Semen cuscutae compound anti-hair loss and hair growth preparation and preparation method thereof

Abstract

The invention provides a semen cuscutae compound anti-hair loss and hair growth preparation and a preparation method thereof, and belongs to the technical field of medicines. The semen cuscutae compound anti-hair-loss hair-growing preparation is prepared from the following raw materials: 3 parts of semen cuscutae, 3 parts of cortex lycii radicis, 8 parts of fructus aurantii, 19-20 parts of fresh radix rehmanniae and 20-21 parts of fresh radix achyranthis bidentatae. By adjusting the ratio of the raw materials and the preparation method, a complex preparation mode of refining honey into pills in a traditional Chinese dodder pill prescription is eliminated, the effective components of the three medicinal materials of dodder, cortex lycii radicis and fructus aurantii are extracted by adopting an ethanol water reflux method, and the effective components of fresh rehmannia and fresh radix achyranthis bidentatae are extracted by adopting a juicing mode. The nutritional value of the Chinese herbal medicines can be fully utilized, the dosage is reduced, the operation convenience is improved, and the application range is expanded. The semen cuscutae compound anti-alopecia hair-growing preparation provided by the invention has good effects of preventing / treating alopecia and promoting hair growth in an alopecia model caused by chronic pressure stress of C57BL / 6 mice.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a preparation for alleviating hair loss induced by chronic stress, promoting hair growth and preventing hair loss, and a preparation method thereof. Background Art

[0002] Corticosterone is a stress hormone in mice, similar to human cortisol. Chronic stress leads to increased corticosterone secretion from the adrenal glands, accompanied by reduced hair growth. Corticosterone causes hair follicle stem cells to enter a prolonged resting phase without regenerating new hair follicles or hair. Normally, hair follicle stem cells are activated during the growth phase to regenerate hair follicles and hair, while during the resting phase, the stem cells are in a quiescent state and hair is more likely to fall out. If stem cells remain quiescent after hair falls out and do not generate new tissue, hair loss will result. Under chronic stress, corticosterone levels increase, which inhibits the expression of Gas6 protein by acting on the glucocorticoid receptors of hair papilla cells. Gas6 protein is a key molecule for activating hair follicle stem cells. Its suppressed expression will cause the AXL receptor on the hair follicle stem cells to fail to be activated, thereby failing to receive the signal to divide, resulting in the inability of hair follicle stem cells to divide, a prolonged resting phase, and ultimately hair loss.

[0003] Similarly, in the human body, cortisol not only affects the growth cycle of hair follicles, but also interferes with the nutritional supply of hair follicles. It can cause the blood vessels of the scalp to constrict, reduce the blood flow of the scalp, and thus reduce the hair follicles' uptake of nutrients, causing the hair follicles to shrink and fall out due to insufficient nutrition. In addition, cortisol also affects the metabolic process of hair follicle cells, increases the level of oxidative stress in hair follicle cells, and leads to damage and death of hair follicle cells. Oxidative stress refers to the imbalance between the oxidation and antioxidant systems in the body. Excessive reactive oxygen species (ROS) will attack cell membranes, proteins and DNA, causing cell damage. Under the action of oxidative stress, the normal physiological functions of hair follicle cells are inhibited, which in turn causes hair loss.

[0004] Chinese herbal medicine has a long history of treating diseases and is a core component of Chinese medicine culture. Chinese medicine theories such as the Yin-Yang and Five Elements theory, and the theory of zang-fu meridians are closely related to the application of Chinese herbal medicine. For example, Chinese medicine believes that the human body is an organic whole that is interconnected through the meridian system. The meridian theory of Chinese herbal medicine is based on the meridian theory. For example, Chuanxiong belongs to the liver, gallbladder, and pericardium meridians. When treating headaches, Chinese medicine chooses medicines based on the meridians to which the headache belongs. If the headache belongs to the liver meridian headache, which manifests as swelling and pain on both sides of the head, Chuanxiong can be used for treatment, because Chuanxiong can dispel wind and cold, promote blood circulation and qi, and transport the drug effect to the lesion through the meridians, thereby relieving headache symptoms. This application method of Chinese herbal medicine reflects the holistic concept and dialectical treatment of Chinese medicine, and is an important carrier of Chinese medicine culture inheritance.

[0005] Many Chinese medical literatures have mentioned the effect of Cuscuta Seed Pills in promoting hair growth. Among them, the "Puji Fang" Volume 50 Head Chapter records: "It can treat yellow and white beards and turn them black, and treat bald beards and hair that do not grow. It comes from Shenghui Fang. Soak the dodder seeds in wine for three days and pound them separately. Take 3 liang of Radix Rehmanniae, 8 liang of dried atractylodes peel with the pulp removed and bran fried, and pound and extract half a liter of juice from fresh Achyranthes bidentata. , crush the raw Rehmannia root and take out half a liter of juice. Grind the above three ingredients into powder, mix with Achyranthes bidentata and Rehmannia root juice to make cakes, dry them and crush them again, mix with honey to make pills as big as sycamore seeds. Take 15 pills each time, with warm wine after breakfast or with cooked rice water at night. Another recipe crushes the raw Rehmannia root into powder, mixes with honey to make pills, take 30 pills on an empty stomach with raw ginger soup every day, gradually increase to 50 pills, avoid taking raw onions, radishes and garlic for a long time.

[0006] Cuscuta Pills have the effects of nourishing the kidney and replenishing qi, nourishing the liver and kidneys, and strengthening the tendons and bones. It is a commonly used prescription for health care. Currently, the research and application of Cuscuta Pills are mostly focused on the effects of tonifying deficiency and strengthening the spleen, promoting blood circulation and removing blood stasis. There has been no research and application on improving hair loss caused by chronic stress.

[0007] The ancient method of preparing Cuscuta Pills is complicated, including making cakes, drying, refining honey into pills, and taking with warm wine, which affects the full extraction of effective ingredients. The finished product is a brown-black thick semi-fluid. The sugar content is high after being refined into pills with honey, which is inconvenient to prepare and carry, and has the disadvantages of high sugar content. Summary of the invention

[0008] In view of the above deficiencies in the prior art, the present invention provides a dodder seed compound anti-hair loss and hair growth preparation and a preparation method thereof, wherein the preparation can reduce hair loss induced by chronic stress, promote hair growth and prevent hair loss.

[0009] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0010] In a first aspect, the present invention provides a dodder seed compound hair loss prevention and growth preparation, the raw materials of which include: 3 parts of dodder seed, 3 parts of lycium bark, 8 parts of aurantium, 19-20 parts of fresh rehmannia root and 20-21 parts of fresh achyranthes bidentata.

[0011] Preferably, the raw materials of the Cuscuta compound hair loss prevention and hair growth preparation include: 3 parts of Cuscuta, 3 parts of Lycium bark, 8 parts of Citrus aurantium, 19.6 parts of fresh Rehmannia root and 20.3 parts of fresh Achyranthes bidentata.

[0012] In a second aspect, the present invention provides a method for preparing the Cuscuta chinensis compound hair loss prevention and hair growth preparation, comprising the following steps:

[0013] soaking powdered Cuscuta seeds, Lycium bark and Fructus Aurantii Immaturus in ethanol water, extracting under reflux conditions, filtering and concentrating to obtain a mixture I;

[0014] Separately squeeze the juice of fresh Rehmannia root and fresh Achyranthes bidentata, filter, combine and concentrate to obtain a mixture II;

[0015] The mixture I and the mixture II are mixed evenly, and freeze-dried to obtain a Cuscuta compound hair loss prevention and hair growth preparation.

[0016] Preferably, the freeze-dried powder obtained by freeze-drying is made into an oral preparation, which includes but is not limited to granules, compressed tablets, and oral liquids.

[0017] Preferably, the content of ethanol in the ethanol water is 60% to 80%.

[0018] Preferably, the amount of the ethanol water is 3 to 5 times the total weight of Cuscuta australis, Cortex Lycii and Fructus Aurantii Immaturus.

[0019] Preferably, the soaking time of the Cuscuta seeds, the Cortex Lycii and the Fructus Aurantii Immaturus in the ethanol water is 30 to 60 min.

[0020] Preferably, the extraction is repeated multiple times, specifically 2 to 3 times, and after each reflux extraction, the mixture is filtered while hot, the filtrates are combined, and concentrated under reduced pressure to obtain a mixture I; the temperature for the reduced pressure concentration is 55 to 60°C.

[0021] Preferably, before squeezing the juice of the fresh Rehmannia root, 0.4 to 0.6 times of water by weight is added; before squeezing the juice of the fresh Achyranthes bidentata root, 1.0 to 1.2 times of water by weight is added.

[0022] Specifically, the preparation method of the Cuscuta australis compound hair loss prevention and hair growth preparation comprises the following steps:

[0023] 1) crushing Cuscuta chinensis, Lycium bark and Fructus Aurantii Immaturus into powder, soaking them in 60% to 80% ethanol water, and then extracting them 2 to 3 times under reflux conditions, filtering them while hot after each reflux extraction, and concentrating the filtrate to obtain a mixture I;

[0024] 2) Add 0.4-0.6 times and 1.0-1.2 times of water to fresh Rehmannia root and fresh Achyranthes bidentata, respectively, squeeze the juice, filter, combine, and concentrate to obtain mixture II;

[0025] 3) Mixing mixture I and mixture II evenly, and freeze-drying to obtain a Cuscuta compound hair loss prevention and hair growth preparation.

[0026] In a third aspect, the present invention provides use of the preparation in preparing a product for preventing and / or treating hair loss caused by chronic stress.

[0027] Specifically, the preparation helps to increase the density and follicle length of the hair on the back of mice; promote the transition of hair from the catagen phase to the growth phase; reduce hair loss induced by chronic stress; reduce the level of stress hormones in skin tissue; restore the level of oxidative stress in the body to normal; reduce hair follicle inflammation by reducing the expression of IL-1β, IL-6, and IL-17A; accelerate the activation of hair follicle stem cells and increase the hair growth cycle by increasing the level of LDH; promote angiogenesis, cell proliferation and differentiation, and inhibit cell apoptosis by regulating the expression levels of VEGFA, IGF-1, and TGF-β2 mRNA; induce hair growth through the Wnt / β-catenin pathway, and downregulate the expression of GSK3β protein and the accumulation and activation of β-catenin and Wnt 5a.

[0028] Compared with the prior art, the present invention is beneficial in that:

[0029] The present invention adjusts the prescription ratio and preparation method of the traditional dodder pill, eliminates the complicated production method, uses the ethanol-water reflux method to extract the effective ingredients of the three medicinal materials of dodder, radix rehmanniae and fructus aurantii, and squeezes the juice of fresh rehmanniae and fresh achyranthes bidentata to extract their effective ingredients. The present invention can make full use of the nutritional value of Chinese herbal medicine, reduce the dosage, improve the convenience of operation, and expand the scope of application. The dodder compound hair loss prevention and hair growth preparation provided by the present invention has a good effect of preventing / treating hair loss and promoting hair growth in the hair loss model caused by chronic stress in C57BL / 6 mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The effect of the preparation of the present invention on the hair loss mouse model induced by corticosterone stress hormone;

[0031] Figure 2 This is a graph of the weight growth rate of mice during the process of the present invention;

[0032] Figure 3 This is a graph showing the relative values ​​of liver and kidney organ indexes of mice in the results of the present invention;

[0033] Figure 4 The results of H&E staining of the back skin of mice during the process of the present invention;

[0034] Figure 5 This is a graph showing the relative thickness of the dermis and epidermis of the mouse back skin during the process of the present invention;

[0035] Figure 6 This is a graph showing the relative value of the hair follicle length on the back of mice during the process of the present invention;

[0036] Figure 7 This is a graph showing the relative values ​​of corticosterone levels in mouse serum in the results of the present invention;

[0037] Figure 8 The effect of the preparation of the present invention on the LDH content in mouse serum;

[0038] Fig. 9 The effect of the preparation of the present invention on the IL-1β content in mouse serum;

[0039] Fig.10 The effect of the preparation of the present invention on the IL-6 content in mouse serum;

[0040] Fig.11 The effect of the preparation of the present invention on the IL-17A content in mouse serum;

[0041] Fig.12 The effect of the preparation of the present invention on the level of ROS in mouse skin tissue;

[0042] Fig.13 The results of the present invention are the results of measuring the mRNA expression levels of VEGFA, IGF-1 and TGF-β2 genes in each treatment group;

[0043] Fig.14 These are the results of measuring the expression levels of GSK3β, P-GSK3β, Wnt 5a and β-catenin proteins in each treatment group in the results of the present invention. DETAILED DESCRIPTION

[0044] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] The invention provides a dodder seed compound hair loss prevention and growth preparation, the raw materials of which include: 3 parts of dodder seeds, 3 parts of lycium bark, 8 parts of fructus aurantii, 19-20 parts of fresh rehmannia root and 20-21 parts of fresh achyranthes bidentata.

[0046] The preparation method of the Cuscuta australis compound hair loss prevention and hair growth preparation comprises the following steps:

[0047] soaking powdered Cuscuta seeds, Lycium bark and Fructus Aurantii Immaturus in ethanol water, extracting under reflux conditions, filtering and concentrating to obtain a mixture I;

[0048] Separately squeeze the juice of fresh Rehmannia root and fresh Achyranthes bidentata, filter, combine and concentrate to obtain a mixture II;

[0049] The mixture I and the mixture II are mixed evenly, and freeze-dried to obtain a Cuscuta compound hair loss prevention and hair growth preparation.

[0050] In some examples, the ethanol content in the ethanol water is 60% to 80%.

[0051] In some examples, the amount of ethanol water used is 3 to 5 times the total weight of Cuscuta australis, Lycium bark and Fructus Aurantii Immaturus.

[0052] In some examples, the soaking time of the Cuscuta chinensis, the Cortex Lycii and the Fructus Aurantii Immaturus in the ethanol water is 30 to 60 min.

[0053] In some examples, the extraction is repeated multiple times, specifically 2 to 3 times, and after each reflux extraction, the mixture is filtered while hot, the filtrates are combined, and the mixture is concentrated under reduced pressure to obtain a mixture I; the temperature of the reduced pressure concentration is 55 to 60 °C.

[0054] In some examples, before squeezing the fresh Rehmannia root, 0.4 to 0.6 times of water by weight is added; before squeezing the fresh Achyranthes bidentata root, 1.0 to 1.2 times of water by weight is added.

[0055] Example 1

[0056] A dodder seed compound hair loss prevention and hair growth preparation, the preparation method comprising the following steps:

[0057] (1) Weigh 112 g, 112 g, 298 g, 750 g, and 750 g of Cuscuta australis, respectively.

[0058] (2) Crush Cuscuta chinensis, Radix Lycii and Fructus Aurantii Immaturus into powder, add 60% ethanol water (3 times the total mass of Cuscuta chinensis, Radix Lycii and Fructus Aurantii Immaturus) to soak for 30 min, extract under reflux for 2 h, repeat the extraction twice, filter with gauze while hot after each reflux extraction, combine the filtrates, and concentrate under reduced pressure at 60 °C to obtain mixture I.

[0059] (3) Wash fresh Rehmannia root and fresh Achyranthes bidentata, add 0.5 times and 1.1 times their weight of water respectively, squeeze the juices respectively, filter, combine, and concentrate under reduced pressure at 60°C to obtain mixture II;

[0060] (4) Mix mixture I and mixture II evenly, freeze-dry them in a freeze dryer to obtain 198 g of freeze-dried powder, and store it at -20°C for future use.

[0061] Comparative Example 1

[0062] Weigh 100 g, 100 g, 267 g, 670 g, and 670 g of Cuscuta, Radix Lycii, Fructus Aurantii Immaturus, and Radix Achyranthis Bidentatae, respectively, add 3 times the total mass of purified water to soak for 2 h, boil and decoct for 30 min, then pour out the liquid and collect it, add 3 times the amount of purified water again, and boil and decoct for 30 min. Combine the medicinal liquids obtained from the two decoctions, concentrate under reduced pressure at 60 °C, and freeze-dry in a freeze dryer to obtain 123 g of freeze-dried powder, which is stored at -20 °C for later use.

[0063] Example 2

[0064] The preventive and therapeutic effects and mechanism of the hair loss prevention and growth preparation of Cuscuta australis compound in a mouse model

[0065] 1. Experimental solution and its preparation method

[0066] Table 1: Drug solution and its preparation method

[0067]

[0068] Note: The above solution should be stored at 4 °C and used within 3 days.

[0069] 2. Experimental Animals

[0070] Ninety 5-week-old female SPF Kunming (C57BL / 6) mice weighing 13-16 g were provided by Hubei Provincial Center for Disease Control and Prevention. Animal experiments were carried out in accordance with internationally recognized standards and in strict compliance with the Guide for the Care and Use of Laboratory Animals and relevant regulations. The animals were housed in polyethylene cages and placed in an environment with room temperature (25 ± 2 °C), natural humidity, and a 12-h light / dark cycle. Standard feed and distilled water were used to feed the animals. Each group of mice was housed in separate cages and adapted to the environment for several days. The experiments were performed after the hair of the mice entered the resting period.

[0071] 3 Experimental methods

[0072] 3.1 Establishment of corticosterone stress hair loss model

[0073] During the feeding period of the experimental mice, except for the blank control group that drank untreated drinking water, the mice in the other groups drank drinking water supplemented with corticosterone. The corticosterone water was changed every 3 days to prevent degradation. Because corticosterone is poorly soluble in water, it was first ground into very fine powder using an agate mortar and then added to drinking water (pre-prepared 0.45% hydroxypropyl-B-cyclodextrin, stirred until no white powder was visible) and ultrasonicated for 30 min.

[0074] 3.2 Animal grouping and drug administration

[0075] After several days of adaptive feeding of mice, all mice were fed every morning and had free access to water. Randomly grouped into blank control group, model group, minoxidil group, high-dose group of Example 1, low-dose group of Example 1, and comparative example 1 group, a total of 6 groups. Each group of 15 mice was randomly divided into 3 cages for feeding, weighing, and marking. The grouping and dosage are shown in Table 2.

[0076] Table 2: Animal experimental groups, irradiation and dosage

[0077]

[0078] Note: - indicates no treatment, + indicates treatment, the solvent is 0.9% saline, 200 μL / mouse

[0079] When the mice entered the resting hair period, most of the hair was removed with an electric shaver in the center of the back according to an area of ​​3 x 4 cm², and then a depilatory cream was applied until the depilatory area was smooth. After depilation, the mice began to drink drinking water containing corticosterone the next day, and began to take drugs, which were taken once a day for 4 weeks before the end of the experiment. The content of corticosterone in drinking water was 50 µg / mL in the first week, 25 µg / mL in the second week, 12.5 µg / mL in the third week, and 6.25 µg / mL in the fourth week.

[0080] 3.3 Liver and kidney organ index

[0081] (1) Body weight: Observe the back skin condition of the mice every day, take photos every 3 days, and measure their body weight once a week.

[0082] (2) Weighing of liver and spleen: After the experiment, the liver and kidney tissues of the mice were removed and weighed, and the organ index was calculated.

[0083] 3.4 Skin tissue pathological staining

[0084] Tissue paraffin embedding: Take skin about 0.5 cm x 0.5 cm from the same position in the center of the back of each mouse. Avoid pulling the skin to cause deformation during sampling. After sampling, immediately immerse the tissue in 4% paraformaldehyde solution for tissue fixation. Fix at 4 ℃ for 48 h, and use conventional methods to dehydrate, embed and slice.

[0085] (1) Section staining

[0086] The cut skin tissue sections need to be dewaxed before histopathological staining. The sections are embedded in paraffin in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, followed by gradient dewaxing and washing. H&E staining is performed.

[0087] (2) Microscope photography

[0088] H&E stained sections were taken from each group and observed under a microscope at 6 times magnification. The thickness of the epidermis at 10 random locations was measured for each section. The thickness of the epidermis and dermis (perpendicular to the length of the subcutaneous fat layer), the morphology, number and length of the hair follicles were mainly observed. The experimental results were analyzed by one-way analysis of variance.

[0089] 3.5 Determination of ROS content in skin tissue

[0090] Weigh about 100 mg of skin tissue, cut it into pieces and transfer it to a homogenizer tube, then add 900 μL of pre-cooled physiological saline, homogenize it on a 4 ℃ homogenizer, transfer it to a low-temperature high-speed centrifuge, centrifuge it at 4 ℃ 12000 g for 15 min, carefully aspirate the supernatant with a sterile syringe, transfer it to another clean EP tube, centrifuge it again at 4 ℃ for 15 min, aspirate the supernatant and place it in an EP tube. If it is not used temporarily, place it at -20 ℃ for standby use. The recommended conditions for the skin homogenizer are SPEED: 7 m / s, Time: 40 s, INTER: 20 s, CYCLE: 4~9 times. Since skin tissue is rich in collagen fibers and has strong toughness and is difficult to grind, it takes longer than ordinary tissue grinding time. The supernatant of the mouse skin tissue homogenate prepared above was used to determine the content of tissue reactive oxygen species (ROS). The determination was performed according to the instructions of the ROS content determination kit, and the specific operation steps are not repeated here.

[0091] Since the skin tissue was homogenized with saline, the protein concentration of the homogenate was determined using a BCA protein concentration assay kit. The operating steps are as follows: Standard microplate protocol (working range 50~2000 μg / mL). First, take 2 μL of sample and add 18 μL PBS solution to dilute 10 times, then add it to a 96-well plate. After adding the sample, add 200 μL BCA working solution to each well, mix on a shaker for 30 seconds, and then incubate in a 37 °C incubator for 30 min. Use an enzyme reader to measure the absorbance at 562 nm. The sample measurement value is subtracted from the average 562 nm measurement value of the blank replicate. By drawing a standard curve, use the standard curve to determine the protein concentration of each unknown sample. The standard curve solution is prepared as shown in Table 3.

[0092] Table 3: Albumin standard solution dilution table

[0093]

[0094] NOTE: If using the curve fitting algorithm associated with the microspectrometer, a four-parameter (quadratic) or best-fit curve will provide more accurate results than a pure linear fit. If manually plotting the results, a point-to-point curve is preferable to a linear fit of the standard points.

[0095] 3.6 Determination of serum IL-1β, IL-6, IL-17A, LDH and corticosterone levels

[0096] Whole blood was collected longitudinally from the orbital vein of mice, kept at room temperature for 2 h, centrifuged at 3000 r / min, 4 °C for 15 min, and the upper serum was carefully aspirated, aliquoted, and stored in a -80 °C refrigerator.

[0097] The levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-17A (IL-17A), lactate dehydrogenase (LDH) and corticosterone (CORT) in serum were determined according to the instructions of the enzyme-linked immunosorbent assay kit. The specific operation steps are not repeated here. IL-1β (MU30369, Bioswamp), IL-6 (MU30044, Bioswamp), IL-17A (MU30386, Bioswamp), LDH (MU30023, Bioswamp).

[0098] 3.7 Real-time fluorescence quantitative RT-PCR

[0099] (1) Extract RNA from skin tissue using tissue disruptor + RNA extraction kit

[0100] Since the skin contains a large amount of RNase, it is particularly easy to degrade and it is difficult to extract RNA. In this paper, a tissue disruptor + RNA extraction kit (RK30120, Abotek) is used to extract RNA from skin tissue. The RNA concentration and quality are measured using an ultra-micro UV-visible photometer (NanoDrop), followed by reverse transcription, and the obtained cDNA is stored in a -80 °C refrigerator for later use. Real-time fluorescence quantitative RT-PCR was used to measure the transcription levels of VEGFA, IGF-1, and TGF-β2 in mouse skin.

[0101] (2) RNA reverse transcription

[0102] The reaction system for RNA reverse transcription is shown in Table 4.

[0103] Table 4: Reverse transcription reaction system

[0104]

[0105] Prepare the reaction mixture according to Table 4, flick the bottom of the tube to mix, and centrifuge for 1 min. Place the reaction mixture in a PCR instrument, react at 42 °C for 60 min, and then heat to 72 °C for 5 min to terminate the reaction. Store the cDNA at -20 °C for later use.

[0106] Primers were designed according to the cDNA sequence in Genbank. The specificity of primers was verified by nucleic acid gel electrophoresis after PCR amplification, and the efficiency of primer amplification was verified by real-time fluorescence quantitative RT-PCR. The appropriate dilution factor of cDNA was first determined through preliminary experiments. An appropriate amount of cDNA stock solution was diluted to 50 ng / μL with high-purity water without nuclease, and eight rows of PCR tubes were placed on an ice box. Each component was added, mixed and centrifuged for 1 min. The PCR tubes were then placed in a real-time fluorescence quantitative PCR instrument. The qPCR system and amplification program are shown in Tables 5 and 6. GAPDH was used as an internal reference to obtain the number of cycles Ct experienced when each group of cDNA amplification reached the threshold, using 2 -ΔΔCt The computational methods were used to analyze the data.

[0107] Table 5: qPCR system

[0108]

[0109] Table 6: qPCR amplification procedure

[0110]

[0111] 3.8 Statistical analysis

[0112] The experiments were repeated three times, and the results were analyzed using Graphpad prism 9 statistical software to calculate the mean and standard deviation. The experimental data were expressed as mean ± SD. t Test methods and one-way analyses of variance (one-way ANOVA) were used to compare data between and / or within two groups.

[0113] 4. Experimental results

[0114] 4.1 Effects of the preparation of the present invention on mice with alopecia induced by corticosterone

[0115] Figure 1 This reflects the effect of the preparation of the present invention on the hair loss mouse model induced by corticosterone stress hormone. Figure 1As shown, at the beginning of the experiment, there was no obvious difference in the back skin of each group of mice in the figure. On the 12th day, the hair of the blank group mice entered the growth phase, and the other groups were in the resting phase or in the early growth phase; on the 20th day, the hair of the blank group mice had grown vigorously, and the minoxidil group of the positive control showed obvious hair growth, while the skin color of the mice in the low-dose group of Example 1 began to darken, indicating that the hair follicles began to enter the growth phase, but the back skin of the mice in the model group and the comparative example 1 group was still pink, indicating that the hair of the mice had not begun to grow; on the 28th day, the hair of the model group and the comparative example 1 group of mice also grew a lot, but the black area was significantly lower than that of the high-dose group of Example 1 and the low-dose group of Example 1, indicating that the preparation of the present invention helps to promote the growth of mouse hair and accelerate the entry of hair follicles into the growth phase.

[0116] 4.2 Organ index results

[0117] Figure 2 Reflects the weight gain rate of mice. Except for the blank control group, the mice in each group showed a phenomenon of weight loss first and then gain. At the end of the second week, the weight of mice in all groups increased; Figure 3 The relative values ​​of liver and kidney indexes of mice were reflected. At the end of the experiment, the organ indexes of mice in each group had no significant difference with those of the blank group, indicating that the preparation of the present invention had no obvious toxicity.

[0118] 4.3 Histological test results

[0119] Figure 4 This is the H&E staining result of the mouse back skin during the process of the present invention. Figure 5 and Figure 6 The changes in back skin thickness, hair follicle length and number during treatment with the preparation of the present invention are shown. Skin thickening is an important sign that hair enters the growth phase. The back skin thickness and hair follicle length were measured and quantified using Case Viewer software. The results are shown in Tables 7 and 8. # P < 0.05 ， ## P < 0.01 ， ### P < 0.001 blank group compared with model group; * P < 0.05 ， ** P <0.01 ， *** P < 0.001 other groups compared with the model group.

[0120] Table 7: Skin thickness of mice during treatment

[0121]

[0122] Note: # P < 0.05, ## P < 0.01, ### P < 0.001 blank group compared with model group; * P < 0.05, ** P < 0.01, *** P < 0.001 other groups compared with the model group.

[0123] Table 8: Hair follicle length of mice during treatment

[0124]

[0125] Note: # P < 0.05, ## P < 0.01, ### P < 0.001 blank group compared with model group; * P < 0.05, ** P < 0.01, *** P < 0.001 other groups compared with the model group.

[0126] Depend on Figures 4 to 6 As shown in Tables 7 and 8, on the 12th day, the hair of the blank group mice was in the growth phase, the epidermal thickness was relatively large, and the hair follicle density was large. At this time, the skin layer of the mice in the model group and the drug administration group was thin, the hair follicles were atrophied and the number was small, the hair papilla cells moved upward, and the hair follicle stem cells were quiescent. On the 19th day, compared with the model group, the low-dose group of Example 1 and the minoxidil group entered the growth phase, the skin thickness and the number of hair follicles increased, and compared with the model group, the skin thickness and the number of hair follicles and the length increased significantly, but the comparative example 1 group did not change significantly. On the 28th day, the vigorous growth of the blank group hair had ended, so the skin thickness decreased, and each group showed a delayed growth phase compared with the blank group; at this time, the mice in the model group and the comparative example 1 group also entered the hair growth phase, and the skin thickened, but the hair follicle length and skin thickness of the embodiment 1 group and the minoxidil group were still significantly different from those of the model group, reflecting the potential effect of the embodiment 1 group preparation of the present invention in promoting hair growth.

[0127] 4.4 Effect of the preparation of the present invention on the levels of corticosterone and LDH in mouse serum

[0128] When the body is under stress, the hypothalamus releases corticotropin-releasing hormone (CRH), which stimulates the anterior pituitary to secrete adrenocorticotropic hormone (ACTH). ACTH then acts on the adrenal cortex to prompt the adrenal glands to secrete glucocorticoids such as cortisol. Cortisol is an important stress hormone that plays a key role in regulating the body's stress response. Excessive cortisol levels can have a negative impact on hair follicles. Studies have shown that cortisol can inhibit the proliferation and differentiation of hair follicle cells by binding to the glucocorticoid receptors on hair follicle cells, causing the hair follicles to enter the resting phase prematurely, leading to hair loss. The main glucocorticoid in humans is cortisol, and the adrenal cortex of mice mainly secretes corticosterone. Figure 7 Graph showing the relative value of corticosterone levels in mouse serum in the results of the present invention. After 28 days of administration, there was no significant difference in the corticosterone levels in the mouse serum of the low-dose group of Example 1 compared with the model group, indicating that the Example 1 preparation of the present invention cannot reduce the serum corticosterone content, and therefore its effect is not achieved by directly reducing the corticosterone content.

[0129] Figure 8 The effect of the preparation of the present invention on the LDH content in mouse serum. The upregulation of lactate content in hair follicle stem cells and LDH activity can activate hair follicle stem cells and promote their proliferation. Studies have found that the absence of lactate dehydrogenase (Ldha) prevents the activation of hair follicle stem cells. On the contrary, promoting the lactate production of hair follicle stem cells genetically through the absence of mitochondrial pyruvate carrier 1 (Mpc1) can accelerate their activation and hair cycle. After 28 days of administration, the LDH in the serum of mice in the low-dose group of Example 1 had a higher level than that in the model group, and the blank group had a lower level than that in the model group, and there was a significant difference. It shows that the preparation of the present invention can activate LDH and has a potential effect in promoting hair growth.

[0130] 4.5 Effect of the preparation of the present invention on the inflammatory response of hair follicles induced by corticosterone

[0131] Fig. 9 , 10 , 11 respectively show the effects of the preparation of the present invention on the levels of IL-1β, IL-6 and IL-17A in mouse serum. These cytokines play a central role in regulating inflammatory and immune responses and are key mediators in chronic inflammatory states.

[0132] IL-17A can act on macrophages, promote the release of inflammatory factors such as IL-1β and IL-6, and exert a pro-inflammatory effect. IL-17A is a cytokine produced by Th17 cells, which plays an important role in promoting inflammation and tissue damage. Blocking IL-17A can make the growth rate of hair follicles in old mice consistent with that of young mice, activate hair follicle stem cells, and promote hair growth. IL-6 is an important inflammatory factor. When hair follicles are damaged or inflamed, the secretion of IL-6 increases. In inflammatory hair loss diseases such as alopecia areata, IL-6 can stimulate the differentiation of initial T cells (Th0) to Th17 and participate in maintaining the balance of Th17 cells. Th17 cells participate in inflammatory response and immune regulation by secreting cytokines such as IL-17A. Excessive Th17 cell response may cause hair follicle damage and aggravate the development of hair loss. IL-1β has been shown to have an inhibitory effect on hair growth in vitro experiments. IL-1β may play a role in the infiltration of inflammatory cells around the hair follicles of patients with alopecia areata, and by promoting the activation and aggregation of inflammatory cells, it attacks the hair follicles and causes hair loss. An appropriate amount of IL-1β may promote hair growth by affecting the activity of hair follicle stem cells. The figure shows that 28 days after administration, the levels of IL-1β, IL-6 and IL-17A in the serum of mice in the low-dose group and the minoxidil group of Example 1 were significantly reduced, and there were significant differences compared with the model group. In summary, the preparation of the present invention can play an important role in reducing skin inflammation and hair growth by reducing the levels of IL-1β, IL-6, and IL-17A. These results support the value of the preparation of the present invention as a therapeutic strategy for treating hair loss induced by chronic stress.

[0133] 4.6 Effect of the preparation of the present invention on the ROS content in mouse skin tissue

[0134] Cortisol affects the metabolic process of hair follicle cells, increases the level of oxidative stress in hair follicle cells, and causes damage and death of hair follicle cells. Oxidative stress refers to the imbalance between the oxidation and antioxidant systems in the body. Excessive reactive oxygen species (ROS) will attack cell membranes, proteins, and DNA, causing cell damage. Under the action of oxidative stress, the normal physiological functions of hair follicle cells are inhibited, which in turn causes hair loss. Fig.12 As shown, 28 days after administration, the ROS content in the mouse skin of the model group was still high due to the influence of corticosterone levels, and the ROS levels of the high-dose and low-dose groups and the minoxidil group of Example 1 were significantly reduced compared with the model group. At this time, the blank group had entered the catagen or even quiescent stage, which was significantly different from the model group. The above results indicate that the preparation of the present invention may inhibit the oxidative stress response of mice with alopecia induced by chronic stress by reducing the activity of reactive oxygen species.

[0135] 4.7 Effect of the preparation of the present invention on the transcription level of growth factors in mouse skin tissue

[0136] Fig.13 These are the results of measuring the mRNA expression levels of VEGFA, IGF-1 and TGF-β2 genes in each treatment group in the results of the present invention.

[0137] Vascular endothelial growth factor (VEGFA), insulin-like growth factor-1 (IGF-1), and transforming growth factor-β2 (TGF-β2) jointly participate in the regulation of hair growth by regulating the proliferation, differentiation, and apoptosis of hair follicle cells and improving the scalp microenvironment. VEGFA is an important angiogenic factor secreted by DPCs and participates in hair growth by promoting angiogenesis around hair follicles and improving local blood supply. Compared with normal hair follicles (HF), VEGFA expression is reduced in HF during alopecia. Studies have shown that VEGFA expression increases significantly during the growth phase of hair follicles and decreases during the resting phase. IGF-1 also plays an important role in hair growth. IGF-1 can stimulate the proliferation and differentiation of hair follicle cells and promote the transformation of hair follicles from the resting phase to the growth phase. It has been reported that IGF-1 signaling affects the development of HF and tissue renewal. In experiments, IGF-1 can prevent HF from entering the regression phase, and its expression peaks during the growth phase of hair follicles, indicating that it has a direct promoting effect on the growth and development of hair follicles. TGF-β family members (such as TGF-β1, TGF-β2 and TGF-β3) have complex regulatory effects in hair growth. TGF-β1 may inhibit hair follicle growth in some cases. TGF-β2 can inhibit the extension of hair shaft and plays an important role in the regression phase. It induces apoptosis of hair follicle epithelial cells, causes regression-like morphological changes in hair follicle cells, and promotes tissue remodeling to make hair follicles enter the resting phase. Hair growth can be promoted by inhibiting the activity of TGF-β2. TGF-β3 has been found to activate hair follicle stem cells and promote hair growth through the secretion of regulatory T cells (Treg).

[0138] like Fig.13 As shown, after 19 days of administration, the low-dose group of Example 1 significantly upregulated the expression of VEGFA and IGF-1 mRNA, which are regulatory factors related to hair growth, and reduced the expression of TGF-β2 mRNA, an inhibitory factor. On the 28th day, since the hair of the mice in the model group also began to grow, the data difference was not obvious. This preliminarily shows that the preparation has a certain effect in promoting the formation of new blood vessels and the proliferation of hair follicle cells in mice, and can inhibit the hair follicles from entering the regression phase.

[0139] In summary, the preparation of the present invention plays an important role in promoting angiogenesis, cell proliferation and differentiation, and inhibiting cell apoptosis by regulating the expression of VEGFA, IGF-1 and TGF-β2. These results further support the potential therapeutic effect of the preparation of the present invention in promoting hair growth.

[0140] 4.8 Effects of the Preparations of the Invention on Protein Levels of the Wnt / β-catenin Signaling Pathway

[0141] Fig.14 These are the results of measuring the expression levels of GSK3β, P-GSK3β, Wnt 5a and β-catenin proteins in each treatment group in the results of the present invention.

[0142] During hair growth, activation of the Wnt signaling pathway can promote the proliferation of hair follicle stem cells and hair regeneration. Studies have shown that the Wnt signaling pathway is very important for the proliferation of hair papilla cells and the induction of hair follicle regeneration, and is one of the important signals regulating the growth and development of hair follicles. Among the Wnt signaling pathways, the classical signaling pathway Wnt / β-catenin is the most studied.

[0143] The core of the Wnt / β-catenin signaling pathway is the β-catenin protein. In the absence of Wnt ligands (such as Wnt5a), β-catenin is phosphorylated by GSK-3β and CK1α and subsequently degraded by the proteasome. This process depends on a degradation complex, including Axin, APC, GSK-3β and CK1. When Wnt ligands (such as Wnt5a) bind to Frizzled receptors and LRP5 / 6 co-receptors, the signaling pathway is activated. Dishevelled protein is recruited to the cell membrane, thereby inhibiting the activity of GSK-3β and preventing the phosphorylation and degradation of β-catenin. Stabilized β-catenin then enters the nucleus, binds to TCF / LEF transcription factors, and activates the expression of downstream target genes.

[0144] Wnt5a is a secreted glycoprotein that acts as a ligand for the Wnt signaling pathway. It activates the Wnt / β-catenin signaling pathway by binding to the Frizzled receptor. Wnt5a plays an important role in cell differentiation, tissue regeneration, and tumorigenesis. GSK-3β is a key regulator in the Wnt / β-catenin signaling pathway. In the absence of Wnt signals, GSK-3β phosphorylates β-catenin, causing it to be degraded by the proteasome. When the Wnt signal is activated, the activity of GSK-3β is inhibited, thereby preventing the phosphorylation of β-catenin, causing it to accumulate in the cytoplasm and enter the nucleus. In addition, GSK-3β is also involved in phosphorylating the LRP5 / 6 receptor, promoting its binding to Axin, thereby further regulating signal transduction.

[0145] From the above, we can see that after Wnt5a binds to the Frizzled receptor, it inhibits the activity of GSK-3β through the Dishevelled protein. GSK-3β is phosphorylated and degraded, so that it cannot bind to β-catenin and then phosphorylate β-catenin, activating β-catenin to enter the cell nucleus and regulate its degradation or stability. Stabilized β-catenin enters the cell nucleus, binds to transcription factors, initiates the transcription of target genes, activates downstream gene expression, and promotes the proliferation of hair follicle cells and hair growth. If this pathway is blocked, the hair follicles will enter the degeneration phase prematurely, leading to hair loss. The interaction between Wnt5a, GSK-3β and β-catenin plays an important role in cell proliferation, differentiation and tissue regeneration. Fig.14 As shown, after 28 days of administration, the phosphorylation of GSK-3β, the expression of Wnt 5a and β-catenin in the low-dose group of the example significantly increased, and the level of GSK-3β decreased, indicating that the preparation of the present invention played an important regulatory role in the Wnt / β-catenin pathway. These results further support the potential therapeutic effect of the preparation of the present invention in promoting hair growth.

[0146] In summary, the present invention adjusts the ratio of raw materials and the preparation method, eliminates the complex preparation method of refining honey into pills in the traditional dodder pill prescription, adopts ethanol-water reflux method to extract the effective ingredients of three medicinal materials, dodder, radix rehmanniae and fructus aurantii, and extracts the effective ingredients of fresh rehmanniae and fresh achyranthes bidentata respectively. The present invention can make full use of the nutritional value of Chinese herbal medicine, reduce the dosage, improve the convenience of operation, and expand the scope of application. The anti-hair loss and hair growth preparation of the dodder compound provided by the present invention has a good effect of preventing / treating hair loss and promoting hair growth in the hair loss model caused by chronic stress in C57BL / 6 mice.

[0147] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A dodder seed compound hair loss prevention and hair growth preparation, characterized in that: The raw materials include: 3 parts of Cuscuta seeds, 3 parts of Lycium bark, 8 parts of Citrus aurantium, 19-20 parts of fresh Rehmannia root and 20-21 parts of fresh Achyranthes bidentata.

2. The anti-hair loss and hair growth preparation of a Cuscuta Semen compound according to claim 1, characterized in that: The raw materials of the dodder seed compound hair loss prevention and hair growth preparation include: 3 parts of dodder seed, 3 parts of lycium bark, 8 parts of aurantium, 19.6 parts of fresh rehmannia root and 20.3 parts of fresh achyranthes bidentata.

3. The method for preparing a hair loss prevention and hair growth preparation of a Cuscuta Semen compound according to claim 1 or 2, characterized in that: The following steps are involved: soaking powdered Cuscuta seeds, Lycium bark and Fructus Aurantii Immaturus in ethanol water, extracting under reflux conditions, filtering and concentrating to obtain a mixture I; Separately squeeze the juice of fresh Rehmannia root and fresh Achyranthes bidentata, filter, combine and concentrate to obtain a mixture II; The mixture I and the mixture II are mixed evenly, and freeze-dried to obtain a Cuscuta compound hair loss prevention and hair growth preparation.

4. The method for preparing a hair loss prevention and hair growth preparation of a Cuscuta Semen compound according to claim 3, characterized in that: The lyophilized powder obtained by freeze-drying is made into oral preparations, which include but are not limited to granules, compressed tablets, and oral liquids.

5. The method for preparing a Cuscuta Semen compound hair loss prevention and hair growth preparation according to claim 3, characterized in that: The content of ethanol in the ethanol water is 60% to 80%.

6. The method for preparing a Cuscuta Semen compound hair loss prevention and hair growth preparation according to claim 3, characterized in that: The amount of the ethanol water used is 3 to 5 times the total weight of Cuscuta australis, Cortex Lycii and Fructus Aurantii Immaturus.

7. The method for preparing a hair loss prevention and hair growth preparation of a Cuscuta Semen compound according to claim 3, characterized in that: The soaking time of the dodder seeds, the bark of Lycium bark and the fructus aurantii in the ethanol water is 30 to 60 minutes.

8. The method for preparing a Cuscuta Semen compound hair loss prevention and hair growth preparation according to claim 3, characterized in that: The extraction is repeated for multiple times, specifically 2 to 3 times. After each reflux extraction, the mixture is filtered while hot, the filtrates are combined, and the mixture is concentrated under reduced pressure to obtain a mixture I. The temperature of the reduced pressure concentration is 55 to 60°C.

9. The method for preparing a Cuscuta Semen compound hair loss prevention and hair growth preparation according to claim 3, characterized in that: Before squeezing the fresh Rehmannia root, 0.4 to 0.6 times of water by weight is added; before squeezing the fresh Achyranthes bidentata root, 1.0 to 1.2 times of water by weight is added.

10. Use of the preparation according to claim 1 or 2 in the preparation of a product for preventing and / or treating hair loss caused by chronic stress.

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