New application of plant-derived alkaline phosphatase expression promoter

Abstract

The present invention relates to a new application of a plant-derived alkaline phosphatase expression promoter, mainly using a plant-derived alkaline phosphatase expression promoter for the preparation of daily chemicals or medicines for hair growth or / and prevention and treatment of hair loss. The present invention uses a plant-derived alkaline phosphatase expression promoter for the preparation of daily chemicals or medicines for preventing and treating hair loss or / and hair growth, so as to achieve the effects of promoting hair growth and preventing hair loss. These alkaline phosphatase expression promoters are activators of the Wnt / β-catenin signaling pathway, promote β-ring catenin expression and signal transduction, increase the expression of AXIN2 gene and LEF-1 gene, interfere with the DKK-1 protein of hair loss, and have the function of starting hair follicle growth and promoting the growth of keratinocytes or hair follicle cells. At the same time, the alkaline phosphatase expression promoter of the present invention selects plant-derived ingredients and has good safety.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a new application of a plant-derived alkaline phosphatase expression promoter. Background Art

[0002] Hair growth is a dynamic process controlled by the hair follicle cycle. In addition to being an integral part of the human appearance, hair has multiple functions, such as protecting the scalp from UV damage, transmitting sensory signals, and maintaining optimal body temperature. The scalp is one of the most important organs of the human body, and alopecia refers to the loss of hair from its growth site, mainly from the scalp.

[0003] Currently, the FDA has approved two drugs, finasteride and minoxidil, for the treatment of hair loss, however, these drugs have limited therapeutic effects and many undesirable side effects, such as loss of interest in sex, difficulty in orgasm, and abnormal ejaculation in patients taking oral finasteride.

[0004] Therefore, how to safely and effectively intervene in hair loss and other conditions is a technical problem that needs to be solved urgently. Summary of the invention

[0005] Based on the above technical problems, one of the purposes of the present invention is to provide a new application of a plant-derived alkaline phosphatase expression promoter, which is mainly used for the preparation of daily chemicals or medicines for hair growth and / or prevention and treatment of hair loss, which is safe and effective.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] Application of a plant-derived alkaline phosphatase expression promoter in the preparation of daily chemicals or medicines for preventing and treating hair loss and / or promoting hair growth.

[0008] In some embodiments, the plant-derived alkaline phosphatase expression promoter comprises a plant water extract, and the plant water extract is selected from one or more of the following: yam water extract, changshan water extract, tangerine peel water extract, vitex leaf water extract, phellodendron water extract, thunbergia water extract, plantain water extract, clematis water extract, qinpi water extract, arborvitae branch water extract, litchi water extract, mulberry branch water extract, swollen node water extract, costus water extract, processed aconite water extract, wild cotton water extract, jujube leaf water extract, catharanthus roseus water extract, quassia water extract, rose water extract, mulberry bark water extract, lychee water extract, mulberry branch water extract, schizonepeta spicate water extract, and loblolly leaf water extract.

[0009] In some embodiments, the plant-derived alkaline phosphatase expression promoter comprises a plant alcohol extract, and the plant alcohol extract is selected from one or more of the following: ethanol extract of Belamcanda chinensis, ethanol extract of Cirsium japonicum, ethanol extract of Andrographis paniculata, ethanol extract of Euphorbia koreana, ethanol extract of Sarcandra chinensis, ethanol extract of Quassima, ethanol extract of Apocynum venetum stem, ethanol extract of Gleditsia sinensis thorn, ethanol extract of Platycladus orientalis branch, ethanol extract of Schisandra chinensis, ethanol extract of Ephedra sinica, water extract of Caulis dahurica, ethanol extract of Notoginseng, ethanol extract of Schizonepeta tenuifolia spicate, ethanol extract of Caulis dahurica, ethanol extract of Jujube leaf, ethanol extract of Hawthorn leaf, ethanol extract of Processed Aconite, ethanol extract of Citrus aurantium, ethanol extract of Stir-fried Lentil, ethanol extract of Costusroot rhizome, ethanol extract of Cotton seed, ethanol extract of Isatis indigotica, ethanol extract of Malt, ethanol extract of Cynanchum indigofera, ethanol extract of Citrus grandis peel, ethanol extract of Litchi, ethanol extract of Thistle and ethanol extract of Sophora japonica.

[0010] In some embodiments, the plant-derived alkaline phosphatase expression promoter comprises a plant compound selected from the group consisting of daidzein, formononetin, calycosin, vitexin, chasteberry acetic acid, vinblastine sulfate, sophora flavescens, a-mangostin, irisin, luteolin, polygalain, formononetin, glycitin, polydatin, formononetin, vinblastine, rhynchophylline, mongoside, irisin, hyperoside, artemisinin, myricetin, daphnetin, glycitein, epimedium One or more of glycosides, irisin, myricetin, isoliquiritigenin, chamomile B, galactoside, codonopsis pilosula, silybin, astilbin, cimicifuga glycosides, a-asarone, epicatechin gallate, hesperitin, irisin, isovitexin, wogonin, paeonol, pinocytogenin, spinosum, cardamomine, isorhamnetin, violacein, mangoside, platycoside D, madder, cyanidin chloride, neohesperidin, guazepaponin and isoquercitrin.

[0011] In some embodiments, the daily chemical product comprises the plant-derived alkaline phosphatase expression promoter and an auxiliary agent.

[0012] In some embodiments, the auxiliary agent comprises one or more of a primary surfactant, a co-surfactant, a thickener, a cosolvent, a pearlescent agent, a conditioner, an anti-dandruff agent, a preservative, a chelating agent, a buffer, a flavor and a pigment.

[0013] In some of the embodiments, the daily chemical product is a gel, a hair lotion, a shampoo, a conditioner, a hair spray or a hair cream.

[0014] In some embodiments, the drug comprises the plant-derived alkaline phosphatase expression promoter and pharmaceutically acceptable excipients.

[0015] In some embodiments, the excipients include at least one of a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a color and flavor regulator, a solvent, a solubilizer, a cosolvent, an emulsifier, an antioxidant, a metal complexing agent, an inert gas, a preservative, a local analgesic, a pH regulator, and an isotonic or isotonic regulator.

[0016] In some embodiments, the drug is in the form of granules, granules, capsules, tablets or ointments.

[0017] Compared with the traditional technical solution, the present invention has the following advantages:

[0018] The present invention uses a plant-derived alkaline phosphatase expression promoter for the preparation of daily chemicals or medicines for preventing and treating hair loss or / and hair growth, thereby achieving the effect of promoting hair growth and preventing hair loss. These alkaline phosphatase expression promoters are activators of the Wnt / β-catenin signaling pathway, promote β-catenin expression and signal transduction, increase the expression of the AXIN2 gene and the LEF-1 gene, interfere with the DKK-1 protein of hair loss, and have the function of starting hair follicle growth and promoting the growth of keratinocytes or hair follicle cells. At the same time, the alkaline phosphatase expression promoter of the present invention selects plant-derived ingredients and has good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The results of cytotoxicity analysis of irisin and different extracts of Rhizoma Coptidis;

[0021] Figure 2 This is the HPLC analysis result of irisin content in Belamcanda chinensis extract;

[0022] Figure 3 The results of the changes in luciferase activity after adding different extracts of Ilex chinensis to HEK 293T cells transfected with pTOPFlash plasmid;

[0023] Figure 4 The results are as follows: the change of luciferase activity after adding irisin in HEK 293T cells transfected with pTOPFlash plasmid;

[0024] Figure 5The results are for β-catenin labeled with green fluorescent protein (GFP) in HEK 293T cells;

[0025] Figure 6 The results verify that irisin can activate the Wnt / β-catenin signaling pathway and allow the β-catenin construct to enter the cell nucleus;

[0026] Figure 7 The results are as follows: the changes in mRNA expression levels after adding different concentrations of irisin in DPC cells;

[0027] Figure 8 This is a picture of mouse vibrissa hair follicles after treatment with irisin. DETAILED DESCRIPTION

[0028] For ease of understanding of the present invention, the present invention will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific implementation methods or embodiments and are not intended to limit the present invention. The optional scope of the term "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of related listed items, including any combination of any two related listed items, any more related listed items, or all related listed items.

[0030] The following examples are provided for the purpose of illustrating various embodiments of the present invention and are not intended to limit the present invention in any way. It will be understood by those skilled in the art that variations and other uses thereof are within the spirit of the present invention as defined by the scope of the claims. Materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0031] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0032] The percentage content involved in the present invention, unless otherwise specified, refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0033] The percentage concentrations involved in the present invention, unless otherwise specified, refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0034] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0035] The present invention provides application of a plant-derived alkaline phosphatase expression promoter in the preparation of daily chemicals or medicines for hair growth and / or prevention and treatment of hair loss.

[0036] "Daily chemicals" Daily chemicals (Everyday chemicals, household chemicals), referred to as daily chemicals. Daily chemicals refer to daily chemicals, which are technological chemical products that people use every day, including shampoo, shower gel, skin care, hair care, cosmetics, etc.

[0037] The plant-derived alkaline phosphatase expression promoter of the present invention comprises a plant water extract, and the plant water extract can be selected from, but is not limited to, one or more of the following plant water extracts: yam water extract, changshan water extract, tangerine peel water extract, vitex leaf water extract, phellodendron water extract, thunbergia water extract, plantain water extract, clematis water extract, qinpi water extract, arborvitae branch water extract, litchi water extract, mulberry branch water extract, swollen joint water extract, costus water extract, processed aconite water extract, wild cotton water extract, jujube leaf water extract, catharanthus roseus water extract, quassia water extract, rose water extract, mulberry bark water extract, lychee water extract, mulberry branch water extract, schizonepeta spicate water extract, and lophatherum gracile leaf water extract.

[0038] The plant-derived alkaline phosphatase expression promoter of the present invention comprises a plant alcohol extract, and the plant alcohol extract can be selected from, but not limited to, one or more of the following plant alcohol extracts: ethanol extract of Belamcanda chinensis, ethanol extract of Cirsium japonicum, ethanol extract of Andrographis paniculata, ethanol extract of Euphorbia koreana, ethanol extract of Sarcandra chinensis, ethanol extract of Quassima, ethanol extract of Apocynum venetum stem, ethanol extract of Gleditsia sinensis thorn, ethanol extract of Platycladus orientalis branch, ethanol extract of Schisandra chinensis, ethanol extract of Ephedra sinica, water extract of Caulis dahurica, ethanol extract of Radix Notoginseng, ethanol extract of Nepeta tenuifolia spicate, ethanol extract of Caulis dahurica, ethanol extract of Jujube leaf, ethanol extract of Hawthorn leaf, ethanol extract of Aconite processed, ethanol extract of Citrus aurantium, ethanol extract of Stir-fried Lentil, ethanol extract of Aucklandia lappa, ethanol extract of Cotton seed, ethanol extract of Radix Isatidis indigotica, ethanol extract of Malt, ethanol extract of Cynanchum indigofera, ethanol extract of Cortex cymoides, ethanol extract of Litchi, ethanol extract of Thistle, and ethanol extract of Sophora japonica.

[0039] The plant-derived alkaline phosphatase expression promoter of the present invention comprises a plant compound, and the plant compound can be selected from, but not limited to, one or more of the following plant compounds: daidzein, formononetin, verbascoside, vitexin, vinblastine acetic acid, vinblastine sulfate, sophora flavescens glycoside, a-mangostin, irisin, luteolin, polygalain, formononetin, glycitin, polydatin, formononetin, vinblastine, rhynchophylline, mongoside, irisin, hyperoside, artemisinin, myricetin , daphnetin, glycitein, icariin, tectoriusin, myricetin, isoliquiritigenin, chamomile B, galactoside, codonopsis pilosula, silybin, cimicifuga glycoside, a-asarone, epicatechin gallate, hesperitin, tectoriusin, isovitexin, baicalin, paeonol, pinotnosin, astilbin, spinosum, cardamomine, isorhamnetin, violacein, mangoside, platycoside D, madder, cyanidin chloride, neohesperidin, guajava saponin H, and isoquercetin.

[0040] In the present invention, "plant water extract", which may also be referred to as "traditional Chinese medicine water extract", can be prepared by the following preparation method: weighing the traditional Chinese medicine, crushing, adding water (solid-liquid ratio of 1:8-1:12), soaking (for example, for 30 minutes), ultrasonic extraction (for example, ultrasonic extraction for 30 minutes), and collecting the filtrate. The preparation method also includes drying the collected filtrate (including but not limited to freeze drying) to prepare dry powder.

[0041] In the present invention, "plant alcohol extract", which may also be referred to as "traditional Chinese medicine alcohol extract", can be prepared by the following preparation method: weigh the Chinese medicinal materials, crush them, add an ethanol-containing extraction solvent (solid-liquid ratio is 1:8-1:12), soak (for example, for 30 minutes), ultrasonically extract (for example, ultrasonically extract for 30 minutes), and collect the filtrate. The preparation method also includes drying the collected filtrate (including but not limited to freeze drying) to prepare a dry powder. In the preparation method, the volume percentage of ethanol in the ethanol-containing extraction solvent can be 50%, 60%, 70%, 80%, 90%, or 100%.

[0042] In the present invention, the daily chemical product comprises the plant-derived alkaline phosphatase expression promoter and an auxiliary agent.

[0043] The auxiliary agent described in the present invention can be selected from, but not limited to, one or more of the following auxiliary agents: primary surfactant, auxiliary surfactant, thickener, cosolvent, pearlescent agent, conditioner, anti-dandruff agent, preservative, chelating agent, buffer, flavor, and pigment.

[0044] In the present invention, the types of the daily chemical products may include, but are not limited to: gel, hair milk, shampoo, conditioner, hair spray or hair cream.

[0045] In the present invention, the medicine comprises the plant-derived alkaline phosphatase expression promoter and pharmaceutically acceptable auxiliary materials.

[0046] In the present invention, the excipients can be selected from, but not limited to, one or more of the following excipients: diluents, wetting agents, adhesives, disintegrants, lubricants, color and flavor regulators, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH regulators, isotonic or isotonic regulators.

[0047] In the present invention, the dosage form can be selected from, including but not limited to, the following dosage forms: granules, granules, capsules, tablets or ointments.

[0048] Example 1

[0049] This embodiment provides a method for preparing a plant extract, including an ultrasonic extraction method, which is specifically performed according to the following operations:

[0050] 1. Weigh 50 g of Chinese medicinal materials and crush them, add 500 mL of deionized water or ethanol (volume percentage ranges from 50% to 100%) and soak for 30 minutes, ultrasonically extract for 30 minutes, centrifuge, collect the supernatant of the extract, filter it through a filter membrane, and place it in a -30°C refrigerator to freeze it into ice cubes.

[0051] 2. Place the obtained ice cubes in a freeze dryer, control the cold trap temperature to -80°C, and grind the product into powder after freeze drying to obtain the Chinese medicine extract.

[0052] Example 2

[0053] In order to evaluate the cytotoxicity of the plant extracts and phytochemical components of the present invention, the present invention takes irisin and Iris chuanxiong extract as examples to detect whether they have cytotoxicity to hair follicle dermal papilla cells and HEK 293T cells, and selects a concentration without toxicity for drug addition. In this example, the MTT method is used to detect the cell survival rate. The specific operation is as follows:

[0054] 1. The hair follicle dermal papilla cells and HEK 293T cells were cultured in DMEM containing 10% (v / v) FBS (fetal bovine serum) and 1% (w / v) P / S (penicillin / streptomycin). The adherent cells at the bottom of the culture dish were digested with 0.25% (w / v) trypsin solution to prepare a cell suspension, which was counted and diluted to a cell concentration of 1×10 4 The suspension was inoculated into a 96-well plate and placed in a CO2 incubator (5% CO2, 37°C) for 24 hours.

[0055] 2. Add plant extracts and phytochemical ingredients respectively ( Figure 1Different extracts of Sichuan belamcanda chinensis and irisin, the main component of Sichuan belamcanda chinensis, are used as examples for illustration). After continuing to culture for 24 hours, the culture medium in the 96-well plate is aspirated, 0.5 mg / mL MTT solution is added, and the plate is placed in an incubator for culture for 4 hours.

[0056] 3. Add DMSO solution and shake on a shaker for 15 minutes, measure the absorbance at 570 nm, and then analyze the cell activity.

[0057] The results are as follows Figure 1 As shown, taking the water extract and alcohol extract of Chuanxionggan and irisin, the main component of Chuanxionggan, as examples, the dermal papilla cells (DCP) of hair follicles and HEK 293T cells did not show a decrease in cell number when the concentration of Chuanxionggan extract was in the range of 0μg / mL-150μg / mL, and the concentration of irisin was in the range of 0μM-200μM. The results show that irisin and different extracts of Chuanxionggan in the present invention will not induce hair follicle cell death within the concentration range, and their safety is reliable.

[0058] Example 3

[0059] In this implementation case, HEK 293T cells were transfected with pTOPFlash (reporter gene plasmid) to screen plant extracts or plant compounds that can activate the Wnt signaling pathway. pTOPFlash (reporter gene plasmid) is a reporter gene plasmid used to detect the level of TCF / LEF transcriptional activity mediated by β-catenin in the Wnt signaling pathway. pTOPFlash has two repeated TCF / LEF binding site sequences, each with three repeated sequences, one for the forward sequence and the other for its reverse complementary sequence, which can detect the transcriptional activity level of TCF / LEF with high sensitivity.

[0060] Specifically, the activity screening is carried out by the following steps:

[0061] 1. HEK293T cells were seeded in 96-well plates at a density of 5,000 cells per well and cultured for 24 hours.

[0062] 2. Use the jetPRIME kit to transfect the pTOPFlash plasmid into HEK293T cells.

[0063] 3. Add plant extracts or plant compounds at a certain concentration to a 96-well plate and culture for 24 hours.

[0064] 4. Perform luciferase assay using PierceTM Firefly Luciferase Glow Assay Kit according to the instructions on the kit. Luminescence reaction is quantified in a GloMax® 96 Microplate Luminometer.

[0065] The Wnt / β-catenin signaling pathway can maintain the hair-inducing properties of DPCs (follicle dermal papilla cells) and trigger hair growth in mice. This screening platform compares the degree of stimulation of the Wnt / β-catenin signaling pathway by different plant extracts or plant compounds by transfecting pTOPFlash plasmids in HEK 293T cells. Using this screening platform, the present invention screened more than 1,000 plant extracts and phytochemicals, and ultimately identified more than 100 Chinese herbal extracts and phytochemicals that can activate the Wnt / β-catenin signaling pathway. The drug screening results are shown in Tables 1 and 2.

[0066] Table 1. Plant extract screening results

[0067]

[0068] Table 2. Plant compound screening results

[0069]

[0070] Through this screening platform, the present invention takes the ethanol extract of Iris chinensis and its main chemical component irisin as an example to further study its mechanism of action in preventing and promoting hair follicles.

[0071] Example 4

[0072] This implementation case uses HPLC high performance liquid chromatography to determine the content of irisin in the ethanol extract of Sichuan iris root. The specific operation is as follows:

[0073] 1. Prepare reference solution

[0074] Take 2.0 mg of irisin reference substance and dissolve it in 100 mL of ethanol aqueous solution with an ethanol content of 70% (v / v).

[0075] 2. Test solution

[0076] The extracts of Ilex sutchuenensis prepared by different extraction methods were prepared to a final concentration of 100 mg / mL and filtered using a 0.45 μm microporous membrane (PTFE).

[0077] 3. Chromatographic system

[0078] The liquid chromatography detection method with a diode array detector was used, the detection wavelength was 266nm, a 4.6×250mm octadecyl bonded silica gel (5μm) filling column was used, and the flow rate was about 1.0mL / min. The elution process is as follows:

[0079] Table 3

[0080]

[0081] 4. Take 10 μL of irisin reference solution and test solution, inject into liquid chromatograph, and record the chromatogram. Determine the retention time of iris peak in the chromatogram of reference solution and the retention time of characteristic peak in the chromatogram of test solution. Under the same liquid chromatography conditions, compare the retention time of iris peak in the chromatogram of reference solution and the retention time of component peak in the chromatogram of test solution. The retention time difference of iris peak in the chromatogram should not exceed 2.0%.

[0082] The results of HPLC fingerprint analysis are as follows Figure 2 As shown in Figure 2, the linear regression equation was used to calculate the content of irisin in the extract of Chuanxiong Gan. The correlation coefficient (r 2 ) was 0.9984, showing a good linear relationship. The linear calibration equation was Y=42.26X+15.413, where Y was the peak area and X was the concentration of irisin. The results showed that the contents of irisin in the water extract, 50% ethanol extract and 70% ethanol extract of Chuanxionggan were 0.63±0.08, 1.71±0.13 and 9.73±1.34 mg / g (n=4), respectively. The irisin content in the 70% ethanol extract was the highest.

[0083] Example 5

[0084] In this implementation case, pTOPFlash (reporter gene plasmid) was transfected into HEK 293T cells to compare the activation degree of different Sichuan Ilex glabra extracts on the Wnt / β-catenin signaling pathway. Specifically, the activity screening was carried out by the following steps:

[0085] 1. Prepare Sichuan Ivy Extract

[0086] Ultrasonic extraction method: weigh 50g of Sichuan Belamcanda chinensis and crush it, add 500mL of deionized water or ethanol to soak for 30 minutes, ultrasonically extract for 30 minutes, centrifuge, collect the supernatant of the extract, filter it through a 0.22μm filter membrane, place it in a -30℃ refrigerator, and freeze it into ice cubes. Place the obtained ice cubes in a freeze dryer, control the cold trap temperature to -90℃, grind the product into powder after freeze drying, and obtain the Chinese medicine extract.

[0087] 2. HEK293T cells were seeded in 96-well plates at a density of 5,000 cells per well and cultured for 24 hours.

[0088] 3. Use the jetPRIME kit transfection reagent to transfect pTOPFlash according to the instructions on the kit.

[0089] 4. Add different Rhizoma Coptidis extracts into a 96-well plate and culture for 24 hours.

[0090] Luciferase assays were performed using the PierceTM Firefly Luciferase Glow Assay Kit according to the kit instructions. Luminescence reactions were quantified in a GloMax® 96 Microplate Luminometer.

[0091] The results of HPLC fingerprint analysis showed that the ethanol extract of Sichuan Shegan contained a large amount of irisin, so the Sichuan Shegan extract was tested in HEK293T cells transfected with pTOPFLASH. The experiment showed that the Sichuan Shegan extract induced an increase in luciferase activity in a dose-dependent manner ( Figure 3 Among them, the 70% ethanol extract of Sichuan Ilex chinensis had the most significant induction effect on luciferase activity, and the luciferase activity increased by about 3 times at a concentration of 150 μg / mL.

[0092] Example 6

[0093] The results of HPLC fingerprint analysis showed that the ethanol extract of Sichuan Iris chinensis contained a large amount of irisin. This implementation case studied the activation degree of irisin at different concentrations on the Wnt / β-catenin signaling pathway by transfecting pTOPFlash (reporter gene plasmid) in HEK 293T cells. Specifically, this implementation case was carried out by the following steps:

[0094] 1. HEK293T cells were seeded at a density of 5,000 cells per well in a 96-well plate and cultured for 24 hours.

[0095] 2. Use jetPRIME kit transfection reagent to transfect pTOPFlash according to the kit instructions.

[0096] 3. Add different concentrations of irisin, DKK-1 and Wnt3a into 96-well plates and culture for 24 hours.

[0097] 4. Perform luciferase assay using PierceTM Firefly Luciferase Glow Assay Kit according to the kit instructions. Luminescence reaction is quantified in a GloMax® 96 Microplate Luminometer.

[0098] The experimental results are as follows Figure 4 As shown in the figure, different concentrations of irisin can increase luciferase activity in a dose-dependent manner. Compared with the control group, 40μM-50μM irisin can induce an increase in luciferase activity by about 80% ( Figure 4DKK-1 is an inhibitor of Wnt / β-catenin signaling. It binds to LRP5 / 6 and can directly prevent Wnt from binding to Wnt frizzled receptors.

[0099] To further investigate the mechanism of irisin acting on the Wnt / β-catenin signaling pathway, the transfected cells were pretreated with DKK-1 for 1 hour before the addition of Wnt3a or irisin. The results showed that after pretreatment with DKK-1, the luciferase activity induced by Wnt3a or irisin was inhibited ( Figure 5 ). Therefore, irisin acts upstream of Wnt / β-catenin signaling or directly binds to Wnt receptors.

[0100] Example 7

[0101] This implementation case proves that irisin can activate the Wnt / β-catenin signaling pathway and allow the β-catenin construct to enter the cell nucleus by transfecting a green fluorescent protein (GFP)-labeled β-catenin construct gene plasmid in HEK 293T cells. Specifically, this implementation case is carried out by the following steps:

[0102] 1. In order to observe the localization of GFP-labeled β-catenin, the cells were cultured on a 10 mm glass coverslip, and HEK 293T cells were transfected with a green fluorescent protein (GFP)-labeled β-catenin gene plasmid as shown in Example 3, and irisin was added to treat the cells.

[0103] 2. Wash twice with PBS, then immediately fix with 4% (v / v) paraformaldehyde in PBS at room temperature for 15 minutes, and then permeabilize the cells with 0.2% (v / v) Triton X-100 in PBS at room temperature for 10 minutes.

[0104] 3. Samples were mounted using ProLong Gold Antifade Mountant with or without DAPI (Thermo Fisher Scientific) and then examined using a Zeiss Cell Discoverer 7 automated microscope (Zeiss, Oberkochen, Germany).

[0105] The accumulation of β-catenin in the cell nucleus is a key step in the Wnt / β-catenin signaling pathway. After translocation to the cell nucleus, β-catenin binds to T cell factor (TCF) and initiates the transcription of downstream target genes. The experimental results showed that after the addition of irisin, the β-catenin-GFP fusion protein in HEK293T cells was able to enter the cell nucleus. Valproic acid (VPA) is known to be a GSK-3β inhibitor that can inhibit the phosphorylation activity of GSK-3β, so it was used as a positive control. Compared with the control group, in the transfected cells treated with VPA or irisin, GFP was localized in the cell nucleus ( Figure 6 ).

[0106] Example 8

[0107] This implementation case uses the qRT-PCR method to study the changes in the mRNA expression levels of downstream genes in the Wnt / β-catenin signaling pathway after irisin treatment, namely, the expression levels of AXIN-associated protein 2 (AXIN2), β-catenin, lymphoid enhancer binding factor-1 (LEF-1), insulin-like growth factor 1 (IGF-1) and alkaline phosphatase (ALP). The activity of ALP in dermal papilla cells reaches the maximum level during the growth phase. During the regression phase, the activity of ALP in dermal papilla cells decreases. IGF-I can stimulate hair follicle growth, maintain the growth phase, and delay the regression phase. Specifically, this implementation case is carried out through the following steps:

[0108] 1. Follicle dermal papilla cells – TERT (DPC) were seeded in 6-well plates and cultured for 24 hours, then different concentrations of irisin were added and cultured for another 48 hours.

[0109] 2. Total RNA was extracted from cultured DPC cells using RNAzol™ reagent (Sigma-Aldrich, St. Louis, MO), and RNA was reverse transcribed using Prime-Script™ RT kit (Takara).

[0110] 3. Use qRT-PCR to detect the expression levels of AXIN2, β-catenin, LEF-1, IGF-1 and ALP.

[0111] The experimental results are as attached Figure 7 As shown in the figure, irisin has a significant activating effect on the Wnt / β-catenin signaling pathway. As the concentration of irisin increases, the expression of alkaline phosphatase ALP increases, thus having a positive effect on hair growth. The expression of IGF-1 increased by more than 2 times after irisin treatment. Wnt3a was used as a positive control.

[0112] Example 9

[0113] This embodiment isolates mouse vibrissa follicles and adds irisin to prove that irisin has the effect of protecting hair follicles and promoting hair growth. Specifically, this embodiment is carried out by the following steps:

[0114] 1. Take the whiskers from the upper lip pad of 4-week-old C57BL / 6 male mice and carefully separate single vibrissa follicles in the growth phase.

[0115] 2. Culture the hair follicles in William's E medium (Sigma-Aldrich) containing 1% (v / v) P / S (penicillin / streptomycin) solution at 37°C and 5% CO2.

[0116] 3. Five hair follicles were isolated from each group. The vibrissa hair follicles were cultured in different concentrations of irisin or 2 μM WAY-316606 for 3 days, and the increase in hair length was measured from day 0 to day 3.

[0117] In order to evaluate the promoting effect of irisin on hair growth, the present invention treated the isolated vibrissa follicles with different concentrations of irisin for 48 hours and measured the hair length during the growth process.

[0118] The results are as follows Figure 8 As shown, hair length after irisin treatment increased significantly in a dose-dependent manner compared to hair follicles in the control group. Hair growth was up to 60% after 3 days of treatment with 50 or 100 µM irisin. WAY 316606, an inhibitor of secreted frizzled-related protein 1 (sFRP-1) that prevents sFRP-1 from interacting with Wnt, was used as a positive control and showed an increase in hair length of approximately 25%.

[0119] The technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. The above-described embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical scheme of the present invention in a specific and detailed manner, but it cannot be understood as a limitation on the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the scope of protection of the present invention.

[0120] It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the attached claims of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. Use of a plant-derived alkaline phosphatase expression promoter in the preparation of daily chemicals or medicines for hair growth and / or prevention and treatment of hair loss; the plant-derived alkaline phosphatase expression promoter is one or more of daidzein, formononetoside, verbascosin, vitex luteolin, vinblastine sulfate, sophora flavescens glycoside, and α-mangostin.

2. The use according to claim 1, characterized in that: The daily chemical product comprises the plant-derived alkaline phosphatase expression promoter and an auxiliary agent.

3. The use according to claim 2, characterized in that: The auxiliary agent comprises one or more of a main surfactant, a co-surfactant, a thickener, a cosolvent, a pearlescent agent, a conditioner, an anti-dandruff agent, a preservative, a chelating agent, a buffer, a flavor and a pigment.

4. The use according to any one of claims 1 to 3, characterized in that: The types of the daily chemical products are gel, hair milk, shampoo, conditioner, hair spray or hair cream.

5. The use according to claim 1, characterized in that: The medicine comprises the plant-derived alkaline phosphatase expression promoter and pharmaceutically acceptable auxiliary materials.

6. The use according to claim 5, characterized in that: The auxiliary materials include at least one of diluents, wetting agents, adhesives, disintegrants, lubricants, color and flavor regulators, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH regulators, and isotonic or isotonic regulators.

7. The use according to any one of claims 1, 5 to 6, characterized in that: The dosage form of the medicine is granules, granules, capsules, tablets or ointments.

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