Formula and preparation method of traditional Chinese medicine anti-hair-loss hair-growing liquid

Abstract

The invention discloses a traditional Chinese medicine anti-hair loss hair growth liquid formula and a preparation method thereof, and particularly relates to the technical field of traditional Chinese medicine hair growth, the traditional Chinese medicine anti-hair loss hair growth liquid formula comprises the following components by mass: 2-5% of a traditional Chinese medicine raw material, 25% of glycerin, 15% of butanediol, 1% of a preservative, and the balance deionized water; the traditional Chinese medicine raw material is clematis or a composition of clematis, cacumen biotae and ginger according to a mass ratio of 2: 2: 1; the preservative is a mixture of p-hydroxyacetophenone and hexanediol according to the mass ratio of 1: 1, the traditional Chinese medicine anti-hair loss and hair growth liquid is not added with chemical agents harmful to human skin, the active ingredients are high in safety performance, the medicines are mutually coordinated, and hair regeneration of a mouse seborrheic alopecia model can be effectively promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine hair growth, and particularly relates to a formula of a traditional Chinese medicine anti-hair loss and hair growth liquid and a preparation method thereof. Background Art

[0002] Hair loss is a common skin disease. Modern medicine clinically divides hair loss into seborrheic (SA), alopecia areata (AA), chemotherapy-induced alopecia, physical alopecia, pathological alopecia, growth stagnation and senile alopecia, among which seborrheic alopecia and alopecia are the most common. Traditional Chinese medicine believes that the cause of hair loss may be related to blood stasis, liver and kidney deficiency, and insufficient qi and blood, which ultimately leads to lack of nutrition in the hair and cause hair loss. In addition, the prevalence of hair loss in men in my country is about 21.3%, and that in women is about 6.0%. The clinical manifestations are a gradual reduction in hair, a receding hairline, and a gradual thinning of the hair diameter until baldness. Nowadays, people are under increasing pressure in life, and the rate of hair loss is also rising. Hair loss has also brought great trouble to people's lives.

[0003] At present, there are Western medicine, traditional medicine and a combination of Chinese and Western medicine for the treatment of hair loss, but the current treatment has not achieved obvious results and the methods are relatively limited. In the prior art, the treatment of hair loss mainly includes surgical treatment and drug treatment. Surgical treatment is to separate and cultivate the healthy hair follicles of the back of the head by surgical methods, and then evenly plant them in the hair loss area to make it grow new hair again. However, the cost of surgical treatment of hair loss is expensive. There are many drugs for the treatment of hair loss. At present, the commonly used Western medicines in clinical practice are finasteride and minoxidil, but the cure rate of these two drugs for hair loss is not high, and it is easy to relapse after stopping the drug. Long-term use may also cause side effects of varying degrees, such as scalp itching, dry hair, dizziness, cold sweats, anorexia, myocardial infarction, etc. Therefore, based on these adverse reactions, people are prompted to choose alternative effective therapeutic drugs with limited side effects, especially Chinese herbal medicines. At the same time, some hair loss products with certain therapeutic effects require a large number of raw materials, and most of them are expensive, which makes the price of hair loss treatment drugs high, making them unavailable to most hair loss patients. In addition, there are shortcomings such as many ingredients and complicated manufacturing processes. Summary of the invention

[0004] In order to solve the above problems, the present invention discloses a formula of a traditional Chinese medicine anti-hair loss and hair growth liquid and a preparation method thereof.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] A formula of a traditional Chinese medicine anti-hair loss and hair growth liquid comprises the following ingredients by mass percentage: 2-5% of traditional Chinese medicine raw materials, 25% of glycerol, 15% of butylene glycol, 1% of a preservative, and the balance is deionized water; the traditional Chinese medicine raw materials are clematis or a combination of clematis, cypress leaves and ginger in a mass ratio of 2:2:1; the preservative is a mixture of p-hydroxyacetophenone and hexylene glycol in a mass ratio of 1:1.

[0007] Preferably, the Clematis is Clematis grown in Tibet.

[0008] Preferably, the butanediol is 1,3-butanediol.

[0009] Preferably, the hexanediol is 1,2-hexanediol.

[0010] A method for preparing a Chinese medicine anti-hair loss and hair growth liquid formula comprises the following steps:

[0011] S1. Rinse the selected clematis, arborvitae leaves and ginger with clean water to remove impurities and dust on the surface;

[0012] S2. Place the cleaned clematis, arborvitae leaves and ginger in a well-ventilated and dry environment to dry naturally, avoiding direct sunlight;

[0013] S3, putting the dried clematis, arborvitae leaves and ginger into a slicer batch by batch, and slicing them for later use;

[0014] S4, weigh the corresponding proportion of Chinese medicine raw materials and put them into a container, then add the proportion of glycerol, butylene glycol and deionized water, heat to 95-100° C., and keep warm at 95-100° C. for 1 hour;

[0015] S5, turning on the cooling water to cool the mixture obtained in step S4 to 50° C. using a cooling water circulation machine, filtering the filtrate with a filter cloth, and adding a proportioned preservative;

[0016] S6. Filter the filtrate added with preservatives obtained in step S5 with a 0.1 μm filter element, and collect the filtrate to obtain the traditional Chinese medicine anti-hair loss and hair growth liquid.

[0017] Preferably, the filter cloth used in step S5 has a mesh size of 50 to 100.

[0018] Preferably, the components of the preservative in step S5 are heated to 50-60° C. to dissolve each other and then added to the filtrate.

[0019] The beneficial effects of the present invention are:

[0020] The drug formula adopted by the present invention is simple and has a wide source, and is convenient to obtain and manufacture. Conventional and easily available herbs are used, and the cost is low. The entire manufacturing method does not add chemical agents that harm human skin, so it can be used for different skin constitutions, has a wide range of applications, and the active ingredients have high safety performance. The drugs are coordinated with each other, effectively promoting hair regeneration without producing side effects. The number of hair follicles can be increased in a short period of time to obtain a better anti-hair loss and hair growth effect, and it is very convenient to be promoted and used on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Comparison photos of mouse hair before and after modeling;

[0022] Figure 2 HE staining of the skin hair follicles of mice in the blank control group and the model group (photographed under a microscope ×100);

[0023] Figure 3 This is a bar graph of serum testosterone levels in AGA model mice;

[0024] Figure 4 This is a bar graph of serum estradiol levels in AGA model mice;

[0025] Figure 5 The hair growth record of the Clematis high-dose group, low-dose group and Chinese medicine compound group on AGA model mice;

[0026] Figure 6 It is a bar graph of hair length of the Clematis high-dose group, low-dose group and Chinese medicine compound group on AGA model mice;

[0027] Figure 7 It is a bar graph of the gross weight of the Clematis high-dose group, low-dose group and Chinese medicine compound group in AGA model mice;

[0028] Figure 8 It is a bar graph of testosterone content in the Clematis high-dose group, low-dose group and Chinese medicine compound group in AGA model mice;

[0029] Fig. 9 It is a bar graph of the estradiol content of the Clematis high-dose group, the low-dose group and the Chinese medicine compound group in AGA model mice;

[0030] Fig.10 HE staining of the hair follicles on the skin of AGA model mice in the Clematis high-dose group, the low-dose group and the Chinese medicine compound group of the present invention (photographed under a microscope ×200);

[0031] Fig.11 It is a bar graph of the number of terminal hairs in the Clematis high-dose group, the low-dose group and the Chinese medicine compound group of the present invention on AGA model mice;

[0032] Fig.12 A bar graph showing the total number of hair follicles in the Clematis high-dose group, the low-dose group and the Chinese medicine compound group of the present invention on AGA model mice;

[0033] Fig.13 This is a bar graph showing the effect of the Clematis chinensis low-dose group of the present invention on the proliferation of HUVEC cells at different dilution multiples;

[0034] Fig.14 This is a bar graph showing the effect of the Chinese medicine compound group of the present invention on the proliferation of HUVEC cells at different dilution multiples;

[0035] Fig.15 This is a bar graph showing the effect of the Clematis chinensis low-dose group of the present invention on the proliferation of HaCaT cells at different dilution multiples;

[0036] Fig.16 This is a bar graph showing the effect of the Chinese herbal compound group of the present invention on the proliferation of HaCaT cells at different dilution multiples. DETAILED DESCRIPTION

[0037] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0038] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0039] A formula of a traditional Chinese medicine anti-hair loss and hair growth liquid comprises the following components by mass percentage: 2-5% of traditional Chinese medicine raw materials, 25% of glycerol, 15% of butylene glycol, 1% of a preservative, and the balance is deionized water; the traditional Chinese medicine raw materials are clematis or a combination of clematis, cypress leaves and ginger in a mass ratio of 2:2:1; the preservative is a mixture of p-hydroxyacetophenone and hexylene glycol in a mass ratio of 1:1.

[0040] The clematis was grown in Tibet, and the arborvitae leaves and ginger were purchased from the traditional Chinese medicine market.

[0041] Butanediol is 1,3-butanediol, purity ≥99.5%, from OQ Chemicals Corporation, USA, CAS No.: 110-63-41.

[0042] Glycerin is cosmetic grade propylene glycol, purity ≥99.5%, Malaysia KLK, CAS number: 56-81-5.

[0043] Hexanediol is 1,2-hexanediol, purity ≥99.7%, Shandong Heya Biotechnology, CAS No.: 6920-22-5.

[0044] p-Hydroxyacetophenone, purity ≥99.0%, Kunshan Shuangyou, CAS No.: 99-93-4.

[0045] A method for preparing a Chinese medicine anti-hair loss and hair growth liquid formula comprises the following steps:

[0046] S1. Rinse the selected clematis, arborvitae leaves and ginger with clean water to remove impurities and dust on the surface;

[0047] S2. Place the cleaned clematis, arborvitae leaves and ginger in a well-ventilated and dry environment to dry naturally, avoiding direct sunlight;

[0048] S3, putting the dried clematis, arborvitae leaves and ginger into a slicer batch by batch, and slicing them for later use;

[0049] S4, weigh the corresponding proportion of Chinese medicine raw materials and put them into a container, then add the proportion of glycerol, butylene glycol and deionized water, heat to 95-100° C., and keep warm at 95-100° C. for 1 hour;

[0050] S5. Turn on the cooling water to cool the mixture obtained in step S4 to 50°C using a cooling water circulation machine (DLSB-20 / 10 from Shanghai Yarong Biochemical Instrument Factory, parameters of the cooling liquid circulation machine: temperature range (°C): -10 to 99°C; temperature stability (°C): ±0.1°C), filter out the filtrate using a 50-100 mesh filter cloth, and add a proportioned preservative;

[0051] S6. Filter the filtrate added with preservatives obtained in step S5 with a 0.1 μm filter element, and collect the filtrate to obtain the traditional Chinese medicine anti-hair loss and hair growth liquid.

[0052] The components of the preservative in step S5 are heated to 50-60° C. to dissolve each other and then added to the filtrate.

[0053] Example 1

[0054] The following components are weighed in percentage by mass: 2% of clematis, 25% of glycerol, 15% of butylene glycol, 1% of preservative, and 57% of deionized water; the clematis is placed in a container; the proportion of glycerol, butylene glycol, and deionized water are then added; the mixture is heated to 100°C and kept at 100°C for 1 hour; the mixture is then cooled to 50°C, the filtrate is filtered out with a 100-mesh filter cloth, and a proportion of preservative is added (the components of the preservative are heated to 50°C and dissolved with each other, and then the filtrate is added); finally, the filtrate with the preservative added is filtered with a 0.1 μm filter element, and the collected product is a low-dose clematis anti-hair loss and hair growth liquid.

[0055] Example 2

[0056] The following components are weighed in percentage by mass: 5% of clematis, 25% of glycerol, 15% of butylene glycol, 1% of preservative, and 54% of deionized water; the clematis is placed in a container; then the proportioned glycerol, butylene glycol, and deionized water are added; the mixture is heated to 100°C and kept at 100°C for 1 hour; the mixture is then cooled to 50°C, the filtrate is filtered out with a 100-mesh filter cloth, and a proportioned preservative is added (the components of the preservative are heated to 50°C and dissolved in each other, and then the filtrate is added); finally, the filtrate with the preservative added is filtered with a 0.1 μm filter element, and the collected product is the clematis high-dose anti-hair loss and hair growth liquid.

[0057] Example 3

[0058] Weigh the following components by mass percentage: 3% of clematis, 25% of glycerol, 15% of butylene glycol, 1% of preservative, and 56% of deionized water, put clematis in a container, then add the proportioned glycerol, butylene glycol, and deionized water, heat to 95°C, and keep warm at 95°C for 1 hour, then cool the mixture to 50°C, filter out the filtrate with a 50-mesh filter cloth, and add the proportioned preservative (the components of the preservative are heated to 60°C and dissolved in each other before adding the filtrate), finally filter the filtrate with the preservative added with a 0.1 μm filter element, and collect the clematis medium-dose anti-hair loss and hair growth liquid. The medium-dose anti-hair loss and hair growth liquid of clematis was used alone for hair loss treatment experiments, and it has a significant effect on promoting hair growth, and does not participate in the experiments of Experimental Examples 1, 2, and 3.

[0059] Example 4

[0060] The following components are weighed in percentage by mass: 2% of clematis, 2% of Platycladus orientalis leaves, 1% of ginger, 25% of glycerol, 15% of butylene glycol, 1% of preservative, and 54% of deionized water; the clematis is placed in a container, and then the proportioned glycerol, butylene glycol, and deionized water are added, and the mixture is heated to 100°C and kept at 100°C for 1 hour; the mixture is then cooled to 50°C, and the filtrate is filtered out with a 100-mesh filter cloth, and a proportioned preservative is added (the components of the preservative are heated to 50°C and dissolved in each other, and then the filtrate is added); finally, the filtrate with the preservative added is filtered with a 0.1 μm filter element, and the collected product is a traditional Chinese medicine compound anti-hair loss and hair growth liquid.

[0061] Efficacy evaluation

[0062] Experimental Example 1: Experimental evaluation of the efficacy of high- and low-dose Clematis anti-hair loss and hair growth liquid and Chinese herbal compound anti-hair loss and hair growth liquid on the seborrheic alopecia model in C57BL / 6JGpt mice

[0063] 1. Experimental Animals

[0064] The experimental animals were 38 male C57BL / 6JGpt, 5 weeks old, weighing 18-20 g, purchased from Jicui Yaokang Biological Co., Ltd. C57BL / 6JGpt mice were raised under normal conditions, with a temperature of 25±2°C and a humidity of 50%-70%, and the experiment began after they had adapted to the breeding environment for 7 days.

[0065] 2. Drug preparation

[0066] The dosage concentrations of the Clematis low-dose group, the Clematis high-dose group and the Chinese medicine compound group were 5 times diluted with pure water based on the anti-hair loss and hair growth liquid of Example 1, Example 2 and Example 4, respectively.

[0067] Minoxidil lotion: The content of active ingredient minoxidil is 2%, prepared by: Dermatology Hospital, Chinese Academy of Medical Sciences.

[0068] Preparation of testosterone propionate solution: Prepare according to the 5mg / kg / d dose required by mice. The specification of testosterone propionate injection is 25mg:1ml (Hangzhou Animal Pharmaceutical Factory). Dilute with peanut oil before injection, according to the ratio of oil to drug 4:1, and the concentration is 5mg / ml. Use a vortex mixer to mix thoroughly during the dilution process.

[0069] 3. Seborrheic alopecia mouse model

[0070] Ten mice were randomly selected as the blank control group, and the remaining mice were fed in separate cages. The blank control group was subcutaneously injected with normal saline (purchased from Shanghai Lianzu Biotechnology Co., Ltd.), and the modeling group was subcutaneously injected with testosterone propionate at a dose of 5 mg / kg / d on the back of the mice parallel to the spine for 5 weeks to establish a mouse model of androgenetic alopecia (AGA).

[0071] The judgment criteria for the successful establishment of the C57BL / 6JGpt mouse seborrheic alopecia animal model are as follows: 1. Compared with the blank control group, the hair of the mice in the model group became greasy and had a tendency to fall off. Figure 1 ; 2. In the HE staining, the number of terminal hair in the model group decreased, the number of vellus hair increased (red arrows indicate terminal hair, black arrows indicate vellus hair), and a few hair follicles were necrotic. Figure 2 ; 3. The testosterone content in the serum of mice after modeling was significantly increased compared with the blank control group (*** indicates P < 0.001), while the serum estradiol content was decreased (* indicates P < 0.05). Figure 3 , 4 If the above three conditions are met at the same time, it means that the seborrheic alopecia model is successfully established.

[0072] 4. Treatment experiment on mice with seborrheic alopecia

[0073] After successful modeling, 25 modeling mice were randomly divided into a high-dose Clematis group, a low-dose Clematis group, a Chinese medicine compound group, a minoxidil positive group, and a model control group, with 5 mice in each group. Depilatory cream was used to remove hair and evenly applied on the back of the mice. The depilatory area was 3 cm × 3 cm. Except for the blank control group, the other groups continued to be injected with testosterone propionate, and the corresponding drugs were applied to the depilatory area. The high-dose Clematis group, the low-dose Clematis group, and the Chinese medicine compound group were applied with 0.2 ml / mouse of Clematis high-dose anti-hair loss and hair growth liquid, low-dose Clematis anti-hair loss and hair growth liquid of Chinese medicine compound, respectively, twice a day; the minoxidil positive group was applied with 0.2 ml / mouse of minoxidil lotion, twice a day; the model control group and the blank control group were continuously applied with PBS solution (Sangon Biotechnology Co., Ltd.) for 18 days.

[0074] 5. Experiment on the efficacy of Chinese herbal extracts on mice with seborrheic alopecia

[0075] 1) Analysis of hair growth in AGA mice

[0076] Hair growth was photographed and scored in each group of mice every day. Figure 5 , it can be seen from the hair growth pictures that the back skin of mice appeared pink after the hair fell off, and with the passage of time, the back skin of the blank control group turned gray on the 5th day, the skin of the minoxidil positive group, the Chinese medicine compound group and the high-dose clematis group gradually turned gray on the 8th day, and the skin of the model control group turned gray on the 10th day, indicating that the hair growth of mice in the blank control group entered the growth phase earliest, while the minoxidil positive group, the Chinese medicine compound group and the high-dose clematis group entered the growth phase earlier than the mice in the seborrheic model control group; when the hair grew to 14 days, the density and length of the back skin hair of the blank control group mice were about half of that of the non-hair-shedding area. When the hair growth reached 18 days, the back hair of the blank control group mice was the longest and densest, followed by the minoxidil positive group, the hair density of the Chinese medicine compound group, the high-dose clematis group and the low-dose clematis group decreased successively, and the back skin hair of the model control group mice was not fully grown.

[0077] On the 18th day of administration, mice were killed by cervical dislocation, and 10 hairs were randomly plucked from each mouse. The average value was taken as the length of new hair of each mouse, and the mean hair length of each group was calculated. SPSS21.0 software one-way ANOVA was used for analysis. Measurement data were expressed as mean ± standard deviation. Indicates, P<0.05 indicates statistically significant difference, compared with the model control group, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001. Figure 6As shown in the figure, compared with the model group, the high-dose Clematis group, the low-dose Clematis group and the Chinese medicine compound group all significantly increased hair length (*P<0.05). Compared with the minoxidil positive group, there was no statistical difference among the groups.

[0078] After the hair length measurement is completed, carefully shave the hair in the modeling area with an animal shaver, collect and save the hair on weighing paper, and then weigh it with a 100,000th balance to record the gross weight value. Figure 7 As shown in the figure, compared with the model control group, the Clematis high-dose group, Clematis low-dose group and the Chinese medicine compound group all significantly increased hair weight (**P<0.01). And compared with the minoxidil positive group, there was no statistical difference among the groups.

[0079] The above results indicate that the high-dose Clematis group, low-dose Clematis group, and Chinese medicine compound group can promote hair growth in AGA mice, and have the same effect as the minoxidil positive drug in increasing hair length and weight.

[0080] 2) Analysis of hormone levels in AGA mice

[0081] 18 days after the mice were smeared on the skin, the eyeballs were removed and blood was collected. The blood was allowed to coagulate at room temperature for 30 minutes, centrifuged at 3000r / min for 10 minutes, and the serum was collected and stored in a -80℃ refrigerator. According to the instructions of the kit, the testosterone (T) and estradiol (E2) in the mouse serum were detected. Compared with the model control group, * indicates P<0.05, and ** indicates P<0.01.

[0082] Rat testosterone ELISA kit, catalog number D751045, was purchased from Sangon Biotechnology Co., Ltd.; mouse estradiol ELISA kit, catalog number D741005, was purchased from Sangon Biotechnology Co., Ltd.

[0083] The results are as follows Figure 8 , 9 Compared with the model control group, there were no significant differences in the testosterone and estradiol levels in the minoxidil positive group, the Clematis high-dose group, and the Clematis low-dose group. This indicates that the above groups did not have an effect on the hormones of AGA model mice. Compared with the model control group, the testosterone content in the Chinese medicine compound group decreased significantly (*P<0.05), while the estradiol content in the Chinese medicine compound group increased significantly (*P<0.05).

[0084] The above results indicate that the Chinese herbal compound group can downregulate the ratio of testosterone to estradiol and inhibit seborrheic alopecia by regulating the hormone levels of mice.

[0085] 3) Analysis of terminal hair number and total hair follicle number in AGA mice

[0086] In order to observe the terminal hair follicles and the total number of hair follicles, HE staining sections were performed on the experimental area of ​​the back of the mice. The cross-section of the hair follicles was photographed under a microscope (×200) (Motic, model ae2000), and 5 fields of view were randomly selected under a microscope (×100). The terminal hair and the total number of hair follicles were counted using Image J software, and the number of hair follicles and terminal hair in different groups was statistically analyzed using SPSS21.0 software one-way ANOVA. Compared with the model control group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001; compared with the minoxidil positive group, # Indicates P < 0.05. Microscope ×200 means the magnification of the eyepiece is 10 times and the objective lens is 20 times; ×100 means the magnification of the eyepiece is 10 times and the objective lens is 10 times.

[0087] The results are as follows Fig.10 , 11 and Fig.12 , Fig.10 The red arrow indicates terminal hair, and the black arrow indicates vellus hair. Compared with the model control group, the number of terminal hair follicles in the minoxidil positive group (***P < 0.001), the Chinese medicine compound group (***P < 0.001), and the Clematis high-dose group (***P < 0.001) increased significantly, and the Clematis low-dose group also increased the number of terminal hair (*P < 0.05). Compared with the minoxidil positive group, there was no significant difference in the number of terminal hair in the Clematis high-dose group and the Chinese medicine compound group. In terms of the total number of hair follicles, compared with the model control group, the minoxidil positive group (**P < 0.01), the Chinese medicine compound group (**P < 0.01), the Clematis high-dose group (*P < 0.05), and the Clematis low-dose group (*P < 0.05) could significantly increase the total number of hair follicles. Compared with the minoxidil positive group, there was no significant difference in the total number of hair follicles among the high-dose Clematis group, low-dose Clematis group and Chinese medicine compound group.

[0088] The above results indicate that the high-dose Clematis group, low-dose Clematis group and Chinese medicine compound group can increase the total number of terminal hair and hair follicles in AGA mice, and the high-dose Clematis group and Chinese medicine compound group have the same effect as the positive drug minoxidil.

[0089] Experimental Example 2: Effects of low-dose Clematis anti-hair loss and hair growth liquid and traditional Chinese medicine compound anti-hair loss and hair growth liquid on HUVEC cell proliferation

[0090] 1. Preparation of experimental drugs

[0091] The Clematis low-dose anti-hair loss and hair growth liquid (Example 1 above) and the Chinese herbal compound anti-hair loss and hair growth liquid (Example 4 above) were diluted in DMEM culture medium in sequence into six concentration gradients of 40, 80, 160, 320, 640, and 1280 times.

[0092] 2. Cell culture conditions

[0093] After taking out the HUVEC cells frozen at -80°C, immediately place them in a 37°C constant temperature water bath and gently shake them to thaw quickly. Then add the cell suspension into a 10ml centrifuge tube containing 5ml complete culture medium, centrifuge at 1000rpm for 5min, discard the supernatant, and culture. When the cells grew to cover 80% to 90% of the bottom of the culture flask, they were subcultured, washed twice with 2 mL of PBS, digested with 1 mL of 0.25% trypsin (Quanshijin Biotechnology Co., Ltd.), and observed under a trinocular upright microscope (McOdy). When the cells became round, 2 mL of complete culture medium was immediately added to terminate the digestion. The cells were gently blown off the wall of the culture flask and transferred to a 10 mL centrifuge tube. After centrifugation at 1000 rpm for 5 min, the supernatant in the centrifuge tube was discarded, 2 mL of complete culture medium was added, and the cell suspension was subcultured to a new t25 culture flask at a ratio of 1:2 to 1:5 after gently blowing. The culture was continued in a 37°C constant temperature and 5% CO2 saturated humidity incubator (Thermo Electron Corporation).

[0094] The complete culture medium contains 89% DMEM high glucose culture medium (Gibco), 10% fetal bovine serum (Sijiqing), and 1% penicillin-streptomycin solution (including 100 U / mL penicillin and 100 μg / mL streptomycin, Sangon Biotechnology Co., Ltd.) by volume.

[0095] 3. HUVEC cell proliferation assay

[0096] HUVEC cells in logarithmic growth were taken and about 3000 cells were inoculated in each well of a 96-well plate. A blank control group (no cells), a cell control group (no drug), and groups of different concentrations of Chinese herbal extracts were set up, with 6 replicate wells in each group.

[0097] After the cells were cultured overnight, the medium was discarded, and the blank control group and the cell control group were added with DMEM medium, and the experimental group was given 100 μL of drug, and cultured in the incubator for 48 h. 10 μL of CCK-8 reagent (Sangon Biotechnology Co., Ltd.) was added to each group of wells and incubated in a 37°C incubator for 1 h to detect the absorbance value at a wavelength of 450 nm (BioTeK EON microplate reader).

[0098] Repeat 3 times, cell proliferation rate = (As-Ab) / (Ac-Ab). As: absorbance of drug administration group, Ab: absorbance of blank control group, Ac: absorbance of cell control group.

[0099] SPSS 21.0 software was used for one-way ANOVA analysis. Measurement data were expressed as mean ± standard deviation. Compared with the cell control group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0100] The experimental results are as follows Fig.13 and Fig.14 Compared with the cell control group, 48 hours after administration, the Clematis low-dose anti-hair loss and hair growth liquid (Example 1) can significantly promote the proliferation of HUVEC cells at concentrations diluted 80 and 160 times (*P < 0.05), and the proliferation-promoting effect begins to decline at a dilution of 320 times; the Chinese herbal compound ingredients can significantly promote the proliferation of HUVEC cells at concentrations diluted 40, 80, 160, and 320 times (***P < 0.001), and still have a proliferation-promoting effect at a concentration diluted 640 times (*P < 0.05).

[0101] The above results show that the low-dose Clematis group and the Chinese medicine compound group can promote the proliferation of vascular endothelial cells (HUVEC). Under the same Clematis content conditions, the Chinese medicine compound group has a better proliferation-promoting effect than the single Clematis group.

[0102] Experimental Example 3: Effects of low-dose Clematis anti-hair loss and hair growth liquid and traditional Chinese medicine compound anti-hair loss and hair growth liquid on the proliferation of HaCaT cells

[0103] 1. Preparation of experimental drugs

[0104] Same as Experiment 2

[0105] 2. Cell culture conditions

[0106] After taking out the HaCaT cells frozen at -80°C, immediately place them in a 37°C constant temperature water bath and gently shake them to thaw quickly. Then add the cell suspension into a 10ml centrifuge tube containing 5ml complete culture medium, centrifuge at 1000rpm for 5min, discard the supernatant, and culture. When the cells grew to cover 80% to 90% of the bottom of the culture flask, they were subcultured, washed twice with 2 mL of PBS, added with 1 mL of 0.25% trypsin (Quanshijin Biotechnology Co., Ltd.), digested in a 37°C incubator for 10 min, and observed under a trinocular upright microscope (McOdy). When the cells became round, 2 mL of complete culture medium was immediately added to terminate the digestion. The cells were gently blown off the wall of the culture flask and transferred to a 10 mL centrifuge tube. After centrifugation at 1000 rpm for 5 min, the supernatant in the centrifuge tube was discarded, 2 mL of complete culture medium was added, and the cell suspension was subcultured to a new t25 culture flask at a ratio of 1:2 to 1:5 after gently blowing evenly, and continued to be cultured in a 37°C constant temperature, 5% CO2, saturated humidity incubator (Thermo Electron Corporation, model 3111).

[0107] The complete culture medium contained 89% DMEM high glucose culture medium (Gibco), 10% fetal bovine serum (Shanghai Jingkai Biotechnology Co., Ltd.), and 1% penicillin-streptomycin solution (including 100 U / mL penicillin and 100 μg / mL streptomycin, Sangon Biotechnology Co., Ltd.) by volume.

[0108] 3. HaCaT cell proliferation experiment

[0109] HaCaT cells in logarithmic growth were taken and about 5000 cells were inoculated in each well of a 96-well plate. A blank control group (no cells), a cell control group (no drug), and groups of different concentrations of Chinese herbal extracts were set up, and each group had 6 replicate wells.

[0110] After the cells were cultured overnight, the medium was discarded, and the blank control group and the cell control group were added with DMEM medium, and the experimental group was given 100 μL of drug, and cultured in the incubator for 48 h. 10 μL of CCK-8 reagent (Sangon Biotechnology Co., Ltd.) was added to each group of wells and incubated in a 37°C incubator for 1 h to detect the absorbance value at a wavelength of 450 nm (BioTeK EON microplate reader).

[0111] The experiment was repeated 3 times, and the cell proliferation rate = (As-Ab) / (Ac-Ab). As: absorbance of the drug administration group, Ab: absorbance of the blank control group, Ac: absorbance of the cell control group.

[0112] SPSS 21.0 software was used for one-way ANOVA analysis. Measurement data were expressed as mean ± standard deviation. Compared with the control group, *P<0.05, **P<0.01, ***P<0.001.

[0113] The results are as follows Fig.15 and Fig.16 Compared with the cell control group, after 48 hours of administration, the proliferation rate of HaCaT in the low-dose Clematis group (Example 1) and the Chinese medicine compound group (Example 4) increased with the increase of concentration within the dilution range of 40 to 320 times, and the cell proliferation was dose-dependent. The low-dose Clematis group and the Chinese medicine compound group had significant proliferation-promoting effects at concentrations of 40, 80, 160, 320, and 640 times dilution; while the Chinese medicine compound group still had a proliferation-promoting effect at a concentration of 1280 times dilution (*P<0.05).

[0114] The above results show that the low-dose Clematis group and the Chinese medicine compound group can promote the proliferation of human immortalized epidermal cells (HaCaT). Under the same Clematis content conditions, the Chinese medicine compound group has a better proliferation-promoting effect than the single Clematis group.

[0115] It should be noted that the above content only illustrates the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications all fall within the protection scope of the claims of the present invention.

Claims

1. A Chinese medicine formula for preventing hair loss and promoting hair growth, characterized in that: The composition comprises the following ingredients by mass percentage: 2-5% of Chinese medicine raw materials, 25% of glycerol, 15% of butylene glycol, 1% of preservative, and the balance is deionized water; the Chinese medicine raw materials are clematis or a combination of clematis, cypress leaves and ginger in a mass ratio of 2:2:1; the preservative is a mixture of p-hydroxyacetophenone and hexylene glycol in a mass ratio of 1:

1.

2. A Chinese medicine anti-hair loss and hair growth liquid formula according to claim 1, characterized in that: The clematis is the clematis produced in Tibet.

3. A Chinese medicine anti-hair loss and hair growth liquid formula according to claim 1, characterized in that: The butanediol is 1,3-butanediol.

4. A Chinese medicine anti-hair loss and hair growth liquid formula according to claim 1, characterized in that: The hexanediol is 1,2-hexanediol.

5. A method for preparing a traditional Chinese medicine anti-hair loss and hair growth liquid formula as claimed in any one of claims 1 to 4, characterized in that: The steps include: S1. Rinse the selected clematis, arborvitae leaves and ginger with clean water to remove impurities and dust on the surface; S2. Place the cleaned clematis, arborvitae leaves and ginger in a well-ventilated and dry environment to dry naturally, avoiding direct sunlight; S3, putting the dried clematis, arborvitae leaves and ginger into a slicer batch by batch, and slicing them for later use; S4, weigh the corresponding proportion of Chinese medicine raw materials and put them into a container, then add the proportion of glycerol, butylene glycol and deionized water, heat to 95-100° C., and keep warm at 95-100° C. for 1 hour; S5, cooling the mixture obtained in step S4 to 50° C. using a cooling water circulation machine, filtering the filtrate with a filter cloth, and adding a proportioned preservative; S6. Filter the filtrate added with preservatives obtained in step S5 with a 0.1 μm filter element, and collect the filtrate to obtain the traditional Chinese medicine anti-hair loss and hair growth liquid.

6. The method for preparing a traditional Chinese medicine anti-hair loss and hair growth liquid formula according to claim 5, characterized in that: The filter cloth used in step S5 is 50-100 meshes.

7. The method for preparing a traditional Chinese medicine anti-hair loss and hair growth liquid formula according to claim 5, characterized in that: The components of the preservative in step S5 are heated to 50-60° C. to dissolve each other and then added to the filtrate.

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