Preparation method of lipidosome containing hair-growing active ingredients

Abstract

The invention provides a preparation method of lipidosome containing hair-growing active ingredients, and belongs to the technical field of hair-growing products. According to the invention, active substances are utilized to regulate hormone level, improve scalp blood circulation, stimulate anti-hair loss factors and promote hair growth in multiple directions, and lipidosome formed by lecithin and oil-soluble active ingredients as raw materials is utilized to integrally entrap the active substances, so that the anti-hair loss effect is improved. The defect of weak transdermal ability caused by large water solubility difference among the active substances is overcome, the stability and bioavailability of the active substances can be improved, and the hair growth and development effects of the prepared lipidosome containing the hair growth active components are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of hair growth products, and in particular to a cobalt metal organic framework material and a preparation method and application thereof. Background Art

[0002] The pace of modern life and work is very fast, and people are under great pressure from all aspects. Similarly, many diseases are increasing day by day. Hair loss is one of them. Both men and women may be distressed by hair loss. Hair loss affects the image and may also indicate the unhealthiness of a certain part of the body. There are usually five reasons for hair loss. The first is endocrine disorders. Insufficient thyroid hormone can also cause hair loss. The second is physical and chemical factors. Frequent hair dyeing and perming can cause hair loss. The third is diseases and drugs. For example, suffering from anemia, malnutrition or skin diseases can cause hair loss. The fourth is long-term lack of vitamins, especially lack of vitamin B. 2 ; The fifth is the lack of trace elements, which will lead to hair loss.

[0003] There are many kinds of hair growth products sold in the market, and their modes of action can be divided into two categories: one is chemical interference hormones, such as finasteride, which has a structure similar to androgen and can inhibit the production of dihydrotestosterone, block the catalytic function of 5α-reductase, and relieve the symptoms of hair loss, but it will cause side effects such as dizziness, headache, edema, skin rash, and will bring hidden dangers of birth of deformed babies when taken. Another example is minoxidil, which can dilate the capillaries of hair follicles, provide sufficient nourishment to the dermal papilla, and relieve the symptoms of hair loss, but it will cause side effects such as redness, swelling, itching, dizziness, chest pain, etc. The other is natural herbal plant ingredients, such as Polygonum multiflorum, ginseng, ginkgo, ginger, etc. Most of the daily chemical products on the market have this kind of plant extract ingredients, but due to the single natural herbal plant ingredients, too low content of active ingredients, poor permeability of water-soluble active ingredients, short product retention time, and difficulty in the active ingredients to fully exert their effects, these products are not effective in preventing hair loss and promoting hair growth. Therefore, providing a hair growth and development product with high utilization rate of active ingredients is a technical problem that needs to be solved urgently in the prior art. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing liposomes containing hair growth active ingredients. The method provided by the present invention can improve the stability and bioavailability of adenosine and active substances including plant extracts, polypeptide compounds, and vitamin compounds, thereby improving the hair growth and development effect of the prepared liposomes containing hair growth active ingredients.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing liposomes containing hair growth active ingredients, comprising the following steps:

[0007] (1) Using lecithin, an oil-soluble active ingredient and a solvent as raw materials to prepare a film;

[0008] (2) dissolving the active substance in a buffer solution to obtain a hydrated solution;

[0009] The hydration liquid is added to the film obtained in step (1), and hydration and dispersion are performed in sequence to obtain liposomes containing hair growth active ingredients.

[0010] The step (1) and the step (2) are both carried out under light-proof conditions.

[0011] Preferably, the oil-soluble active ingredient in step (1) includes one or more of cholesterol, oil-soluble plant extracts, ceramides, oil-soluble vitamins and other active substances with hair growth effects; the solvent includes one or more of vegetable oils, ethanol, polyols, polyethers, polyglycerols and polyethylene glycol solvents.

[0012] Preferably, in step (1), the mass ratio of lecithin to oil-soluble active ingredient is (0.1-10): (0.01-10).

[0013] Preferably, the step (1) specifically comprises: mixing lecithin, an oil-soluble active ingredient and a solvent, and then performing rotary evaporation and vacuum drying in sequence to obtain a film.

[0014] Preferably, the temperature of the rotary evaporation is 28-43° C., and the rotation speed of the rotary evaporation is 20-50 rpm.

[0015] Preferably, the active substance in step (2) includes one or more of adenosine, compound plant extracts, polypeptides, polyphenols, amino acids, minerals, proteins, vitamin compounds and other anti-hair loss and hair growth active compounds.

[0016] Preferably, the polypeptide is tripeptide-1, tripeptide-1 copper, tripeptide-34 copper, alanine / histidine / lysine polypeptide copper HCl, tripeptide-10 citrulline, palmitoyl tripeptide-38, biotin tripeptide-1, palmitoyl dipeptide-5 diaminobutyryl hydroxythreonine, tetrapeptide-3, tetrapeptide-4, acetyl tetrapeptide-3, acetyl tetrapeptide-11, myristoyl tetrapeptide-12, palmitoyl tetrapeptide-7, palmitoyl tetrapeptide-20 , palmitoyl pentapeptide-4, myristoyl pentapeptide-4, myristoyl pentapeptide-17, hexapeptide-3, hexapeptide-10, hexapeptide-11, hexapeptide-12, myristoyl hexapeptide-16, palmitoyl hexapeptide-12, acetyl hexapeptide, acetyl octapeptide, palmitoyl octapeptide, myristoyl octapeptide, octapeptide-2, acetyl octapeptide-2, nonapeptide-1, decapeptide-4, acetyl decapeptide-3, oligopeptide-41, oligopeptide-74;

[0017] The protein substance is at least one of recombinant collagen, hydrolyzed collagen, silk protein, hydrolyzed keratin, recombinant fibronectin, recombinant elastin, recombinant mussel viscose, mussel viscose, and silk fibroin;

[0018] The composite plant extract is at least one of saw palmetto extract, Platycladus orientalis leaf extract, Ligustrum lucidum fruit extract, Swertia vulgaris extract, Trifolium pratense extract, ginger root extract, Artemisia argyi extract, Geranium herb extract, Centella asiatica extract, Polygonum multiflorum extract, Sophora flavescens extract, Panax notoginseng extract, Angelica sinensis extract, and Miscanthus variegatus extract.

[0019] Preferably, the other anti-hair loss and hair growth active compound is at least one of diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, gallic acid, caffeine and taxifolin.

[0020] Preferably, the hydration in step (2) comprises: performing a first heat treatment at 35-45° C. until the film is completely wetted and falls off, and then performing a second heat treatment at 35-45° C. with stirring for 20-50 minutes.

[0021] Preferably, the dispersion in step (2) is carried out by ultrasound, stirring, extrusion or homogenization.

[0022] The present invention also provides liposomes containing hair growth active ingredients prepared by the preparation method described in the above technical scheme.

[0023] The present invention provides a method for preparing a liposome containing a hair growth active ingredient. First, lecithin, an oil-soluble active ingredient and a solvent are used as raw materials to prepare a film; the active substance is dissolved in a buffer solution to obtain a hydration liquid, which is then added to the film for hydration and dispersion to obtain a liposome containing a hair growth active ingredient, and the preparation steps are all performed under light-proof conditions. The present invention regulates hormone levels through active substances, improves scalp blood circulation, stimulates anti-hair loss factors, and promotes hair growth in all directions. The liposomes formed by using lecithin and oil-soluble active ingredients as raw materials are integrated to encapsulate active substances (including at least one of adenosine, composite plant extracts, polypeptides, protein substances, water-soluble vitamin compounds and other anti-hair loss active compounds), overcome the disadvantage of weak transdermal ability caused by large differences in water solubility between various active substances, can improve the stability and bioavailability of adenosine and active substances, and significantly improve the hair growth effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a TEM image of the liposome containing the hair growth active ingredient prepared in Example 1 of the present invention;

[0025] Figure 2 This is a particle size distribution diagram of the liposome containing the hair growth active ingredient prepared in Example 1 of the present invention;

[0026] Figure 3 This is a 24-hour cumulative drug release rate variation trend diagram of the liposome solution containing the hair growth active ingredient prepared in Example 1 of the present invention;

[0027] Figure 4 This is a graph showing the change of the zeta potential of the liposome containing the hair growth active ingredient prepared in Example 1 of the present invention at room temperature and 4°C over time, wherein: Figure 4 a in the middle is the first day, Figure 4 B is the 15th day, room temperature, Figure 4 The middle c is the 15th day, 4℃, Figure 4 The d in the table is the 30th day, room temperature. Figure 4 Middle e is day 30, 4°C;

[0028] Figure 5 Fluorescence images of 3T3 cells after culturing the liposomes containing the hair growth active ingredient prepared in Example 1 of the present invention for 24 hours and 48 hours;

[0029] Figure 6 Fluorescence images of HaCat cells after culturing the liposomes containing the hair growth active ingredient prepared in Example 1 of the present invention for 24 hours and 48 hours;

[0030] Figure 7 This is a graph showing the cumulative drug release amount versus time during the transdermal experiment of the liposome containing the hair growth active ingredient prepared in Example 1 of the present invention;

[0031] Figure 8 This is a graph showing the cumulative drug release rate versus time during the transdermal experiment of the liposome containing the hair growth active ingredient prepared in Example 1 of the present invention;

[0032] Fig. 9 The fluorescence images of the transdermal experiment of the liposome solution containing the hair growth active ingredient prepared in Example 1 of the present invention on pig skin at 1h, 6h and 12h, wherein rhodamine B is red fluorescence and pig skin tissue is blue fluorescence;

[0033] Fig.10 The following are the physical pictures and dermoscopic pictures of the mice in group A in the animal experiment of the present invention without growth intervention, among which: Fig.10 The middle left picture is the actual picture. Fig.10 The middle right image is a dermoscopic image;

[0034] Fig.11 The actual pictures of mice in groups B, C, D, E and F from the 1st to the 28th day of drug administration in the animal experiment of the present invention;

[0035] Fig.12 These are dermoscopic images of mice in groups B, C, D, E and F in the animal experiment of the present invention from the 1st week to the 4th week of administration;

[0036] Fig.13 This is a bar graph of testosterone concentrations of mice in groups C, D, E and F of the animal experiment of the present invention on the 28th day of administration;

[0037] Fig.14 This is a bar graph of estradiol concentrations of mice in groups C, D, E and F of the animal experiment of the present invention on the 28th day of administration;

[0038] Fig.15 These are HE staining sections of Group A, Group B, Group C, Group D, Group E and Group F in the present invention. DETAILED DESCRIPTION

[0039] The present invention provides a method for preparing liposomes containing hair growth active ingredients, comprising the following steps:

[0040] (1) using lecithin, an oil-soluble active ingredient and a solvent as raw materials to prepare a film;

[0041] (2) dissolving the active substance in a buffer solution to obtain a hydrated solution;

[0042] The hydration liquid is added to the film obtained in step (1), and hydration and dispersion are performed in sequence to obtain liposomes containing hair growth active ingredients.

[0043] The step (1) and the step (2) are both carried out under light-proof conditions.

[0044] In the present invention, unless otherwise specified, the raw materials used are conventional commercial products in the art.

[0045] The invention uses lecithin, oil-soluble active ingredients and solvent as raw materials to prepare the film.

[0046] In the present invention, the lecithin is preferably one or more of egg yolk lecithin and soybean lecithin.

[0047] In the present invention, the oil-soluble active ingredients preferably include one or more of cholesterol, oil-soluble plant extracts, ceramides, oil-soluble vitamins and other active substances with hair growth effects; the solvent preferably includes one or more of vegetable oils, ethanol, polyols, polyethers, polyglycerols, and polyethylene glycol solvents.

[0048] In the present invention, the mass ratio of lecithin to oil-soluble active ingredient is preferably (0.1-10):(0.01-10), more preferably (2-8):(0.5-8), and more preferably (3-7):1.

[0049] In the present invention, the preparation of the film preferably comprises: dissolving lecithin and oil-soluble active ingredients in a solvent, and then sequentially performing ultrasonic treatment, rotary evaporation and vacuum drying to obtain a film.

[0050] The present invention has no special restrictions on the power and time of ultrasound, as long as each component is dissolved and mixed uniformly.

[0051] In the present invention, the temperature of the rotary evaporation is preferably 28-43°C, more preferably 30-40°C; the speed of the rotary evaporation is preferably 20-50rpm, more preferably 25-45rpm. The present invention controls the temperature and speed of the rotary evaporation within the above range, which is conducive to uniform mixing of the components and forming a uniform film, thereby improving the encapsulation rate of the prepared liposomes for the active substance and improving the hair growth and development effect of the prepared liposomes containing hair growth active ingredients.

[0052] In the present invention, the temperature of the vacuum drying is preferably 50° C. The present invention removes the residual solvent by vacuum drying, which is beneficial to the subsequent hydration and promotes the complete encapsulation of the active substance by the liposome.

[0053] The present invention dissolves the active substance in a buffer solution to obtain a hydration solution.

[0054] In the present invention, the active substance preferably includes one or more of adenosine, compound plant extracts, polypeptides, polyphenols, amino acids, minerals, proteins, vitamin compounds and other hair loss prevention and hair growth active compounds.

[0055] In the present invention, the polypeptide is preferably tripeptide-1, tripeptide-1 copper, tripeptide-34 copper, alanine / histidine / lysine polypeptide copper HCl, tripeptide-10 citrulline, palmitoyl tripeptide-38, biotin tripeptide-1, palmitoyl dipeptide-5 diaminobutyryl hydroxythreonine, tetrapeptide-3, tetrapeptide-4, acetyl tetrapeptide-3, acetyl tetrapeptide-11, myristoyl tetrapeptide-12, palmitoyl tetrapeptide-7, palmitoyl tetrapeptide- 20. At least one of palmitoyl pentapeptide-4, myristoyl pentapeptide-4, myristoyl pentapeptide-17, hexapeptide-3, hexapeptide-10, hexapeptide-11, hexapeptide-12, myristoyl hexapeptide-16, palmitoyl hexapeptide-12, acetyl hexapeptide, acetyl octapeptide, palmitoyl octapeptide, myristoyl octapeptide, octapeptide-2, acetyl octapeptide-2, nonapeptide-1, decapeptide-4, acetyl decapeptide-3, oligopeptide-41, and oligopeptide-74.

[0056] In the present invention, the protein substance is preferably at least one of recombinant collagen, hydrolyzed collagen, silk protein, hydrolyzed keratin, recombinant fibronectin, recombinant elastin, recombinant mussel viscose, mussel viscose, and silk fibroin.

[0057] In the present invention, the composite plant extract is preferably at least one of saw palmetto extract, Platycladus orientalis leaf extract, Ligustrum lucidum fruit extract, deer sprout extract, Trifolium pratense extract, ginger root extract, wormwood extract, geranium extract, Centella asiatica extract, Polygonum multiflorum extract, Sophora flavescens extract, Panax notoginseng extract, Angelica sinensis extract, and Miscanthus angustifolia extract.

[0058] In the present invention, the water-soluble vitamin compound is preferably vitamin A, vitamin A derivatives, vitamin B 5 , Vitamin B 7 , vitamin C and at least one of them.

[0059] In the present invention, the other active compound for preventing hair loss and promoting hair growth is preferably at least one of diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, gallic acid, caffeine and taxifolin.

[0060] In the present invention, the buffer is preferably a PBS buffer; the pH value of the PBS buffer is preferably 7-8, more preferably 7.2-7.6, and further preferably 7.4; the concentration of the PBS buffer is preferably 0.6-0.15M, more preferably 0.7-0.12M.

[0061] In the present invention, the ratio of the mass of adenosine to the volume of PBS buffer is preferably 1 mg: (60-120) mL.

[0062] In the present invention, the mass ratio of the lecithin to the adenosine, plant extract, polypeptide compound, vitamin compound and co-active compound is preferably (0.1-10): (0.1-1): (5-50): (1×10 -6 ~1):(1×10 -3 ~10):(1×10 -3 ~10).

[0063] In the present invention, the ratio of the mass of the active substance to the sum of the mass of the lecithin and the oil-soluble active ingredient (referred to as the drug-lipid ratio) is 1:(0.5-14), more preferably 1:(1-12). The present invention controls the drug-lipid ratio within the above range to increase the encapsulation efficiency of the prepared liposome.

[0064] After obtaining the hydrating liquid and the film, the present invention adds the hydrating liquid to the film, and sequentially performs hydration and dispersion to obtain liposomes containing hair growth active ingredients.

[0065] In the present invention, the hydration preferably includes: a first heat treatment at 35-45° C. until the film is completely wetted and detached, and then a second heat treatment at 35-45° C. with stirring for 20-50 minutes.

[0066] After the hydration is completed, the present invention preferably allows the hydrated product to stand and cool, then disperses it in an ice bath, and then filters it to obtain the liposome containing the hair growth active ingredient.

[0067] In the present invention, the cooling by standing is preferably reduced to room temperature. In the present invention, the dispersion method is preferably ultrasound, stirring, extrusion or homogenization. In the present invention, the power of the ultrasound is preferably 200-250W, more preferably 220w; the time of the ultrasound is preferably 6-15min, more preferably 10min; the working mode of the ultrasound is preferably working for 3s and pausing for 3s.

[0068] In the present invention, the pore size of the filter membrane used for the filtration is preferably 0.18 to 0.25 μm, more preferably 0.20 to 0.23 μm, more preferably 0.22 μm; the number of filtrations is preferably 2 to 5 times.

[0069] In the present invention, the preparation of the film and the preparation of the liposome containing the hair growth active ingredient are preferably carried out under light-proof conditions.

[0070] The present invention utilizes active substances including adenosine, composite plant extracts, polypeptides, protein substances, water-soluble vitamin compounds and other hair loss prevention and hair growth active compounds, as well as oil-soluble active ingredients to regulate hormone levels, improve scalp blood circulation, stimulate anti-hair loss factors, and achieve multi-directional promotion of hair growth. The active substances are integrated into liposomes formed by using lecithin and oil-soluble active ingredients as raw materials, thereby overcoming the disadvantage of weak transdermal ability caused by large differences in water solubility among the active substances, improving the stability and bioavailability of the active substances, and significantly improving the hair growth and growth effect of the prepared liposomes containing the hair growth active ingredients.

[0071] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a 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.

[0072] Example 1

[0073] The preparation method of liposomes containing hair growth active ingredients comprises the following steps:

[0074] (1) dissolving egg yolk lecithin and cholesterol in 80 mL of anhydrous ethanol, ultrasonically dissolving and mixing, and then rotary evaporating at 35° C. and 30 rpm until the anhydrous ethanol is almost evaporated, then increasing the speed at the same temperature until the anhydrous ethanol is evaporated, and then placing in a vacuum drying oven at 50° C. and vacuum drying overnight to obtain a film;

[0075] The mass ratio of the egg yolk lecithin to cholesterol is 6:1; the sum of the mass of the egg yolk lecithin and cholesterol is 100 mg;

[0076] (2) 1 mg of adenosine, 2 mg of Platycladus orientalis leaf extract, 2 mg of Ligustrum lucidum fruit extract, 2 mg of Swertia japonica extract, 10.5 mg of biotin tripeptide, 0.5 mg of myristoyl pentapeptide-4, 1 mg of vitamin C, and 0.33 mg of taxifolin were dissolved in 100 mL of 0.1 M PBS buffer having a pH value of 7.4 to obtain a hydrated solution;

[0077] The hydration liquid is added to the film obtained in step (1), and a first heat treatment is performed at 40° C. until the film is completely wetted and falls off, and then a second heat treatment is performed at 40° C. for 30 min while stirring to complete hydration, and then the film is allowed to stand and cool to room temperature, and then ultrasonicated in an ice bath to obtain a suspension;

[0078] The power of the ultrasound is 220W; the time of the ultrasound is 10 minutes; the working mode of the ultrasound is: work for 3 seconds and pause for 3 seconds;

[0079] The suspension is filtered three times using a filter membrane with a pore size of 0.22 μm to obtain liposomes (solution) containing hair growth active ingredients;

[0080] The mass ratio of the active substance to the liposome (i.e. the sum of the masses of egg yolk phosphatidylcholine and cholesterol) is 1:12; and both step (1) and step (2) are carried out under light-proof conditions.

[0081] Examples 2, 3 and 4

[0082] Liposomes containing hair growth active ingredients were prepared according to the method of Example 1, except that the total amount of egg yolk lecithin and cholesterol in Examples 2, 3 and 4 was 120 mg, and the mass ratios of egg yolk lecithin to cholesterol in Examples 2, 3 and 4 were 4:1, 5:1 and 6:1, respectively.

[0083] Examples 5, 6 and 7

[0084] Liposomes containing hair growth active ingredients were prepared according to the method of Example 1, except that the mass ratio of egg yolk lecithin to cholesterol in Examples 5, 6 and 7 was 5:1, and the total amounts of egg yolk lecithin and cholesterol in Examples 5, 6 and 7 were 80 mg, 100 mg and 120 mg, respectively.

[0085] Examples 8, 9 and 10

[0086] Liposomes containing hair growth active ingredients were prepared according to the method of Example 1, except that the mass ratio of egg yolk lecithin to cholesterol in Examples 8, 9 and 10 was 5:1, the total amount of egg yolk lecithin and cholesterol was 120 mg, and the rotary evaporation temperatures in step (1) in Examples 8, 9 and 10 were 30° C., 35° C. and 40° C., respectively.

[0087] Examples 11 to 15

[0088] Liposomes containing hair growth active ingredients were prepared according to the method of Example 1, except that in Examples 11 to 15, the mass ratio of egg yolk lecithin to cholesterol was 5:1, the total amount of egg yolk lecithin and cholesterol was 120 mg, and the mass ratio of the sum of the mass of adenosine and active substance in step (2) of Examples 10 to 14 to the mass of liposomes (i.e., the sum of the mass of egg yolk lecithin and cholesterol) (referred to as drug-lipid ratio) was 1:1, 1:2, 1:4, 1:6 and 1:12, respectively.

[0089] 1. Characterization and performance testing of liposomes

[0090] (1)TEM

[0091] Prepare a 2% phosphotungstic acid dye solution and adjust the pH to 6.0-7.0; drop a drop of the liposome solution containing the hair growth active ingredient prepared in Example 1 on a copper mesh, let it stand until the surface is slightly dry, then add a 2.0% phosphotungstic acid solution for negative staining for 15 minutes, absorb the excess dye solution with filter paper, and dry naturally; use a transmission electron microscope to photograph and observe the morphology of the liposome at a low voltage, and obtain a TEM image of the liposome containing the hair growth active ingredient prepared in Example 1 as shown in FIG. Figure 1 As shown by Figure 1 It can be seen that the liposomes containing hair growth active ingredients prepared in Example 1 have regular morphology, uniform size, are mostly round, have good dispersibility, and the active substances can form a relatively stable liposome structure with the membrane material, with a particle size of about 100 nm.

[0092] (2) Determination of encapsulation efficiency

[0093] The free drug content (i.e., the total amount of adenosine and active substances) in the liposome containing the hair growth active ingredient was determined by ultrafiltration. 5 ml of the prepared liposome solution containing the hair growth active ingredient was taken into a 3 kD ultrafiltration tube using a pipette and centrifuged at 5000 rpm for 10 min. The solution in the ultrafiltration tube was taken, and 0.1 M PBS buffer was used as a blank control, and its absorbance at 260 nm was measured using an ultraviolet spectrophotometer. Another 1 mL of the prepared liposome containing the hair growth active ingredient was taken, and the volume was adjusted to 5 mL with anhydrous ethanol, and its absorbance at 260 nm was also measured. According to the standard curve regression equation, W and W were obtained respectively. 总 and W 游 .

[0094] EE(%)=(W 总 -W 游 ) / W 总 ×100%;

[0095] Where: W 总 is the amount of the total drug encapsulated; W 游 is the amount of free drug, and EE (%) is the encapsulation efficiency.

[0096] The results of the above encapsulation efficiency measurements are shown in Tables 1 to 3 below.

[0097] Table 1 Statistics of the encapsulation efficiency of liposomes containing hair growth active ingredients prepared in Examples 2 to 10

[0098] Example The mass ratio of egg yolk lecithin and cholesterol Encapsulation rate% 2 4:1 88.10 3 5:1 89.42 4 6:1 88.72 Example Total amount of egg yolk lecithin and cholesterol (mg) Encapsulation rate% 5 80 84.84 6 100 84.41 7 120 89.42 Example Rotary evaporation temperature ℃ Encapsulation rate% 8 30 88.01 9 35 89.42 10 40 87.35

[0099] Table 2 Statistics of the encapsulation efficiency of liposomes containing hair growth active ingredients prepared in Examples 11 to 15

[0100]

[0101]

[0102] In order to further explore the effects of the mass ratio of egg yolk lecithin to cholesterol (A), the total amount of egg yolk lecithin to cholesterol (B), and the rotary evaporation temperature (C) on the encapsulation efficiency of the liposomes containing the hair growth active ingredient prepared by the present invention, an orthogonal experiment was designed; 3 different levels were selected for each factor, and an L9 (33) orthogonal experiment was performed. According to the same method as Example 1, Examples 16 to 23 were set to prepare liposomes containing the hair growth active ingredient, and the differences from Example 1 are shown in Table 3 below. The encapsulation efficiencies of Examples 16 to 23 measured by the above-mentioned ultrafiltration method are shown in Table 3 below.

[0103] Table 3 Statistics of the encapsulation efficiency of liposomes containing hair growth active ingredients prepared in Examples 1, 16 to 23

[0104] Example A B C Encapsulation rate% Example 16 4:1 80mg 30℃ 85.61 Embodiment 17 14:1 100mg 35℃ 84.84 Embodiment 18 4:1 120mg 40℃ 85.16 Embodiment 19 5:1 80mg 30℃ 86.06 Embodiment 20 5:1 100mg 35℃ 84.41 Embodiment 21 5:1 120mg 40℃ 79.21 Embodiment 22 6:1 80mg 30℃ 87.75 Example 1 6:1 100mg 35℃ 89.42 Embodiment 23 6:1 120mg 40℃ 84.12

[0105] (3) Particle size measurement

[0106] The particle size and dispersion index (PDI) of the liposome containing the hair growth active ingredient prepared in Example 1 were measured by a laser particle size analyzer, and the particle size distribution diagram of the liposome containing the hair growth active ingredient prepared in Example 1 was obtained as shown in FIG. Figure 2 As shown by Figure 2 It can be seen that the average particle size of the liposome containing the hair growth active ingredient prepared in Example 1 is 157 nm, the PDI is 0.360, and the dispersibility is good.

[0107] (4) Drug release evaluation

[0108] The preparation method of blank liposomes comprises the following steps: dissolving egg yolk lecithin and cholesterol in 80 mL of anhydrous ethanol, dissolving and mixing by ultrasonication, and then performing rotary evaporation at 35° C. and 30 rpm until the anhydrous ethanol is almost evaporated, and then increasing the speed at the same temperature until the anhydrous ethanol is evaporated, and then placing the liposome in a vacuum drying oven at 50° C. and vacuum drying overnight to obtain a film;

[0109] The mass ratio of the egg yolk lecithin to cholesterol is 6:1; the sum of the mass of the egg yolk lecithin and cholesterol is 100 mg;

[0110] A first heat treatment is performed at 40° C. until the film is completely wetted and falls off, and then a second heat treatment is performed at 40° C. for 30 minutes with stirring to complete hydration, and then the film is allowed to stand and cool to room temperature, and then ultrasonicated in an ice bath to obtain a suspension; the ultrasonic power is 220 W; the ultrasonic time is 10 minutes; the ultrasonic working mode is: work for 3 seconds and pause for 3 seconds;

[0111] The suspension was filtered three times using a filter membrane with a pore size of 0.22 μm to obtain blank liposomes (solution).

[0112] Take 1 mL of the liposome solution containing hair growth active ingredients prepared in Example 1 and 1 mL of the blank liposome prepared by the above method, respectively, place them in 1 KDa dialysis bags, and place them in 50 mL centrifuge tubes, add 20 mL of 0.1 M PBS buffer solution with a pH of 7.4 as a release medium, place the centrifuge tubes in a 37°C constant temperature water bath oscillator, speed 100r, take 1 mL of the release medium at 0.5, 1, 2, 4, 6, 8, 12, 24h, respectively, and add 1 mL of the medium, detect the absorbance, and calculate the cumulative drug release rate according to the following formula.

[0113] Permeability K = (Q / m) × 100%;

[0114] Wherein, m is the mass of the active substance in the liposome containing the hair growth active ingredient prepared in Example 1 in the sampling medium, and Q is the mass of the active substance detected in the sampling medium;

[0115] The 24-hour cumulative release rate variation trend diagram of the liposome solution containing the hair growth active ingredient prepared in Example 1 is as follows: Figure 3 As shown. Figure 3 It can be seen that the cumulative drug release rate of the liposome solution containing the hair growth active ingredient prepared in Example 1 is 72.25% over 24 hours.

[0116] (4) Stability evaluation

[0117] Method 1: Take 3 portions of the liposomes containing the hair growth active ingredient prepared in Example 1, place them at 4°C and room temperature for 30 days, respectively, and take samples on the 0th, 15th and 30th days of storage to observe the appearance properties, and use ultrafiltration to measure the change in encapsulation efficiency, and calculate the permeability; take 1 mL of the above liposome solution and dilute it to 5 mL in a 3kD centrifuge tube, 5000 rpm, centrifuge for 10 min, use ultraviolet to measure the absorbance at 260 nm, and calculate the permeability.

[0118] Permeability (%) = (EE 起始时 -EE 储藏一定时间后 ) / EE 起始时 ×100;

[0119] Among them, EE 起始时 is the encapsulation efficiency on day 0 at the start, EE 储藏一定时间后 It is the encapsulation rate after storage for a certain number of days.

[0120] The permeability of the liposome solution containing the hair growth active ingredient prepared in Example 1 was detected and varied over time. The average permeability was calculated and the results were as follows: the average permeability of the liposome containing the hair growth active ingredient prepared in Example 1 was 4.39% at room temperature after 15 days, and was 62.07% at 4° C. after 15 days; the average permeability at room temperature after 30 days was 3.03%, and the average permeability at 4° C. after 30 days was 73.04%, indicating that the liposome solution containing the hair growth active ingredient prepared in the present invention has good permeability at both room temperature and 4° C.

[0121] Method 2: Place the liposome solution at 4°C and room temperature respectively, take samples on the 0th, 15th and 30th days to observe the appearance and measure its zeta potential.

[0122] The zeta potential of the liposomes prepared in Example 1 at room temperature and 4°C was measured and the time variation was shown in the figure below: Figure 4 As shown by Figure 4 It can be seen that the zeta potential detection results show that the liposomes prepared in Example 1 are relatively stable at room temperature and 4°C.

[0123] 2. Biosafety Evaluation of Liposomes

[0124] Evaluation of cytotoxicity CalceinAM / PI

[0125] The liposome solution containing the hair growth active ingredient prepared in Example 1 and the control group were set up; 3T3 cells were cultured at 3×10 5 The cells were inoculated on a 24-well plate and cultured for 24 h, and 5 μL of the liposome solution was added, and a group of blank controls were set for each well plate. The cells were cultured for another 24 h, and then the cells were treated according to the CalceinAM / PI cell activity and cytotoxicity detection kit method. The cells were photographed and observed under a laser confocal microscope to obtain the fluorescence image of the 3T3 cells stained with Calcein-AM after culturing with the liposome containing the hair growth active ingredient prepared in Example 1 for 24 h as shown in the figure below. Figure 5 As shown;

[0126] According to the above method, after adding the above liposome solution, the fluorescence image of 3T3 cells stained with Calcein-AM after culturing with the liposome containing the hair growth active ingredient prepared in Example 1 for 48 hours was obtained as shown in the following figure: Figure 5 shown.

[0127] According to the above method, after adding the above liposome solution, the fluorescence images of the HaCat cells stained with Calcein-AM after culturing the liposome containing the hair growth active ingredient prepared in Example 1 for 24 hours and 48 hours were obtained. Figure 6 shown.

[0128] Depend on Figure 5and Figure 6 It can be seen that both 3T3 cells and HaCat cells were stained with green fluorescence by Calcein-AM. Compared with the control group, after adding the liposome solution prepared in Example 1, the cells proliferated significantly, indicating that the liposome containing the hair growth active ingredient prepared in Example 1 had no obvious toxicity to fibroblasts and epidermal cells.

[0129] 3. Transdermal Experiment

[0130] The skin of the pig ear was taken, and the subcutaneous fat and cartilage were scraped off. The pig skin was soaked in physiological saline for half an hour before transdermal operation. The receiving chamber was filled with 0.1M PBS solution, and a small rotor was added. The pig skin was clamped between the receiving chamber and the donor chamber to avoid bubbles. Then 2mL of the liposome solution containing the hair growth active ingredient prepared in Example 1 was added to the donor chamber, and the corresponding concentration of adenosine was used as a control group. The transdermal experiment was carried out at 37°C and 300rpm. At the specified time points of 1h, 2h, 4h, 6h, 8h, 12h, 24h, and 48h, 0.2mL of liquid was taken from the receiving chamber (and an equal volume of PBS solution was added). The absorbance value was measured in the same way as before, and the cumulative drug release amount and cumulative drug release rate were calculated. The specific test results are shown in Table 4, and the cumulative drug release amount of the liposome containing the hair growth active ingredient prepared in Example 1 during the transdermal experiment was obtained as shown in the figure below: Figure 7 As shown in FIG. 1 , and the cumulative drug release rate of the liposome containing the hair growth active ingredient prepared in Example 1 during the transdermal experiment changes over time as shown in FIG. Figure 8 As shown. Figure 7 and Figure 8 It can be seen that the liposome containing the hair growth active ingredient prepared in Example 1 has a very good transdermal effect.

[0131] Table 4 Data statistics during the transdermal experiment of liposomes containing hair growth active ingredients prepared in Example 1

[0132]

[0133]

[0134] According to the above method, the pig skin was stained with rhodamine B at 1h, 6h and 12h of the transdermal experiment, and the fluorescence images of the transdermal experiment of the liposome solution containing the hair growth active ingredient prepared in Example 1 on the pig skin at 1h, 6h and 12h were obtained as shown in FIG. Fig. 9 As shown, Rhodamine B is red fluorescence and pig skin tissue is blue fluorescence. Fig. 9 It can be seen that at 1 hour of the transdermal experiment, the liposomes containing the hair growth active ingredient prepared in Example 1 had passed through the epidermis of the pig skin and reached the dermis; at 6 hours of the transdermal experiment, the liposomes gradually diffused through the dermis; at 12 hours of the transdermal experiment, the pig skin had completely turned red, and the liposomes diffused completely to the subcutaneous tissue.

[0135] 4. Animal Experiments

[0136] (1) Mouse preparation: 30 male C57BL / 6 mice (6-8 weeks old, body weight 20-25 g) were raised in an SPF animal breeding room at a room temperature of 22-23°C and a humidity of about 50%, with normal drinking water and food. The experiment was conducted after three days of adaptive feeding. The C57BL / 6 mice were then randomly divided into five groups, namely A, B, C, D, E, and F, with 5 mice in each group.

[0137] (2) Solution preparation

[0138] Testosterone propionate solution: Take 250 mg of testosterone propionate powder, fully dissolve it in 5 mL of anhydrous ethanol, add it into soybean oil and shake it thoroughly. After mixing, it becomes a light yellow transparent oily liquid, and the concentration of testosterone propionate modeling agent is 5 mg / mL;

[0139] Active composition solution: 20 mL of aqueous solution with a mass concentration of 0.78% of active composition;

[0140] Liposome solution I: prepared according to the method of Example 1, 20 mL of liposome solution containing hair growth active ingredients with a mass concentration of active substances of 0.78%;

[0141] Liposome solution II: 20 mL of a liposome solution containing a hair growth active ingredient with a mass concentration of 0.3% prepared according to the method of Example 1;

[0142] (3) Animal drug treatment

[0143] The five groups of mice were fed or treated as follows:

[0144] Blank group A: conventional feeding, no intervention measures;

[0145] Control group B: back hair removal and injection of saline and application of saline;

[0146] Model Group

[0147] Group C: back hair removal and injection of testosterone propionate solution and application of normal saline;

[0148] Group D: back hair removal and injection of testosterone propionate solution and application of adenosine solution;

[0149] Group E: back hair removal and injection of testosterone propionate solution and application of liposome solution I;

[0150] Group F: back hair removal and injection of testosterone propionate solution and application of liposome solution II;

[0151] The specific steps are as follows:

[0152] The hair on the back of the mice was shaved, with an area of ​​about 2cm×3cm. The remaining hair was removed with a depilatory cream, and it was confirmed that the hair growth of the mice was in the resting phase (the skin appeared pink). 24 hours after depilation, 0.2mL of normal saline was injected into the depilated area on the back of mice in group B; 0.2mL of 5mg / mL testosterone propionate dilution was injected into the depilated area on the back of mice in groups C, D, E, and F once a day. 30 minutes after subcutaneous injection of testosterone propionate solution on the back, the drugs were applied to the corresponding parts of the back of the mice with a cotton swab: group B was applied with normal saline, and groups C, D, E, and F were applied with corresponding drugs. The drugs were administered once a day, with a dose of 0.4mL, for 28 consecutive days. During the experiment, the mice ate and drank normally.

[0153] (4) Observation of mouse status and hair loss after administration

[0154] General status observation: observe and take photos every day, record the skin color changes of mice, and record the skin conditions of the back observation area of ​​each group of mice, including the basic physiological characteristics of the skin and color changes;

[0155] Hair density: Use a dermatoscope to photograph the skin and hair follicles of a fixed area on the back of the test mice twice a week. When photographing, the dermatoscope lens is completely in contact with the scalp and kept vertical. Record the skin color changes and the hair length of the mice under the dermatoscope; (focus on observing whether the hair follicles have positive peritrichous signs and yellow dot signs)

[0156] Determination of testosterone and estradiol content: After 28 days of sampling, blood was collected from all test mice retro-orbitally, centrifuged at 4°C (13000r / min, 10min), and placed in a -80°C refrigerator for later use; the ELISA method was used to test the testosterone and estradiol content in mouse serum. The specific results are shown in Table 5.

[0157] HE staining: 4 weeks after administration, five groups of mice were killed by cervical dislocation, and the skin of the observation area of ​​about 2 cm × 2 cm of the mice was taken for routine tissue dehydration, paraffin embedding, sectioning, and HE staining (hematoxylin-eosin staining). The dermis thickness, number of hair follicles and morphological changes of each sample were observed under a microscope; three fields of view of the cross-section of the sample section were randomly selected for observation and the average hair follicle number density in the field of view was calculated.

[0158] The actual photos and dermoscopic images of the uninterrupted growth of mice in group A in the above animal experiment are as follows Fig.10 As shown, Fig.10 The left picture is a real picture, and the right picture is a dermoscopic picture. In the above animal experiment, the real pictures of the mice in groups B, C, D, E and F from the first day to the 28th day of administration are as follows Fig.11 In the above animal experiment, the dermoscopic images of the mice in groups B, C, D, E and F from the first to the fourth week of administration are shown in Fig.12As shown. Fig.10 , 11 Compared with 12, it can be seen that the encapsulated liposomes (liposomes containing hair growth active ingredients) can reduce the phenomenon of hair loss in mice and have the effect of reducing hair loss.

[0159] Table 5 Serum testosterone (T) and estradiol (E) of mice in groups C, D, E and F on day 28 of administration 2 ) Concentration statistics

[0160] Group T(ng / mL) <![CDATA[E 2 (ng / mL)]]> <![CDATA[T / E 2 ]]> C 0.794 0.376 2.111 D 0.89 0.521 1.71 E 0.851 0.507 1.678 F 0.814 0.479 1.697

[0161] The bar graph of serum testosterone (T) concentrations of mice in groups C, D, E, and F on day 28 of administration is shown in Figure 2. Fig.13 As shown, estradiol (E 2 ) concentration bar graph is as follows Fig.14 As shown. Fig.13 , Fig.14 As shown in Table 5, compared with the model group C, the T and E2 values ​​of groups D, E, and F increased, but T / E 2 The value is reduced, indicating that the liposome can reduce the phenomenon of hair loss in mice and has the effect of reducing hair loss.

[0162] The HE staining of group A, group B, group C, group D, group E and group F was shown in the following figure. Fig.15 As shown by Fig.15 It can be seen that the encapsulated liposomes prepared by the present invention (liposomes containing hair growth active ingredients) increase the thickness of the dermis and the number of hair follicles.

[0163] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing liposomes containing hair growth active ingredients, characterized in that: The following steps are involved: (1) Using lecithin, an oil-soluble active ingredient and a solvent as raw materials to prepare a film; (2) dissolving the active substance in a buffer solution to obtain a hydrated solution; The hydration liquid is added to the film obtained in step (1), and hydration and dispersion are performed in sequence to obtain liposomes containing hair growth active ingredients. The step (1) and the step (2) are both carried out under light-proof conditions.

2. The preparation method according to claim 1, characterized in that The oil-soluble active ingredients in step (1) include one or more of cholesterol, oil-soluble plant extracts, ceramides, oil-soluble vitamins and other active substances with hair growth effects; the solvent includes one or more of vegetable oils, ethanol, polyols, polyethers, polyglycerols and polyethylene glycol solvents.

3. The preparation method according to claim 1, characterized in that In the step (1), the mass ratio of lecithin to the oil-soluble active ingredient is (0.1-10): (0.01-10).

4. The preparation method according to claim 1, characterized in that The step (1) comprises: mixing lecithin, an oil-soluble active ingredient and a solvent, and then performing rotary evaporation and vacuum drying in sequence to obtain a film.

5. The preparation method according to claim 1, characterized in that The temperature of the rotary evaporation is 28-43° C., and the rotation speed of the rotary evaporation is 20-50 rpm.

6. The preparation method according to claim 1, characterized in that The active substance in step (2) includes one or more of adenosine, compound plant extracts, polypeptides, polyphenols, amino acids, minerals, proteins, vitamin compounds and other anti-hair loss and hair growth active compounds.

7. The preparation method according to claim 6, characterized in that The polypeptides are tripeptide-1, tripeptide-1 copper, tripeptide-34 copper, alanine / histidine / lysine polypeptide copper HCl, tripeptide-10 citrulline, palmitoyl tripeptide-38, biotin tripeptide-1, palmitoyl dipeptide-5 diaminobutyryl hydroxythreonine, tetrapeptide-3, tetrapeptide-4, acetyl tetrapeptide-3, acetyl tetrapeptide-11, myristoyl tetrapeptide-12, palmitoyl tetrapeptide-7, palmitoyl tetrapeptide-20, palmitoyl tetrapeptide-3, palmitoyl tetrapeptide-4, palmitoyl tetrapeptide-3, palmitoyl tetrapeptide-11, palmitoyl tetrapeptide-12, palmitoyl tetrapeptide-7, palmitoyl tetrapeptide-20, palmitoyl tetrapeptide-3 ... At least one of palmitoyl pentapeptide-4, myristoyl pentapeptide-4, myristoyl pentapeptide-17, hexapeptide-3, hexapeptide-10, hexapeptide-11, hexapeptide-12, myristoyl hexapeptide-16, palmitoyl hexapeptide-12, acetyl hexapeptide, acetyl octapeptide, palmitoyl octapeptide, myristoyl octapeptide, octapeptide-2, acetyl octapeptide-2, nonapeptide-1, decapeptide-4, acetyl decapeptide-3, oligopeptide-41, and oligopeptide-74; The protein substance is at least one of recombinant collagen, hydrolyzed collagen, silk protein, hydrolyzed keratin, recombinant fibronectin, recombinant elastin, recombinant mussel viscose, mussel viscose, and silk fibroin; The composite plant extract is at least one of saw palmetto extract, Platycladus orientalis leaf extract, Ligustrum lucidum fruit extract, Swertia vulgaris extract, Trifolium pratense extract, ginger root extract, Artemisia argyi extract, Geranium herb extract, Centella asiatica extract, Polygonum multiflorum extract, Sophora flavescens extract, Panax notoginseng extract, Angelica sinensis extract, and Miscanthus variegatus extract. The other anti-hair loss and hair growth active compounds are at least one of diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, gallic acid, caffeine and taxifolin.

8. The preparation method according to claim 1, characterized in that The hydration in step (2) includes: performing a first heat treatment at 35-45° C. until the film is completely wetted and falls off, and then performing a second heat treatment at 35-45° C. with stirring for 20-50 minutes.

9. The preparation method according to claim 1, characterized in that The dispersion method in step (2) is ultrasound, stirring, extrusion or homogenization.

10. Liposome containing hair growth active ingredient prepared by the preparation method according to any one of claims 1 to 9.

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