Anti-hair loss spray containing leech extract

Abstract

The invention relates to the technical field of daily chemical products, and discloses a leech extract anti-hair loss spray which comprises the following raw material components in percentage by weight: 1.00%-5.00% of leech extract, 84.00%-90.00% of deionized water, 3.00%-4.00% of butanediol, 0.00%-0.00% of 1, 3-butanediol, 0.00%-0.00% of 1, 3-butanediol, 0.00%-0.00% of 1, 3-butanediol, 0.00%-0.00% The traditional Chinese medicine composition is prepared from the following components in percentage by weight: 6.00%-8.00% of 1, 2-pentanediol, 0.50%-0.80% of pantothenic acid, 0.40%-0.60% of lactobacillus, 1.00%-2.00% of propylene glycol, 0.20%-0.40% of PEG-40, 0.20%-0.60% of hydrogenated castor oil, 0.50%-0.80% of a radix notoginseng extract and 0.02%-0.06% of cedar wood oil. The active ingredients of the poecilobdella manillensis extract can well promote the growth of vascular epidermis factors, improve the scalp environment, prevent hair follicle blockage, promote cell proliferation of hair follicle bulbs and more remarkably promote activation of hair follicle tissues, so that the effects of preventing hair loss and fixing hair are achieved, and meanwhile, the use is safe, mild and non-irritating.

Description

Technical Field

[0001] The invention belongs to the technical field of daily chemical products, and particularly relates to a leech extract anti-hair loss spray. Background Art

[0002] Common hair loss is mainly caused by the hair follicles being in the growth phase and the resting phase, but the hair follicles in each phase are in a dynamic equilibrium state. The hair follicles of patients with pathological hair loss shrink and cannot enter the growth phase, resulting in gradually thinning hair. In the early stage of hair loss, consumers first think of hair care products and pay more attention to ingredients. Chinese herbal ingredients such as ginger and Polygonum multiflorum are more popular. Although these ingredients have certain effects, they are slow to take effect. At present, most of the drugs and cosmetics used to promote hair growth on the market are chemical synthetics, such as the FDA-approved topical drug minoxidil for the treatment of hair loss. Minoxidil is an over-the-counter drug (OTC) and is also the only OTC drug professionally used to treat hair loss. However, it has many side effects, such as local skin redness, swelling, burning pain, peeling, and crazy hair loss. Therefore, the market needs an anti-hair loss spray that is easy to use, high concentration, high efficiency, safe, and effective.

[0003] PoecilobdellamanillensisLesson. is a traditional herbal medicine for promoting blood circulation and removing blood stasis. Modern research shows that leeches contain hirudin, amino acids, fatty acids, trace elements and other ingredients, and have pharmacological effects such as anticoagulation, inhibition of platelet aggregation, antithrombosis, lowering blood lipids, and anti-tumor. Hirudin is the most active and most studied ingredient among the many active ingredients extracted from leeches and their salivary glands. It is a small molecule protein (peptide) composed of 65-66 amino acids. Leech extract has a certain promoting effect on hair growth.

[0004] Hirudin is a natural product polypeptide extracted from natural leeches. It has low toxicity and few toxic side effects. The product is administered by smearing, which is more acceptable to patients. Since hirudin is a polypeptide, it is soluble in water but easily inactivated and deteriorated in water, and leech powder has poor solubility in water, and has the disadvantages of being unstable in water, easily inactivated, easily deteriorated, and having a fishy smell at room temperature. Leech extracts are used in many fields of cosmetics, such as whitening, removing spots, reducing dark circles, etc., but are rarely used to prevent hair loss and promote hair growth. Leech extracts are currently mostly used to solve patients' poor coagulation function and promote wound healing. There are no reports on preventing hair loss and promoting hair growth. Therefore, a stable and effective leech extract anti-hair loss spray is proposed, which can overcome the shortcomings of hirudin and make the product easy to use while maintaining activity. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art and solve the shortcomings of the current leech powder, such as poor solubility in water, instability in water, easy inactivation, easy deterioration, and fishy smell at room temperature, the present invention provides a leech extract anti-hair loss spray.

[0006] To achieve the above object, the present invention provides the following technical solutions: A leech extract anti-hair loss spray comprises the following raw material components calculated by weight percentage: Leech extract 1.00%-5.00%, deionized water 84.00%-90.00%, butylene glycol 3.00%-4.00%, 1,2-pentanediol 6.00%-8.00%, pantothenic acid 0.50%-0.80%, lactobacillus 0.40%-0.60%, propylene glycol 1.00%-2.00%, PEG-40 0.20%-0.40%, hydrogenated castor oil 0.20%-0.60%, Panax notoginseng extract 0.50%-0.80%, cedar wood oil 0.02%-0.06%.

[0007] Preferably, the leech extract anti-hair loss spray comprises the following raw material components calculated by weight percentage: Leech extract 5.00%, deionized water 84.20%, butylene glycol 3.50%, 1,2-pentanediol 6.90%, pantothenic acid 0.75%, lactobacillus 0.50%, propylene glycol 1.20%, PEG-40 0.30%, hydrogenated castor oil 0.50%, Panax notoginseng extract 0.60%, cedar wood oil 0.05%.

[0008] Preferably, the leech extract anti-hair loss spray comprises the following raw material components calculated by weight percentage: Leech extract 1.00%, deionized water 88.20%, butylene glycol 3.50%, 1,2-pentanediol 6.90%, pantothenic acid 0.75%, lactobacillus 0.50%, propylene glycol 1.20%, PEG-40 0.30%, hydrogenated castor oil 0.50%, Panax notoginseng extract 0.60%, cedar wood oil 0.05%.

[0009] A method for preparing a leech extract anti-hair loss spray comprises the following steps: S1. Add deionized water, butylene glycol, pantothenic acid, and lactobacillus to a mixing drum and stir evenly, then add 1,2-pentanediol and stir to dissolve completely, then add leech extract and stir until completely dissolved to prepare a pre-soak; S2. Add propylene glycol, PEG-40, hydrogenated castor oil, Panax notoginseng extract to another mixing drum and stir evenly, and then add cedar wood oil and stir evenly to form a formula liquid; S3. The pre-soak prepared in step S1 and the formula solution prepared in step S2 are mixed and stirred to prepare a leech extract anti-shedding spray solution; S4. Sterilize the leech extract anti-shedding spray solution prepared in step S3; S5. The sterilized leech extract anti-hair loss spray solution is dispensed into spray bottles under vacuum and aseptic conditions to prepare a leech extract anti-hair loss spray.

[0010] Preferably, in step S1 and step S2, the stirring speed of the raw materials is 250 rpm, and the stirring time is 20 min.

[0011] Preferably, the preparation of the leech extract comprises the following steps: (1) grinding and crushing the Philippine leech, and using a 12-mesh sieve to obtain a powder; (2) The insoluble macromolecular substances are removed from the powder by filtering, and the macromolecular components with a molecular weight between 1000Da and 10000Da are obtained by using a semipermeable membrane; the 1000Da-10000Da here refers to the range of molecular weight, which means that the components retained after filtration are macromolecular components with a molecular weight between 1000 and 10000 Daltons.

[0012] Dalton is the unit of molecular weight, 1Da is equivalent to the mass of a hydrogen atom. The active ingredients in leech extract are within this range. This method can be used to enrich and extract the required active ingredients.

[0013] (3) Sequentially purifying and separating the macromolecular components by ion exchange resin, gel chromatography, and fermentation and enzymatic digestion to obtain an active leech extract solution; (4) The active leech extract solution is filtered through an ultrafiltration tube to obtain macromolecular components with a molecular weight between 1000 Da and 5000 Da, and the active leech extract solution is freeze-dried to obtain a light yellow freeze-dried powder, which is sterilized by irradiation and then sealed and dried at room temperature for future use.

[0014] Compared with the prior art, the technical effects and advantages of the present invention are: The leech extract anti-hair loss spray is mainly composed of a solution formed by mixing leech extract with deionized water, butylene glycol, 1,2-pentanediol, pantothenic acid, lactobacillus, propylene glycol, PEG-40, hydrogenated castor oil, Panax notoginseng extract, and cedar wood oil. The active ingredients of the leech extract have a good effect of promoting the growth of vascular epidermal factors, improving the scalp environment, preventing hair follicle blockage, promoting the proliferation of hair follicle bulb cells, and more significantly promoting the activation of hair follicle tissue, thereby achieving the effect of preventing hair loss and fixing hair. At the same time, it is safe, gentle and non-irritating to use.

[0015] The anti-hair loss spray prepared from leech extract is a product with the potential to prevent hair loss, consolidate and grow hair for head health care. Its active ingredients are different from existing hair growth chemicals. The leech extract used in the present invention is an extract from natural insects and involves the field of natural small molecule compounds. It is not only safe and effective, but also easy to industrialize and produce, and can subsequently achieve better economic and social benefits.

[0016] In traditional technology, leech powder has poor solubility in water, is unstable, and the effective ingredients are easily inactivated, and the penetration and absorption after acting on the skin surface are poor. In the preparation of leech extract anti-hair loss spray, active polypeptides with a molecular weight of 1000-5000Da are extracted from leeches through fermentation and low-temperature extraction technology, and then freeze-dried, sterilized and purified. This overcomes the shortcomings of traditional leech extracts that are unstable in water, easy to inactivate, easy to deteriorate, and have a fishy smell at room temperature. The processed leech extract can retain the high activity of the effective ingredients and has better stability. When applied to the anti-hair loss spray, it can be quickly absorbed after use, thereby promoting the growth of vascular epidermal factors in the head, reducing oil secretion, and strengthening hair roots, thereby achieving the effect of preventing hair loss and solidifying hair.

[0017] The leech extract anti-hair loss spray prepared by the invention is easy to use and can be sprayed on the scalp and massaged until absorbed. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effect of different concentrations of leech extracts on the proliferation of primary human dermal papilla cells; Figure 2 The effect of different concentrations of leech extracts on the migration of primary human dermal papilla cells in the present invention; Figure 3 The migration rate of human primary dermal papilla cells to different concentrations of leech extracts in the present invention; Figure 4 The effect of different concentrations of leech extracts on VEGF secretion in primary human dermal papilla cells; Figure 5 The hair growth of mice in Examples 1 and 2 and the control group of the present invention; Figure 6 is the length of new hair of mice in Examples 1 and 2 and the control group of the present invention; Figure 7 This is a longitudinal section of the skin at the administration site of mice in Examples 1 and 2 and the control group of the present invention (X100); Figure 8 It is a schematic diagram of the human safety evaluation results of Examples 1 and 2 and the control group in the present invention. DETAILED DESCRIPTION

[0019] To make the present invention easier to understand, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Obviously, the examples described are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by professional and technical personnel in the field; unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the present invention can be purchased on the market or can be prepared by existing methods.

[0020] The following is combined with Figure 1-8 To further explain this application in detail, A leech extract anti-hair loss spray comprises the following raw material components calculated by weight percentage: Leech extract 1.00%-5.00%, deionized water 84.00%-90.00%, butylene glycol 3.00%-4.00%, 1,2-pentanediol 6.00%-8.00%, pantothenic acid 0.50%-0.80%, lactobacillus 0.40%-0.60%, propylene glycol 1.00%-2.00%, PEG-40 0.20%-0.40%, hydrogenated castor oil 0.20%-0.60%, Panax notoginseng extract 0.50%-0.80%, cedar wood oil 0.02%-0.06%.

[0021] A method for preparing a leech extract anti-hair loss spray comprises the following steps: S1. Add deionized water, butylene glycol, pantothenic acid, and lactobacillus to a mixing drum and stir evenly, then add 1,2-pentanediol and stir to dissolve completely, then add leech extract and stir until completely dissolved to prepare a pre-soak; S2. Add propylene glycol, PEG-40, hydrogenated castor oil, Panax notoginseng extract to another mixing drum and stir evenly, and then add cedar wood oil and stir evenly to form a formula liquid; S3. The pre-soak prepared in step S1 and the formula solution prepared in step S2 are mixed and stirred to prepare a leech extract anti-shedding spray solution; S4. Sterilize the leech extract anti-shedding spray solution prepared in step S3; S5. The sterilized leech extract anti-hair loss spray solution is dispensed into spray bottles under vacuum and aseptic conditions to prepare a leech extract anti-hair loss spray.

[0022] Specifically, in step S1 and step S2, the stirring speed of the raw materials is 250 rpm, and the stirring time is 20 min.

[0023] Specifically, the preparation of the leech extract comprises the following steps: (1) grinding and crushing the Philippine leech, and using a 12-mesh sieve to obtain a powder; (2) filtering the powder to remove insoluble macromolecular substances, and using a semipermeable membrane to obtain macromolecular components with a molecular weight between 1000Da and 10000Da; (3) Sequentially purifying and separating the macromolecular components by ion exchange resin, gel chromatography, and fermentation and enzymatic digestion to obtain an active leech extract solution; (4) The active leech extract solution is filtered through an ultrafiltration tube to obtain macromolecular components with a molecular weight between 1000 Da and 5000 Da, and the active leech extract solution is freeze-dried to obtain a light yellow freeze-dried powder, which is sterilized by irradiation and then sealed and dried at room temperature for future use. Example

[0024] A leech extract anti-hair loss spray comprises the following raw material components calculated by weight percentage: Leech extract 5.00%, deionized water 84.20%, butylene glycol 3.50%, 1,2-pentanediol 6.90%, pantothenic acid 0.75%, lactobacillus 0.50%, propylene glycol 1.20%, PEG-40 0.30%, hydrogenated castor oil 0.50%, Panax notoginseng extract 0.60%, cedarwood oil 0.05%. The leech extract anti-hair loss spray prepared from the above raw materials is used as Test Example 1; Example

[0025] A leech extract anti-hair loss spray comprises the following raw material components calculated by weight percentage: Leech extract 1.00%, deionized water 88.20%, butylene glycol 3.50%, 1,2-pentanediol 6.90%, pantothenic acid 0.75%, lactobacillus 0.50%, propylene glycol 1.20%, PEG-40 0.30%, hydrogenated castor oil 0.50%, Panax notoginseng extract 0.60%, cedarwood oil 0.05%. The leech extract anti-hair loss spray prepared from the above raw materials is used as test example 2; Design control group NC, the anti-hair loss spray in the control group has the following raw material components calculated by weight percentage: Deionized water 89.20%, butylene glycol 3.50%, 1,2-pentanediol 6.90%, pantothenic acid 0.75%, lactobacillus 0.50%, propylene glycol 1.20%, PEG-40 0.30%, hydrogenated castor oil 0.50%, Panax notoginseng extract 0.60%, cedar wood oil 0.05%.

[0026] In order to further illustrate the application or effect of the present invention, the following specific cases are used to demonstrate the effectiveness and practicality of the present invention.

[0027] (a) The leech extract was tested to determine whether it could promote the proliferation of human primary dermal papilla cells (HDPCs).

[0028] Digest the logarithmic phase cells with trypsin, pipette and blow until the cells are suspended, count the cells, and after the cell suspension is prepared, gently mix and add 100μL of cell suspension to each well, that is, the number of cells implanted is 5000 / well. After 24 hours of cell attachment, add drugs (leech extract with a mass concentration of 62.5-2000μg / mL and positive control: minoxidil (Minoxidil is a drug commonly used to treat hair loss) 10μg / mL) for 48 hours. After the drug action time is over, remove 10μL of the supernatant, immediately add 10μL of MTT solution (MTT is the abbreviation of ureate (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium), MTT solution is used as a chemical reagent in cell viability and cell proliferation experiments) and place it in the incubator for further culture for 4 hours. After the living cells reacted fully with the MTT solution, the supernatant was discarded, 150 μL DMSO (DMSO is the abbreviation of dimethylsulfoxide) was added to each well, and the cells were shaken at a low speed for 10 minutes on a shaker to fully dissolve the crystals. The absorbance of each well was measured at OD (optical density) 570 nm.

[0029] like Figure 1 As shown, human primary dermal papilla cells were incubated with leech extracts at mass concentrations of 62.5-2000 μg / mL. At 2000 μg / mL, cell proliferation increased by 17.8%, which was more obvious than the positive control group.

[0030] Figure 1 In the figure, NC is the control group without adding hirudin, which is used to compare the effects of different concentrations of hirudin on the proliferation of primary human dermal papilla cells.

[0031] Leech extracts at concentrations of 2000μg / mL, 1000μg / mL and 500μg / mL can promote the proliferation of human primary dermal papilla cells, as shown by a significantly higher cell proliferation rate than that of the blank control group. Leech extracts have the activity of promoting the proliferation of human primary dermal papilla cells in a dose-dependent manner.

[0032] The study provides scientific evidence that leech extract can promote the proliferation of human dermal papilla cells at the cellular level. This is conducive to further studying its potential application in the field of hair growth, determining the effective concentration range of leech extract in promoting the proliferation of human dermal papilla cells, providing a reference for subsequent product development, laying the foundation for further clarifying the mechanism of action of leech extract in the mechanism of hair growth, providing support for the development of new hair care products, and providing experimental data support for evaluating the potential of leech extract in the field of hair loss treatment, which will help promote its transformation in clinical applications.

[0033] (b) To detect whether leech extract can promote the migration of human primary dermal papilla cells (HDPCs).

[0034] First, use a marker pen to evenly draw horizontal lines on the back of the 6-well plate with a ruler, about every 0.5-1cm, and cross the holes horizontally. At least 5 lines are crossed in each hole; use cells in the logarithmic growth phase, trypsinize to make a single cell suspension, count the cells with a hemocytometer, and inoculate 5×105 cells / well in a 6-well culture plate to ensure that the cells can grow full the next day. The final total volume of culture medium in each well is 2mL, and the cells are cultured in a 37℃, 5% CO2 incubator for 24h; after the cells are full, use the tip of the gun to compare with the ruler, and try to scratch perpendicular to the horizontal line on the back. The tip of the gun should be vertical and not tilted (it is best to use the same tip between different wells); after the line is drawn, wash the cells 3 times with PBS (PBS is the abbreviation of Phosphate Buffered Saline), remove the scratched cells, and add serum-free culture medium; take samples and take photos at 0, 6, 12, 24h, and 48h.

[0035] like Figure 2 Figure 3 As shown, 2000 μg / mL of leech extract and the positive control minoxidil both have the ability to promote cell migration without damaging the cell structure.

[0036] Leech extract has the effect of promoting the migration of HDPCs cells. This is because the migration ability of HDPCs cells may be significantly enhanced in the culture medium containing leech extract. Compared with the control group cells without leech extract, leech extract contains some active ingredients that can stimulate HDPCs cells to produce more chemokines and adhesion molecules, thereby promoting cell migration and motility. This effect of promoting HDPCs migration is related to the potential application value of leech extract in processes such as hair growth and skin regeneration. Further research on this will help reveal the possible mechanism of action of leech extract in the treatment of hair and skin diseases.

[0037] (c) The relative expression of intracellular growth factor VEGFA was measured after human primary dermal papilla cells were treated with leech extract.

[0038] HFDPCs (Human Follicle Dermal Papilla Cells) cells were inoculated in 6-well plates at a density of 5×105 cells / well, 1.8 mL per well, and cultured for 24 hours. The culture medium was discarded, and the leech extract stock solution was diluted according to the concentration gradient. The drug concentration gradient was designed to be 2000μg / mL, 1000μg / mL and 500μg / mL, and the positive drug was minoxidil at a concentration of 10μM. 1.8mL of culture medium was added to each well of the blank control group, and 1.8mL of different drug samples were added to each well of the positive control group and the experimental group for 24 hours. Cell RNA was extracted according to the kit, and the extracted RNA was prepared according to the instructions of the HiScriptIIQRTSuperMixforqPCR kit to prepare a system with a total volume of 20μL reaction. The sample was vortexed to mix, centrifuged briefly, and reacted in a PCR instrument at 42°C for 2 minutes to remove gDNA; 1 μg of total RNA was reverse transcribed into cDNA under the conditions of 37°C for 15 minutes and 85°C for 5 seconds, and finally the reverse transcription product was used for fluorescent quantitative PCR (polymerase chain reaction) reaction.

[0039] like Figure 4 As shown in the figure, the VEGF (Vascular Endothelial Growth Factor) in human primary dermal papilla cells incubated with 2000μg / mL, 1000μg / mL and 500μg / mL leech extracts were all higher than those in the blank control group. Leech extracts promote the proliferation of human primary dermal papilla cells and the secretion of VEGF, and have a certain potential to promote hair growth.

[0040] Leech extract may significantly promote the relative expression of VEGFA in human primary dermal papilla cells. This means that leech extract will stimulate HDPCs cells to produce more VEGFA and increase the expression level inside the cells. VEGFA is an important angiogenic factor that plays an important role in angiogenesis, tissue repair and regeneration. Therefore, leech extract promotes the expression of VEGFA in HDPCs cells, which helps to improve angiogenesis and tissue repair ability. This effect of promoting the expression of VEGFA in HDPCs cells may be related to some active ingredients in leech extract. These active ingredients enhance the synthesis and release of intracellular VEGFA by regulating cell signal transduction pathways, thereby affecting cell proliferation, migration and angiogenesis.

[0041] (d) Animal experiments Thirty-two healthy C57BL / 6 mice with normal hair were routinely fed for 1 week to adapt to the environment. The hair in the 2×3cm2 area on the back of the mouse was carefully shaved with a shaver, and a cotton swab was dipped in 10% depilatory cream and applied to the shaved area. After 2 minutes, the hair was wiped with warm water for depilation. After 2 days of routine feeding, mice with smooth and undamaged depilatory areas were selected as successful modeling models. The mice with successful modeling were randomly divided into 4 groups (n=8 / group), namely solvent group, minoxidil positive control group and sample group. 200μL / mouse / day was administered percutaneously for three consecutive weeks, and the hair growth in the depilatory area of ​​the mice was recorded during the experiment.

[0042] The growth of hair on the back of mice Figure 5 As shown, Figure 5 Experimental Example 1 is Example 1, and Experimental Example 2 is Example 2. The hair growth period of C57BL / 6 mice is accompanied by the synthesis of skin melanin. The production of melanin in the skin is closely related to the hair cycle. The skin changes from dark gray to black during hair growth, and it is light pink during the resting period. Before administration, the skin of the mouse depilatory site was light pink. On the fourth day of administration, the positive control group and the administration site of Examples 1 and 2 began to turn gray and black. On the seventh day, the backs of the mice in Experimental Examples 1, 2 and the positive control group began to grow fine short hairs. On the 14th day of administration, the backs of the mice showed a central diffuse growth of black and shiny new hair. On the 21st day, the hair of the mice in the positive control group and Experimental Examples 1 and 2 groups had basically grown, while the solvent control group was not full.

[0043] On the 21st day of administration, 9 newly grown hairs were randomly plucked from the administration area on the back of the mice, and the average length of the hairs was measured. Figure 6 As shown, Figure 6 Experimental Example 1 is Example 1, and Experimental Example 2 is Example 2. The hair length of the solvent control group was (9.0267±0.4278) mm; the hair length of the positive control group was (10.2811±0.1091) mm; the hair length of the Example 1 group was (10.4778±0.0454) mm; and the hair length of the Example 1 group was (10.6733±0.0865) mm. Compared with the solvent control group, the new hair length of the mice in the positive control group and Experimental Examples 1 and 2 groups was longer and significantly different (P<0.0001).

[0044] The skin tissue of the dorsal area of ​​the mice was sliced ​​and stained with H&E. Figure 7 The pictures show that the hair follicles of the newly grown hair in the positive control group and Example 1 and Example 2 groups extend to the deep subcutaneous tissue and have formed a complete hair follicle structure, while the hair follicles in the solvent control group reach the junction of the dermis and epidermis. In addition, we observed that the hair bulbs were obviously dyed black, indicating that the hair follicles were in the growth phase.

[0045] As described above, the use of leech extracts (Examples 1 and 2) can promote hair growth in the hair loss area on the back of mice, while no obvious hair growth was observed in the solvent control group. After the fourth day of administration, the backs of mice in the positive control group and Examples 1 and 2 began to appear gray-black, and short fine hair began to grow on the seventh day. On the 14th day of administration, new black and shiny hair with a central diffusion type appeared on the back of the mice. The measurement results of the length of new hair in the administration area on the back of the mice showed that the hair length of the positive control group and Examples 1 and 2 was significantly longer than that of the solvent control group, and there was a significant difference. The histological section and staining results showed that the hair follicles of the positive control group and Examples 1 and 2 extended to the deep subcutaneous tissue to form a complete hair follicle structure, while the hair follicles of the solvent control group only reached the junction of the dermis and the epidermis, indicating that the leech extract can promote the development and growth of hair follicles.

[0046] (e) Human safety evaluation test In order to facilitate the interpretation of the results, the test site is selected as the normal skin of the arm, the patch protective paper is removed, and after the samples are dripped, the patch is firmly and flatly attached from bottom to top and gently pressed a few times with the palm of the hand to expel the air. 48 hours after application, the patch is first removed. To avoid the possible reaction caused by the patch pressing the skin, the results should be observed at least 30 minutes after the patch is removed. Negative reaction means no change in the skin. The results are determined according to the recommendations of ICDRG (international contact dermatitis research group).

[0047] like Figure 8 As shown, Figure 8 Experimental Example 1 is Example 1, and Experimental Example 2 is Example 2. All experimental groups and blank control groups are negative, and there is no change in the skin. It can be seen that the anti-hair loss spray containing leech extract has a certain safety and will not cause allergic reactions on normal skin. According to the standards of ICDRG, a negative reaction means that there is no adverse reaction or irritation on the skin. Therefore, it can be inferred that leech extract has good safety in the human body as a component of anti-hair loss spray, and can be used in daily skin care and anti-hair loss products.

[0048] The leech extract in the leech extract anti-hair loss spray contains a series of ingredients, including deionized water, butylene glycol, 1,2-pentanediol, pantothenic acid, lactobacillus, propylene glycol, PEG-40, hydrogenated castor oil, Panax notoginseng extract, cedarwood oil, etc. These ingredients work together to promote hair growth and prevent hair loss.

[0049] The active ingredients in leech extract nourish hair follicles and stimulate hair growth. These ingredients enhance hair growth by promoting blood circulation in hair follicles, providing nutrients and promoting cell metabolism. Experimental results show that leech extract can promote the proliferation and migration of primary human dermal hair papilla cells. This shows that leech extract increases hair density and growth rate by activating the proliferation and migration ability of cells. Leech extract will prompt dormant hair follicles to enter the growth phase, thereby increasing the growth cycle and length of hair. The active ingredients in leech extract have antioxidant and anti-inflammatory effects, which help protect hair follicles from damage and improve the health of hair.

[0050] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A leech extract anti-hair loss spray, characterized in that: The following raw material components are included in weight percentage: Leech extract 1.00%-5.00%, deionized water 84.00%-90.00%, butylene glycol 3.00%-4.00%, 1,2-pentanediol 6.00%-8.00%, pantothenic acid 0.50%-0.80%, lactobacillus 0.40%-0.60%, propylene glycol 1.00%-2.00%, PEG-40 0.20%-0.40%, hydrogenated castor oil 0.20%-0.60%, Panax notoginseng extract 0.50%-0.80%, cedar wood oil 0.02%-0.06%.

2. The leech extract anti-hair loss spray according to claim 1, characterized in that: The following raw material components are included in weight percentage: Leech extract 5.00%, deionized water 84.20%, butylene glycol 3.50%, 1,2-pentanediol 6.90%, pantothenic acid 0.75%, lactobacillus 0.50%, propylene glycol 1.20%, PEG-40 0.30%, hydrogenated castor oil 0.50%, Panax notoginseng extract 0.60%, cedar wood oil 0.05%.

3. The leech extract anti-hair loss spray according to claim 1, characterized in that: The following raw material components are included in weight percentage: Leech extract 1.00%, deionized water 88.20%, butylene glycol 3.50%, 1,2-pentanediol 6.90%, pantothenic acid 0.75%, lactobacillus 0.50%, propylene glycol 1.20%, PEG-40 0.30%, hydrogenated castor oil 0.50%, Panax notoginseng extract 0.60%, cedar wood oil 0.05%.

4. A method for preparing the leech extract anti-hair loss spray according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Add deionized water, butylene glycol, pantothenic acid, and lactobacillus to a mixing drum and stir evenly, then add 1,2-pentanediol and stir to dissolve completely, then add leech extract and stir until completely dissolved to prepare a pre-soak; S2. Add propylene glycol, PEG-40, hydrogenated castor oil, Panax notoginseng extract to another mixing drum and stir evenly, and then add cedar wood oil and stir evenly to form a formula liquid; S3. The pre-soak prepared in step S1 and the formula solution prepared in step S2 are mixed and stirred to prepare a leech extract anti-shedding spray solution; S4. Sterilize the leech extract anti-shedding spray solution prepared in step S3; S5. The sterilized leech extract anti-hair loss spray solution is dispensed into spray bottles under vacuum and aseptic conditions to prepare a leech extract anti-hair loss spray.

5. The method for preparing a leech extract anti-hair loss spray according to claim 4, characterized in that: In the step S1 and step S2, the stirring speed of the raw materials is 250 rpm, and the stirring time is 20 min.

6. The method for preparing a leech extract anti-hair loss spray according to claim 4, characterized in that: The preparation of the leech extract comprises the following steps: (1) grinding and crushing the Philippine leech, and using a 12-mesh sieve to obtain a powder; (2) filtering the powder to remove insoluble macromolecular substances, and using a semipermeable membrane to obtain macromolecular components with a molecular weight between 1000Da and 10000Da; (3) Sequentially purifying and separating the macromolecular components by ion exchange resin, gel chromatography, and fermentation and enzymatic digestion to obtain an active leech extract solution; (4) The active leech extract solution is filtered through an ultrafiltration tube to obtain macromolecular components with a molecular weight between 1000 Da and 5000 Da, and the active leech extract solution is freeze-dried to obtain a light yellow freeze-dried powder, which is sterilized by irradiation and then sealed and dried at room temperature for future use.

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