specifically, the present invention relates to new use of glycycoumarin in the preparation of a product for preventing hair loss or promoting hair growth. Glycycoumarin is derived from a glycycoumarin extract extracted and purified from a licorice raw material. The present invention also relates to the glycycoumarin extract and a composition comprising the extract. The present invention demonstrates for the first time that glycycoumarin can effectively prevent hair loss and promote the proliferation and differentiation of hair follicle cells, has the effect of significantly preventing hair loss and promoting hair growth, has no cytotoxicity, and has high safety, thereby filling a technical gap of the substance in the fields of hair loss prevention and hair growth. The glycycoumarin extract of the present invention can be industrially produced and prepared, and the method is simple, highly efficient, cost-effective, and controllable, providing favorable conditions for wide application of glycycoumarin.

Description

Licorice coumarin extract and its composition and applications for preventing hair loss or promoting hair growth. Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a glycyrrhizin extract, composition, and application for preventing hair loss or promoting hair growth. Background Technology

[0002] Hair loss refers to the phenomenon of hair falling out due to various reasons, encompassing both physiological and pathological hair loss. Normal physiological hair loss stems from the hair's own growth cycle; losing 50-100 hairs per day is within the normal range, and new hair will gradually grow in. Pathological hair loss, however, has complex causes, including genetic factors, hormonal imbalances, nutritional deficiencies, chronic stress and anxiety, unhealthy lifestyle habits, fungal and bacterial infections, head trauma or surgery, or exposure to certain chemicals, leading to congenital or acquired hair loss. Currently, hair loss is an increasingly serious problem. Data shows that over 250 million people in my country suffer from hair loss, approximately 163 million men and 88 million women. Furthermore, the hair loss population is becoming increasingly younger, with a significantly higher proportion of people experiencing hair loss before the age of 30 compared to the previous generation. Over 50% of patients report that hair loss severely impacts their quality of life. Therefore, research and development to address hair loss is urgently needed.

[0003] Currently, there are many treatments for hair loss, each with its own advantages and disadvantages. In terms of drug treatment, anti-androgen drugs such as finasteride (for men) and spironolactone (for women) can be used to treat androgenetic alopecia. Combined with topical minoxidil, they can improve hair loss. However, finasteride may have side effects such as decreased libido and requires long-term use; hair loss may recur once discontinued. Minoxidil may initially worsen hair loss, and some people may experience mild scalp itching and redness. For alopecia areata, topical minoxidil, and if necessary, topical corticosteroids such as halometasone ointment, or oral prednisone, can promote hair growth. However, long-term use of corticosteroids may cause skin atrophy, hirsutism, and steroid-dependent dermatitis. For tinea capitis caused by fungal infection, antifungal drugs such as terbinafine and ketoconazole are needed, along with disinfection of related items. Antifungal drugs may cause damage to organs such as the liver, and the treatment period is relatively long. In surgical treatment, autologous hair transplantation is an effective option for treating severe and permanent hair loss, significantly improving appearance. However, the surgery is expensive and carries risks such as infection and low follicle survival rate, and postoperative recovery takes time. Alternatively, fractional laser treatment can induce hair growth by promoting local blood flow and angiogenesis; however, this method requires multiple treatments, the effects are short-lived, and the treatment process may be accompanied by pain and discomfort.

[0004] Licorice is a perennial herb belonging to the genus Glycyrrhiza in the family Leguminosae. It is a traditional Chinese medicine. The Chinese Pharmacopoeia includes three varieties as sources of medicinal licorice: Ural licorice (Glycyrrhiza uralensis Fisch.), inflated licorice (Glycyrrhiza inflata Bat.), and smooth licorice (Glycyrrhiza glabra L.). Due to its dual nature as both food and medicine, it is listed as a traditional Chinese medicine with both medicinal and edible properties by the National Health Commission. Licorice is used to treat symptoms such as spleen and stomach weakness, fatigue, palpitations, shortness of breath, cough with excessive phlegm, abdominal and limb spasms and pain, carbuncles and boils, and to alleviate the toxicity and harshness of other drugs.

[0005] It is evident that existing medications for treating hair loss cannot meet market demands and urgently require further improvement. The development of a new glycyrrhizin coumarin extract, its composition, and its application for preventing hair loss or promoting hair growth, enabling a safe, reliable, and effective solution to hair loss, has become a pressing goal for the industry.

[0006] Summary of the Invention

[0007] One of the technical problems to be solved by the present invention is to provide the use of glycyrrhizin in the preparation of products for preventing hair loss or promoting hair growth.

[0008] Another technical problem to be solved by the present invention is to provide a glycyrrhizin extract for preventing hair loss or promoting hair growth.

[0009] Another technical problem to be solved by the present invention is to provide a composition for preventing hair loss or promoting hair growth.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0011] This invention provides the use of glycyrrhizin in the preparation of products for preventing hair loss or promoting hair growth.

[0012] Furthermore, glycyrrhizin can promote the production of VEGF, increase the expression of β-catenin gene, and reduce the expression of DKK1 gene, TGF-β2 gene and AR gene, so as to prevent androgenetic alopecia or promote hair growth.

[0013] Furthermore, the product is a pharmaceutical or cosmetic product, and the content of glycyrrhizin in the pharmaceutical or cosmetic product is 0.01~2%, more preferably 0.2~1%.

[0014] Furthermore, the glycyrrhizin is derived from a glycyrrhizin extract purified from licorice raw materials, wherein the purity of glycyrrhizin in the glycyrrhizin extract is ≥10%.

[0015] The present invention also provides a glycyrrhizin coumarin extract for preventing hair loss or promoting hair growth, wherein the glycyrrhizin coumarin extract is obtained by extraction and purification of licorice raw material, and the purity of glycyrrhizin coumarin in the glycyrrhizin coumarin extract is ≥10%.

[0016] Furthermore, the extraction and purification method for the glycyrrhizin extract is as follows:

[0017] Q1: Select licorice raw materials, dry them to remove moisture, and cut the licorice into thin slices or crush them;

[0018] Q2: Add sliced ​​or crushed licorice to an extraction vessel and extract by reflux with 5-20 mL / g of ethyl acetate;

[0019] Q3: Combine the extracts and concentrate them into a paste. The resulting extract paste is then redissolved in ethyl acetate.

[0020] Q4: Add silica gel to the ethyl acetate reconstituted solution obtained in Q3. The amount of silica gel is 1 / 30 to 1 / 5 of the extract. After stirring, let it stand for 30 to 120 minutes. Take the supernatant and concentrate it. Dissolve it in a mixed solvent of ethyl acetate and n-hexane, wherein the content of ethyl acetate in the mixed solvent of ethyl acetate and n-hexane is 30 to 60%.

[0021] Q5: The mixed solution from Q4 was wet-coated onto a silica gel column and eluted with a hexane-ethyl acetate system. The ratio of hexane to ethyl acetate during elution was 100:0, 80:20, and 70:30, respectively. The eluents were collected in fractions and analyzed by high performance liquid chromatography. The eluents of different purities of glycyrrhizin were combined and concentrated under reduced pressure to obtain glycyrrhizin extracts of different purities.

[0022] Furthermore, in step Q1, the licorice used is from Korla, Xinjiang and Zhangye, Gansu, and the slice thickness is 0.5-4 mm; in step Q2, the extraction time of ethyl acetate is 30-120 min, the extraction temperature is 60-90℃, and the extraction is performed 2-3 times; in step Q3, the amount of ethyl acetate used for redissolving is 6-15 mL / g; in step Q4, the silica gel added is 300-400 mesh chromatography silica gel, and the amount of ethyl acetate-n-hexane mixed solvent used is 5-10 mL / g; in step Q5, the sample loading volume on the silica gel column is 1 / 15-1 / 40 of the amount of silica gel used, wherein the washing is performed with 100:0 n-hexane and ethyl acetate for 3-4 column volumes, with 80:20 n-hexane and ethyl acetate for 5-6 column volumes, and with 70:30 n-hexane and ethyl acetate for 10-12 column volumes.

[0023] Furthermore, in step Q5, the eluent with a glycyrrhizin purity ≥85% is combined, concentrated under reduced pressure to form an extract, and dissolved in one or more solvents selected from petroleum ether, diethyl ether, methyl tert-butyl ether, n-pentane, ethyl acetate, n-hexane, cyclohexane, dichloromethane, and ethanol. Then, glycyrrhizin crystals are obtained by evaporation crystallization or cooling crystallization. After pulverization, glycyrrhizin powder is obtained, wherein the purity of glycyrrhizin is ≥98%.

[0024] The present invention also provides a composition for preventing hair loss or promoting hair growth, the composition comprising the above-mentioned glycyrrhizin extract for preventing hair loss or promoting hair growth.

[0025] Furthermore, the composition is a topical medicine or cosmetic, wherein the content of glycyrrhizin in the composition is 0.01-2%, more preferably 0.2-1%, and it also includes physiologically acceptable excipients. The composition is in the form of a solution, ointment, plaster, gel, cream, lotion, liniment, tincture, emulsion, or patch.

[0026] With this design, the present invention has at least the following advantages:

[0027] 1. This invention demonstrates through experiments that glycyrrhizin can promote VEGF production, increase β-catenin gene expression, and decrease the expression levels of DKK1, TGF-β2, and AR genes. Specifically, it is the first time that glycyrrhizin has been shown to promote hair follicle angiogenesis, regulate the hair follicle growth cycle, promote hair follicle stem cell differentiation, enhance hair follicle cell survival and anti-apoptosis capabilities, regulate the cytokine and growth factor network surrounding the hair follicle, reduce inhibition of the Wnt / β-catenin signaling pathway, reduce hair follicle cell apoptosis, reduce the arrest of the hair follicle cell growth cycle, and reduce sebaceous gland cell proliferation and sebum secretion. Therefore, it can effectively prevent hair loss and promote hair follicle cell proliferation and differentiation, achieving the goals of preventing hair loss and promoting hair growth. In other words, this invention provides a new use for glycyrrhizin in preventing hair loss or promoting hair growth, with a clear mechanism, definite efficacy, no cytotoxicity, and high safety, filling a technological gap in the field of hair loss prevention and hair regrowth.

[0028] 2. The glycyrrhizin of this invention is obtained by direct extraction and purification from licorice raw material. By controlling the purification steps, glycyrrhizin extracts of different purity levels can be obtained according to requirements, providing an industrial production process for the widespread application of glycyrrhizin. The preparation method is simple, the extraction efficiency is high, and the process cost is low and controllable, which can provide favorable conditions for further research and widespread application of glycyrrhizin.

[0029] Attached Figure Description

[0030] The above is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0031] Figure 1 shows the effect of each group on the VEGF content in hair papilla cells in the hair papilla cell anti-hair loss activity test of Example 6 of the present invention.

[0032] Figure 2 shows the effect of each group on the expression of the DKK1 gene in dermal papilla cells during the anti-hair loss activity test of dermal papilla cells in Example 6 of the present invention.

[0033] Figure 3 shows the effect of each group on the expression of TGF-β2 gene in hair papilla cells in the hair papilla cell anti-hair loss activity test of Example 6 of the present invention.

[0034] Figure 4 shows the effect of each group on the expression of AR gene in dermal papilla cells in the hair loss prevention activity test of dermal papilla cells in Example 6 of the present invention.

[0035] Figure 5 shows the effect of each group on the expression of β-catenin gene in dermal papilla cells in the anti-hair loss activity test of dermal papilla cells in Example 6 of the present invention.

[0036] Figure 6 shows the hair growth on the backs of mice in each group 12 days after administration of the drug in Example 7 of the present invention.

[0037] Detailed Implementation

[0038] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the embodiments shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0039] Example 1

[0040] A glycyrrhizin coumarin extract, the preparation method of which includes the following steps:

[0041] (1) Raw material pretreatment: Select licorice from Zhangye, Gansu, dry it, and cut it into 1mm thin slices.

[0042] (2) Extraction: Take 5 kg of the treated licorice and add it to the extraction tank. Add 50 L of ethyl acetate and heat under reflux for extraction at 80 °C. Extract three times and combine the extracts. The first extraction time is 60 min, and the second and third extraction times are 75 min.

[0043] (3) Concentration: The extract obtained in (2) above is concentrated into a paste to obtain crude licorice extract. The paste is redissolved in 6 mL / g ethyl acetate.

[0044] (4) Impurity removal: Add silica gel of 1 / 20 of the weight of the extract to the ethyl acetate solution obtained in (3) above. The silica gel is 300-400 mesh chromatography silica gel. After stirring, let stand for 60 min, take the supernatant, concentrate the supernatant into an extract, and dissolve it with a mixed solvent of ethyl acetate-n-hexane. The amount of mixed solvent used is 8 mL / g, and the content of ethyl acetate in the mixed solvent of ethyl acetate-n-hexane is 40%. The column sample is obtained.

[0045] (5) Silica gel column separation: The sample obtained in (4) above is separated by wet silica gel column separation. The silica gel is 300-400 mesh chromatography silica gel with a column diameter-to-height ratio of 1:8. The sample loading amount is 1 / 30 of the silica gel volume. First, wash with n-hexane-ethyl acetate system for 3-4 column volumes, where the volume fraction of ethyl acetate in the n-hexane-ethyl acetate system is 0%; then wash with n-hexane-ethyl acetate system for 5-6 column volumes, where the volume fraction of ethyl acetate in the n-hexane-ethyl acetate system is 20%; finally, wash with n-hexane-ethyl acetate system for 10-12 column volumes, where the volume fraction of ethyl acetate in the n-hexane-ethyl acetate system is 30%. Collect the eluent in segments according to the method of collecting one bucket for each column volume.

[0046] Meanwhile, high performance liquid chromatography was used to detect glycyrrhizin in each eluent. The eluents with glycyrrhizin content ≥10% were combined and concentrated under reduced pressure to obtain a glycyrrhizin extract with a purity ≥20%.

[0047] The chromatographic conditions for the high-performance liquid chromatograph are as follows: C 18 The chromatographic column was 4.6 × 250 mm and 5 μm. Mobile phase A was methanol and mobile phase B was pure water. The elution was carried out with a gradient of 70% A to 90% A at a flow rate of 1 mL / min. The column temperature was 25 °C, the injection volume was 10 μL, and the detection wavelength was 254 nm.

[0048] Example 2

[0049] A glycyrrhizin coumarin extract, the preparation method of which includes the following steps:

[0050] (1) Raw material pretreatment: Licorice from Korla, Xinjiang was selected, dried, and pulverized.

[0051] (2) Extraction: Take 10 kg of the treated licorice and add it to the extraction tank. Add 100 L of ethyl acetate and heat under reflux for extraction at 75 °C. Extract three times and combine the extracts. The first extraction time is 60 min, and the second and third extraction times are 75 min.

[0052] (3) Concentration: The extract obtained in (2) above is concentrated into a paste to obtain crude licorice extract. The paste is redissolved in 10 mL / g ethyl acetate.

[0053] (4) Impurity removal: Add silica gel of 1 / 10 of the weight of the extract to the ethyl acetate solution obtained in (3) above. The silica gel is 300-400 mesh chromatography silica gel. After stirring, let stand for 60 min, take the supernatant, concentrate the supernatant into an extract, and dissolve it with a mixed solvent of ethyl acetate-n-hexane. The amount of mixed solvent used is 8 mL / g, and the content of ethyl acetate in the mixed solvent of ethyl acetate-n-hexane is 40%. The column sample is obtained.

[0054] (5) Silica gel column separation: The sample obtained in (4) above is wet-coated onto a silica gel column for separation. The silica gel is 300-400 mesh chromatography silica gel with a column diameter-to-height ratio of 1:8. The sample loading amount is 1 / 25 of the silica gel volume. First, wash with a hexane-ethyl acetate system for 3-4 column volumes, where the volume fraction of ethyl acetate in the hexane-ethyl acetate system is 0%; then wash with a hexane-ethyl acetate system for 5-6 column volumes, where the volume fraction of ethyl acetate in the hexane-ethyl acetate system is 20%; finally, wash with a hexane-ethyl acetate system for 10-12 column volumes, where the volume fraction of ethyl acetate in the hexane-ethyl acetate system is 30%. Collect the eluent in segments according to the method of collecting one bucket for each column volume.

[0055] Meanwhile, high performance liquid chromatography was used to detect glycyrrhizin in each eluent. The eluents with glycyrrhizin content ≥40% were combined and concentrated under reduced pressure to obtain a glycyrrhizin extract with a purity ≥60%.

[0056] Example 3

[0057] A glycyrrhizin coumarin extract, the preparation method of which includes the following steps:

[0058] (1) Raw material pretreatment: Select licorice from Zhangye, Gansu, dry it, and cut it into 1mm thin slices.

[0059] (2) Extraction: Take 10 kg of the treated licorice and add it to the extraction tank. Add 100 L of ethyl acetate and heat under reflux for extraction at 80 °C. Extract three times and combine the extracts. The first extraction time is 60 min, and the second and third extraction times are 75 min.

[0060] (3) Concentration: The extract obtained in (2) above is concentrated into a paste to obtain crude licorice extract. The paste is redissolved in 10 mL / g ethyl acetate.

[0061] (4) Impurity removal: Add 1 / 20 of the weight of the extract to the ethyl acetate solution obtained in (3) above. The silica gel is 300~400 mesh chromatography silica gel. After stirring, let stand for 90 min, take the supernatant, concentrate the supernatant into an extract, and dissolve it with a mixed solvent of ethyl acetate-n-hexane. The amount of mixed solvent used is 6 mL / g, and the content of ethyl acetate in the mixed solvent of ethyl acetate-n-hexane is 50%. The column sample is obtained.

[0062] (5) Silica gel column separation: The sample obtained in (4) above is separated by wet silica gel column separation. The silica gel is 300-400 mesh chromatography silica gel with a column diameter-to-height ratio of 1:8. The sample loading amount is 1 / 20 of the silica gel volume. First, wash with n-hexane-ethyl acetate system for 3-4 column volumes, where the volume fraction of ethyl acetate in the n-hexane-ethyl acetate system is 0%; then wash with n-hexane-ethyl acetate system for 5-6 column volumes, where the volume fraction of ethyl acetate in the n-hexane-ethyl acetate system is 20%; finally, wash with n-hexane-ethyl acetate system for 10-12 column volumes, where the volume fraction of ethyl acetate in the n-hexane-ethyl acetate system is 30%. Collect the eluent in segments according to the method of collecting one bucket for each column volume.

[0063] Meanwhile, high performance liquid chromatography was used to detect glycyrrhizin in each eluent. The eluents with a glycyrrhizin content ≥60% were combined and concentrated under reduced pressure to obtain a glycyrrhizin extract with a purity ≥80%.

[0064] Example 4

[0065] A glycyrrhizin coumarin extract, the preparation method of which includes the following steps:

[0066] (1) Raw material pretreatment: Select licorice from Korla, Xinjiang, dry the moisture, and cut the licorice into 1mm thin slices.

[0067] (2) Extraction: Take 10 kg of the treated licorice and add it to the extraction tank. Add 100 L of ethyl acetate and heat under reflux for extraction at 80 °C. Extract three times and combine the extracts. The first extraction time is 60 min, and the second and third extraction times are 75 min.

[0068] (3) Concentration: The extract obtained in (2) above is concentrated into a paste to obtain crude licorice extract. The paste is redissolved in 10 mL / g ethyl acetate.

[0069] (4) Impurity removal: Add silica gel of 1 / 20 of the weight of the extract to the ethyl acetate solution obtained in (3) above. The silica gel is 300~400 mesh chromatography silica gel. After stirring, let stand for 90 min, take the supernatant, concentrate the supernatant into an extract, and dissolve it with a mixed solvent of ethyl acetate-n-hexane. The amount of mixed solvent used is 8 mL / g, wherein the content of ethyl acetate in the mixed solvent of ethyl acetate-n-hexane is 50%, and the column sample is obtained.

[0070] (5) Silica gel column separation: The sample obtained in (4) above is wet-coated onto a silica gel column for separation. The silica gel is 300-400 mesh chromatography silica gel with a column diameter-to-height ratio of 1:8. The sample loading amount is 1 / 25 of the silica gel volume. First, wash with a hexane-ethyl acetate system for 3-4 column volumes, where the volume fraction of ethyl acetate in the hexane-ethyl acetate system is 0%; then wash with a hexane-ethyl acetate system for 5-6 column volumes, where the volume fraction of ethyl acetate in the hexane-ethyl acetate system is 20%; finally, wash with a hexane-ethyl acetate system for 10-12 column volumes, where the volume fraction of ethyl acetate in the hexane-ethyl acetate system is 30%. Collect the eluent in segments according to the method of collecting one bucket for each column volume.

[0071] Meanwhile, high performance liquid chromatography was used to detect glycyrrhizin in each eluent. The eluents with glycyrrhizin content ≥85% were combined and concentrated under reduced pressure to obtain a glycyrrhizin extract with a purity ≥95%.

[0072] The glycyrrhizin extract with a purity ≥95% was dissolved in ethyl acetate, evaporated and crystallized to obtain a glycyrrhizin extract with a purity ≥98%. After vacuum low-temperature drying and pulverization, a glycyrrhizin extract powder with a purity ≥98% was obtained and named NWT-GU-P01.

[0073] Example 5: Hair papilla cytotoxicity test

[0074] 1. Reagents and Materials

[0075] MSCM culture medium (Gibco), PBS (Solepro), MTT (Sigma), DMSO (Sigma).

[0076] 2. Instruments

[0077] CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), microplate reader (BioTek, Epoch).

[0078] 3. Cell lines

[0079] The cells used in this test were dermal papilla cells, batch number: 210727, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0080] 4. Sample to be tested

[0081] Sample group: NWT-GU-P01, with test concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.5625 μg / mL, and 0.78125 μg / mL.

[0082] Solvent control group: MSCM culture medium.

[0083] Positive control group: MSCM culture medium containing 10% DMSO.

[0084] Zeroing group: MSCM culture medium (cell-free).

[0085] 5. Experimental Methods

[0086] Hair papilla cells in the exponential growth phase were seeded into 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator. Drug administration was initiated when the cell seeding rate reached 50-60% in the 96-well plates. For the solvent control group, 200 μL of culture medium was added to each well; for the positive control group, 200 μL of culture medium containing 10% DMSO was added to each well; for the sample group, 200 μL of culture medium containing the corresponding concentration of NWT-GU-PO1 was added to each well; for the zeroing group, no cells were seeded, only 200 μL of cell culture medium was added. After drug administration, the 96-well plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. After 24 hours of cell incubation, the supernatant was discarded, and MTT working solution (0.5 mg / mL) was added, followed by incubation at 37°C in the dark for 4 hours. After incubation, the supernatant was discarded, and 150 µL of DMSO was added to each well. The OD value was read at 490 nm, and cell viability was calculated using the following formula:

[0087]

[0088] 6. Experimental Results

[0089] The MTT assay is a method for detecting cell viability and growth, and the measured OD value is directly proportional to cell activity. Table 1 shows the detection results obtained by the MTT assay in this embodiment.

[0090] Table 1. MTT assay results of glycyrrhizin coumarin extract

[0091]

[0092] As shown in Table 1, the survival rate of dermal papilla cells was greater than 90% when the concentration of NWT-GU-P01 in the sample group was below 6.25 μg / mL, indicating that glycyrrhizin NWT-GU-P01 did not show significant cytotoxicity to dermal papilla cells in the concentration range of 6.25 μg / mL.

[0093] Example 6: Hair papilla cell anti-hair loss activity test

[0094] 1. Reagents and Materials

[0095] MSCM culture medium (Gibco), PBS (Solepro), dihydrotestosterone (Sigma), minoxidil (Sigma), AG RNAex Pro Reagent (Abcam), reverse transcription kit (Abcam), fluorescent dye (Abcam), VEGF ELISA kit (Abcam).

[0096] 2. Instruments

[0097] CO2 incubator (Thermo, 150I), ultra-clean workbench (Sujing Antai, SW-CJ-1F), microplate reader (BioTek, Epoch), ordinary PCR instrument (Bori), real-time PCR instrument (Roche, lightcycler 480 II).

[0098] 3. Cell lines

[0099] The cells used in this test were dermal papilla cells, batch number: 210727, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0100] 4. Sample to be tested

[0101] Sample group: NWT-GU-P01, with test concentrations of 1.5 μg / mL, 3 μg / mL, and 6 μg / mL.

[0102] Blank control group: MSCM culture medium.

[0103] Negative control group: MSCM culture medium containing 800 nM dihydrotestosterone.

[0104] Positive control group: MSCM culture medium containing 800 nM dihydrotestosterone and 500 μM minoxidil.

[0105] 5. Experimental Methods

[0106] Hair papilla cells in the exponential growth phase were seeded into 6-well plates and incubated overnight at 37°C with 5% CO2. When the cell deposition rate reached 80-90%, drug administration was performed, with three replicates per group. For the blank control group, 2 mL of culture medium was added to each well; for the negative control group, 2 mL of culture medium containing 800 nM dihydrotestosterone was added to each well; for the positive control group, 2 mL of culture medium containing 800 nM dihydrotestosterone and 500 μM minoxidil was added to each well; and for the sample group, 2 mL of culture medium containing 800 nM dihydrotestosterone and the corresponding concentration of NWT-GU-PO1 was added to each well. After drug administration, the 6-well plates were incubated at 37°C with 5% CO2 for 24 hours.

[0107] ELISA test: After incubation, collect the culture medium into centrifuge tubes. After collection, freeze the samples for ELISA detection at -80°C and perform detection and analysis according to the ELISA kit instructions.

[0108] Gene expression detection: After incubation, discard the old solution, wash twice with PBS, add 1 mL of AG RNAex Pro Reagent to each well, lyse the cells by pipetting, and collect the samples. Extract RNA, reverse transcribe it into cDNA, and perform quantitative real-time PCR detection. Calculate the results using the 2-ΔΔCT method.

[0109] Results and statistical analysis: GraphPad Prism was used for plotting, and results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0110] 6. Experimental Results

[0111] Dihydrotestosterone (DHT) and testosterone both bind to androgen receptors (AR), but DHT's affinity is five times that of testosterone. When DHT binds to AR, it shortens the hair growth cycle, causing hair in the anagen phase to gradually thin, hair follicles to miniaturize or even atrophy, leading to hair loss. This process mainly involves the Wnt signaling pathway—the first signal for hair follicle development—and various cytokines involved in cell growth, such as TGF-β and VEGF. By stimulating dermal papilla cells with DHT and detecting changes in androgen receptor (AR), Wnt signaling pathway (DKK1, β-catenin), TGF-β2, and VEGF levels, the anti-hair loss efficacy of the tested samples can be evaluated.

[0112] Figure 1 shows the effect of each group on VEGF content in dermal papilla cells in this embodiment. Figure 2 shows the effect of each group on DKK1 gene expression in dermal papilla cells in this embodiment. Figure 3 shows the effect of each group on TGF-β2 gene expression in dermal papilla cells in this embodiment. Figure 4 shows the effect of each group on AR gene expression in dermal papilla cells in this embodiment. Figure 5 shows the effect of each group on β-catenin gene expression in dermal papilla cells in this embodiment.

[0113] 7. Results Analysis

[0114] As shown in Figure 1, the DHT model group differed significantly from the control group, indicating successful modeling. At a concentration of 1.5 μg / mL, the NWT-GU-P01 sample group increased the content of vascular endothelial growth factor (VEGF) by 10.8%. Furthermore, at concentrations of 3 and 6 μg / mL, the NWT-GU-P01 sample group showed a positive correlation with the increase in VEGF content, with increases of 17.8% and 26.9%, respectively. At a concentration of 6 μg / mL, the NWT-GU-P01 sample group's increase in VEGF content was close to that of the positive control group. This indicates that glycyrrhizin NWT-GU-P01 can significantly increase VEGF content.

[0115] As shown in Figure 2, the DHT model group differed significantly from the control group, indicating successful model establishment. At a concentration of 1.5 μg / mL, the NWT-GU-P01 sample group downregulated DKK1 gene expression by 23.6%. Furthermore, at concentrations of 3 and 6 μg / mL, the NWT-GU-P01 sample group showed a positive correlation with DKK1 gene expression downregulation, with downregulation rates of 33.6% and 42.9%, respectively, both exceeding the downregulation rate of the positive control group. This indicates that glycyrrhizin NWT-GU-P01 can significantly reduce DKK1 gene expression.

[0116] As shown in Figure 3, the DHT model group differed significantly from the control group, indicating successful model establishment. At a concentration of 1.5 μg / mL, the NWT-GU-P01 sample group downregulated TGF-β2 gene expression by 19.7%. Furthermore, at concentrations of 3 and 6 μg / mL, the NWT-GU-P01 sample group showed a positive correlation with TGF-β2 gene expression downregulation, with downregulation rates of 33.3% and 44.9%, respectively. The downregulation rate of the NWT-GU-P01 sample group at a concentration of 6 μg / mL even exceeded that of the positive control group. This indicates that glycyrrhizin NWT-GU-P01 can significantly reduce TGF-β2 gene expression.

[0117] As shown in Figure 4, the DHT model group differed significantly from the control group, indicating successful model establishment. At a concentration of 1.5 μg / mL, the NWT-GU-P01 sample group downregulated AR gene expression by 20.9%. Furthermore, at concentrations of 3 and 6 μg / mL, the NWT-GU-P01 sample group showed a positive correlation with AR gene expression downregulation, with downregulation rates of 21.4% and 53.3%, respectively. The downregulation rate of the NWT-GU-P01 sample group at a concentration of 6 μg / mL was close to that of the positive control group. This indicates that glycyrrhizin NWT-GU-P01 can significantly reduce AR gene expression.

[0118] As shown in Figure 5, the DHT model group differed significantly from the control group, indicating successful modeling. At a concentration of 1.5 μg / mL, the NWT-GU-P01 sample group increased β-catenin gene expression by 34.9%. Furthermore, at concentrations of 3 and 6 μg / mL, the NWT-GU-P01 sample group showed a positive correlation with β-catenin gene expression, with increases of 77.3% and 88.3%, respectively, both exceeding the increase rate of the positive control group. This indicates that glycyrrhizin NWT-GU-P01 can significantly increase β-catenin gene expression.

[0119] In summary, glycyrrhizin NWT-GU-P01 can increase VEGF levels, downregulate the expression levels of DKK1, TGF-β2, and AR genes, and upregulate the expression level of β-catenin gene, thus exhibiting significant anti-hair loss effects.

[0120] Example 7: Activity test of DHT-induced hair loss in mice

[0121] 1. Reagents and Materials

[0122] Dihydrotestosterone (DHT), minoxidil, corn oil, rosin, beeswax, isoflurane, and SPF maintenance diet for rats and mice (Beijing Keao Xieli Feed Co., Ltd.).

[0123] 2. Laboratory animals

[0124] Six-week-old male SPF-grade C57BL / 6J mice were purchased from Beijing Spaford Laboratory Animal Technology Co., Ltd. The mice were housed in a barrier environment with alternating light and dark cycles for 12 hours, with free access to food and water.

[0125] 3. Experimental Grouping

[0126] 1) Blank control group: Hair removal, NWT-GU-P01 solvent applied to the skin;

[0127] 2) Model group: Hair removal, intraperitoneal injection of DHT (1 mg / day), and application of NWT-GU-P01 solvent to the skin;

[0128] 3) Positive control group: hair removal, intraperitoneal injection of DHT (1 mg / day), and topical application of 5% minoxidil;

[0129] 4) 0.1% NWT-GU-P01 administration group: hair removal, intraperitoneal injection of DHT (1 mg / day), and skin application of 0.1% NWT-GU-P01;

[0130] 5) 0.2% NWT-GU-P01 administration group: hair removal, intraperitoneal injection of DHT (1 mg / day), and skin application of 0.2% NWT-GU-P01;

[0131] 6) 0.5% NWT-GU-P01 administration group: hair removal, intraperitoneal injection of DHT (1 mg / day), and skin application of 0.5% NWT-GU-P01;

[0132] 7) 1% NWT-GU-P01 administration group: hair removal, intraperitoneal injection of DHT (1 mg / day), and skin application of 1% NWT-GU-P01;

[0133] 8) 2% NWT-GU-P01 administration group: hair removal, intraperitoneal injection of DHT (1 mg / day), and skin application of 2% NWT-GU-P01.

[0134] 4. Experimental Methods

[0135] After 7 days of acclimatization feeding, the mice were randomly divided into 8 groups of 7 mice each. The mice were anesthetized with isoflurane, and after the skin on their backs was shaved, a 1:1 mixture of rosin and wax, which had been melted and stored at 50°C, was evenly applied to their backs. After the mixture cooled, the mice were dehaired. Once no significant abnormal reaction was observed in the mice's skin, the modeling process began.

[0136] DHT was dissolved in corn oil to prepare a 10 mg / mL DHT solution. Except for the control group, mice in other groups were injected intraperitoneally with 0.1 mL (1 mg) DHT in the morning to establish an androgenic alopecia model. According to the experimental group, solvent, 5% minoxidil or the corresponding concentration of NWT-GU-P01 were applied to the hair-avoiding area once a day for 17 consecutive days.

[0137] Starting from the date of administration, the back of the mice was photographed once on days 0, 3, 6, 9, 12, 15, and 17.

[0138] 5. Experimental Results

[0139] Androgenetic alopecia is a condition characterized by thinning and loss of hair caused by the effects of testosterone on hair follicles. Dihydrotestosterone (DHT)-induced androgenetic alopecia in mice is a common preparation method. This method involves injecting DHT to mimic the physiological process of androgenetic alopecia in humans, while simultaneously treating the test substance to evaluate its effects on preventing hair loss and promoting hair growth.

[0140] Figure 6 shows the hair growth on the backs of mice in each group 12 days after administration of the drug in this embodiment.

[0141] 6. Results Analysis

[0142] As shown in Figure 6, compared with the solvent control group, the hair growth in the dihydrotestosterone (DHT) model group was significantly reduced, indicating successful modeling. Compared with the DHT model group, the hair growth in the minoxidil (positive control) group was significantly restored. Compared with the DHT model group, the NWT-GU-P01 administration group showed varying degrees of hair growth recovery at test concentrations of 0.1%, 0.2%, 0.5%, 1%, and 2%, exhibiting a dose-related relationship within the 0.1% to 0.5% concentration range. At a test concentration of 1%, the level of hair growth recovery was comparable to or slightly lower than at the 0.5% test concentration. However, at a high concentration of 2%, the level of hair growth recovery actually decreased. This indicates that the optimal effective concentration of glycyrrhizin NWT-GU-P01 is 0.2%–1%.

[0143] In summary, in a DHT-induced mouse alopecia model, glycyrrhizin NWT-GU-P01 showed significant efficacy in promoting hair regeneration, with the optimal effective concentration being 0.2-1%.

[0144] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, or alterations made by those skilled in the art using the disclosed technical content shall fall within the protection scope of the present invention.

Claims

1. Use of glycyrrhizin in the preparation of products for preventing hair loss or promoting hair growth.

2. The use according to claim 1, characterized in that, Glycyrrhizin can promote the production of VEGF, increase the expression of β-catenin gene, and reduce the expression of DKK1 gene, TGF-β2 gene and AR gene, so as to prevent androgenetic alopecia or promote hair growth.

3. The use according to claim 2, characterized in that, The product is a pharmaceutical or cosmetic product, and the content of glycyrrhizin in the pharmaceutical or cosmetic product is 0.01~2%.

4. The use according to claim 1, characterized in that, Glycyrrhizin is derived from a glycyrrhizin extract purified from licorice raw material, wherein the purity of glycyrrhizin in the glycyrrhizin extract is ≥10%.

5. A glycyrrhizin coumarin extract for preventing hair loss or promoting hair growth, characterized in that, The glycyrrhizin coumarin extract is obtained by extraction and purification of licorice raw material, and the purity of glycyrrhizin in the glycyrrhizin coumarin extract is ≥10%.

6. The glycyrrhizin coumarin extract for preventing hair loss or promoting hair growth according to claim 5, characterized in that, The extraction and purification method of the glycyrrhizin coumarin extract is as follows: Q1: Select licorice raw materials, dry them to remove moisture, and cut the licorice into thin slices or crush them; Q2: Add sliced ​​or crushed licorice to an extraction vessel and extract by reflux with 5-20 mL / g of ethyl acetate; Q3: Combine the extracts and concentrate them into a paste. The resulting extract paste is then redissolved in ethyl acetate. Q4: Add silica gel to the ethyl acetate reconstituted solution obtained in Q3. The amount of silica gel is 1 / 30 to 1 / 5 of the extract. After stirring, let it stand for 30 to 120 minutes. Take the supernatant and concentrate it. Dissolve it in a mixed solvent of ethyl acetate and n-hexane, wherein the content of ethyl acetate in the mixed solvent of ethyl acetate and n-hexane is 30 to 60%. Q5: The mixed solution from Q4 was wet-coated onto a silica gel column and eluted with a hexane-ethyl acetate system. The ratio of hexane to ethyl acetate during elution was 100:0, 80:20, and 70:30, respectively. The eluents were collected in fractions and analyzed by high performance liquid chromatography. The eluents of different purities of glycyrrhizin were combined and concentrated under reduced pressure to obtain glycyrrhizin extracts of different purities.

7. The glycyrrhizin coumarin extract for preventing hair loss or promoting hair growth according to claim 6, characterized in that, In step Q1, the thickness of the licorice slices is 0.5-4 mm; in step Q2, the extraction time of ethyl acetate is 30-120 min, the extraction temperature is 60-90℃, and the extraction is repeated 2-3 times; in step Q3, the amount of ethyl acetate used for redissolving is 6-15 mL / g; in step Q4, the silica gel added is 300-400 mesh chromatography silica gel, and the amount of ethyl acetate-n-hexane mixed solvent used is 5-10 mL / g; in step Q5, the sample loading volume on the silica gel column is 1 / 15-1 / 40 of the amount of silica gel used, wherein, 3-4 column volumes are washed with 100:0 n-hexane and ethyl acetate, 5-6 column volumes are washed with 80:20 n-hexane and ethyl acetate, and 10-12 column volumes are washed with 70:30 n-hexane and ethyl acetate.

8. The glycyrrhizin coumarin extract for preventing hair loss or promoting hair growth according to claim 7, characterized in that, In step Q5, the eluent with a glycyrrhizin purity ≥85% is combined, concentrated under reduced pressure to form an extract, and dissolved in one or more solvents selected from petroleum ether, diethyl ether, methyl tert-butyl ether, n-pentane, ethyl acetate, n-hexane, cyclohexane, dichloromethane, and ethanol. Then, glycyrrhizin crystals are obtained by evaporation crystallization or cooling crystallization. After pulverization, glycyrrhizin powder is obtained, wherein the purity of glycyrrhizin is ≥98%.

9. A composition for preventing hair loss or promoting hair growth, characterized in that, Including the glycyrrhizin extract for preventing hair loss or promoting hair growth as described in any one of claims 5 to 8.

10. The composition for preventing hair loss or promoting hair growth according to claim 9, characterized in that, The composition is a topical medicine or cosmetic, and the content of glycyrrhizin in the composition is 0.01-2%, and it also includes physiologically acceptable excipients. The composition is in the form of a solution, ointment, plaster, gel, cream, lotion, liniment, tincture, emulsion or patch.

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