Non-injectable and non-implantable hair-growing, hair-growing and hair-loss-preventing external skin-growing oil preparation as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly discloses a non-injectable and non-implantable hair-growing, hair-growing and hair-loss-preventing external skin-growing oil preparation as well as a preparation method and application thereof. The preparation method of the external skin-generating oil preparation comprises the following steps: (1) preparing freeze-dried powder of a skin-generating oil alcohol extract: respectively taking dry powder of gynura procumbens, pericarpium citri reticulatae viride, astragalus membranaceus, angelica sinensis, frankincense, raw malt, rheum officinale and golden cypress and borneol according to a certain weight part ratio, adding the dry powder and borneol into a container, adding an ethanol solution with the volume fraction of 40-90% into the container, soaking, carrying out flash extraction, and drying to obtain the freeze-dried powder of the skin-generating oil alcohol extract; and concentrating the filtrate, and freeze-drying. (2) preparing a skin-generating oil preparation: dissolving the skin-generating oil alcohol extract freeze-dried powder and Span 60 in edible vegetable oil to prepare an oil phase; and dropwise adding the oil phase into a water phase obtained by dissolving Tween-60 and glycerol in ultrapure water, stirring, shearing at a high speed, and homogenizing at a high pressure, thereby obtaining the product. The external skin-generating oil preparation disclosed by the invention has the comprehensive effects of growing hair, generating hair and preventing alopecia.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a non-injection and non-implantable external skin oil preparation for hair growth, hair regrowth and hair loss prevention, and its preparation method and application. Background Art

[0002] Hair loss can be classified into physiological hair loss and pathological hair loss according to clinical manifestations. Physiological hair loss is a normal physiological phenomenon in which hair naturally enters the telogen and exogen phases and falls out normally, usually without affecting health. Pathological hair loss is a common disease clinically characterized by a large amount of abnormal hair loss, sparse hair, and receding hairline caused by various reasons, and it shows a trend of getting younger, affecting the emotions and mental health of patients and leading to a decline in their quality of life. Among them, 1) androgenetic alopecia is the most common pathological hair loss, which is mainly caused by genetic factors and high levels of androgens in the body. In men, it is manifested as a gradual recession of the hairline on the top of the head or forehead, while in women, it is mostly thinning of the hair on the top of the head; 2) immune deficiency alopecia is caused by the attack of the autoimmune system on hair follicles and is common in alopecia areata; 3) traumatic alopecia is usually caused by physical, chemical or thermal factors that damage the scalp and hair, such as overuse of curling irons, hair dyes or tight hairstyles; 4) nutritional alopecia is related to unbalanced diet or lack of certain key nutrients, such as insufficient iron, zinc, vitamin D and protein; 5) drug-induced alopecia is a side effect caused by taking certain drugs, such as chemotherapy drugs, antidepressants and antihypertensive drugs, etc.

[0003] Preventing and treating hair loss is a worldwide medical problem involving multiple factors such as genetics, environment and psychology. Chemical drug treatments for hair loss are effective, such as minoxidil and finasteride, but they have many adverse reactions when used for a long time, including skin irritation, headache and sexual dysfunction, etc. Hair transplantation surgery (by microsurgery to remove healthy hair follicle tissue, which is cultured and then transplanted to the bald or hair loss area of the patient. After the hair follicles survive, new hair will grow and will not fall out or necrosis again) is an effective treatment method, but it is expensive, has high technical requirements and a long postoperative recovery period, making it difficult to popularize among the general population. Platelet-rich plasma therapy (PRP) refers to using platelet concentrates extracted from blood and then injecting them into the scalp through microneedles to convert them into various inflammatory regulatory factors and growth factors, and providing a large amount of nutrients for hair, promoting the proliferation and differentiation of dermal papilla cells, improving local blood perfusion, promoting collagen synthesis, increasing the survival rate of hair follicles, and thus promoting hair growth (the effect of using PRP alone to treat hair loss is not very good and can only be used as an adjuvant treatment method in combination with other treatment methods to improve the treatment efficiency). Laser treatment, as an emerging minimally invasive therapy, stimulates hair follicles through low-energy lasers to promote hair growth, but its effect varies from person to person and the equipment cost is relatively high.

[0004] Different from Western medicine prevention and treatment, traditional Chinese medicine believes that hair loss is mainly related to the liver, kidneys, qi, and blood. "Huangdi Neijing" mentions that "when qi and blood are strong, kidney qi is robust, and when kidney qi is robust, the bone marrow is full, so the hair is black and shiny; when qi and blood are weak, kidney qi is weak, and when kidney qi is weak, the bone marrow dries up, so the hair turns white and falls out." In addition to the influence of the liver, kidneys, qi, and blood, hair loss is also easily affected by emotional factors. For example, stress, insomnia, depression, anxiety, etc. can all lead to hair loss. In addition, bad habits such as excessive drinking, spicy foods, and staying up late will damage the liver and kidney functions, and thus affect hair growth. Moreover, seasonal changes (dry climate in autumn and winter) also make hair more vulnerable to damage and shedding. Of course, the prevention and treatment of hair loss with traditional Chinese medicine also has the characteristics of high safety and has received extensive attention from scholars at home and abroad. For example, traditional Chinese medicines such as angelica and wolfberry are considered to have the effects of nourishing the liver and kidneys, promoting blood circulation to remove blood stasis, and helping to improve hair loss symptoms. In recent years, the research on the combination of traditional Chinese medicine and modern technology has been continuously deepened, and there are more opportunities to newly develop oral traditional Chinese medicines and topical preparations for hair loss, providing more choices for hair loss patients, but the curative effect is limited. Summary of the Invention

[0005] Regarding the above-mentioned chemical drugs such as minoxidil and finasteride in the prior art, they have certain effects in hair growth and hair retention, but their long-term use has obvious side effect problems that are difficult to avoid (the common side effects of minoxidil include increased heart rate, arrhythmia, skin flushing caused by reflex sympathetic excitement, weight gain and lower extremity edema caused by water and sodium retention, and hair hyperplasia that is more obvious on the face, arms, and back; the side effects of finasteride mainly include decreased libido, impotence, decreased ejaculation, as well as breast tenderness and / or enlargement, skin allergic reactions, testicular pain, liver function disorders, etc.):

[0006] The first object of the present invention is to provide a non-injectable and non-implantable topical skin oil preparation for hair nourishing, hair growth, and hair loss prevention. The topical skin oil preparation is obtained by substantially adjusting the early topical skin oil formula (CN202410557338.0) of the inventor's team for diseases such as hair loss, hair sparseness, and hair fineness.

[0007] The second object of the present invention is to provide a preparation method for the above-mentioned topical skin oil preparation for hair nourishing, hair growth, and hair loss prevention.

[0008] The third object of the present invention is to provide the medical use of the above-mentioned topical skin oil preparation for hair nourishing, hair growth, and hair loss prevention.

[0009] The inventive concept of the present invention is as follows:

[0010] Based on the pathological characteristics of pathological alopecia, the applicant believes that it is mainly related to the liver, kidney, qi and blood. Therefore, the applicant points out that for the prevention and treatment of pathological alopecia, priority should be given to using the prescription drug "Shengfu Oil Preparation" of "tonifying qi, nourishing blood and filling essence, promoting blood circulation to remove stasis and dredging collaterals" under the condition of sufficient intake of nutrients, and achieving the effects of hair growth, hair regrowth and anti - hair loss quickly in a non - injection and non - implantation external application mode.

[0011] To achieve the first object, the present invention adopts the following technical measures:

[0012] An external Shengfu oil preparation for hair growth, hair regrowth and anti - hair loss, wherein the Shengfu oil preparation is an aqueous - oil mixed preparation of the ethanol extract of Shengfu oil, and the solute of the Shengfu oil preparation is the freeze - dried powder of the ethanol extract of Shengfu oil; the content of the ethanol extract of Shengfu oil in the Shengfu oil preparation is not less than 10mg / ml and not more than 50mg / ml, and the preferred content is 30mg / ml. The ethanol extract of Shengfu oil is prepared by ethanol extraction from 9 raw materials with the following weight - part ratio (preferably, extracted by soaking in an ethanol solution with a volume fraction of 40% - 90% for 2 - 3h): Gynura procumbens (Lour.) Merr. 20 - 50 parts by weight, Pericarpium Citri Reticulatae Viride 20 - 50 parts by weight, Astragalus membranaceus (Fisch.) Bge. 20 - 50 parts by weight, Angelica sinensis (Oliv.) Diels 5 - 15 parts by weight, Olibanum 5 - 15 parts by weight, Fructus Hordei Germinatus 5 - 15 parts by weight, Rheum officinale Baill. 5 - 15 parts by weight, Phellodendron amurense Rupr. 5 - 15 parts by weight, Borneol 3 - 8 parts by weight.

[0013] Furthermore, the preparation method of the freeze - dried powder of the ethanol extract of Shengfu oil is: take the dry powders of 8 raw materials of Shengfu oil and borneol according to a certain weight - part ratio and add them into a container, then add an ethanol solution with a volume fraction of 40% - 90% into the container, soak for 2 - 3h, perform flash extraction 3 - 5 times, 5min each time, concentrate the filtrate, and freeze - dry to obtain the freeze - dried powder of the ethanol extract of Shengfu oil.

[0014] To achieve the second object, the present invention adopts the following technical measures:

[0015] A preparation method of the above - mentioned external Shengfu oil preparation for hair growth, hair regrowth and anti - hair loss, and its steps are as follows:

[0016] (1) Preparation of freeze-dried powder of ethanol extract of Shengfu oil: Take 9 raw materials of Shengfu oil according to a certain weight ratio (the weight parts of each raw material are: Gynura procumbens 20 - 50 weight parts, Pericarpium Citri Reticulatae Viride 20 - 50 weight parts, Astragalus membranaceus 20 - 50 weight parts, Angelica sinensis 5 - 15 weight parts, Olibanum 5 - 15 weight parts, Fructus Hordei Germinatus 5 - 15 weight parts, Rheum palmatum 5 - 15 weight parts, Phellodendron amurense 5 - 15 weight parts, Borneol 3 - 8 weight parts), a total of 500 g. Except for borneol, the other drugs are ground finely (passed through a 200-mesh sieve) and placed in a 10 L stainless steel cup, added with 3 L - 7 L of ethanol with a volume fraction of 40% - 90%, soaked for 2 - 3 h, extracted by flash extraction 3 - 5 times, 5 min each time, and the filtered supernatant is quickly concentrated by a multi-stage flash evaporator and then freeze-dried to obtain the freeze-dried powder of ethanol extract of Shengfu oil (the yield is 11.8% - 13.7%).

[0017] (2) Preparation of Shengfu oil preparation: Dissolve the freeze-dried powder of ethanol extract of Shengfu oil and Span 60 in edible vegetable oil to prepare an oil phase, and according to the volume ratio of the oil phase to the water phase of 1:5 - 10, drop the oil phase into the water phase prepared by dissolving Tween 60 and glycerol in ultrapure water, stir and then carry out high-speed shearing and high-pressure homogenization to obtain; the zeta potential and particle size distribution of the obtained Shengfu oil preparation are uniform; its particle size distribution is 141 - 825 nm, and there is no multiple scattering phenomenon, and the average particle size is 300 ± 2 nm; the average zeta potential is -45 mV - -39.1 mV. Among them, the edible vegetable oil is selected from at least one of olive oil, sesame seed oil and camellia oil. Preferably, the steps for preparing the Shengfu oil preparation in step (2) are:

[0018] 1) Dissolve the freeze-dried powder of ethanol extract of Shengfu oil and Span 60 in edible vegetable oil to prepare an oil phase; the final concentration of Span 60 is 1% - 5% (W / V; g / ml); 2) Dissolve Tween 60 and glycerol in ultrapure water to prepare a water phase; the final concentration of Tween 60 is 1% - 5% (V / V); the final concentration of glycerol is 5% - 15% (V / V); 3) Under the condition of a constant temperature water bath at 60 °C, drop the oil phase prepared in step 1) into the water phase prepared in step 2), and stir at 3000 r / min for 3 min; the volume ratio of the oil phase to the water phase is 1:5 - 10; 4) Carry out high-speed shearing at 10000 rpm for 2 min, and then carry out high-pressure homogenization 6 times at 1000 bar to obtain the water-oil mixed preparation of ethanol extract of Shengfu oil.

[0019] To achieve the third object, the present invention also adopts the following technical measures:

[0020] Use of the above-mentioned external Shengfu oil preparation or the external Shengfu oil preparation prepared by the above-mentioned method in the preparation of external drugs for preventing or treating alopecia, especially in the preparation of external drugs with comprehensive functions of hair growth, hair nourishing and anti-alopecia without injection and implantation.

[0021] The hair loss includes physiological hair loss and pathological hair loss. The types of hair loss include hair absence, thinning and / or fineness, and the hair is preferably hair on the head.

[0022] The principles for formulating the traditional Chinese medicine raw materials in the present invention are as follows:

[0023] Gynura procumbens, Pericarpium Citri Reticulatae Viride, and Astragalus membranaceus replenish qi and promote qi movement, serving as the monarch drugs; Angelica sinensis, Olibanum, and Hordeum vulgare L. var. germinatum nourish blood and replenish essence, serving as the minister drugs; Rheum palmatum, Phellodendron amurense, etc. cool the blood and dispel pathogenic factors, removing stasis and dredging collaterals, serving as the assistant drugs; Borneol and vegetable oil (using their viscosity to cover the scalp and hair, maintaining the moisture of the hair in the form of an oil film and preventing hair dryness), serving as the guiding drug. All the drugs together can quickly moisten the skin, promote the growth of microvessels and improve blood circulation, thereby prolonging the growth phase of hair follicles, improving the catagen phase of hair follicles, shortening the telogen phase of hair follicles, and increasing the number of hair follicles, thus significantly promoting hair growth, thickening, and straightening in a short time.

[0024] Compared with the prior art, the present invention has the following advantages and effects:

[0025] The external skin-nourishing oil preparation of the present invention has a more significant hair growth effect, a larger number of hair follicles, and a more obvious blood vessel improvement effect compared with minoxidil; compared with finasteride, its hair retention effect is better. Generally speaking, the external skin-nourishing oil preparation of the present invention has the comprehensive effects of promoting hair growth, generating new hair, and preventing hair loss. In addition, the external skin-nourishing oil preparation of the present invention also has the following advantages: ① low drug cost, no irritation, no toxic and side effects, and no expensive costs and potential pain brought to subjects by other methods such as injecting stem cells and hair transplantation; ② extremely simple operation (only a thin layer needs to be applied to the scalp and hair roots, without applying to the whole hair, for example, dropping an appropriate amount of the skin-nourishing oil preparation on the skin and hair roots at the hair absence or sparse area, and then quickly spreading it), and it can reach the hair roots through local drug delivery. Description of the Drawings

[0026] Figure 1 It is a characterization diagram of the particle size and potential of the skin-nourishing oil preparation prepared for Examples 1 and 2.

[0027] Figure 2 It is an impact diagram of the skin-nourishing oil preparation prepared for Example 1 on the hair growth in the hair loss area of the hair catagen model mice induced by testosterone propionate.

[0028] Figure 3 It is an impact diagram of the skin-nourishing oil preparation prepared for Example 1 on the skin tissue structure in the hair loss area of the hair catagen model mice induced by testosterone propionate (H&E staining diagram).

[0029] Figure 4 It is an impact diagram of the skin-nourishing oil preparation prepared for Example 1 on the skin tissue structure in the hair loss area of the hair catagen model mice induced by testosterone propionate (immunohistochemical staining diagram of CD31 protein).

[0030] Figure 5 Effect of the skin - promoting oil preparation prepared in Example 1 on the skin tissue structure of the hair telogen model mice induced by testosterone propionate (immunohistochemical staining map of CD34 protein).

[0031] Figure 6 Figure showing the effect of the skin - promoting oil preparation prepared in Example 2 on the hair growth in the depilated area of the hair growth - phase model mice induced by cyclosporin A.

[0032] Figure 7 Effect of the skin - promoting oil preparation prepared in Example 2 on the skin tissue structure of the hair growth - phase model mice induced by cyclosporin A (H&E staining map).

[0033] Figure 8 Effect of the skin - promoting oil preparation prepared in Example 2 on the skin tissue structure of the hair growth - phase model mice induced by cyclosporin A (immunohistochemical staining map of CD31 protein).

[0034] Figure 9 Effect of the skin - promoting oil preparation prepared in Example 2 on the skin tissue structure of the hair growth - phase model mice induced by cyclosporin A (immunohistochemical staining map of CD34 protein).

[0035] Figure 10 Figure showing the effect of the skin - promoting oil preparation prepared in Example 1 on the hair growth in the shaved area of the hair resting - phase model mice induced by cyclophosphamide.

[0036] Figure 11 Effect of the skin - promoting oil preparation prepared in Example 1 on the skin tissue structure of the hair resting - phase model mice induced by cyclophosphamide (H&E staining map). Detailed implementation manners

[0037] Next, the applicant will further elaborate on the technical solutions of the present invention in combination with the attached drawings and specific examples. It should be understood that the following content should not limit the protection scope of the present invention in any way.

[0038] In the embodiments of the present invention, the dry powders of 8 traditional Chinese medicine materials such as Gynura procumbens (Lour.) Merr., Pericarpium Citri Reticulatae Viride, Astragalus membranaceus (Fisch.) Bunge, Angelica sinensis (Oliv.) Diels, Olibanum, Fructus Hordei Germinatus, Rheum palmatum L., and Phellodendron amurense Rupr. are all passed through a 200 - mesh sieve.

[0039] Example 1: Preparation and characterization of the skin - promoting oil preparation

[0040] 1) Preparation of the skin - promoting oil preparation

[0041] Preparation of freeze-dried powder of ethanol extract of Shengyu Oil: Take 426 g of raw materials of Shengyu Oil (100 g of dry powder of Gynura procumbens, Pericarpium Citri Reticulatae Viride, and Astragalus membranaceus each, 30 g of dry powder of Angelica sinensis, Olibanum, and Hordeum vulgare L. var. germinatum each, 10 g of dry powder of Rheum palmatum and Phellodendron amurense each, and 16 g of Borneol), stir evenly and place in a 5 L stainless steel cup, add 3 L of 90 v / v% ethanol, soak for 3 h, perform flash extraction 3 times, 5 min each time, and quickly concentrate the filtered supernatant through a multi-stage flash evaporator, and then obtain the freeze-dried powder of ethanol extract of Shengyu Oil (50.2 g; the yield is 11.8%) through freeze-drying.

[0042] Formulation of Shengyu Oil preparation: ① Dissolve the freeze-dried powder of ethanol extract of Shengyu Oil and Span 60 in olive oil to prepare the oil phase, and the final concentration of Span 60 is 5% (W / V). ② Dissolve Tween 60 and glycerol in ultrapure water to prepare the water phase, the final concentration of Tween 60 is 5% (V / V), and the final concentration of glycerol is 5% (V / V). ③ Under the condition of a constant temperature water bath at 60 °C, drop the oil phase into the water phase, stir at 3000 r / min for 3 min, and the volume ratio of the oil phase to the water phase is 1:5. ④ Perform high-speed shearing at 10000 r / min for 2 min, and then perform high-pressure homogenization 6 times at 1000 bar to obtain the water-oil mixed preparation of ethanol extract of Shengyu Oil, briefly recorded as Shengyu Oil preparation, and the content of ethanol extract of Shengyu Oil in the obtained Shengyu Oil preparation is equal to 30 mg / ml.

[0043] 2) Characterization of particle size and zeta potential of Shengyu Oil preparation

[0044] When measuring the particle size of Shengyu Oil preparation, dilute the sample with ultrapure water so that its count rate is between 200 - 300 Kcps (1,000 counts per second), and then measure the particle size and polydispersity index with a laser particle size analyzer, scattering angle 90°, temperature 25 °C; when measuring the zeta potential, adjust the conductivity of Shengyu Oil preparation to 50 S / cm with 0.9% (w / w) sodium chloride solution. As Figure 1 shown in (A, B) below, the particle size of the Shengyu Oil preparation prepared in Example 1 is distributed in the range of 141 - 825 nm, its average particle size is about 296.4 nm, and there is no multiple scattering phenomenon; moreover, its zeta potential is evenly distributed, with an average of -39.1 mV.

[0045] Example 2: Preparation and Characterization of Shengyu Oil Preparation

[0046] 1) Preparation of Shengyu Oil preparation

[0047] Preparation of freeze-dried powder of ethanol extract of Shengyu Oil: Take 515 g of raw materials of Shengyu Oil (100 g of dry powder of Gynura procumbens, Pericarpium Citri Reticulatae Viride, and Astragalus membranaceus each, 50 g of dry powder of Angelica sinensis, Olibanum, and Hordeum vulgare L. var. germinatum each, 25 g of dry powder of Rheum palmatum and Phellodendron amurense each, and 15 g of Borneol), stir evenly, place in a 5 L stainless steel cup, add 4 L of 40 v / v% ethanol, soak for 3 h, perform flash extraction 5 times, 5 min each time, and quickly concentrate the filtered supernatant through a multi-stage flash evaporator, and then obtain the freeze-dried powder of ethanol extract of Shengyu Oil (70.6 g; the yield is 13.7%) through freeze-drying.

[0048] Preparation of Shengyu Oil preparation: ① Dissolve the freeze-dried powder of ethanol extract of Shengyu Oil and Span 60 in olive oil to prepare the oil phase, and the final concentration of Span 60 is 1% (W / V). ② Dissolve Tween 60 and glycerol in ultrapure water to prepare the water phase, the final concentration of Tween 60 is 1% (V / V), and the final concentration of glycerol is 15% (V / V). ③ Under the condition of a constant temperature water bath at 60 °C, drop the oil phase into the water phase, stir at 3000 r / min for 3 min, and the volume ratio of the oil phase to the water phase is 1:10. ④ Perform high-speed shearing at 10000 r / min for 2 min, and then perform high-pressure homogenization 6 times at 1000 bar to obtain the water-oil mixed preparation of ethanol extract of Shengyu Oil, simply recorded as Shengyu Oil preparation, and the content of ethanol extract of Shengyu Oil in the obtained Shengyu Oil preparation is equal to 30 mg / ml.

[0049] 2) Characterization of the particle size and zeta potential of Shengyu Oil preparation

[0050] When measuring the particle size of Shengyu Oil preparation, dilute the sample with ultrapure water so that its count rate is between 200 - 300 Kcps (1,000 counts per second), and then measure the particle size and polydispersity index with a laser particle size analyzer, scattering angle 90°, temperature 25 °C; when measuring the zeta potential, adjust the conductivity of Shengyu Oil preparation to 50 S / cm with 0.9% (w / w) sodium chloride solution. As Figure 1 shown in (C, D) below, the particle size distribution of the Shengyu Oil preparation prepared in Example 2 is in the range of 141 - 825 nm, the average particle size is about 304 nm, and there is no multiple scattering phenomenon; moreover, its zeta potential is evenly distributed, with an average of -42.6 mV.

[0051] Example 3 Effect of the Shengyu Oil preparation prepared in Example 1 on hair growth in a mouse hair catagen model induced by testosterone propionate

[0052] 1. Grouping and administration

[0053] Twenty 5-week-old SPF-grade male C57 mice (18 - 20 g; provided by Hubei Experimental Animal Center and pre-fed in a professional animal house for one week) were randomly and evenly divided into a blank control group, a testosterone propionate (TP) model group, an SFO group, a finasteride (Fina) group, and a minoxidil group, with 4 mice in each group. After the mice were anesthetized (with 10% chloral hydrate solution), hot-melt mixture of rosin and paraffin (1:1) was used to depilate the back of the mice. Only normal saline was applied to the back of the mice in the blank control group, and the mice in the other groups were subcutaneously injected with 0.1 ml of testosterone propionate solution (5 mg / kg; Macklin) once a day for 14 consecutive days. After each injection of testosterone propionate solution, 0.5 ml of skin-nourishing oil preparation (prepared in Example 1) was applied to the depilated area of the mice in the SFO group, 0.2 ml (0.5 mg / kg; Hangzhou Conba Pharmaceutical Co., Ltd.) of finasteride solution was intragastrically administered to the finasteride group, and 0.5 ml of 5% minoxidil foam (Zhejiang Sansheng Mandi Pharmaceutical Co., Ltd.) was sprayed on the depilated area of the mice in the minoxidil group.

[0054] 2. Detection of hair growth in mice

[0055] On the 1st, 5th, 10th, and 14th days, under the conditions of the same light source and unified green background, a Sony α300 single-lens reflex macro camera was used to record the hair growth on the back of the mice, and the hair growth photos on the back of the mice were scored according to the hair growth scoring criteria in the following table (Table 1).

[0056] Table 1. Hair growth scoring criteria for the back of mice

[0057]

[0058] 3. Histopathological staining of skin tissue in the depilated area of mice

[0059] After the last photo was taken on the 14th day, all the mice were sacrificed by cervical dislocation. The skin tissue in the depilated area was removed, wrapped with gauze, and fixed in formalin solution for 24 h. Then, steps such as rinsing with running water, dehydration, paraffin embedding, sectioning, and staining (H&E and immunohistochemical staining) were carried out. Finally, the changes in the skin tissue structure in the depilated area were observed under an optical microscope.

[0060] 4. Experimental results:

[0061] (1) Changes in hair growth in the depilated area of mice

[0062] As Figure 2 shown, on the first day of the experiment, the back skin of the mice in each group was in a uniformly rosy state and without hair coverage, and the baseline data had good consistency (mean 0.0 ± 0.0, P > 0.05), meeting the requirements of experimental standardization.

[0063] After 5 days of intervention, significant graying changes appeared in the dorsal skin of mice in the blank control group (3.0±0.0). Compared with the blank control group, no obvious hair growth was observed in the model group (0.0±0.0, P<0.001). Compared with the model group, mice in the Shenfu Oil group (1.8±0.3, P<0.05) showed obvious signs of hair follicle activation, and mice in the minoxidil group (0.8±0.3, P>0.05) also showed signs of hair follicle activation, while the finasteride group (0.3±0.3, P>0.05) had less obvious activation signs.

[0064] On the 10th day of intervention, normal hair growth was observed in the blank control group (6.0±0.0), while only a slight increase in hair density was seen in the model group (3.0±0.0, P<0.001). Compared with the model group, the hair growth scores of mice in the Shenfu Oil group (5.0±0.4, P<0.05) and the finasteride group (4.3±0.3, P<0.05) were significantly up-regulated, while the hair growth score of mice in the minoxidil group (3.5±0.3, P>0.05) did not increase significantly.

[0065] After 14 days of intervention, mice in the blank control group returned to the state before hair removal (7.0±0.0), while hair growth in the hair removal area of the model group was still slow (4.3±0.3, P<0.01). Compared with the model group, the hair growth scores of mice in the Shenfu Oil group (6.8±0.3, P<0.01), the minoxidil group (6.0±0.4, P>0.05) and the finasteride group (5.8±0.3, P>0.05) showed an upward trend, and the Shenfu Oil group had the best hair growth effect.

[0066] (2) H&E staining of the skin tissue in the hair removal area of mice

[0067] As Figure 3 shown, the dorsal skin structure of mice in the blank control group was intact, the hair follicle morphology was normal, and the proportions of hair follicles in the growth phase (5.33±1.45), regression phase (1.67±0.88) and resting phase (16.33±0.88) were in line with the physiological state. The following changes were observed in the dorsal skin of mice in the model group (indicating successful construction of the alopecia model): (1) complete absence of hair follicles in the growth phase (0.00±0.00, P<0.01), (2) an upward trend in the number of hair follicles in the regression phase (4.67±0.33, P>0.05) and (3) a significant decrease in the number of hair follicles in the resting phase (6.67±0.88, P<0.001).

[0068] Compared with the model group, the dorsal skin of mice in the Shenfu Oil group showed the following changes (indicating that it significantly promoted the hair follicles to enter the growth phase and inhibited regression): (1) The number of hair follicles in the growth phase increased significantly to (12.00±1.15, P<0.001), (2) The hair follicles in the catagen phase completely disappeared (0.00±0.00, P<0.01), and (3) The number of hair follicles in the telogen phase decreased to 4.33±0.88 (P>0.05). Compared with the model group, the hair follicle status of the dorsal skin of mice in the finasteride group and the minoxidil group also improved significantly. Among them, the number of hair follicles in the growth phase in the finasteride group was 7.00±1.73 (P<0.001), and the number of hair follicles in the growth phase in the minoxidil group was 8.33±0.67 (P<0.001), but both were significantly lower than that in the Shenfu Oil group. It is worth mentioning that the number of hair follicles in the telogen phase in the minoxidil group (14.00±1.15, P<0.001) was significantly higher than that in other treatment groups, suggesting that it may prolong the quiescent state of hair follicles.

[0069] (3) Immunohistochemical staining of CD31 in the skin tissue of the hair removal area of mice

[0070] CD31 can promote angiogenesis, regulate the microenvironment of hair follicle stem cells, participate in inflammation regulation and tissue repair, and its activation will have a positive impact on the hair growth process.

[0071] As Figure 4 shown, compared with the blank control group (100.0%±3.0%), the expression level of CD31 protein in the model group decreased significantly (46.4%±7.9%, P<0.05). Compared with the model group, the expression levels of CD31 protein in the skin tissue of the hair removal area of mice in the finasteride group (134.4%±7.9%, P<0.001), the minoxidil group (159.2%±6.1%, P<0.001), and the Shenfu Oil group (185.84%±23.99%, P<0.001) all increased significantly, and the promotion effect of the Shenfu Oil group was the best (which also suggests that the Shenfu Oil preparation can promote hair growth by increasing the expression of CD31 protein).

[0072] (4) Immunohistochemical staining of CD34 in the skin tissue of the hair removal area of mice

[0073] CD34 is expressed in the bulge area of hair follicles and the dermal sheath. It can maintain the hair follicle stem cell pool, promote the cyclic regeneration of hair follicles, as well as cell proliferation and differentiation, and participate in tissue repair and hair follicle regeneration.

[0074] As Figure 5As shown, compared with the blank control group (100.0% ± 4.2%), the expression level of CD34 protein in the skin tissue of the depilated area of the model group mice showed a downward trend (37.3% ± 6.3%, P < 0.05). Compared with the model group, the expression levels of CD34 protein in the skin tissue of the depilated area of the finasteride group (237.3% ± 13.6%, P < 0.001), the minoxidil group (259.2% ± 21.7%, P < 0.001), and the shengfu oil group (322.7% ± 17.2%, P < 0.001) were all significantly increased, and the promotion effect of the shengfu oil group was the best (which also indicates that the shengfu oil preparation can promote hair growth by regulating the CD34 signal).

[0075] Example 4 Effect of the shengfu oil preparation prepared in Example 2 on hair growth in a mouse hair growth phase model induced by cyclosporin A

[0076] 1. Grouping and administration

[0077] Twenty 7-week-old SPF-grade C57 male mice (18 - 20 g; provided by the Hubei Experimental Animal Center and pre-fed in a professional animal house for one week) were randomly and evenly divided into a blank control group, a cyclosporin A (CsA) model group, an SFO group, a finasteride (Fina) group, and a minoxidil group, with 4 mice in each group. After the mice were anesthetized (with 10% chloral hydrate solution), hot-melt mixture depilation treatment with rosin paraffin (1:1) was performed on their backs. Only normal saline was applied to the backs of the blank control group, and the mice in the other groups were gavaged with 0.2 ml of cyclosporin A solution (50 mg / kg; Macklin) once a day for 10 consecutive days. After each gavage of the cyclosporin A solution, 0.5 ml of the shengfu oil preparation (prepared in Example 2) was applied to the depilated area of the SFO group, 0.2 ml (0.5 mg / kg) of finasteride solution was gavaged to the finasteride group, and 0.5 ml of 5% minoxidil foam was sprayed on the depilated area of the minoxidil group.

[0078] 2. Detection of mouse hair growth

[0079] On the 1st, 5th, and 10th days, under the same light source and unified green background conditions, a Sony α300 single-lens reflex macro camera was used to record the hair growth on the backs of the mice, and the hair growth photos of the backs of the mice were scored according to the hair growth scoring criteria in Table 1.

[0080] 3. Histopathological staining of the skin tissue of the depilated area of the mice

[0081] After the last photograph was taken on the 10th day, all mice were sacrificed by cervical dislocation. The skin tissue in the depilated area was removed, wrapped with gauze, and fixed in formalin solution for 24 h. Subsequently, steps such as running water rinsing, dehydration, paraffin embedding, sectioning, and staining (H&E and immunohistochemical staining) were carried out. Finally, the changes in the skin tissue structure in the depilated area were observed under an optical microscope.

[0082] 4. Experimental results

[0083] (1) Hair growth changes in the depilated area of mice

[0084] As Figure 6 shown, on the first day of the experiment, the back skin of mice in each group was uniformly ruddy and hairless, and the baseline data had good consistency (mean 0.0 ± 0.0, P>0.05), meeting the requirements of experimental standardization.

[0085] After 5 days of intervention, there was no change in the skin of the depilated area on the back of mice in the blank control group (0.0 ± 0.0), while a gray area was seen on the back of the model group (0.8 ± 0.3, P<0.05), indicating that the hair growth model was successfully constructed. Compared with the model group, mice in the Shenfu oil group (1.0 ± 0.0, P>0.05) and the minoxidil group (0.5 ± 0.3, P>0.05) showed signs of hair follicle activation, while the finasteride group showed no activation signs (0.0 ± 0.0, P<0.05).

[0086] After 10 days of intervention, the hair coverage rate in the depilated area on the back of mice in all groups increased: the hair growth score of mice in the blank control group was (1.3 ± 0.3), while the hair growth in the model group was faster (2.3 ± 0.3, P<0.01). Compared with the model group, there were differences in the hair growth scores (hair coverage) in the depilated area on the back of mice in the Shenfu oil group (4.3 ± 0.3, P<0.001), the minoxidil group (3.3 ± 0.5, P<0.01), and the finasteride group (1.8 ± 0.3, P>0.05), among which the effect of the Shenfu oil group was the most obvious.

[0087] (2) H&E staining of skin tissue in the depilated area of mice

[0088] As Figure 7As shown, the skin structure of the depilated area on the back of the mice in the blank control group was intact, the hair follicle morphology was normal, and the numbers of hair follicles in the growth, regression, and resting phases were 3.33 ± 0.33, 3.33 ± 0.88, and 9.33 ± 1.20, respectively. The following changes were observed in the back skin of the mice in the model group (indicating successful construction of the hair growth model): (1) The number of hair follicles in the growth phase increased significantly (7.67 ± 0.33, P < 0.05), (2) the number of hair follicles in the regression phase decreased (2.33 ± 0.67, P > 0.05), and (3) the number of hair follicles in the resting phase decreased (6.33 ± 0.33, P > 0.05).

[0089] Compared with the model group, the following changes were observed in the back skin of the mice in the Shenfu Oil group (indicating its significant and continuous promotion of hair follicles into the growth phase): (1) The number of hair follicles in the growth phase increased significantly (12.67 ± 2.19, P < 0.001), (2) the hair follicles in the regression phase completely disappeared (0.33 ± 0.33, P > 0.05), and (3) the number of hair follicles in the resting phase decreased (5.00 ± 1.53, P > 0.05).

[0090] Compared with the model group, the number of hair follicles in the growth phase in the back skin tissue of the mice in the Minoxidil group was 4.33 ± 1.20 (P > 0.05), the number of hair follicles in the regression phase decreased to 3.33 ± 0.88 (P > 0.05), and the number of hair follicles in the resting phase increased significantly (14.00 ± 1.15, P < 0.001), suggesting that the Minoxidil group promoted hair follicles into the resting phase to prepare for the next growth phase. In the Finasteride group, the number of hair follicles in the growth phase in the back skin tissue of the mice was (4.33 ± 1.45, P > 0.05), the number of hair follicles in the regression phase increased (7.67 ± 0.67, P < 0.01), and the difference in the number of hair follicles in the resting phase was not significant (5.67 ± 0.33, P > 0.05), which corroborated that Finasteride was an anti - hair loss drug rather than a hair growth drug.

[0091] (3) Immunohistochemical staining of CD31 in the skin tissue of the depilated area of the mice

[0092] As Figure 8 shown, compared with the blank control group (100.0% ± 14.4%), the expression level of CD31 protein in the skin tissue of the depilated area on the back of the mice in the model group showed a significant up - regulation (199.6% ± 9.8%, P < 0.05). Compared with the model group, the expression level of CD31 protein in the skin tissue of the depilated area on the back of the mice in the Shenfu Oil group (314.0% ± 23.4%, P < 0.05) showed a significant up - regulation, and the expression levels of CD31 protein in the skin of the depilated area on the back of the mice in the Finasteride group (162.2% ± 17.3%, P > 0.05) and the Minoxidil group (248.8% ± 30.9%, P > 0.05) showed an upward trend.

[0093] (4) Immunohistochemical staining of CD34 in the skin tissue of the depilated area of mice

[0094] As Figure 9 shown, compared with the blank control group (100.0% ± 6.9%), the expression level of CD34 protein in the skin tissue of the depilated area of mice in the model group showed a significant increase (196.5% ± 5.1%, P < 0.01). Compared with the model group, after treatment with the minoxidil group (290.8% ± 32.5%, P < 0.01) and the shengfu oil group (287.0% ± 8.0%, P < 0.05), the expression level of CD34 protein in the skin tissue of the depilated area of mice showed a significant increase, and the improvement effect of the shengfu oil group was more stable. The expression level of CD34 protein in the skin tissue of the finasteride group (258.6% ± 14.4%, P > 0.05) in the depilated area showed an upward trend compared with the model group.

[0095] Example 5 Effect of the shengfu oil preparation prepared in Example 1 on hair growth in a cyclophosphamide-induced telogen model of mice

[0096] 1. Grouping and administration

[0097] Twenty 4-week-old SPF-grade C57 female mice (12 - 15 g; provided by Hubei Experimental Animal Center and pre-fed in a professional animal house for one week) were randomly and evenly divided into a blank control group, a cyclophosphamide (CYP) model group, an SFO group, a finasteride (Fina) group, and a minoxidil group, with 4 mice in each group. After the mice were anesthetized (10% chloral hydrate solution), three-quarters of the hair on their backs was removed using an electric hair clipper. Only normal saline was applied to the backs of the mice in the blank control group, and the mice in the other groups were intraperitoneally injected with 0.2 ml of cyclophosphamide solution (120 mg / kg; Macklin) only on the first day. Subsequently, 0.5 ml of the shengfu oil preparation (prepared in Example 1) was applied to the shaved area of the mice in the SFO group every day, 0.2 ml (0.5 mg / kg) of finasteride solution was administered by gavage to the finasteride group, and 0.5 ml of 5% minoxidil foam was sprayed on the shaved area of the minoxidil group for 9 consecutive days.

[0098] 2. Detection of hair growth in mice

[0099] On days 1, 5, and 9, under the conditions of the same light source and a unified green background, a Sony α300 single-lens reflex macro camera was used to record the hair growth in the shaved area on the backs of the mice. The ImageJ software was used to calculate the hair retention rate in the shaved area on the backs of the mice on day 9 (to ensure the accuracy of subsequent analysis, a scale was placed near the mice being photographed each time). The calculation method is briefly described as follows: (1) Set the shaved area on the back of the mouse to 100%; (2) Then set the part with hair in the shaved area to black (hair retention), and the part without hair in the shaved area to white (hair loss); (3) Set the proportion of the white area in the shaved area as the hair loss rate, then the proportion of the black area on the back of the mouse in the shaved area is the hair retention rate of the mouse (100% - hair loss rate).

[0100] 3. Histopathological staining of the skin tissue in the shaved area of the mice

[0101] After the last photograph was taken on day 9, all the mice were sacrificed by cervical dislocation. The skin tissue in the shaved area was removed, wrapped with gauze, and fixed in formalin solution for 24 h. Then, steps such as rinsing with running water, dehydration, paraffin embedding, sectioning, and staining (H&E and immunohistochemical staining) were carried out. Finally, the changes in the skin tissue structure in the shaved area were observed under an optical microscope.

[0102] 4. Experimental results

[0103] (1) Changes in hair growth in the shaved area of the mice

[0104] As Figure 10 shown, on the first day of the experiment, there were no significant differences in the general appearance of the hair in the shaved areas of the mice in each experimental group.

[0105] On the 5th day of intervention, the mice in the blank control group lost hair slowly at the normal telogen hair loss rate, while there were no obvious changes in hair loss in the model group compared with the blank control group. In contrast, the degree of hair loss in the Shenfu Oil group and the finasteride group was similar to that in the model group, while there was no obvious hair loss in the shaved area on the backs of the mice in the minoxidil group compared with the first day.

[0106] On the 9th day of intervention, there were obvious differences in the hair retention status among the experimental groups: (1) The hair retention rate in the shaved area on the back of the mice in the blank control group was (0.71% ± 0.06%); while the hair retention rate of the mice in the model group decreased significantly (0.52% ± 0.02%, P < 0.001; indicating that the telogen model was successfully established); (2) Compared with the model group, the hair retention rates of the mice in the Shenfu Oil group (0.74% ± 0.01%, P < 0.001) and the finasteride group (0.65% ± 0.01%, P < 0.05) were significantly up-regulated, while there was no obvious change in the hair retention rate of the mice in the minoxidil group (0.62% ± 0.02%, P > 0.05).

[0107] (2) H&E staining of the skin tissue in the shaved area of mice

[0108] As Figure 11 shown, the skin structure of the back of mice in the blank control group was intact, the hair follicle morphology was normal, and the proportions of hair follicles in the growth phase (5.00 ± 0.58), regression phase (3.33 ± 0.33), and resting phase (6.33 ± 0.67) were in line with the physiological state. However, the following changes occurred in the hair follicles of the skin in the shaved area of the back of mice in the model group: (1) The hair follicles in the growth phase were almost completely absent (1.33 ± 0.88, P < 0.05); (2) The number of hair follicles in the regression phase showed an upward trend (5.67 ± 0.33, P > 0.05); and (3) The number of hair follicles in the resting phase increased abnormally (15.67 ± 0.88, P < 0.001). The above data indicate that cyclophosphamide successfully induced the construction of a resting phase model (characterized by inhibition of the growth phase and prolongation of the resting phase).

[0109] Compared with the model group, the data of the hair follicles of the skin in the shaved area of the back of mice in the Shengfu oil group showed that: (1) The number of hair follicles in the growth phase increased significantly, returning to 9.00 ± 0.58 (P < 0.001); (2) The hair follicles in the regression phase almost disappeared (1.00 ± 0.58, P < 0.01); and (3) The number of hair follicles in the resting phase decreased significantly, to 5.33 ± 0.88 (P < 0.001). The above data indicate that the Shengfu oil preparation can restore the number of hair follicles in the growth phase and prevent hair follicles from entering the resting phase. In the finasteride group, the number of hair follicles in the growth phase only increased slightly (2.33 ± 0.67, P > 0.05), but was significantly lower than that in the Shengfu oil group; compared with the model group, both the regression phase (3.67 ± 1.35, P > 0.05) and the resting phase (11.33 ± 1.20, P < 0.01; indicating that finasteride has limited improvement on the induction of hair follicles into the resting phase by cyclophosphamide) were down-regulated. In addition, although the number of hair follicles in the growth phase in the minoxidil group showed a partial recovery trend compared with the model group (3.33 ± 1.20), there were no differences in the number of hair follicles in the regression phase (6.33 ± 0.88) and the resting phase (14.67 ± 1.45) compared with the model group.

Claims

1. An external skin nourishing oil preparation for hair growth, hair regeneration and anti - hair loss, characterized in that, The skin-healing oil preparation is an aqueous-oil mixed preparation of the ethanol extract of skin-healing oil, and the content of the ethanol extract of skin-healing oil in the skin-healing oil preparation is not less than 10 mg / ml and not more than 50 mg / ml; The preparation method of the skin-healing oil preparation is as follows: dissolving the freeze-dried powder of the ethanol extract of skin-healing oil and Span 60 in edible vegetable oil to prepare an oil phase, and dropping the oil phase into the aqueous phase prepared by dissolving Tween 60 and glycerol in ultrapure water according to the volume ratio of the oil phase to the aqueous phase of 1:5 - 10, and stirring, followed by high-speed shearing and high-pressure homogenization to obtain the aqueous-oil mixed preparation of the ethanol extract of skin-healing oil; The preparation method of the freeze-dried powder of the ethanol extract of skin-healing oil is as follows: taking the dry powders of 8 raw materials of skin-healing oil and borneol according to a certain weight part ratio and adding them into a container, then adding an ethanol solution with a volume fraction of 40% - 90% into the container, soaking for 2 - 3 h, followed by flash extraction, concentrating the filtrate, and freeze-drying to obtain the freeze-dried powder of the ethanol extract of skin-healing oil; The 8 raw materials of skin-healing oil are Gynura procumbens (Lour.) Merr., Pericarpium Citri Reticulatae Viride, Astragalus membranaceus (Fisch.) Bunge, Angelica sinensis (Oliv.) Diels, Olibanum, Fructus Hordei Germinatus, Rheum officinale Baill., and Phellodendron amurense Rupr.; the weight part ratios of each raw material and borneol are as follows: 20 - 50 parts by weight of Gynura procumbens (Lour.) Merr., 20 - 50 parts by weight of Pericarpium Citri Reticulatae Viride, 20 - 50 parts by weight of Astragalus membranaceus (Fisch.) Bunge, 5 - 15 parts by weight of Angelica sinensis (Oliv.) Diels, 5 - 15 parts by weight of Olibanum, 5 - 15 parts by weight of Fructus Hordei Germinatus, 5 - 15 parts by weight of Rheum officinale Baill., 5 - 15 parts by weight of Phellodendron amurense Rupr., and 3 - 8 parts by weight of borneol.

2. The external skin healing oil preparation according to claim 1, characterized in that, The content of the ethanol extract of skin-healing oil in the skin-healing oil preparation is 30 mg / ml.

3. The external skin-healing oil preparation according to claim 1, characterized in that, The edible vegetable oil is selected from at least one of olive oil, sesame oil, and camellia oil.

4. The external skin-healing oil preparation according to claim 1, characterized in that, The relationship between the total mass of the 9 raw materials of skin-healing oil and the dosage of the ethanol solution with a volume fraction of 40% - 90% is 500 g:(3 - 7) L.

5. The external skin healing oil preparation according to claim 1, characterized in that, The dry powders of the 8 herbs, namely Gynura procumbens (Lour.) Merr., Pericarpium Citri Reticulatae Viride, Astragalus membranaceus (Fisch.) Bunge, Angelica sinensis (Oliv.) Diels, Olibanum, Fructus Hordei Germinatus, Rheum officinale Baill., and Phellodendron amurense Rupr., all pass through a 200-mesh sieve.

6. The preparation method of the external skin-healing oil preparation according to any one of claims 1 to 5, characterized in that, The preparation method of the skin-healing oil preparation is as follows: (1) Preparation of the freeze-dried powder of the ethanol extract of skin-healing oil: taking the dry powders of 8 raw materials of skin-healing oil and borneol according to a certain weight part ratio and adding them into a container, then adding an ethanol solution with a volume fraction of 40% - 90% into the container, soaking for 2 - 3 h, performing flash extraction 3 - 5 times, 5 min each time, concentrating the filtrate, and freeze-drying to obtain the freeze-dried powder of the ethanol extract of skin-healing oil; (2) Preparation of the skin-healing oil preparation: 1) Dissolving the freeze-dried powder of the ethanol extract of skin-healing oil and Span 60 in edible vegetable oil to prepare an oil phase; the final concentration of Span 60 is 1% - 5% W / V; 2) Dissolving Tween 60 and glycerol in ultrapure water to prepare an aqueous phase; the final concentration of Tween 60 is 1% - 5% V / V; the final concentration of glycerol is 5% - 15% V / V; 3) Under the condition of a constant temperature water bath at 60 °C, dropping the oil phase prepared in step 1) into the aqueous phase prepared in step 2), and stirring at 3000 r / min for 3 min; the volume ratio of the oil phase to the aqueous phase is 1:5 - 10; 4) High-speed shear for 2 min under the condition of 10,000 rpm, and then high-pressure homogenize 6 times under the condition of 1000 bar to obtain the water-oil mixed preparation of the ethanol extract of Shengyu oil.

7. The preparation method according to claim 6, characterized in that, The zeta potential and particle size distribution of the obtained Shengyu oil preparation are uniform; its particle size distribution is 141-825 nm, and there is no multiple scattering phenomenon, and the average particle size is 300±2 nm; the average zeta potential is -45 mV to -39.1 mV.

8. Use of the external Shengyu oil preparation according to any one of claims 1 to 5 or the external Shengyu oil preparation prepared by the method according to any one of claims 6 to 7 in the preparation of an external drug for preventing or treating alopecia; particularly in the preparation of a non-injected and non-implanted external drug for promoting hair growth, growing hair and preventing hair loss.

9. The application according to claim 8, wherein The alopecia includes physiological alopecia and pathological alopecia.

10. The application according to claim 8, characterized in that, The types of alopecia include hair loss, thinning and / or softness.

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