A pharmaceutical preparation for promoting hair growth and a preparation method thereof

Abstract

The present invention relates to the technical field of topical hair preparations, specifically to a pharmaceutical preparation for promoting hair growth and a method for preparing the same. The pharmaceutical preparation comprises, by weight, 0.1-0.5 parts of exosomes, 0.2-0.7 parts of collagen, and 2.5-5 parts of a natural plant extract. The pharmaceutical preparation can stimulate hair growth, improve hair quality, and thicken hair; it can also stimulate hair follicle regeneration, increasing the number of hair follicles.

Description

Technical Field

[0001] The present invention relates to the technical field of external hair pharmaceutical preparations, and in particular to a pharmaceutical preparation for promoting hair growth and a preparation method thereof. Background Art

[0002] Hair loss refers to the loss of hair. Normally lost hair is in the catagen and telogen phases. A constant balance between catagen and new growth maintains a normal hair count. Hair loss can have a significant impact on people's psychology and daily lives.

[0003] Exosomes secreted by cells contain a variety of growth factors, such as stem cell factor (SCF), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and keratinocyte growth factor (KGF). After entering the target cells, exosomes can interact with the cells and mediate intercellular signal transduction. These factors can not only repair damaged hair follicles and activate the growth of hair follicle stem cells, but also promote hair growth and improve hair quality.

[0004] There are increasing reports on the use of plant extracts, including those from traditional Chinese medicine, to treat hair loss. These plant-derived ingredients are relatively safe, effective, and easy to use. Traditional Chinese herbal extracts offer the advantages of easy access, simple production processes, and low cost. They also have good compatibility with many cosmetic ingredients. These plant extracts often promote hair growth, improve hair quality, stimulate hair follicle regeneration, enhance hair follicle vitality, and have antioxidant, anti-inflammatory, and antibacterial properties.

[0005] Collagen has a strong affinity for protein molecules on the hair surface, binding to the hair surface through physical adsorption. Collagen is resistant to rinsing, and its affinity increases with increasing molecular weight. Low-molecular-weight collagen can penetrate the hair cortex to nourish the hair. Supplementing hair with collagen can promote smoothness and shine. Collagen is an essential component for maintaining healthy skin and hair, helping to enhance hair's elasticity and toughness. Collagen also promotes blood circulation and improves nutrient supply to the scalp, thereby reducing hair loss. Furthermore, collagen can stimulate the growth of hair follicles, promoting hair growth.

[0006] However, in existing studies, there are few reports on the combined use of exosomes with collagen and natural plant extracts in topical hair preparations; therefore, providing a method for combining exosomes with collagen and natural plant extracts for the preparation of hair growth preparations with excellent effects is one of the technical problems that need to be solved. Summary of the Invention

[0007] The purpose of the present invention is to prepare a hair external-use pharmaceutical preparation, more specifically a pharmaceutical preparation for promoting hair growth, which can promote the generation of hair follicle cells and stimulate hair follicles to enter the growth phase, thereby significantly improving the problem of hair loss; helping hair loss patients to use a topical excipient treatment method, combining prevention and treatment, reducing the pain of patients during the treatment process, and shortening the treatment time.

[0008] On the one hand, the pharmaceutical preparation for promoting hair growth of the present invention involves three raw materials: the first raw material is exosomes produced by cell culture; the second component is collagen; and the third component is natural plant extracts.

[0009] Furthermore, the pharmaceutical preparation comprises the following raw materials in parts by mass: 0.1-0.5 parts of exosomes, 0.2-0.7 parts of collagen and 2.5-5 parts of natural plant extracts.

[0010] Furthermore, the natural plant extracts include extracts of Polygonum multiflorum, ginger, Platycladus orientalis leaves and mulberries.

[0011] Furthermore, the exosomes are obtained by umbilical cord crushing, primary culture, subculture and separation.

[0012] Furthermore, the method for preparing exosomes comprises the following steps:

[0013] Step (1), fragmentation: clean the umbilical cord, peel off the amniotic membrane and arteries and veins, remove Wharton's jelly, and cut into pieces;

[0014] Step (2), primary culture: placing the obtained fragments in a vessel containing culture medium for culture;

[0015] Step (3), subculture: When the cell confluence in step (2) reaches 68-85%, discard the culture medium and rinse the cell growth surface; add a trypsin aqueous solution to obtain a mixture, then add the culture medium to form a single cell suspension, centrifuge, discard the supernatant, add the culture medium to resuspend the cells, and subculture;

[0016] Step (4), separation: collecting the culture supernatant when the cells are passaged to the third generation, centrifuging to remove cell debris; filtering the supernatant, collecting the filtrate, and freeze-drying to obtain the exosomes.

[0017] Furthermore, the collagen is type I collagen. The collagen can be selected as type I collagen α1 (COL1a1) recombinant protein, Shanghai Boersen Biotechnology Co., Ltd.

[0018] Furthermore, the mass ratio of the exosomes to collagen is 1:1-1.5.

[0019] Furthermore, the preparation method of the natural plant extract comprises the following steps:

[0020] Step 1: Weigh dried Polygonum multiflorum, ginger, Platycladus orientalis leaves, and mulberries, crush them separately, and mix them in the following proportions: 1-3 parts of Polygonum multiflorum, 0.5-2.5 parts of ginger, 1-2.5 parts of Platycladus orientalis leaves, and 1.2-3.5 parts of mulberries to obtain a mixed powder;

[0021] Step 2: Add water to the mixed powder of step 1 and boil it to obtain a boiled material;

[0022] Step 3: Cooling the decoction, adding ethanol to obtain a mixed solution, adjusting the ethanol concentration in the mixed solution to 45-65 wt%, heating and stirring, cooling, filtering, and concentrating to obtain a concentrate;

[0023] Step 4: adding the concentrate to an extractant consisting of an organic phase and an aqueous phase for extraction, then pouring out the aqueous phase, concentrating the extracted organic phase, and removing the organic solvent to obtain a concentrate, which is the natural plant extract.

[0024] Furthermore, in step 1, the particle size selected after crushing is 0.05-1 mm.

[0025] Furthermore, in step 2, the mass ratio of water to mixed powder is 10-28:1.

[0026] Furthermore, in step 2, the decocting temperature is 75-90°C.

[0027] Furthermore, in step 2, the decocting time is 5-10 hours.

[0028] Furthermore, in step 2, the decoction is performed by adding water to the volume before decoction every 30 minutes.

[0029] Furthermore, in step 3, the decoction is cooled to 15-30°C.

[0030] Furthermore, in step 3, the temperature of heating and stirring is 45-55°C.

[0031] Furthermore, in step 3, the heating and stirring speed is 60-500 rpm.

[0032] Furthermore, in step 3, the heating and stirring time is 10-30 hours.

[0033] Furthermore, in step 3, filtration is performed using a filter membrane with a pore size of 0.1-2 microns.

[0034] Furthermore, in step 3, the concentration is concentrated under reduced pressure.

[0035] Furthermore, in step 3, the concentration temperature is 55-60°C

[0036] Furthermore, in step 3, the mixture is concentrated to 0.3-0.8 times the mass of the powder.

[0037] Furthermore, in step 4, the mass ratio of the extractant to the concentrate is 6-20:1.

[0038] Furthermore, in step 4, the volume ratio of the organic phase to the aqueous phase in the extractant is 2.5-4:1.

[0039] Furthermore, in step 4, the organic phase is composed of organic solvents ethyl acetate and cyclohexane in a volume ratio of 4-6:1.

[0040] Furthermore, in step 4, the aqueous phase is a 0.2-0.55 wt% calcium chloride aqueous solution.

[0041] Furthermore, in step 4, the extraction temperature is 35-45°C.

[0042] Furthermore, in step 4, the extraction time is 4-7 hours.

[0043] Furthermore, in step 4, the concentration is concentrated under reduced pressure.

[0044] Furthermore, in step 4, the concentration temperature is 46-52°C.

[0045] Furthermore, in step 4, when no organic solvent component of the organic phase is detected in the concentrate, concentration is stopped to obtain a concentrate, which is the natural plant extract.

[0046] Furthermore, the preparation method of the natural plant extract comprises the following steps:

[0047] Step 1: Weigh dried Polygonum multiflorum, ginger, Platycladus orientalis leaves, and mulberries, crush them separately, and mix them in the following proportions: 1-3 parts of Polygonum multiflorum, 0.5-2.5 parts of ginger, 1-2.5 parts of Platycladus orientalis leaves, and 1.2-3.5 parts of mulberries to obtain a mixed powder;

[0048] Step 2: Add 10-28 times the mass of water to the mixed powder of step 1, and boil at 75-90° C. for 5-10 hours to obtain a boiled material;

[0049] Step 3: Cool the decoction, add ethanol to obtain a mixed solution, adjust the ethanol concentration in the mixed solution to 45-65 wt%, heat and stir for 10-30 hours, cool, filter, and concentrate under reduced pressure at 55-60° C. to 0.3-0.8 times the weight of the mixture powder to obtain a concentrate;

[0050] Step 4: adding the concentrate to an extractant composed of an organic phase and an aqueous phase in a volume ratio of 2.5-4:1 (6-20 times the mass), extracting at 35-45° C. for 4-7 hours, then decanting the aqueous phase, and concentrating the extracted organic phase under reduced pressure at 46-52° C. to remove the organic solvent. When no organic solvent component of the organic phase is detected in the concentrate, concentrating is stopped to obtain a concentrate, which is the natural plant extract;

[0051] The organic phase is composed of organic solvents ethyl acetate and cyclohexane in a volume ratio of 4-6:1, and the aqueous phase is a 0.2-0.55 wt% calcium chloride aqueous solution.

[0052] On the other hand, the present invention provides a method for preparing the pharmaceutical preparation, comprising the step of mixing the raw materials.

[0053] On the other hand, the present invention also provides a method of preparing the pharmaceutical preparation into a dressing external-use pharmaceutical product.

[0054] On the other hand, the present invention also provides a method of preparing the pharmaceutical preparation into a gel-like external-use pharmaceutical product, wherein the gel-like external-use pharmaceutical product is composed of: 4-10% of the above-mentioned pharmaceutical preparation, 0.4-1% carbomer 940, 2-7% glycerol, 1.5-5% butylene glycol, 0.02-0.1% EDTA-2Na, 0.4-0.8% triethanolamine and the remainder water in terms of mass percentage.

[0055] Beneficial effects:

[0056] The pharmaceutical preparation prepared by combining exosomes with collagen and natural plant extracts can effectively stimulate hair growth, improve hair quality, and make hair thicker; it can also promote the recovery of hair follicles, regenerate hair follicles, and increase the number of hair follicles.

[0057] After the pharmaceutical preparation of the present invention is diluted to 10 wt%, the hair length of the mouse test area reaches 6.14±0.67 mm, the number of hair follicles reaches 46.2±4.9, and the hair diameter reaches 12.04±1.34 μm after 20 days.

[0058] The use of exosomes and collagen in the pharmaceutical preparation of the present invention can improve hair growth and enhance hair follicle vitality. The exosomes and collagen used in the pharmaceutical preparation of the present invention can mutually promote the increase in hair growth rate, improve hair quality, and enhance hair follicle vitality. When the mass ratio of exosomes to collagen in the pharmaceutical preparation of the present invention is 1:1-1.5, the resulting preparation has better effects.

[0059] The present invention uses natural plant extracts to further enhance the hair growth effect of the preparation. When extracting the plants, Polygonum multiflorum, ginger, Platycladus orientalis leaves and mulberries are decocted in water, and then ethanol is added to adjust the ethanol concentration to 45-65wt%. The mixture is heated and stirred for extraction, concentrated, and then extracted using an extractant obtained by combining an oil phase consisting of ethyl acetate and cyclohexane with a water phase consisting of a calcium chloride aqueous solution. The extract obtained by the extraction method of the present invention can significantly enhance the effects of the preparation on promoting hair growth, improving hair quality and activating hair follicle vitality.

[0060] The present invention uses 45wt%-65wt% of the plant extract obtained by extraction, which can make the preparation have better hair growth effect.

[0061] The present invention uses a combination of ethyl acetate and cyclohexane to extract the active ingredients from plants, synergistically and efficiently dissolving and extracting the hair-growth active ingredients, resulting in a more effective hair-growth preparation. Furthermore, when the ratio of ethyl acetate to cyclohexane in the organic phase is 4-6:1, the preparation exhibits even greater effectiveness. However, when other organic phases are used to extract the plant extract, replacing the organic phase of the present invention, the resulting preparations exhibit inferior hair-growth effectiveness.

[0062] The hair growth promoting pharmaceutical preparation of the present invention can promote the generation of hair follicle cells and stimulate hair follicles to enter the growth phase, thereby significantly improving the problem of hair loss; it helps patients with hair loss by combining prevention and treatment with a smear excipient treatment method, thereby alleviating the pain of patients during the treatment process and shortening the treatment time. DETAILED DESCRIPTION

[0063] The present invention will be described below in conjunction with specific embodiments, and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments are for illustrating the present invention, rather than for limiting the present invention.

[0064] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0065] 1. Preparation of exosomes: Step (1) Fragmentation: The umbilical cord of a healthy full-term caesarean section infant was washed with normal saline, the amniotic membrane and arteries and veins of the umbilical cord were peeled off, Wharton's jelly was removed, and the umbilical cord was cut into pieces of approximately 1 mm in size;

[0066] Step (2), primary culture: the obtained fragments were placed in a vessel containing DMEM culture medium and cultured at 37°C and 5% CO2;

[0067] Step (3), subculture: When the cell confluence of the primary culture in step (2) reaches 70%, the DMEM culture medium is discarded, PBS buffer is added to rinse the cell growth surface, and the washing solution is discarded; then a 0.25wt% trypsin aqueous solution is added, and the trypsin is shaken so that the bottom of the vessel is evenly covered with the trypsin to obtain a mixture, 10 times the volume of the mixture is added to the DMEM culture medium, and the mixture is repeatedly pipetted to form a single cell suspension, centrifuged at 1500rpm for 8 minutes, the supernatant is discarded, and the cells are resuspended in DMEM culture medium at a density of 1.5×10 5 cels were subcultured at 37°C and 5% CO2;

[0068] Step (4), separation: collecting the culture supernatant when the cells were cultured to the third generation, centrifuging at 4°C and 2000g for 35 min to remove cell debris; filtering the supernatant after centrifugation with a 0.22 μm filter, collecting the filtrate, and freeze-drying to obtain the exosomes.

[0069] 2. Preparation of natural plant extracts

[0070] 1. Extract 1: Prepare as follows:

[0071] Step 1: Weigh dried Polygonum multiflorum, ginger, Platycladus orientalis leaves, and mulberries, crush them separately, and select a particle size between 0.05 and 1 mm after crushing. Mix 2.5 parts of Polygonum multiflorum, 1.8 parts of ginger, 2.2 parts of Platycladus orientalis leaves, and 2.5 parts of mulberries in parts by mass to obtain a mixed powder;

[0072] Step 2: Add 18 times the mass of water to the mixed powder obtained in step 1, and boil at 80°C for 6 hours, adding water every 30 minutes to the volume before boiling to obtain a boiled material;

[0073] Step 3: The decoction was cooled to 28° C., ethanol was added to obtain a mixed solution, the ethanol concentration in the mixed solution was adjusted to 52.5 wt %, the mixture was heated to 52° C., stirred at 100 rpm for 12 hours, cooled to 28° C., filtered through a 0.5 μm pore size filter membrane, and concentrated under reduced pressure at 58° C. to 0.45 times the weight of the mixture powder to obtain a concentrate;

[0074] Step 4: adding the concentrate to an extractant composed of an organic phase and an aqueous phase in a volume ratio of 3.5:1 (12 times the mass), extracting at 40° C. for 5.2 hours, then decanting the aqueous phase, and concentrating the extracted organic phase under reduced pressure at 50° C. to remove the organic solvent. When no organic solvent component of the organic phase is detected in the concentrate, concentrating is stopped to obtain a concentrate, which is the natural plant extract;

[0075] The organic phase is composed of organic solvents ethyl acetate and cyclohexane in a volume ratio of 5:1, and the aqueous phase is a 0.38 wt% calcium chloride aqueous solution.

[0076] Extract 2: Prepare as follows:

[0077] Step 1: Weigh dried Polygonum multiflorum, ginger, Platycladus orientalis leaves, and mulberries, crush them separately, and select a particle size between 0.05 and 1 mm after crushing. Mix 1.8 parts of Polygonum multiflorum, 1.2 parts of ginger, 1.7 parts of Platycladus orientalis leaves, and 3.5 parts of mulberries in parts by mass to obtain a mixed powder;

[0078] Step 2: Add 20 times the mass of water to the mixed powder obtained in step 1, and boil at 82°C for 5.5 hours, adding water every 30 minutes to the volume before boiling to obtain a boiled material;

[0079] Step 3: The decoction was cooled to 25° C., ethanol was added to obtain a mixed solution, the ethanol concentration in the mixed solution was adjusted to 45 wt %, the mixture was heated to 50° C., stirred at 80 rpm for 15 hours, cooled to 26° C., filtered through a 0.22 μm pore size filter membrane, and concentrated under reduced pressure at 58° C. to 0.4 times the weight of the mixture powder to obtain a concentrate;

[0080] Step 4: adding the concentrate to an extractant composed of an organic phase and an aqueous phase in a volume ratio of 3:1 (15 times the mass), extracting at 42° C. for 5.5 hours, then decanting the aqueous phase, and concentrating the extracted organic phase under reduced pressure at 50° C. to remove the organic solvent. When no organic solvent component of the organic phase is detected in the concentrate, concentrating is stopped to obtain a concentrate, which is the natural plant extract;

[0081] The organic phase is composed of organic solvents ethyl acetate and cyclohexane in a volume ratio of 4:1, and the aqueous phase is a 0.25 wt% calcium chloride aqueous solution.

[0082] Extract 3: Prepare as follows:

[0083] Step 1: Weigh dried Polygonum multiflorum, ginger, Platycladus orientalis leaves, and mulberries, crush them separately, and select a particle size between 0.05 and 1 mm after crushing. Mix 2.5 parts of Polygonum multiflorum, 1.8 parts of ginger, 2.2 parts of Platycladus orientalis leaves, and 2.5 parts of mulberries in parts by mass to obtain a mixed powder;

[0084] Step 2: Add 18 times the mass of water to the mixed powder obtained in step 1, and boil at 80°C for 6 hours, adding water every 30 minutes to the volume before boiling to obtain a boiled material;

[0085] Step 3: The decoction was cooled to 28° C., ethanol was added to obtain a mixed solution, the ethanol concentration in the mixed solution was adjusted to 20 wt %, the mixture was heated to 52° C., stirred at 100 rpm for 12 hours, cooled to 28° C., filtered through a 0.5 μm pore size filter membrane, and concentrated under reduced pressure at 58° C. to 0.45 times the weight of the mixture powder to obtain a concentrate;

[0086] Step 4: adding the concentrate to an extractant composed of an organic phase and an aqueous phase in a volume ratio of 3.5:1 (12 times the mass), extracting at 40° C. for 5.2 hours, then decanting the aqueous phase, and concentrating the extracted organic phase under reduced pressure at 50° C. to remove the organic solvent. When no organic solvent component of the organic phase is detected in the concentrate, concentrating is stopped to obtain a concentrate, which is the natural plant extract;

[0087] The organic phase is composed of organic solvents ethyl acetate and cyclohexane in a volume ratio of 5:1, and the aqueous phase is a 0.38 wt% calcium chloride aqueous solution.

[0088] Extract 4: Prepare as follows:

[0089] Step 1: Weigh dried Polygonum multiflorum, ginger, Platycladus orientalis leaves, and mulberries, crush them separately, and select a particle size between 0.05 and 1 mm after crushing. Mix 2.5 parts of Polygonum multiflorum, 1.8 parts of ginger, 2.2 parts of Platycladus orientalis leaves, and 2.5 parts of mulberries in parts by mass to obtain a mixed powder;

[0090] Step 2: Add 18 times the mass of water to the mixed powder obtained in step 1, and boil at 80°C for 6 hours, adding water every 30 minutes to the volume before boiling to obtain a boiled material;

[0091] Step 3: The decoction was cooled to 28° C., ethanol was added to obtain a mixed solution, the ethanol concentration in the mixed solution was adjusted to 52.5 wt %, the mixture was heated to 52° C., stirred at 100 rpm for 12 hours, cooled to 28° C., filtered through a 0.5 μm pore size filter membrane, and concentrated under reduced pressure at 58° C. to 0.45 times the weight of the mixture powder to obtain a concentrate;

[0092] Step 4: adding the concentrate to an extractant composed of an organic phase and an aqueous phase in a volume ratio of 3.5:1 (12 times the mass), extracting at 40° C. for 5.2 hours, then decanting the aqueous phase, and concentrating the extracted organic phase under reduced pressure at 50° C. to remove the organic solvent. When no organic solvent component of the organic phase is detected in the concentrate, concentrating is stopped to obtain a concentrate, which is the natural plant extract;

[0093] The organic phase is an organic solvent ethyl acetate, and the aqueous phase is a 0.38 wt% calcium chloride aqueous solution.

[0094] Extract 5: Prepare as follows:

[0095] Step 1: Weigh dried Polygonum multiflorum, ginger, Platycladus orientalis leaves, and mulberries, crush them separately, and select a particle size between 0.05 and 1 mm after crushing. Mix 2.5 parts of Polygonum multiflorum, 1.8 parts of ginger, 2.2 parts of Platycladus orientalis leaves, and 2.5 parts of mulberries in parts by mass to obtain a mixed powder;

[0096] Step 2: Add 18 times the mass of water to the mixed powder obtained in step 1, and boil at 80°C for 6 hours, adding water every 30 minutes to the volume before boiling to obtain a boiled material;

[0097] Step 3: The decoction was cooled to 28° C., ethanol was added to obtain a mixed solution, the ethanol concentration in the mixed solution was adjusted to 52.5 wt %, the mixture was heated to 52° C., stirred at 100 rpm for 12 hours, cooled to 28° C., filtered through a 0.5 μm pore size filter membrane, and concentrated under reduced pressure at 58° C. to 0.45 times the weight of the mixture powder to obtain a concentrate;

[0098] Step 4: adding the concentrate to an extractant composed of an organic phase and an aqueous phase in a volume ratio of 3.5:1 (12 times the mass), extracting at 40° C. for 5.2 hours, then decanting the aqueous phase, and concentrating the extracted organic phase under reduced pressure at 50° C. to remove the organic solvent. When no organic solvent component of the organic phase is detected in the concentrate, concentrating is stopped to obtain a concentrate, which is the natural plant extract;

[0099] The organic phase is an organic solvent cyclohexane, and the aqueous phase is a 0.38 wt% calcium chloride aqueous solution.

[0100] Extract 6: Prepare as follows:

[0101] Step 1: Weigh dried Polygonum multiflorum, ginger, Platycladus orientalis leaves, and mulberries, crush them separately, and select a particle size between 0.05 and 1 mm after crushing. Mix 2.5 parts of Polygonum multiflorum, 1.8 parts of ginger, 2.2 parts of Platycladus orientalis leaves, and 2.5 parts of mulberries in parts by mass to obtain a mixed powder;

[0102] Step 2: Add 18 times the mass of water to the mixed powder obtained in step 1, and boil at 80°C for 6 hours, adding water every 30 minutes to the volume before boiling to obtain a boiled material;

[0103] Step 3: The decoction was cooled to 28° C., ethanol was added to obtain a mixed solution, the ethanol concentration in the mixed solution was adjusted to 52.5 wt %, the mixture was heated to 52° C., stirred at 100 rpm for 12 hours, cooled to 28° C., filtered through a 0.5 μm pore size filter membrane, and concentrated under reduced pressure at 58° C. to 0.45 times the weight of the mixture powder to obtain a concentrate;

[0104] Step 4: adding the concentrate to an extractant composed of an organic phase and an aqueous phase in a volume ratio of 3.5:1 (12 times the mass), extracting at 40° C. for 5.2 hours, then decanting the aqueous phase, and concentrating the extracted organic phase under reduced pressure at 50° C. to remove the organic solvent. When no organic solvent component of the organic phase is detected in the concentrate, concentrating is stopped to obtain a concentrate, which is the natural plant extract;

[0105] The organic phase is composed of organic solvents ethyl acetate and cyclohexane in a volume ratio of 2:1, and the aqueous phase is a 0.38 wt% calcium chloride aqueous solution.

[0106] Extract 7: Prepare as follows:

[0107] Step 1: Weigh dried Polygonum multiflorum, ginger, Platycladus orientalis leaves, and mulberries, crush them separately, and select a particle size between 0.05 and 1 mm after crushing. Mix 2.5 parts of Polygonum multiflorum, 1.8 parts of ginger, 2.2 parts of Platycladus orientalis leaves, and 2.5 parts of mulberries in parts by mass to obtain a mixed powder;

[0108] Step 2: Add 18 times the mass of water to the mixed powder obtained in step 1, and boil at 80°C for 6 hours, adding water every 30 minutes to the volume before boiling to obtain a boiled material;

[0109] Step 3: The decoction was cooled to 28° C., ethanol was added to obtain a mixed solution, the ethanol concentration in the mixed solution was adjusted to 52.5 wt %, the mixture was heated to 52° C., stirred at 100 rpm for 12 hours, cooled to 28° C., filtered through a 0.5 μm pore size filter membrane, and concentrated under reduced pressure at 58° C. to 0.45 times the weight of the mixture powder to obtain a concentrate;

[0110] Step 4: adding the concentrate to an extractant composed of an organic phase and an aqueous phase in a volume ratio of 3.5:1 (12 times the mass), extracting at 40° C. for 5.2 hours, then decanting the aqueous phase, and concentrating the extracted organic phase under reduced pressure at 50° C. to remove the organic solvent. When no organic solvent component of the organic phase is detected in the concentrate, concentrating is stopped to obtain a concentrate, which is the natural plant extract;

[0111] The organic phase is composed of organic solvents ethyl acetate and cyclohexane in a volume ratio of 8:1, and the aqueous phase is a 0.38 wt% calcium chloride aqueous solution.

[0112] Extract 8: Prepare as follows:

[0113] Step 1: Weigh dried Polygonum multiflorum, ginger, Platycladus orientalis leaves, and mulberries, crush them separately, and select a particle size between 0.05 and 1 mm after crushing. Mix 2.5 parts of Polygonum multiflorum, 1.8 parts of ginger, 2.2 parts of Platycladus orientalis leaves, and 2.5 parts of mulberries in parts by mass to obtain a mixed powder;

[0114] Step 2: Add 18 times the mass of water to the mixed powder obtained in step 1, and boil at 80°C for 6 hours, adding water every 30 minutes to the volume before boiling to obtain a boiled material;

[0115] Step 3: The decoction was cooled to 28° C., ethanol was added to obtain a mixed solution, the ethanol concentration in the mixed solution was adjusted to 52.5 wt %, the mixture was heated to 52° C., stirred at 100 rpm for 12 hours, cooled to 28° C., filtered through a 0.5 μm pore size filter membrane, and concentrated under reduced pressure at 58° C. to 0.45 times the weight of the mixture powder to obtain a concentrate;

[0116] Step 4: adding the concentrate to an extractant composed of an organic phase and an aqueous phase in a volume ratio of 3.5:1 (12 times the mass), extracting at 40° C. for 5.2 hours, then decanting the aqueous phase, and concentrating the extracted organic phase under reduced pressure at 50° C. to remove the organic solvent. When no organic solvent component of the organic phase is detected in the concentrate, concentrating is stopped to obtain a concentrate, which is the natural plant extract;

[0117] The organic phase is an organic solvent chloroform, and the aqueous phase is a 0.38 wt% calcium chloride aqueous solution.

[0118] 3. Pharmaceutical preparations that promote hair growth

[0119] The compositions of each preparation are shown in Table 1 below, with raw materials measured by mass. Exosomes were prepared as described above; collagen was recombinant type I collagen α1 (COL1 a1), Catalog No. BES20423RP, available from Shanghai Boersen Biotechnology Co., Ltd.; and natural plant extracts were extracts 1-8 described above. The mass ratio of exosomes to collagen is indicated as the K value.

[0120] Table 1: Raw material composition of each preparation

[0121] Serial number / copy Preparation 1 Preparation 2 Preparation 3 Preparation 4 Preparation 5 Preparation 6 exosomes 0.45 0.40 0 1.07 0.22 0.75 collagen 0.62 0.50 1.07 0 0.85 0.32 Extract 1 3.43 3.15 3.43 3.43 3.43 3.43 K value 1:1.38 1:1.25 / / 1:3.86 1:0.43

[0122] Among them, the extract used in preparations 1-6 is extract 1; then extracts 2-8 are used to replace extract 1 in preparation 1, and the resulting preparations are named preparations 7-13. Then the above preparations 1-13 are subjected to subsequent performance tests.

[0123] 4. Performance Testing

[0124] The above preparation was diluted with an ethylene glycol aqueous solution to a dilution solution with a mass concentration of 10%, and then a performance test was performed; wherein the mass concentration of the ethylene glycol aqueous solution was 25%.

[0125] SPF-grade C57BL / 6J male mice, weighing approximately 23-25 ​​grams, were randomly divided into the following groups: blank control, model control, positive control, and formulations 1-13, with 8 mice per group. Mice in each group were anesthetized with isoflurane, and the same 3 cm × 3 cm test area was removed. A 12 mg / mL dihydrotestosterone solution in corn oil was prepared. Mice in all groups, except the blank control, were intraperitoneally injected with 0.1 mL of this solution for 20 consecutive days to establish a seborrheic alopecia model. The positive control group was then treated with 0.5 mg of a 6% minoxidil solution in ethylene glycol (30% ethylene glycol) applied to the depilated area once daily in the morning and evening. Mice in formulations 1-13 were also treated with 0.5 mg of the same solution applied to the depilated area once daily in the morning and evening. The blank and model control groups were similarly treated with an equal volume of normal saline. After 20 days, hair growth in the test area of ​​each group of mice was observed and statistically analyzed to determine the average length and diameter of newly grown hair. The mice were then sacrificed, and skin tissue from the test area was collected for HE staining to calculate the average number of hair follicles. Specific results for the average length, diameter, and number of newly grown hair follicles are shown in Table 2.

[0126] Table 2: Average length, diameter and number of hair follicles

[0127]

[0128]

[0129] Based on the results of these tests, the increases in average hair length, diameter, and number of hair follicles in each preparation group and positive control group compared to the model control group were calculated. The results are shown in Table 3. Here, average increase in new hair length (mm) = average length of new hair in the preparation group or positive control group minus average length of new hair in the model control group; average increase in number of new hair follicles (units) = average number of new hair follicles in the preparation group or positive control group minus average number of new hair follicles in the model control group; and average increase in new hair diameter (μm) = average diameter of new hair in the preparation group or positive control group minus average diameter of new hair in the model control group.

[0130] Table 3: Improvement in the preparation group compared to the model control group

[0131]

[0132]

[0133] The test results in Tables 2-3 show that the preparation prepared by combining exosomes with collagen and natural plant extracts can effectively stimulate hair growth, improve hair quality, and make hair thicker; it can also promote the recovery of hair follicles, regenerate hair follicles, and increase the number of hair follicles.

[0134] The test results of the preparations in Tables 2-3 show that after the pharmaceutical preparation of the present invention is diluted to 10 wt%, the hair length of the mouse test area reaches 6.14±0.67 mm, the number of hair follicles reaches 46.2±4.9, and the hair diameter reaches 12.04±1.34 μm after 20 days.

[0135] The use of exosomes and collagen in the pharmaceutical preparations of the present invention can enhance hair growth and boost hair follicle vitality. The test results for Formulations 1, 3, and 4 in Tables 2-3 demonstrate that the exosomes and collagen used in the pharmaceutical preparations of the present invention can mutually promote increased hair growth, improved hair quality, and enhanced hair follicle vitality. Comparison of Formulations 5-6 with Formulations 1, 2, and 7 demonstrates that a mass ratio of exosomes to collagen in the pharmaceutical preparations of the present invention of 1:1-1.5 yields even better results.

[0136] The present invention uses natural plant extracts to further enhance the hair growth effect of the preparation. When extracting the plants, Polygonum multiflorum, ginger, Platycladus orientalis leaves and mulberries are decocted in water, and then ethanol is added to adjust the ethanol concentration to 45-65wt%. The mixture is heated and stirred for extraction, concentrated, and then extracted using an extractant obtained by combining an oil phase consisting of ethyl acetate and cyclohexane with an aqueous phase consisting of a calcium chloride aqueous solution. The extract obtained by the extraction method of the present invention can significantly enhance the effect of the preparation in promoting hair growth, improving hair quality and activating hair follicle vitality, which can be specifically determined by comparing preparations 1, 2, 7 and 8-13.

[0137] Comparison between preparations 1 and 8 shows that the present invention uses 45 wt% to 65 wt% of the plant extract to achieve a better hair growth effect.

[0138] The test results for Preparations 1, 9, and 13 indicate that the present invention's use of a combination of ethyl acetate and cyclohexane for extracting plant active ingredients synergistically and efficiently dissolves and extracts the hair-growth active ingredients, resulting in a more effective hair-growth effect. Furthermore, when the ratio of ethyl acetate to cyclohexane in the organic phase is 4-6:1, the effect is even greater. However, when other organic phases are used to extract plant extracts instead of the organic phase of the present invention, the resulting preparations exhibit inferior hair-growth effects.

[0139] 5. Application

[0140] Hair growth gel, mass percentage: 7.5% of the above preparation 1, 0.54% carbomer 940, 3% glycerol, 2% 1,3-butylene glycol, 0.05% EDTA-2Na, 0.54% triethanolamine and the balance water.

[0141] Preparation method: Carbomer 940 was mixed with water, stirred at 60 rpm for 0.3 hour, the above-mentioned preparation 1, glycerol, 1,3-butylene glycol, EDTA-2Na and triethanolamine were added, and stirring was continued for 0.5 hour to obtain the hair growth gel.

[0142] Volunteers with hair loss were selected to use the hair growth gel prepared from the pharmaceutical preparation 1 of the present invention. The method of use was to apply it to the hair loss area and then spread it evenly. The dosage was 1 g / time, once in the morning and once in the evening every day, for 3 consecutive months.

[0143] The hair growth promoting pharmaceutical preparation of the present invention can promote the generation of hair follicle cells and stimulate hair follicles to enter the growth phase, thereby significantly improving the problem of hair loss; it helps patients with hair loss by combining prevention and treatment with a smear excipient treatment method, thereby alleviating the pain of patients during the treatment process and shortening the treatment time.

[0144] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A pharmaceutical preparation for promoting hair growth, characterized in that: The pharmaceutical preparation is measured in parts by mass, and the raw materials are: 0.1-0.5 parts of exosomes, 0.2-0.62 parts of collagen and 2.5-5 parts of natural plant extracts; The preparation method of the natural plant extract comprises: Step 1: Weigh dried Polygonum multiflorum, ginger, Platycladus orientalis leaves, and mulberries, crush them separately, and mix them in the following proportions: 1-3 parts of Polygonum multiflorum, 0.5-2.5 parts of ginger, 1-2.5 parts of Platycladus orientalis leaves, and 1.2-3.5 parts of mulberries to obtain a mixed powder; Step 2: Add water to the mixed powder of step 1 and boil it to obtain a boiled material; Step 3: Cooling the decoction, adding ethanol to obtain a mixed solution, adjusting the ethanol concentration in the mixed solution to 45-65 wt%, heating and stirring, cooling, filtering, and concentrating to obtain a concentrate; Step 4: adding the concentrate to an extractant consisting of an organic phase and an aqueous phase for extraction, then pouring out the aqueous phase, concentrating the extracted organic phase, and removing the organic solvent to obtain a concentrate, which is the natural plant extract; The organic phase is composed of organic solvents ethyl acetate and cyclohexane in a volume ratio of 4-6:1, and the aqueous phase is a 0.2-0.55wt% calcium chloride aqueous solution; The exosomes are obtained by umbilical cord crushing, primary culture, subculture and separation; The collagen is type I collagen; The mass ratio of the exosomes to collagen is 1:1-1.5; In step 4, the mass ratio of the extractant to the concentrate is 6-20:

1.

2. The pharmaceutical preparation according to claim 1, characterized in that In step 2, the decocting time is 5-10 hours.

3. The pharmaceutical preparation according to claim 1, characterized in that In step 3, the heating and stirring time is 10-30 hours.

4. A method for preparing the pharmaceutical preparation according to any one of claims 1 to 3, characterized in that: The process includes mixing the raw materials.

Images