Alopecia-preventing plant extract, and preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of plant extracts, in particular to an alopecia-preventing plant extract, and a preparation method therefor and the use thereof. Raw materials for preparing the plant extract comprise the following components in parts by weight: 4-8 parts of ginger root, 17-22 parts of Platycladi cacumen, 0.6-1.2 parts of Trifolium pratense leaves and 0.6-1.2 parts of Artemisiae argyi folium. Provided in the present invention are an alopecia-preventing plant extract, and a preparation method therefor and the use thereof. The plant composition has a synergistic effect, and can achieve an alopecia-preventing effect via multiple pathways and multiple targets. The action mechanisms mainly comprise enhancing the autophagic activity of dermal papilla cells, promoting the proliferation of dermal papilla cells, and promoting the blood circulation of the head. By means of the mechanisms, the vitality of dermal papilla cells is enhanced, and the degeneration of follicle cells is delayed, so that the problem of alopecia is fundamentally solved. In addition, the plant composition provided in the present invention also has a significant oil control effect, and realizes the alopecia-preventing effect and a refreshing and comfortable feeling during use by means of inhibiting scalp oil secretion.

Description

Anti-hair loss plant extract, preparation method and application thereof Technical Field

[0001] The present invention relates to the technical field of plant extracts, in particular to an anti-hair loss plant extract and a preparation method and application thereof. Background Art

[0002] Hair loss is a common clinical condition, categorized by alopecia areata, seborrheic alopecia, chemotherapy-induced alopecia, and senile alopecia, with seborrheic alopecia and alopecia areata being the most common. Currently, anti-hair loss products on the market have a limited mechanism of action, making it difficult to address both the symptoms and the root cause.

[0003] The most effective Western medicines for preventing hair loss are minoxidil and finasteride, but long-term use can have significant side effects. Finasteride can cause allergic reactions such as itching, rashes, and facial swelling. Its use by women can lead to birth defects and sexual dysfunction in infants. Minoxidil can cause severe facial and limb hypertrichosis in women, and can also cause irritation reactions such as dryness, scaling, itching, and redness. It can also cause adverse reactions in certain cardiovascular systems. Plant-derived anti-hair loss ingredients are safe and non-toxic, but their efficacy is generally less than ideal due to poor formulation or inappropriate preparation processes. Plant extracts can also have stability issues. Summary of the Invention

[0004] The present invention provides a hair loss prevention plant extract, a preparation method and application thereof, in order to solve the problems of side effects caused by Western medicine treatments for hair loss in related technologies and unsatisfactory hair loss prevention effects of plants.

[0005] In one aspect, the present invention provides an anti-hair loss plant extract, wherein the raw materials for preparing the plant extract include ginger root, Platycladus orientalis leaf, Trifolium repens leaf, and Artemisia argyi leaf.

[0006] In a possible implementation, the raw materials for preparing the plant extract include, by weight: 4-8 parts of ginger root, 17-22 parts of Platycladus orientalis leaves, 0.6-1.2 parts of Trifolium pratense leaves, and 0.6-1.2 parts of Artemisia argyi leaves.

[0007] On the other hand, the present invention provides a method for preparing an anti-hair loss plant extract, comprising the following steps: a. extracting ginger root to obtain a ginger extract.

[0008] b. Extract Platycladus orientalis leaves to obtain Platycladus orientalis leaf extract.

[0009] c. Mix the extracts obtained in step a and step b, and then add the clover leaf extract and the wormwood leaf extract.

[0010] In one possible implementation, the ginger root extraction method in step a is as follows: a-1. The ginger root is placed in an extraction tank, 1,3-propylene glycol is added, and cyclic extraction is performed at 85-90° C., that is, a pipe is provided outside the extraction tank, and the solvent circulates through the pipe.

[0011] a-2. After 1 hour of cyclic extraction, the extract passes through a condenser, is cooled to 50-60°C, and is passed into a neutral alumina column at a flow rate of 1-2 BV / h.

[0012] a-3. The effluent from the alumina column flows back to the extraction tank and is circulated and extracted again.

[0013] a-4. After three cycles of extraction, collect the liquid that passes through the alumina column and filter it to obtain the ginger extract.

[0014] In one possible implementation, the method for extracting Platycladus orientalis leaves in step b is as follows: b-1, Platycladus orientalis leaves and water are mixed in a weight ratio of 1:10.

[0015] b-2. Adjust the pH of the solvent to 9-10.

[0016] b-3. After ultrasonic extraction for 20-30 minutes, filter, collect the filtrate, and adjust the pH to 6-7.

[0017] b-4. Load the filtrate onto a D101 macroporous resin column, elute with 1,3-propylene glycol, and collect the eluate to obtain the Platycladus orientalis leaf extract.

[0018] In one possible implementation, the extraction methods of red clover and mugwort in step c are the same, including the following steps: c-1, the plant raw materials red clover leaves and mugwort leaves are mixed with water in a weight ratio of 1:15, and extracted at 90-95°C for 2h.

[0019] c-2. The extract was loaded onto a D101 macroporous resin column and eluted with water, 30% ethanol, and 70% ethanol in sequence. The 70% ethanol eluate was collected and the solvent was recovered to obtain the clover leaf extract and the artemisia argyi leaf extract.

[0020] On the other hand, the present invention also provides an application of an anti-hair loss plant extract, which can be used in head care products, and can also be used in skin oil control and acne prevention products.

[0021] In step b-3, an ultrasonic extraction and analysis system is provided for monitoring the quality of the solvent during the ultrasonic extraction process. The ultrasonic extraction and analysis system includes an extraction state acquisition module, an extraction state analysis module, a solvent state acquisition module, a solvent state analysis module, an ultrasonic extraction database, and a quality analysis judgment module.

[0022] The extraction state acquisition module is used to obtain the power and frequency of the ultrasonic extraction instrument and the image of the ultrasonic probe on the ultrasonic extraction instrument at each preset time point, and obtain the distance between the ultrasonic probe and the bottom of the solvent container through image analysis. The extraction state acquisition module is also used to obtain the parameters of the bubbles generated around the ultrasonic probe at each preset time point.

[0023] The extraction state analysis module obtains the ultrasonic extraction compliance by reading the power and frequency of the ultrasonic extraction instrument at each preset time point, the distance between the ultrasonic probe and the bottom of the solvent container, and the parameters of the bubbles generated around the ultrasonic probe, and records it as τ.

[0024] The solvent state acquisition module is used to obtain the temperature and pH value of the solvent at each preset time point, which are respectively recorded as T h 、P h , h represents the hth preset time point, h=1,2,…,m.

[0025] The solvent state analysis module is used to obtain the temperature and pH value of the solvent at each preset time point to analyze and obtain the solvent state compliance, which is recorded as ω.

[0026] The ultrasonic extraction database is used to store the standard power and frequency of the ultrasonic extraction instrument when the solvent is ultrasonically extracted, the reference distance between the ultrasonic probe of the ultrasonic extraction instrument and the bottom of the container, the reference parameters for the ultrasonic probe of the ultrasonic extraction instrument to generate bubbles, and the reference temperature and reference pH value when the solvent is ultrasonically extracted.

[0027] The quality analysis and judgment module is used to obtain the ultrasonic extraction quality coefficient through ultrasonic extraction compliance and solvent state compliance analysis, compare the ultrasonic extraction quality coefficient with the ultrasonic extraction quality coefficient threshold, and then analyze whether the ultrasonic extraction operation of the solvent meets the requirements.

[0028] The parameters of the bubbles generated around the ultrasonic probe include bubble density, the size of each bubble, and the distance between each bubble and the ultrasonic probe. The bubble density obtained at each preset time point is recorded as ρ h , the distance between each bubble and the ultrasonic probe obtained at each preset time point is recorded as D h i , i represents the i-th bubble, i=1,2,…,r, using the formula: Get the discrete degree of bubbles at each preset time point χ h , where r represents the number of bubbles, D 参考 Indicates the reference distance of the bubble from the ultrasonic probe; by obtaining the size of each bubble at each preset time point and screening the bubbles whose size is larger than the reference size of the bubble, they are recorded as abnormal bubbles, and by dividing the number of abnormal bubbles at each preset time point by the total number of bubbles, the abnormal bubble ratio at each preset time point is obtained, which is recorded as αh ; Through the formula: The bubble parameter conformity index is obtained, where m represents the number of preset time points, ρ 参考 represents the reference density of the bubble, e represents a constant, and ε1 represents the correction coefficient of the bubble parameter conforming to the exponential; through the formula: Get the coincidence index of the ultrasonic instrument, where m represents the number of preset time points, W h , Hz h are the power and frequency of the ultrasonic extraction instrument at the hth preset time point, W 标准 Indicates the standard power of ultrasonic instruments, Hz 标准 It represents the standard frequency of ultrasonic instruments, and ε2 represents the correction coefficient of the ultrasonic instrument’s compliance index; through the formula: The ultrasound probe distance coincidence index is obtained, where m represents the number of preset time points, μ 参考 Indicates the reference distance between the ultrasonic probe and the bottom of the solvent container, μ h represents the distance between the ultrasonic probe and the bottom of the solvent container at the hth preset time point, and ε3 represents the correction coefficient of the ultrasonic probe distance compliance index. The ultrasonic extraction compliance is obtained by adding the bubble parameter compliance index, the ultrasonic instrument compliance index, and the ultrasonic probe distance compliance index.

[0029] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The present invention provides an anti-hair loss plant extract and its preparation method and application. The plant composition has a synergistic effect, and achieves an anti-hair loss effect through multiple pathways and multiple targets. The mechanism of action mainly includes enhancing the autophagic activity of hair papilla cells, promoting hair papilla cell proliferation, and promoting blood circulation in the head. Through these mechanisms, the vitality of hair papilla cells is enhanced, the degeneration of hair follicle cells is delayed, and the problem of hair loss is fundamentally solved. In addition, the plant composition provided by the present invention also has a significant oil control effect, and achieves an anti-hair loss effect and a refreshing and comfortable use feeling by inhibiting scalp oil secretion.

[0030] The anti-hair loss plant extract provided by the present invention uses ginger root as one of its raw materials. Ginger root contains a large amount of starch and a small amount of tannin. Extraction with water will extract a large amount of starch, which brings great difficulties to subsequent filtration and purification. In order to avoid the influence of starch, the present invention uses 1,3-propylene glycol for extraction. The ability of polyols to penetrate plant cells is weak. To solve this problem and improve the extraction rate of effective ingredients, the present invention adopts a cyclic extraction method. The extract passes through neutral alumina at medium temperature, tannins are adsorbed, and other active ingredients are retained in the extract. The extraction process is simple and efficient. The prepared ginger extract has good stability, does not change color during storage, and no precipitation is generated.

[0031] The ultrasonic extraction and analysis system provided by the present invention comprehensively analyzes the extraction state and solvent state of the ultrasonically extracted solvent, and then determines whether the ultrasonic extraction operation of the solvent meets the requirements, and then determines whether the quality of the Platycladus orientalis leaf extract meets the requirements, thereby increasing the preparation effect of the anti-hair loss plant extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 is a graph showing one of the test results of enhancing the autophagy activity of dermal papilla cells provided by an embodiment of the present invention (stained by immunofluorescence staining)

[0033] FIG2 is a second test result diagram of enhancing the autophagy activity of dermal papilla cells provided by an embodiment of the present invention (stained using the MDC staining method).

[0034] FIG3 is a third diagram of the test results of enhancing the autophagy activity of dermal papilla cells provided by an embodiment of the present invention.

[0035] FIG4 is a graph showing the results of a cell type 17 collagen expression test provided in an embodiment of the present invention.

[0036] FIG. 5 is one of the test result diagrams of promoting the proliferation of dermal papilla cells provided by an embodiment of the present invention.

[0037] FIG. 6 is a second diagram showing the test results of promoting the proliferation of dermal papilla cells according to an embodiment of the present invention.

[0038] FIG. 7 is a third diagram showing the test results of promoting the proliferation of dermal papilla cells according to an embodiment of the present invention.

[0039] FIG8 is a fourth diagram showing the test results of promoting the proliferation of dermal papilla cells provided by an embodiment of the present invention.

[0040] FIG9 is one of the test result diagrams of the cell lipid production test provided in an embodiment of the present invention.

[0041] FIG. 10 is a second diagram showing the test results of the cell lipid production test provided in an embodiment of the present invention.

[0042] FIG11 is a diagram showing the oil control effect test results according to an embodiment of the present invention.

[0043] FIG12 is a connection diagram of the ultrasonic extraction and analysis system of the present invention. DETAILED DESCRIPTION

[0044] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described below, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0045] A method for preparing a hair loss prevention plant extract. The raw materials for preparing the plant extract include, by weight, 6 parts of ginger root, 20 parts of Platycladus orientalis leaves, 1 part of Trifolium pratense leaves, and 1 part of Artemisia argyi. The preparation steps include: a. extracting the ginger root to obtain a ginger extract.

[0046] b. Extract Platycladus orientalis leaves to obtain Platycladus orientalis leaf extract.

[0047] c. Mix the extracts obtained in step a and step b, add the red clover leaf extract and the wormwood leaf extract, stir at 55-60° C. until a uniform solution is formed, and filter through a 0.22 μm polypropylene membrane to obtain the product.

[0048] The ginger root extraction method is as follows: a-1. The ginger root is put into an extraction tank, 1,3-propylene glycol is added at a weight ratio of 1:10, and cyclic extraction is carried out at 85-90° C., that is, a pipe is provided outside the extraction tank, and the solvent circulates through the pipe.

[0049] a-2. After 1 hour of cyclic extraction, the extract passes through a condenser and is cooled to 50-60°C. The cooled extract is passed through a neutral alumina column at a flow rate of 1-2 BV / h, preferably 1.5 BV / h, wherein the weight of the alumina is 40% of the weight of the ginger root.

[0050] a-3. The effluent from the alumina column flows back to the extraction tank and is circulated and extracted again.

[0051] a-4. After three cycles of extraction, the liquid passing through the alumina column was collected and filtered through a 0.22 μm polypropylene membrane to obtain the ginger extract.

[0052] The extraction method of Platycladus orientalis leaves is as follows: b-1, Platycladus orientalis leaves and water are mixed in a weight ratio of 1:10.

[0053] b-2. Adjust the pH of the solvent to 9-10.

[0054] b-3. After 20-30 min of ultrasonic extraction at 500 W, filter the solution through a 5 μm polypropylene membrane, collect the filtrate, and adjust the pH to 6-7.

[0055] b-4. Load the filtrate onto a D101 macroporous resin column, elute with 1,3-propylene glycol, and collect the eluate to obtain the Platycladus orientalis extract, wherein the amount of resin used is 50% of the weight of the Platycladus orientalis leaves, and the amount of 1,3-propylene glycol used is 3 times the weight of the Platycladus orientalis leaves.

[0056] The extraction methods of red clover leaves and mugwort leaves are the same, comprising the following steps: c-1, mixing the plant raw materials red clover leaves and mugwort leaves with water in a weight ratio of 1:15, and extracting at 90-95° C. for 2 hours.

[0057] c-2. The extract is loaded onto a D101 macroporous resin column and eluted with water (3 times the weight of the plant raw material), 30% ethanol, and 70% ethanol, in that order. The 70% ethanol eluate is collected and the solvent is recovered to obtain a clover leaf extract and an artemisia argyi leaf extract, wherein the amount of resin used is 50% of the weight of the plant raw material.

[0058] Referring to Figure 12, step b-3 is provided with an ultrasonic extraction and analysis system for monitoring the quality of the solvent during the ultrasonic extraction process. The ultrasonic extraction and analysis system performs a comprehensive analysis on the extraction state and solvent state of the ultrasonically extracted solvent, and then determines whether the ultrasonic extraction operation of the solvent meets the requirements. The ultrasonic extraction and analysis system includes an extraction state acquisition module, an extraction state analysis module, a solvent state acquisition module, a solvent state analysis module, an ultrasonic extraction database, and a quality analysis and judgment module; the ultrasonic extraction database is respectively connected to the extraction state analysis module, the solvent state analysis module, and the quality analysis and judgment module, the quality analysis and judgment module is respectively connected to the extraction state analysis module and the solvent state analysis module, the extraction state acquisition module is connected to the extraction state analysis module, and the solvent state acquisition module is connected to the solvent state analysis module. By judging whether the ultrasonic extraction operation of the solvent meets the requirements, a basis can be provided for judging the quality of the Platycladus orientalis leaf extract, thereby increasing the intelligence and accuracy of the preparation of the Platycladus orientalis leaf extract, and thereby improving the preparation effect of the anti-hair loss plant extract.

[0059] The ultrasonic extraction database is used to store the standard power and frequency of the ultrasonic extraction instrument when the solvent is ultrasonically extracted, the reference distance between the ultrasonic probe of the ultrasonic extraction instrument and the bottom of the container, the reference parameters for the ultrasonic probe of the ultrasonic extraction instrument to generate bubbles, and the reference temperature and reference pH value when the solvent is ultrasonically extracted.

[0060] The extraction state acquisition module is used to obtain the power and frequency of the ultrasonic extraction instrument and the image of the ultrasonic probe on the ultrasonic extraction instrument at each preset time point, and obtain the distance between the ultrasonic probe and the bottom of the solvent container through image analysis. The extraction state acquisition module is also used to obtain the parameters of the bubbles generated around the ultrasonic probe at each preset time point. The multiple data collection of the present invention can increase the accuracy of ultrasonic extraction state acquisition, and the power and frequency of the ultrasonic extraction instrument can reflect whether the ultrasonic extraction instrument is in a standard working state. The distance between the ultrasonic probe and the bottom of the solvent container can directly reflect the extraction state of the ultrasonic extraction operation. The parameters of the bubbles generated around the ultrasonic probe can reflect the impact of the bubbles generated during the ultrasonic extraction operation. The acquisition of the above data can increase the accuracy of the ultrasonic extraction state analysis of the present invention.

[0061] The extraction state analysis module obtains the ultrasonic extraction compliance by reading the power and frequency of the ultrasonic extraction instrument at each preset time point, the distance between the ultrasonic probe and the bottom of the solvent container, and the parameters of the bubbles generated around the ultrasonic probe, and records it as τ.

[0062] The solvent state acquisition module is used to obtain the temperature and pH value of the solvent at each preset time point, which are respectively recorded as T h 、P h , h represents the hth preset time point, h=1,2,...,m.

[0063] The solvent state analysis module is used to obtain the temperature and pH value of the solvent at each preset time point to analyze the solvent state compliance and record it as ω. T 标准 represents the reference temperature of the solvent, P 标准 The present invention increases the accuracy of quality analysis of the preparation of Platycladus orientalis leaf extract by referring to the influence of solvent state temperature and pH value on the preparation of Platycladus orientalis leaf extract.

[0064] The parameters of the bubbles generated around the ultrasonic probe include bubble density, bubble size, and the distance between each bubble and the ultrasonic probe. The bubble density obtained at each preset time point is recorded as ρ h , the distance between each bubble and the ultrasonic probe obtained at each preset time point is recorded as D h i , i represents the i-th bubble, i=1,2,...,r, using the formula: Get the discrete degree of bubbles at each preset time point χ h , where r represents the number of bubbles, D 参考Indicates the reference distance of the bubble from the ultrasonic probe; by obtaining the size of each bubble at each preset time point and screening the bubbles whose size is larger than the reference size of the bubble, they are recorded as abnormal bubbles, and by dividing the number of abnormal bubbles at each preset time point by the total number of bubbles, the abnormal bubble ratio at each preset time point is obtained, which is recorded as α h ; Through the formula: The bubble parameter conformity index is obtained, where m represents the number of preset time points, ρ 参考 represents the reference density of the bubble, e represents a constant, and ε1 represents the correction coefficient of the bubble parameter conforming to the exponential; through the formula: Get the coincidence index of the ultrasonic instrument, where m represents the number of preset time points, W h , Hz h are the power and frequency of the ultrasonic extraction instrument at the hth preset time point, W 标准 Indicates the standard power of ultrasonic instruments, Hz 标准 It represents the standard frequency of ultrasonic instruments, and ε2 represents the correction coefficient of the ultrasonic instrument’s compliance index; through the formula: The ultrasound probe distance coincidence index is obtained, where m represents the number of preset time points, μ 参考 Indicates the reference distance between the ultrasonic probe and the bottom of the solvent container, μ h represents the distance between the ultrasonic probe and the bottom of the solvent container at the hth preset time point, and ε3 represents the correction coefficient for the ultrasonic probe distance compliance index. The ultrasonic extraction compliance index is calculated by adding the bubble parameter compliance index, the ultrasonic instrument compliance index, and the ultrasonic probe distance compliance index. The present invention comprehensively analyzes the impact of bubbles on the ultrasonic extraction process by analyzing the density of bubbles generated by the ultrasonic probe, the distance between the bubbles and the ultrasonic probe, and the size of abnormal bubbles. It is understood that the bubble density can be obtained through ultrasonic imaging or echo amplitude measurement. These methods are known techniques and are therefore not further elaborated herein.

[0065] The quality analysis and judgment module is used to obtain the ultrasonic extraction quality coefficient by multiplying the ultrasonic extraction compliance and the solvent state compliance by the corresponding weight ratio, and compare the ultrasonic extraction quality coefficient with the ultrasonic extraction quality coefficient threshold. If the ultrasonic extraction quality coefficient is greater than or equal to the ultrasonic extraction quality coefficient threshold, the ultrasonic extraction operation of the solvent meets the requirements, otherwise it does not meet the requirements.

[0066] Performance evaluation

[0067] d. Evaluation of Cell Autophagy

[0068] Principle: In this experiment, a dermal papilla cell stress model was established through serum starvation culture. Immunofluorescence staining and monodansylcadaverine (MDC) staining were used to qualitatively analyze the cell autophagy markers LC3 II protein and autophagosomes, respectively. Image Pro Plus software was used to quantitatively analyze the fluorescence intensity to test the autophagic efficacy of the samples.

[0069] d-1, HHDPC cells (hair papilla cells) were cultured at 0.8*10 5 Cells were seeded into 24-well plates at 100 cells / well and cultured in complete medium (CM) (MSCM medium + 5% FBS + 1% MSCGS + 1% double antibody (PS)) for 24 h. The cells were allowed to adhere to the plate and then replaced with BM (serum-free medium: MSCM medium + 1% double antibody (PS)) for serum starvation. The normal culture group still used CM and was cultured for another 12 h before treatment with the test substance.

[0070] d-2, chloroquine was prepared into 60 mM (millimoles per liter) using deionized water. The sample prepared in Example (named AP) was prepared into three concentrations of 0.1%, 0.2%, and 0.4%. The positive control group was CM or 50 μM (micromoles per liter) of chloroquine. The original culture medium in the 24-well plate was discarded, and the plate was washed once with PBS. 500 μL of the prepared samples of different concentrations were added to the cell plate per well and incubated in a 37°C, 5% CO2 constant temperature incubator for 2 h, 4 h, 12 h, 24 h, and 48 h. Negative and positive controls were set for each cell plate, and at least 3 replicates were used for each concentration group.

[0071] After incubation, the culture medium was discarded, and the cells were gently washed twice with PBS. 500 μL of cold formaldehyde was added to each well and fixed at 4°C for 10 minutes. The formaldehyde was discarded, and the cells were gently washed twice with PBS. 500 μL of 0.2% Triton-X (a non-ionic surfactant) was added, and the cells were permeabilized at room temperature for 15 minutes. 3% BSA, primary antibody solution, and secondary antibody solution were then added, and the cells were fluorescently stained. The primary antibody was incubated overnight, and the secondary antibody was incubated at room temperature for 1 hour. DAPI stain was added, the slides were mounted, and the staining was observed and photographed under a fluorescence microscope. The fluorescence intensity was analyzed using Image Pro Plus software. It should be noted that the primary antibody solution refers to the solution containing the LC3B antibody, and the secondary antibody is the solution containing the anti-rabbit IgG antibody.

[0072] d-3, chloroquine was prepared into 60 mM (millimoles per liter) using deionized water, and the example samples were prepared into three concentrations of 0.1%, 0.2%, and 0.4%. The positive control was CM or 50 μM (micromoles per liter) of chloroquine. The original culture medium in the 24-well plate was discarded, and the plates were washed once with PBS. 500 μL of the prepared samples at different concentrations were added to the cell plates per well, and the plates were incubated in a 37°C, 5% CO2 constant temperature incubator for 24 h. Negative and positive control groups were set for each cell plate, and at least 3 replicates were used for each concentration group.

[0073] After the cell culture, wash the plate twice with 1× washing solution, add at least 500 μL of MDC fluorescent dye to each well and incubate in the dark for 30 minutes. After staining, wash again with 1× washing solution twice, fix the cells with tissue fixative for 10-15 minutes, seal the slides, and photograph under a fluorescence microscope.

[0074] The conclusion shows that: the blue fluorescence in Figure 1 represents the cell nucleus after DAPI staining, the red dot fluorescence in Figure 1 represents LC3II, and the green fluorescence in Figure 2 represents the autophagosome after MDC staining. Combining Figures 1-3, it can be seen that 0.1% to 0.4% concentration of AP can increase the expression of LC3 II protein, an autophagy marker, in dermal papilla cells under serum starvation culture for 12h to 48h, increase the number of autophagosomes, and enhance cell autophagy activity, which has potential anti-hair loss effect.

[0075] e. Cell type 17 collagen expression test

[0076] Experimental principle: This experiment established a dermal papilla cell stress model through serum-free culture, and used immunofluorescence staining to detect changes in COL 17 expression to test the efficacy of the sample.

[0077] e-1, HHDPC cells were cultured at 0.8*10 5 Cells were seeded into 24-well plates and cultured in complete medium for 24 h to allow the cells to adhere to the plate.

[0078] e-2. The example sample (named AP) was diluted with serum-free culture medium to prepare three concentrations of 0.1%, 0.2%, and 0.4%. The positive control was complete culture medium. The original culture medium in the 24-well plate was discarded, and the plate was washed once with PBS. 500 μL of the prepared samples of different concentrations were added to each well of the cell plate and incubated in a constant temperature incubator at 37°C and 5% CO2 for 48 hours. Negative and positive control groups were set up for each cell plate, and at least 3 replicates were used for each concentration group.

[0079] e-3. After incubation, discard the culture medium, wash twice with PBS, add 500 μL of cold formaldehyde to each well and fix at 4°C for 10 min, then add 500 μL of 0.2% Triton-X, permeabilize at room temperature for 15 min, then add 3% BSA, primary antibody solution, and secondary antibody solution respectively, and perform fluorescent staining on the cells. Incubate with primary antibody overnight and secondary antibody at room temperature for 1 h, add DAPI staining solution, seal the slides, observe the staining under a fluorescence microscope and take pictures.

[0080] The conclusion shows that: as shown in Figure 4, AP at a concentration of 0.1% to 0.4% can significantly increase the expression of type 17 collagen in hair papilla cells, and has potential anti-hair loss efficacy.

[0081] f. Cell proliferation assay

[0082] Experimental Principle: Detecting cell proliferation and the expression of various growth factors represents the activity of dermal papilla cells, which are closely related to hair follicle health. This experiment established a dermal papilla cell stress model through serum-free culture. Neutral red staining and enzyme-linked immunosorbent assay (ELISA) were used to detect changes in cell proliferation and expression of the cytokines VEGF and FGF-7, respectively, to test the efficacy of the samples.

[0083] f-1, HHDPC cells were cultured at 2*10 4 Cells were seeded into 96-well plates and cultured in complete medium for 24 h to allow the cells to adhere to the plate.

[0084] f-2. Minoxidil was prepared to 50 mM using 50% ethanol. The example sample (named AP) was diluted 2-fold to a concentration of 0.06% to 1%. The positive control was complete culture medium or minoxidil with a final concentration of 5 μM. The original culture medium in the 96-well plate was discarded, and the plate was washed once with PBS. 200 μL of the prepared samples of different concentrations were added to each well of the cell plate and incubated in a constant temperature incubator at 37°C and 5% CO2 for 72 h. Negative and positive control groups were set for each cell plate, and each concentration group was replicated at least three times.

[0085] f-3. After the incubation, add 200 μL of preheated neutral red dye (33 μg / mL) to each well of the cell plate and incubate for 3 hours. Discard the culture medium and add 150 μL of eluent. After gentle shaking at room temperature for 10 minutes, measure the absorbance at 540 nm on a microplate reader. Collect the cell supernatant and detect the expression levels of VEGF (vascular endothelial growth factor) and FGF-7 (fibroblast growth factor 7) according to the instructions of the kit. Calculate their relative expression levels based on the standard curve.

[0086] The results showed that, as shown in Figure 8, AP at concentrations of 0.06% to 0.25% significantly promoted dermal papilla cell proliferation. As shown in Figure 5, at a concentration of 0.25%, cell viability increased to 138.28% compared to the negative control. As shown in Figure 6, VEGF expression increased by 264.41%. As shown in Figure 7, FGF-7 expression increased by 123.21%, demonstrating potential anti-hair loss efficacy.

[0087] g. Cellular lipid production assay

[0088] Experimental principle: This experiment uses LA to induce Seb-E6E7 cells with lipid production ability, and the intracellular lipid content is detected by Oil Red O staining to evaluate the efficacy of the sample.

[0089] g-1. Take cells in the logarithmic phase, discard the culture medium, wash once with PBS, add 0.25% trypsin and place in a cell culture incubator for 4 minutes, terminate digestion with complete culture medium, centrifuge at 1000 rpm for 3 minutes, resuspend the cells, count them, and adjust the cell concentration to 0.6×10 5 100 μL of the suspension was added to each well of a 96-well plate. The middle well was selected for cell suspension loading, and 200 μL of PBS buffer was added to the surrounding wells. The cells were incubated in a 37°C, 5% CO2 incubator for 24 h.

[0090] g-2. LA (linoleic acid) was prepared to a concentration of 0.05% using complete medium and shaken to form an emulsion. The example sample (designated AP) was prepared to concentrations of 4%, 1%, and 0.5% using complete medium. 100 μL of complete medium was added to each well of the negative control group, 50 μL of complete medium and 50 μL of LA were added to each well of the model group, and 50 μL of the test substance and 50 μL of LA were added to each well of the test substance group, for a total volume of 200 μL per well. The remaining 100 μL was filled with complete medium. Three replicate wells were set up for each concentration group and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0091] g-3. Mix 0.5% Oil Red O stock solution and deionized water in a ratio of 3:2, filter with filter paper, and place at room temperature for 10 minutes to obtain the Oil Red O working solution, which should be used up within 4 hours. Carefully and gently discard the culture medium from the cell plate, add 50μL Oil Red O working solution, stain for 10 minutes, discard the staining solution, gently wash the cells 4 times with 40% isopropanol, and then add 50μL deionized water. Observe under an inverted microscope and take pictures for preservation. After taking pictures, discard the liquid in the cell plate, add 200μL isopropanol to each well to dissolve for 5 minutes, and detect the absorbance at 492nm.

[0092] The conclusion shows: as shown in Figure 10, the red dots represent lipid droplets after Oil Red O staining. Under the stimulation of LA, sebaceous gland cells Seb-E6E7 can induce lipid synthesis by activating the PPARγ signaling pathway, which ultimately leads to oily skin. As shown in Figure 9, 0.13% to 1% AP can significantly reduce the number of large and small lipid droplets under LA-induced state and regulate lipid synthesis. Compared with LA, the relative oil content dropped from 100% to a maximum of 43.5%. Therefore, the example sample has potential oil control effect.

[0093] h. Clinical anti-hair loss efficacy test 1

[0094] h-1. Subject selection: 26 healthy subjects aged 20 to 60 years, both male and female.

[0095] h-2. Basic shampoo and basic essence were used during the wash-out period and the recovery period, and shampoo containing 1% of the example sample and essence containing 2% of the example sample were used during the test period.

[0096] h-3. Test environment: Constant temperature and humidity chamber (temperature 20-22°C, relative humidity 40-60% RH). The subjects can be evaluated and tested after 20-30 minutes of acclimatization.

[0097] h-4. After the subjects were informed of the risks, they were asked about their hair loss situation, and basic shampoo and basic essence were distributed. The usage of the products was explained. After the 2-week washout period, test shampoo and essence were distributed. Follow-up visits were conducted 2 weeks and 4 weeks after use to collect all lost hair, recover the test shampoo and essence, and enter the recovery period. During the follow-up visits, some subjects had their hair shaved in designated areas of the scalp, and photos were taken with a dermatoscope. Any discomfort and adverse reactions during use were also recorded.

[0098] h-5. Product usage plan: Take 2 pumps of shampoo each time, rub your hair with foam and massage for 2 minutes, then rinse with clean water; take 2 pumps of shampoo again and repeat the previous steps. After wiping half dry with a towel, use the essence applicator to apply it to the entire scalp, massage for 2 minutes, and blow dry with a hair dryer.

[0099] h-6. Dermatoscope examination of hair follicles: Subjects with severe hair loss (more than 100 hairs lost per shampooing) were selected from the scalp with severe hair loss, their hair was shaved, and photos were taken using a scalp dermatoscope. The same location was used for each photo, and the hair was shaved again and photos were taken until the end of the study.

[0100] The conclusion shows that dermatoscope photographs were taken of the severely hair-loss areas (CF area on the top of the head) of three subjects who lost more than 100 hair roots during the washout period. As shown in Figure 11, the red digital marks indicate that the hair in the hair follicles has become thicker / increased, and the yellow digital marks indicate new hair. Compared with the washout period, the hair in the hair follicles at the same position showed a trend of thickening and increasing in number during the 2-week test period, with occasional new hair. This trend was more obvious during the 4-week test period.

[0101] Clinical Anti-Hair Loss Efficacy Test 2

[0102] The clinical anti-hair loss efficacy was tested by Guangzhou Leadpu Testing Technology Co., Ltd. according to the "Cosmetic Safety Technical Specifications (2015 Edition)". The test product was a serum containing 2.0% of the sample from the example, and the control product was a serum without the sample from the example. A total of 30 subjects (all female, aged 19 to 59 years, with an average age of 40.8 years) were included in the test product group. A total of 30 subjects (1 male and 29 female, aged 18 to 56 years, with an average age of 41.2 years) were included in the control product group. Subjects used the serum every other day, applying 7-8 ml of the serum evenly to the scalp each time. The number of hair loss cases was recorded before use of the serum and after 4, 8, and 12 weeks of use.

[0103] Table 1

[0104] As can be seen from Table 1, after 4 weeks, 8 weeks, and 12 weeks of external application of the essence containing 2.0% of the sample of the embodiment, the amount of hair loss was significantly reduced, with the reduction rates of 19.9%, 37.8%, and 53.8%, respectively. In contrast, the amount of hair loss in the control group remained almost unchanged before and after the use of the essence. The experimental results show that the anti-hair loss plant composition provided by the present invention has a significant anti-hair loss effect.

[0105] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An anti-hair loss plant extract, characterized in that: The raw materials for preparing the plant extract include ginger root, arborvitae leaves, clover leaves and mugwort leaves.

2. The anti-hair loss plant extract according to claim 1, characterized in that: The raw materials for preparing the plant extract include, by weight, 4 to 8 parts of dried ginger root, 17 to 22 parts of Platycladus orientalis leaves, 0.6 to 1.2 parts of Trifolium repens leaves, and 0.6 to 1.2 parts of Artemisia argyi leaves.

3. A method for preparing the anti-hair loss plant extract according to claim 1 or 2, characterized in that: The following steps are involved: a. Extract ginger root to obtain ginger root extract; b. extracting Platycladus orientalis leaves to obtain Platycladus orientalis leaf extract; c. Mix the extracts obtained in step a and step b, and then add the clover leaf extract and the wormwood leaf extract.

4. The method for preparing an anti-hair loss plant extract according to claim 3, wherein: The extraction method of the ginger root extract in step a is: a-1. Put ginger root into the extraction tank, add 1,3-propylene glycol, and cycle extraction at 85-90℃; a-2. After 1 hour of cyclic extraction, the extract passes through a condenser, is cooled to 50-60°C, and is passed into a neutral alumina column at a flow rate of 1-2 BV / h; a-3. The effluent from the alumina column flows back to the extraction tank and is circulated and extracted again; a-4. After three cycles of extraction, collect the liquid that passes through the alumina column and filter it to obtain the ginger root extract.

5. The method for preparing an anti-hair loss plant extract according to claim 3, wherein: The extraction method of Platycladus orientalis leaves in step b is: b-1. Mix Platycladus orientalis leaves and water in a weight ratio of 1:10; b-2. Adjust the pH of the solvent to 9-10; b-3. After ultrasonic extraction for 20-30 minutes, filter, collect the filtrate, and adjust the pH to 6-7; b-4. Load the filtrate onto a macroporous resin column, elute with 1,3-propylene glycol, and collect the eluate to obtain the Platycladus orientalis leaf extract.

6. The method for preparing an anti-hair loss plant extract according to claim 3, characterized in that: The extraction methods of the red clover leaves and the wormwood leaves in step c are the same, comprising the following steps: c-1. The plant raw materials, Trifolium repens leaves and Artemisia argyi leaves, were mixed with water at a weight ratio of 1:15 and extracted at 90-95°C for 2 hours; c-2. The extract is loaded onto a macroporous resin column and eluted with water, 30% ethanol, and 70% ethanol in sequence. The 70% ethanol eluate is collected and the solvent is recovered to obtain the clover leaf extract and the artemisia argyi leaf extract.

7. Use of the anti-hair loss plant extract according to any one of claims 1 to 3, characterized in that: It is used in head care products.

8. The use of the anti-hair loss plant extract according to claim 7, characterized in that: It is also used in skin oil-control and acne-control products.

9. The method for preparing an anti-hair loss plant extract according to claim 5, characterized in that: In step b-3, an ultrasonic extraction and analysis system is provided for monitoring the quality of the solvent during the ultrasonic extraction process. The ultrasonic extraction and analysis system includes an extraction state acquisition module, an extraction state analysis module, a solvent state acquisition module, a solvent state analysis module, an ultrasonic extraction database, and a quality analysis and judgment module; The extraction state acquisition module is used to obtain the power and frequency of the ultrasonic extraction instrument and the image of the ultrasonic probe on the ultrasonic extraction instrument at each preset time point, and obtain the distance between the ultrasonic probe and the bottom of the solvent container through image analysis. The extraction state acquisition module is also used to obtain the parameters of the bubbles generated around the ultrasonic probe at each preset time point; The extraction state analysis module obtains the ultrasonic extraction compliance by reading the power and frequency of the ultrasonic extraction instrument at each preset time point, the distance between the ultrasonic probe and the bottom of the solvent container, and the parameters of the bubbles generated around the ultrasonic probe, and records it as τ; The solvent state acquisition module is used to obtain the temperature and pH value of the solvent at each preset time point, which are respectively recorded as T h 、P h , h represents the hth preset time point, h=1,2,...,m; The solvent state analysis module is used to obtain the temperature and pH value of the solvent at each preset time point to analyze the solvent state compliance, and record it as ω; The ultrasonic extraction database is used to store the standard power and frequency of the ultrasonic extraction instrument when the solvent is ultrasonically extracted, the reference distance between the ultrasonic probe of the ultrasonic extraction instrument and the bottom of the container, the reference parameters for the ultrasonic probe of the ultrasonic extraction instrument to generate bubbles, and the reference temperature and reference pH value when the solvent is ultrasonically extracted; The quality analysis and judgment module is used to obtain the ultrasonic extraction quality coefficient through ultrasonic extraction compliance and solvent state compliance analysis, compare the ultrasonic extraction quality coefficient with the ultrasonic extraction quality coefficient threshold, and then analyze whether the ultrasonic extraction operation of the solvent meets the requirements.

10. The method for preparing an anti-hair loss plant extract according to claim 9, characterized in that: The parameters of the bubbles generated around the ultrasonic probe include bubble density, the size of each bubble, and the distance between each bubble and the ultrasonic probe. The bubble density obtained at each preset time point is recorded as ρ h , the distance between each bubble and the ultrasonic probe obtained at each preset time point is recorded as D h i , i represents the i-th bubble, i=1,2,...,r, using the formula: Get the discrete degree of bubbles at each preset time point χ h , where r represents the number of bubbles, D 参考 Indicates the reference distance of the bubble from the ultrasonic probe; by obtaining the size of each bubble at each preset time point and screening the bubbles whose size is larger than the reference size of the bubble, they are recorded as abnormal bubbles, and by dividing the number of abnormal bubbles at each preset time point by the total number of bubbles, the abnormal bubble ratio at each preset time point is obtained, which is recorded as α h ; Through the formula: The bubble parameter conformity index is obtained, where m represents the number of preset time points, ρ 参考 represents the reference density of the bubble, e represents a constant, and ε1 represents the correction coefficient of the bubble parameter conforming to the exponential; through the formula: Get the coincidence index of the ultrasonic instrument, where m represents the number of preset time points, W h , Hz h are the power and frequency of the ultrasonic extraction instrument at the hth preset time point, W 标准 Indicates the standard power of ultrasonic instruments, Hz 标准 It represents the standard frequency of ultrasonic instruments, and ε2 represents the correction coefficient of the ultrasonic instrument’s compliance index; through the formula: The ultrasound probe distance coincidence index is obtained, where m represents the number of preset time points, μ 参考 Indicates the reference distance between the ultrasonic probe and the bottom of the solvent container, μ h represents the distance between the ultrasonic probe and the bottom of the solvent container at the hth preset time point, and ε3 represents the correction coefficient of the ultrasonic probe distance compliance index. The ultrasonic extraction compliance is obtained by adding the bubble parameter compliance index, the ultrasonic instrument compliance index, and the ultrasonic probe distance compliance index.

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