Snakehead mucus polypeptide compound for promoting hair growth and preparation method of snakehead mucus polypeptide compound
The snakehead fish mucus polypeptide complex, through enzymatic hydrolysis and nano-encapsulation, addresses stability and absorption issues of existing hair loss treatments, achieving superior hair growth promotion and safety.
Patent Information
- Application Number
- CN202411377348.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2024-12-17
AI Technical Summary
Current hair loss treatments, such as minoxidil, suffer from stability issues and poor transdermal absorption of peptides, leading to limited effectiveness and potential side effects.
A snakehead fish mucus polypeptide complex is prepared by enzymatic hydrolysis and nano-encapsulation, using lipid components to enhance stability and absorption, with dopamine coating for adhesion and eumelanin-like structure for better skin penetration.
The snakehead fish mucus polypeptide complex demonstrates improved stability, enhanced skin penetration, and significant promotion of hair growth, outperforming minoxidil in hair growth promotion and safety.
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Figure CN119139446A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of drugs for promoting hair growth, and in particular to a snakehead fish mucus polypeptide complex for promoting hair growth and a preparation method thereof. Background Art
[0002] Hair growth involves the growth and differentiation of hair follicles, including dermal papilla cells and epithelial cells. The life cycle of hair follicles is divided into three stages: growth phase, degeneration and resting phase. Under normal circumstances, hair follicles shrink after the growth phase, and the hair shaft falls off during the resting phase. The life span of hair in the early growth phase is about 2-6 years, after which it enters the resting phase and falls off, and grows about 0.3mm per day.
[0003] Hair loss or alopecia is a disease in which hair is lost. Hair loss during alopecia is not limited to the head and can occur anywhere on the body. Although alopecia itself is not life-threatening, it can cause severe psychological stress related to appearance. Currently, research on hair growth therapy focuses on direct local treatment. With the increasing prominence of social pressure, environmental pollution and other issues, the incidence of alopecia is increasing year by year and is showing a trend of younger people.
[0004] Currently, minoxidil is a drug commonly used in clinical practice to treat hair loss. Topical application of 2% minoxidil can make most male and female patients with androgenetic alopecia grow visible hair / fine hair, but side effects such as scalp inflammation and increased facial hair may occur after administration.
[0005] In recent years, active peptides have been used to solve hair loss and promote hair regeneration, and have received attention from dermatologists and beauty and hairdressing experts in various countries. However, adding peptide ingredients directly to prescriptions not only has poor stability, but the peptides cannot be smoothly absorbed through the skin barrier, limiting the effectiveness of the peptides.
[0006] Snakehead mucus is a type of active substance rich in mucopolysaccharides, glycoproteins, immunoglobulins, enzymes, etc., secreted by mucus cells located in the skin epithelium of snakehead fish. The mucus secreted by snakehead fish widely covers the surface of the fish body and constitutes the first gateway for the fish body to directly contact the outside world. As the first barrier for aquatic animals to contact the outside world, it can also protect the fish body from invasion by pathogens, parasites and viruses, etc. It is the first line of defense for the fish itself to resist foreign invasion and the immune system.
[0007] If peptides are extracted from black fish mucus and used in hair growth preparations, a product with good stability, easy transdermal absorption and small feed amount can be prepared, which has excellent application prospects. Summary of the invention
[0008] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a snakehead fish mucus polypeptide complex for promoting hair growth and a preparation method thereof.
[0009] A snakehead mucus polypeptide complex for promoting hair growth comprises raw materials by mass: 1-4 parts of pretreated mucus polypeptide, 1-2 parts of lecithin, 1-2 parts of cholesterol, 20-40 parts of squalane, 0.01-0.024 parts of dopamine hydrochloride, 1-2 parts of polyethylene glycol, and 0.1-1 parts of emulsifier; the pretreated mucus polypeptide is prepared by the following steps: mucus is taken from the surface of a fresh snakehead fish body, purified by centrifugal microfiltration, ultrafiltration and freeze drying, water is added and mixed evenly, the pH value of the system is adjusted to 9.5-10.0, a composite protease preparation is added, enzymolysis is carried out at 40-55°C for 4-8 hours, centrifugation is performed, the supernatant is taken, and vacuum freeze drying is performed.
[0010] Preferably, the mucus taken from the surface of the fresh snakehead fish is evenly mixed with water in a volume ratio of 1:1-4.
[0011] Preferably, the composite protease preparation comprises: papain, neutral protease, alkaline protease.
[0012] More preferably, the enzymatic activity of papain is 4-6×10 4 U / g, the activity of neutral protease is 8-9×10 4 U / g, the activity of alkaline protease is 6-8×10 4 U / g.
[0013] Preferably, the emulsifier is sorbitan fatty acid ester and / or polyoxyethylene sorbitan fatty acid ester.
[0014] The method for preparing the black fish mucus polypeptide complex for promoting hair growth comprises the following steps:
[0015] S1. Add the pretreated mucus polypeptide into deionized water and stir evenly to obtain preform a;
[0016] S2, adding lecithin and cholesterol to squalane and stirring evenly, adding preform a dropwise thereto under stirring, and continuing stirring for 1-2 hours after the addition is complete to obtain preform b;
[0017] S3, adding dopamine hydrochloride aqueous solution, polyethylene glycol, and emulsifier into deionized water and stirring evenly to obtain preform c;
[0018] S4. Add preform b dropwise to preform c under stirring and stir for 10-20 minutes. High pressure homogenization for 10-30 seconds. Add sodium hydroxide solution and stir at 40-50°C for 1-2 hours. Cool to room temperature, centrifuge and retain by ultrafiltration tube.
[0019] Preferably, in S3, the concentration of the dopamine hydrochloride aqueous solution is 1-1.2 mg / mL.
[0020] Preferably, in S4, the pressure of high-pressure homogenization is 6-10 MPa.
[0021] Preferably, in S4, the concentration of the sodium hydroxide solution is 0.5-1.2 mol / L.
[0022] Preferably, in S4, the molecular weight of the ultrafiltration tube is 5 kDa and 10 kDa.
[0023] Beneficial effects:
[0024] The invention extracts proteins and polypeptides from black fish mucus and hydrolyzes them by enzymes, dissolving the mucus polypeptides in the inner phase of the multiple emulsion, and uses high-pressure homogenization to reduce the particle size to form nanoparticles, which are then coated on the surface of the nanoparticles by self-oxidation of dopamine under alkaline conditions. The nanoparticles not only have excellent adhesion and increase the retention time, but also are easier to be absorbed by the scalp due to their similar structure to eumelanin, thereby greatly promoting the absorption of the polypeptides.
[0025] The invention is simple to prepare, can not only realize the nano-encapsulation of black fish mucus polypeptide, but also has good stability, excellent skin penetration and skin retention, and can significantly promote hair growth. Through in vitro tissue culture experiments, it is found that the invention can significantly promote the proliferation of human dermal papilla cells, resist oxidative stress, thereby promoting the production of hair follicles, accelerating hair growth, and inhibiting hair degeneration and shedding; compared with 2% minoxidil, the invention is significantly better than minoxidil in promoting hair growth.
[0026] The present invention adopts black fish mucus polypeptide for promoting hair growth, and utilizes lipid components to encapsulate the black fish mucus polypeptide in a nano inclusion body, so that the black fish mucus polypeptide has good stability, high safety, and is easy to be absorbed through the skin. Compared with before nano inclusion, the accumulation amount of active polypeptide after nano inclusion in the skin is higher, and the same feed amount can achieve a better effect of promoting hair growth. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is a comparison chart of the particle size change rate before and after the black fish mucus polypeptide complexes obtained in Examples 1-5 and Comparative Example 1 were stored at room temperature for 30 days.
[0028] Figure 2 The following is the curve of polypeptide content change of the black fish mucus polypeptide complex obtained in Examples 1-5 and Comparative Example 1 in the accelerated test.
[0029] Figure 3 This is a comparison chart of the transdermal performance of the pretreated mucus polypeptide obtained in Example 5, the black fish mucus polypeptide complex obtained in Example 5, and the black fish mucus polypeptide complex obtained in Comparative Example 1.
[0030] Figure 4 This is a comparison chart of cell viability among NC group, PC group, A group, B group, and C group.
[0031] Figure 5 This is a comparison chart of hair regeneration scores among the NC group, PC group, A group, B group, and C group. DETAILED DESCRIPTION
[0032] The present invention will be further explained below in conjunction with specific embodiments.
[0033] Example 1
[0034] A black fish mucus polypeptide complex for promoting hair growth comprises raw materials: 10 g of pretreated mucus polypeptide, 10 g of lecithin, 10 g of cholesterol, 200 g of squalane, 0.1 g of dopamine hydrochloride, 10 g of polyethylene glycol and 1 g of Span 20.
[0035] The pretreated mucus polypeptide was prepared by the following steps: mucus was removed from the surface of fresh snakehead fish, centrifuged at 5000rpm for 4min, microfiltered with a 0.4μm microfiltration membrane, and then a 10kDa ultrafiltration membrane was used to retain proteins with a molecular weight greater than 10kDa, then freeze-dried and purified, and then mixed evenly with water at a volume ratio of 1:1, the pH value of the system was adjusted to 9.5-10.0, a composite protease preparation was added, enzymolysis was performed at 40℃ for 4h, centrifuged, the supernatant was taken, and vacuum freeze-dried.
[0036] The compound protease preparation includes: enzyme activity of 4×10 4 U / g papain, enzyme activity is 8×10 4 U / g of neutral protease, enzyme activity is 6×10 4 U / g of alkaline protease.
[0037] The method for preparing the black fish mucus polypeptide complex for promoting hair growth comprises the following steps:
[0038] S1. Add the pretreated mucus polypeptide to 50 g of deionized water and stir evenly to obtain preform a;
[0039] S2, adding lecithin and cholesterol to squalane and stirring evenly, adding preform a dropwise thereto under stirring at a stirring speed of 500 r / min, and continuing stirring for 1 h after the addition is complete to obtain preform b;
[0040] S3, adding a 1 mg / mL dopamine hydrochloride aqueous solution, polyethylene glycol, and Span 20 to 1000 g of deionized water and stirring evenly to obtain a preform c;
[0041] S4. Add preform b dropwise into preform c under stirring, stir at 1000 r / min for 10 min, homogenize at 6 MPa for 10 s, add 50 g of 0.5 mol / L sodium hydroxide solution, continue stirring at 1000 r / min for 1 h, keep stirring at 40°C, cool to room temperature, centrifuge, filter with a 10 kDa ultrafiltration tube, and then retain with a 5 kDa ultrafiltration tube.
[0042] Example 2
[0043] A black fish mucus polypeptide complex for promoting hair growth comprises raw materials including: 40 g of pretreated mucus polypeptide, 20 g of lecithin, 20 g of cholesterol, 400 g of squalane, 0.24 g of dopamine hydrochloride, 20 g of polyethylene glycol and 10 g of Tween 40.
[0044] The pretreated mucus polypeptide was prepared by the following steps: mucus was removed from the surface of fresh snakehead fish, centrifuged at 7000rpm for 6min, microfiltered with a 0.4μm microfiltration membrane, and then a 10kDa ultrafiltration membrane was used to retain proteins with a molecular weight greater than 10kDa, then freeze-dried and purified, and then mixed evenly with water at a volume ratio of 1:4, the pH value of the system was adjusted to 9.5-10.0, a composite protease preparation was added, enzymolysis was performed at 55°C for 8h, centrifuged, the supernatant was taken, and vacuum freeze-dried.
[0045] The compound protease preparation includes: enzyme activity of 6×10 4 U / g papain, enzyme activity is 9×10 4 U / g of neutral protease, enzyme activity is 8×10 4 U / g of alkaline protease.
[0046] The method for preparing the black fish mucus polypeptide complex for promoting hair growth comprises the following steps:
[0047] S1. Add the pretreated mucus polypeptide to 100 g of deionized water and stir evenly to obtain preform a;
[0048] S2, adding lecithin and cholesterol to squalane and stirring evenly, adding preform a dropwise thereto under stirring at a stirring speed of 1000 r / min, and continuing stirring for 2 h after the addition is complete to obtain preform b;
[0049] S3, adding a 1.2 mg / mL dopamine hydrochloride aqueous solution, polyethylene glycol, and Tween 40 to 2000 g of deionized water and stirring evenly to obtain a preform c;
[0050] S4. Add preform b to preform c under stirring, stir at 2000 r / min for 20 min, homogenize at 10 MPa for 30 s, add 100 g of 1.2 mol / L sodium hydroxide solution, continue stirring at 2000 r / min for 2 h, keep stirring at 50°C, cool to room temperature, centrifuge, filter with a 10 kDa ultrafiltration tube, and then intercept with a 5 kDa ultrafiltration tube.
[0051] Example 3
[0052] A black fish mucus polypeptide complex for promoting hair growth comprises raw materials including: 20 g of pretreated mucus polypeptide, 17 g of lecithin, 12 g of cholesterol, 350 g of squalane, 0.14 g of dopamine hydrochloride, 17 g of polyethylene glycol and 3 g of Span 60.
[0053] The pretreated mucus polypeptide was prepared by the following steps: mucus was removed from the surface of fresh snakehead fish, centrifuged at 6000rpm for 5min, microfiltered with a 0.4μm microfiltration membrane, and then a 10kDa ultrafiltration membrane was used to retain proteins with a molecular weight greater than 10kDa, then freeze-dried and purified, and then mixed evenly with water at a volume ratio of 1:3, the pH value of the system was adjusted to 9.5-10.0, a composite protease preparation was added, enzymolysis was performed at 45℃ for 7h, centrifuged, the supernatant was taken, and vacuum freeze-dried.
[0054] The compound protease preparation includes: enzyme activity of 4.5×10 4 U / g papain, enzyme activity is 8.8×10 4 U / g of neutral protease, and the enzyme activity was 6.5×10 4 U / g of alkaline protease.
[0055] The method for preparing the black fish mucus polypeptide complex for promoting hair growth comprises the following steps:
[0056] S1. Add the pretreated mucus polypeptide to 90 g of deionized water and stir evenly to obtain preform a;
[0057] S2, adding lecithin and cholesterol to squalane and stirring evenly, adding preform a dropwise thereto under stirring at a stirring speed of 700 r / min, and continuing stirring for 2 h after the addition is complete to obtain preform b;
[0058] S3, adding a 1.05 mg / mL dopamine hydrochloride aqueous solution, polyethylene glycol, and Span 60 to 1800 g of deionized water and stirring evenly to obtain a preform c;
[0059] S4. Add preform b dropwise into preform c under stirring, stir at 1300 r / min for 18 min, homogenize at 7 MPa for 25 s, add 70 g of 1 mol / L sodium hydroxide solution, continue stirring at 1300 r / min for 100 min, keep stirring at 42°C, cool to room temperature, centrifuge, filter with 10 kDa ultrafiltration tube, and retain with 5 kDa ultrafiltration tube.
[0060] Example 4
[0061] A black fish mucus polypeptide complex for promoting hair growth comprises raw materials including: 30 g of pretreated mucus polypeptide, 13 g of lecithin, 18 g of cholesterol, 250 g of squalane, 0.2 g of dopamine hydrochloride, 13 g of polyethylene glycol and 7 g of Span 80.
[0062] The pretreated mucus polypeptide was prepared by the following steps: mucus was removed from the surface of fresh snakehead fish, centrifuged at 6000rpm for 5min, microfiltered with a 0.4μm microfiltration membrane, and then a 10kDa ultrafiltration membrane was used to retain proteins with a molecular weight greater than 10kDa, then freeze-dried and purified, and then mixed evenly with water at a volume ratio of 1:2, the pH value of the system was adjusted to 9.5-10.0, a composite protease preparation was added, enzymolysis was performed at 50℃ for 5h, centrifuged, the supernatant was taken, and vacuum freeze-dried.
[0063] The compound protease preparation includes: enzyme activity of 5.5×10 4 U / g papain, enzyme activity is 8.2×10 4 U / g of neutral protease, enzyme activity is 7.5×10 4 U / g of alkaline protease.
[0064] The method for preparing the black fish mucus polypeptide complex for promoting hair growth comprises the following steps:
[0065] S1. Add the pretreated mucus polypeptide to 70 g of deionized water and stir evenly to obtain preform a;
[0066] S2, adding lecithin and cholesterol to squalane and stirring evenly, adding preform a dropwise thereto under stirring at a stirring speed of 900 r / min, and continuing stirring for 1 h after the addition is complete to obtain preform b;
[0067] S3, adding a 1.15 mg / mL dopamine hydrochloride aqueous solution, polyethylene glycol, and Span 80 to 1200 g of deionized water and stirring evenly to obtain a preform c;
[0068] S4. Add preform b dropwise into preform c under stirring, stir at 1700 r / min for 12 min, homogenize at 9 MPa for 15 s, add 90 g of 0.6 mol / L sodium hydroxide solution, continue stirring at 1700 r / min for 80 min, keep stirring at 48°C, cool to room temperature, centrifuge, filter with 10 kDa ultrafiltration tube, and retain with 5 kDa ultrafiltration tube.
[0069] Example 5
[0070] A black fish mucus polypeptide complex for promoting hair growth comprises raw materials including: 25 g of pretreated mucus polypeptide, 15 g of lecithin, 15 g of cholesterol, 300 g of squalane, 0.17 g of dopamine hydrochloride, 15 g of polyethylene glycol and 5 g of Tween 80.
[0071] The pretreated mucus polypeptide was prepared by the following steps: mucus was removed from the surface of fresh black carp, centrifuged at 6000rpm for 5min, microfiltered with a 0.4μm microfiltration membrane, and then a 10kDa ultrafiltration membrane was used to retain proteins with a molecular weight greater than 10kDa, then freeze-dried and purified, and then mixed evenly with water at a volume ratio of 1:2.5, the pH value of the system was adjusted to 9.5-10.0, a composite protease preparation was added, enzymolysis was carried out at 47℃ for 6h, centrifuged, the supernatant was taken, and vacuum freeze-dried.
[0072] The compound protease preparation includes: enzyme activity of 5×10 4 U / g papain, enzyme activity is 8.5×10 4 U / g of neutral protease, enzyme activity is 7×10 4 U / g of alkaline protease.
[0073] The method for preparing the black fish mucus polypeptide complex for promoting hair growth comprises the following steps:
[0074] S1. Add the pretreated mucus polypeptide to 80 g of deionized water and stir evenly to obtain preform a;
[0075] S2, adding lecithin and cholesterol to squalane and stirring evenly, adding preform a dropwise thereto under stirring at a stirring speed of 800 r / min, and continuing stirring for 1.5 h after the addition is complete to obtain preform b;
[0076] S3, adding a 1.1 mg / mL dopamine hydrochloride aqueous solution, polyethylene glycol, and Tween 80 to 1500 g of deionized water and stirring evenly to obtain a preform c;
[0077] S4. Add preform b dropwise into preform c under stirring, stir at 1500 r / min for 15 min, homogenize at 8 MPa for 20 s, add 80 g of 0.8 mol / L sodium hydroxide solution, continue stirring at 1500 r / min for 90 min, keep stirring at 45°C, cool to room temperature, centrifuge, filter with 10 kDa ultrafiltration tube, and retain with 5 kDa ultrafiltration tube.
[0078] Comparative Example 1
[0079] A black fish mucus polypeptide complex for promoting hair growth comprises raw materials including: 25 g of pretreated mucus polypeptide, 15 g of lecithin, 15 g of cholesterol, 300 g of squalane, 0.17 g of dopamine hydrochloride, 15 g of polyethylene glycol and 5 g of Tween 80.
[0080] The pretreated mucus polypeptide was prepared by the following steps: mucus was removed from the surface of fresh black carp, centrifuged at 6000rpm for 5min, microfiltered with a 0.4μm microfiltration membrane, and then a 10kDa ultrafiltration membrane was used to retain proteins with a molecular weight greater than 10kDa, then freeze-dried and purified, and then mixed evenly with water at a volume ratio of 1:2.5, the pH value of the system was adjusted to 9.5-10.0, a composite protease preparation was added, enzymolysis was carried out at 47℃ for 6h, centrifuged, the supernatant was taken, and vacuum freeze-dried.
[0081] The compound protease preparation includes: enzyme activity of 5×10 4 U / g papain, enzyme activity is 8.5×10 4 U / g of neutral protease, enzyme activity is 7×10 4 U / g of alkaline protease.
[0082] The method for preparing the black fish mucus polypeptide complex for promoting hair growth comprises the following steps:
[0083] S1. Add the pretreated mucus polypeptide to 80 g of deionized water and stir evenly to obtain preform a;
[0084] S2, adding lecithin and cholesterol to squalane and stirring evenly, adding preform a dropwise thereto under stirring at a stirring speed of 800 r / min, and continuing stirring for 1.5 h after the addition is complete to obtain preform b;
[0085] S3, adding a 1.1 mg / mL dopamine hydrochloride aqueous solution, polyethylene glycol, and Tween 80 to 1500 g of deionized water and stirring evenly to obtain a preform c;
[0086] S4. Add preform b dropwise into preform c under stirring, stir at 1500 r / min for 15 min, homogenize at 8 MPa pressure for 20 s, centrifuge, filter with an ultrafiltration tube with a molecular weight of 10 kDa, and then retain with an ultrafiltration tube with a molecular weight of 5 kDa.
[0087] The black fish mucus polypeptide complexes obtained in Examples 1-5 and Comparative Example 1 were placed in a sealed container at room temperature for 30 days, and it was found that no aggregation, precipitation, or stratification occurred in any of the samples. The particle sizes of the samples in each group before and after placement were measured, and the particle size change rate was calculated.
[0088] Particle size change rate = sample particle size after placement ÷ sample particle size before placement × 100%
[0089] like Figure 1 As shown, the particle size of the black fish mucus polypeptide complex obtained in Examples 1-5 did not change significantly, and its particle size change rate was better than that of Comparative Example 1 (P < 0.05); but there was no significant difference among the five (P > 0.05), which confirmed that the black fish mucus polypeptide complex obtained in Examples 1-5 had good stability at room temperature. The particle size change rate of the black fish mucus polypeptide complex obtained in Example 5 was the smallest.
[0090] The black fish mucus polypeptide complexes obtained in Examples 1-5 and Comparative Example 1 were subjected to accelerated tests, as follows:
[0091] The samples were placed in a constant temperature and humidity chamber (40±2°C, 75±5% RH) for 6 months, and the polypeptide content in each group of samples was detected by HPLC at the 1st, 2nd, 3rd and 6th month to evaluate its stability.
[0092] like Figure 2 As shown, the content of the black fish mucus polypeptide complex obtained in Examples 1-5 did not change much, which was better than that in Comparative Example 1 (P<0.05); and the content of the black fish mucus polypeptide complex obtained in Example 5 had the smallest change.
[0093] Since Example 5 has the best stability among Examples 1-5, Example 5 was used for further subsequent experiments.
[0094] The black fish mucus polypeptide complex obtained in Example 5 was sent for inspection and the material composition was analyzed, as shown in the following table:
[0095]
[0096]
[0097] The vertical Franz diffusion cell method was used to evaluate the transdermal performance of the pretreated mucus polypeptide obtained in Example 5 (marked as Group A), the black fish mucus polypeptide complex obtained in Example 5 (marked as Group B), and the black fish mucus polypeptide complex obtained in Comparative Example 1 (marked as Group C). The ex vivo skin of the abdomen of SD rats was fixed between the receiving chamber and the donor chamber of the diffusion cell, and 1 g of the sample was taken on the skin surface of the donor chamber. The effective diffusion area was 3.14 cm 2 , add physiological saline as receiving solution to the receiving pool, remove all bubbles to make the dermis side completely contact with the receiving solution, and stir and diffuse at 32℃ and 300r / min. Take 0.5mL of receiving solution after 24h. Use HPLC to determine the concentration of polypeptide in the receiving solution.
[0098] At the end of the transdermal absorption test, the ex vivo skin was completely removed, rinsed with normal saline, dried at room temperature, crushed, added with 4 mL of methanol, and ultrasonically extracted for 10 min each time, repeated 3 times, the extracts were combined, filtered through a 0.45 μm microporous membrane, and the skin retention amount was determined by HPLC.
[0099] like Figure 3 As shown, the in vitro cumulative transdermal amount and cumulative skin retention amount of the black fish mucus polypeptide complex obtained in Example 5 were better than those of the pretreated mucus polypeptide obtained in Example 5 and the black fish mucus polypeptide complex obtained in Comparative Example 1 (P<0.05).
[0100] The effects of the test samples on the proliferation of human dermal papilla cells are as follows:
[0101] Human dermal papilla cells (hDPCs) were purchased from Shanghai Mouyuan Biotechnology Co., Ltd. hDPCs were added to 1% penicillin and 10% fetal bovine serum to prepare DMEM culture medium and incubated at 37°C with 5% CO 2 Culture for 24 hours.
[0102] hDPCs were plated at 3 × 10 3 Cells were seeded at a density of 100 μl / well in a 96-well plate and incubated at 37°C with 5% CO 2 The pretreated mucus polypeptide obtained in Example 5 (marked as Group A), the black fish mucus polypeptide complex obtained in Example 5 (marked as Group B) and the black fish mucus polypeptide complex obtained in Comparative Example 1 (marked as Group C) were diluted to 125 ppm, and 100 μL of diluent was added to each well; the above-mentioned DMEM culture medium (NC group), minoxidil (PC group) and triton X-100 were used as negative control, positive control and blank control, respectively; each group was incubated at 37°C and 5% CO 2 Incubate for 24h.
[0103] After the incubation was completed, the culture medium was removed, 100 μL of CCK8 (10%, v / v) was added to each well, and the cells were incubated at 37° C. for 4 h. The absorbance at a wavelength of 570 nm was measured using an ELISA reader.
[0104] Cell viability = absorbance of this group ÷ absorbance of blank control group × 100%
[0105] The cell viability of the NC group was adjusted to 100% as a reference, and the remaining groups were reported as relative values. Figure 4 As shown, the PC group, as a positive control, effectively promoted the cell viability of hDPCs (P < 0.05); and relative to the NC group, the pretreated mucus polypeptide obtained in Example 5, the black fish mucus polypeptide complex obtained in Example 5, and the black fish mucus polypeptide complex obtained in Comparative Example 1 could all promote the cell viability of hDPCs (P < 0.05).
[0106] However, the pretreated mucus polypeptide obtained in Example 5 had a better promoting effect on hDPCs cell viability than minoxidil (P < 0.05); the black fish mucus polypeptide complex obtained in Example 5 had a similar promoting effect on hDPCs cell viability as minoxidil, with no significant difference (P > 0.05).
[0107] Further animal hair regeneration tests were conducted as follows:
[0108] Five-week-old healthy C57BL / 6 male mice were used for modeling. Before the experiment, all mice were adapted to feeding for 7 days. After all mice were anesthetized, paraffin-rosin was used to remove the hair on their backs, and the removal area was about 3×4 cm. They were randomly divided into 6 groups: the NC group was smeared with distilled water, the PC group was smeared with minoxidil (2.0%, W / V), and the A, B, and C groups were smeared with the pretreated mucus polypeptide obtained in Example 5, the black fish mucus polypeptide complex obtained in Example 5, and the black fish mucus polypeptide complex obtained in Comparative Example 1 (all diluted with water to 20 g / L) for 21 days.
[0109] During the control experiment, photos of the mouse back skin were taken with a digital camera. Hair regrowth scores were quantified using the ImageJ program.
[0110] Hair regrowth was scored based on differences in back skin color as follows:
[0111]
[0112]
[0113] The hair regrowth score was calculated by measuring the ratio of the hair regrowth area to the total area, and the area ratio of each group was multiplied by the sum of the scores to get the average. Therefore, the minimum value of the hair regrowth score is 0 and the maximum value is 3.
[0114] Total hair regeneration score = (level 1 area × 0 + level 2 area × 1 + level 3 area × 2 + level 4 area × 3) ÷ 12 cm 2
[0115] like Figure 5 As shown, the total hair regeneration scores of groups A, B, C and PC were better than those of group NC (P < 0.05), indicating that the interventions in each group could promote hair regeneration; and the total hair regeneration score of group B was the highest, which was better than that of groups A, C and PC (P < 0.05), indicating that the black fish mucus polypeptide complex obtained in Example 5 had the best effect on hair regeneration.
[0116] The reason why the total hair regeneration score of Group A was lower than that of Group B may be that the pretreated mucus polypeptide obtained in Example 5 used in Group A had poor transdermal effect and its effective active ingredients were difficult to be absorbed by the skin.
[0117] The applicant believes that the reason for the above results is that the present invention extracts proteins and polypeptides from black fish mucus and hydrolyzes them by enzymes, dissolving mucus polypeptides in the internal phase of the multiple emulsion, and uses high-pressure homogenization to reduce the particle size to form nanoparticles, and then uses dopamine to self-oxidize and coat the surface of the nanoparticles under alkaline conditions, which not only has excellent adhesion and increases the retention time, but also is easier to be absorbed by the scalp due to its similar structure to eumelanin, greatly promoting the absorption of polypeptides. The black fish mucus polypeptide is then wrapped in a nano inclusion using lipid components, which not only makes the black fish mucus polypeptide stable, highly safe, and easy to be absorbed through the skin, but also has a higher accumulation of active polypeptides in the skin after nano encapsulation than before nano encapsulation, and the same amount of feed can achieve a better effect of promoting hair growth.
[0118] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A snakehead fish mucus polypeptide complex for promoting hair growth, characterized in that: The raw materials include, by mass: 1-4 parts of pretreated mucus polypeptide, 1-2 parts of lecithin, 1-2 parts of cholesterol, 20-40 parts of squalane, 0.01-0.024 parts of dopamine hydrochloride, 1-2 parts of polyethylene glycol, and 0.1-1 parts of emulsifier; The pretreated mucus polypeptide is prepared by the following steps: mucus is taken from the surface of fresh snakehead fish, purified by centrifugal microfiltration, ultrafiltration and freeze drying, water is added and mixed evenly, the pH value of the system is adjusted to 9.5-10.0, a composite protease preparation is added, enzymolysis is carried out at 40-55°C for 4-8 hours, centrifuged, the supernatant is taken, and vacuum freeze drying is performed.
2. The black fish mucus polypeptide complex for promoting hair growth according to claim 1, characterized in that: The mucus taken from the surface of fresh snakehead fish is purified by centrifugal microfiltration, ultrafiltration and freeze-drying, and then evenly mixed with water in a volume ratio of 1:1-4.
3. The black fish mucus polypeptide complex for promoting hair growth according to claim 1, characterized in that: The composite protease preparation includes: papain, neutral protease, and alkaline protease.
4. The black fish mucus polypeptide complex for promoting hair growth according to claim 3, characterized in that: The enzymatic activity of papain is 4-6×10 4 U / g, the activity of neutral protease is 8-9×10 4 U / g, the activity of alkaline protease is 6-8×10 4 U / g.
5. The black fish mucus polypeptide complex for promoting hair growth according to claim 1, wherein the emulsifier is sorbitan fatty acid ester and / or polyoxyethylene sorbitan fatty acid ester.
6. A method for preparing a snakehead fish mucus polypeptide complex for promoting hair growth as claimed in any one of claims 1 to 5, characterized in that: The steps include: S1. Add the pretreated mucus polypeptide into deionized water and stir evenly to obtain preform a; S2, adding lecithin and cholesterol to squalane and stirring evenly, adding preform a dropwise thereto under stirring, and continuing stirring for 1-2 hours after the addition is complete to obtain preform b; S3, adding dopamine hydrochloride aqueous solution, polyethylene glycol, and emulsifier into deionized water and stirring evenly to obtain preform c; S4. Add preform b dropwise to preform c under stirring and stir for 10-20 minutes. High pressure homogenization for 10-30 seconds. Add sodium hydroxide solution and stir at 40-50°C for 1-2 hours. Cool to room temperature, centrifuge and retain by ultrafiltration tube.
7. The method for preparing the black fish mucus polypeptide complex for promoting hair growth according to claim 6, characterized in that: In S3, the concentration of the dopamine hydrochloride aqueous solution is 1-1.2 mg / mL.
8. The method for preparing the black fish mucus polypeptide complex for promoting hair growth according to claim 6, characterized in that: In S4, the pressure of high-pressure homogenization is 6-10MPa.
9. The method for preparing the black fish mucus polypeptide complex for promoting hair growth according to claim 6, characterized in that: In S4, the concentration of the sodium hydroxide solution is 0.5-1.2 mol / L.
10. The method for preparing the black fish mucus polypeptide complex for promoting hair growth according to claim 6, characterized in that: In S4, the molecular weight of the ultrafiltration tube is 5kDa and 10kDa.