Ozonized leech polypeptide liposome cosmetic for alopecia and preparation method of ozonized leech polypeptide liposome cosmetic

Abstract

The invention discloses an ozonized leech polypeptide lipidosome cosmetic for alopecia and a preparation method of the ozonized leech polypeptide lipidosome cosmetic. On the basis of the original patent'stable and efficient ozonized leech polypeptide liposome as well as the preparation method and application thereof ', the ozonized leech polypeptide liposome is used as a core active component, a formula and a preparation process are further optimized, and an oil control active matter (0.5-2% of zinc pyrithione), an antifungal component (0.1-0.5% of clomidazole), a scalp conditioner (1-3% of panthenol) and the like are compounded, so that the ozonized leech polypeptide liposome is prepared. By optimizing a formula system and adopting a two-step dispersing-homogenizing process, the special cosmetic for seborrheic alopecia, which is high in encapsulation efficiency, strong in transdermal absorption and capable of resisting inflammation, controlling oil and growing hair in a synergistic manner, is prepared.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetics, in particular to an ozonated hirudo peptide liposome cosmetic for hair loss and a preparation method. By compounding synergistic active ingredients and optimizing the preparation process, the present application solves the problems of insufficient oil control, poor antifungal effect, and weak hair follicle regeneration ability of existing products. BACKGROUND

[0002] Seborrheic alopecia is a common hair health problem closely related to excessive sebum secretion, abnormal proliferation of Malassezia, and chronic inflammation of hair follicles. In the prior art, patent CN118047829A discloses a hirudo peptide extraction method, but does not involve ozonated modification, liposome delivery, and anti-inflammatory hair growth applications; patent CN104072644A proposes a zinc pyrithione compounding technology, but the use of zinc pyrithione alone is highly irritating and has low transdermal efficiency; literature reports that climbazole can inhibit Malassezia, but the compatibility and stability of climbazole with polypeptide liposomes have not been studied.

[0003] The prior art has the following problems: 1. Single active ingredient cannot synergistically regulate androgen, microbial, and inflammatory pathways; 2. Oil control ingredients (such as zinc salts) have poor transdermal absorption and can easily cause dry scalp; 3. Antifungal ingredients have poor compatibility with the liposome system, which can easily lead to activity loss.

[0004] The present application uses the "ozonated hirudo peptide liposome" claimed in the applicant's self-developed and applied "stable and efficient ozonated hirudo peptide liposome and its preparation method and use" patent (application number: 2025105914017) as the main formula raw material. Through further research, zinc pyrithione, climbazole panthenol, and hydrogenated lecithin are compounded, and a two-step dispersion-homogenization process is used to achieve efficient delivery and synergistic effect of active ingredients, filling the gap in seborrheic alopecia multi-target care technology. SUMMARY

[0005] The present application aims to provide an ozonated hirudo peptide liposome cosmetic for hair loss and a preparation method to overcome the shortcomings of the prior art. The cosmetic of the present application achieves the synergistic effect of anti-inflammatory, oil control, and hair growth by optimizing the formula and preparation process, effectively improving the problem of seborrheic alopecia.

[0006] 1. Technical solution design (1) Formula optimization: ozonated hirudo peptide liposome (anti-inflammatory-hair growth) as the core, compounded with zinc pyrithione (oil control), climbazole (antifungal), panthenol (barrier repair), and hydrogenated lecithin (emulsifying agent) with a mass percentage of 5-15% liposome, 0.5-2% zinc pyrithione, 0.1-0.5% climbazole, 1-3% panthenol, and 1-3% hydrogenated lecithin.

[0007] Preferably, the ozone-oxidized leech polypeptide liposome is added in the cosmetic in an amount of 10%, zinc pyrithione is 1%, climbazole is 0.3%, panthenol is 2%, and hydrogenated lecithin is 2%.

[0008] (2) Process innovation: two-step dispersion-homogenization method is adopted, first, the liposome and hydrogenated lecithin (stabilizer) are dispersed, then the fat-soluble active substance is added, to avoid mutual interference of the components and ensure the stability of the system.

[0009] 2. Preparation method (1) Preparation of ozone-oxidized leech polypeptide liposome ① Co-solvent system: the mass ratio of leech polypeptide with a molecular weight of 1000-3000 Da prepared from artificially bred leeches or leeches of the genus Hirudinaria or Philodina to oleic acid with a purity of ≥98% is 1:3, oleic acid is weighed and dissolved in ethanol (mass-volume ratio g:mL is 1:25), leech polypeptide is dissolved in purified water (mass-volume ratio g:mL is 1:25), and ultrasonic mixing (100 W) is performed for 5 minutes, 0.5% polysorbate 80 is added, and stirring is maintained to uniformly disperse, and the pH is adjusted to 7.0 with a phosphate buffer; ② Ozonation: ozone (concentration 15 mg / L, flow rate 1.0 L / min) is introduced into the co-solvent system, and 254 nm ultraviolet light irradiation (power 30 W) is simultaneously performed, and the reaction is carried out at 10°C for 60 minutes, and 0.1% sodium thiosulfate is added to quench the ozone; ③ Lipidization: soybean lecithin and cholesterol are dissolved in ethanol at a mass ratio of 4:1, a lipid film is prepared by rotary evaporation, and the ozone-oxidized mixture is hydrated at 45°C for 2 hours, ultrasonic treatment (power 300 W) is performed for 5 minutes, 0.22 μm filter membrane is used for filtration, 5% mannitol freeze-drying protectant is added, and freeze-drying is performed to obtain a liposome powder.

[0010] (2) Preparation of cosmetic ① Hydrogenated lecithin is dispersed in purified water (60°C), and ozone-oxidized leech polypeptide liposome is added, and homogenization (5000 rpm, 10 minutes) is performed; ② Zinc pyrithione and climbazole are dissolved in propylene glycol (5%), and the mixture liquid of step (1) is slowly added, and stirring and dispersion are performed; ③ Panthenol and preservative (phenoxanol 0.5%) are added, citric acid is used to adjust the pH to 5.5-6.0, homogenization (8000 rpm, 5 minutes) is performed, and cooling and filling are performed.

[0011] 3. Significant progress and substantial technical effects Compared with the prior art, the present application has the following significant progress and substantial technical effects: (1) Simplified preparation process: the ozone-oxidized leech polypeptide liposome is prepared by one-step method, which significantly simplifies the preparation process and reduces the production cost.

[0012] (2) Improve the utilization rate of active ingredients: the obtained liposomes have high encapsulation efficiency, can effectively protect active ingredients, and improve their utilization rate on the skin.

[0013] (3) Enhance anti-inflammatory, oil control and hair growth effects: by optimizing the formula design, zinc pyrithione and climbazole are wrapped by liposomes, the transdermal absorption rate and antibacterial activity are significantly improved; the sulfonic acid group of ozonated leech peptide, azelaic acid synergistically inhibit the NF-κB pathway, panthenol promotes hair papilla cell proliferation, and the hair regeneration cycle is shortened by 38.7%; the synergistic effect of anti-inflammatory, oil control and hair growth is achieved, and the problem of seborrheic alopecia is effectively improved.

[0014] (4) Improve stability: the combination of liposomes, hydrogenated lecithin and propylene glycol isolates the influence of the external environment on active ingredients. In the 3-month accelerated test, the encapsulation rate of cosmetics remains 78%, the pH is stable, the microbial limit is qualified, and there is no obvious change in appearance, which significantly improves the stability of the product. DETAILED DESCRIPTION

[0015] The application will be further described below with reference to specific examples. Those skilled in the art can understand that these examples are only used to illustrate the application, and do not limit the scope of the application in any way. Any changes or substitutions made on the basis of the teaching of the application are within the scope of protection of the application.

[0016] Example 1: Ozonated leech peptide and oleic acid (1) Ozonated leech peptide: After homogenization of Hirudo nipponia, enzymatic hydrolysis (trypsin, pH 8.0, 37°C, 4 hours), ultrafiltration, the filtrate was collected and purified by Sephadex G-25 column, and freeze-dried to obtain a polypeptide with a molecular weight of 1000-3000 Da.

[0017] (2) Oleic acid: commercially available standard oleic acid with a purity of 98.5% was purchased.

[0018] Example 2: Preparation of ozonated leech peptide liposome (1) 1000 g of ozonated leech peptide prepared in Example 1 (molecular weight 1000-3000 Da) was dissolved in 25000 mL of purified water; (2) 3000 g of oleic acid prepared in Example 1 (purity ≥98%) was dissolved in 75000 mL of ethanol; (3) Ultrasonic mixing (100 W) for 5 minutes, adding 0.5% polysorbate 80, and adjusting the pH to 7.0 with phosphate buffer; (4) Pass ozone (concentration 15 mg / L, flow rate 1.0 L / min), and simultaneously irradiate with 254 nm ultraviolet light (30 W) at 10°C for 60 minutes; (5) Add 0.1% sodium thiosulfate to quench ozone; (6) Dissolve soybean lecithin and cholesterol (4:1) in ethanol, rotary evaporate to form lipid film, and hydrate with the oxidation mixture at 45°C for 2 hours; (7) Ultrasonic treatment (300 W) for 5 minutes, pass through a 0.22 μm filter, add 5% mannitol and freeze-dry to obtain a liposome powder.

[0019] Example 3: Preparation of a cosmetic product (1) Take 100 g of ozonated leech polypeptide liposomes prepared in Example 2, 10 g of zinc pyrithione, 3 g of climbazole, 20 g of panthenol, 20 g of hydrogenated lecithin, 50 g of propylene glycol, 5 g of phenoxyethanol, and the balance is purified water; (2) Disperse the hydrogenated lecithin in purified water at 60°C, add the liposomes, and homogenize (5000 rpm, 10 minutes); (3) Dissolve the zinc pyrithione and climbazole in propylene glycol, slowly add the mixture in step (2), and stir to disperse; (4) Add the panthenol and phenoxyethanol, adjust the pH to 5.8 with citric acid, homogenize (8000 rpm, 5 minutes), and cool and fill.

[0020] Example 4: Preparation of a control cosmetic product The formulation does not contain ozonated leech polypeptide liposomes, and the remaining formulation materials and amounts, as well as the preparation scheme, are the same as in Example 3.

[0021] Example 5: Preparation of a traditional emulsion (1) Free ozonated leech polypeptide: prepare the oxidation mixture according to steps (1)-(5) in Example 2, directly freeze-dry, and do not perform lipidization.

[0022] (2) Traditional emulsion: take 100 g of free ozonated leech polypeptide to replace the ozonated leech polypeptide liposomes, and the remaining formulation materials and amounts, as well as the preparation scheme, are the same as in Example 3.

[0023] In order to further illustrate the technical effects of the present application, the cosmetic product of Example 3, the control cosmetic product of Example 4, and the traditional emulsion of Example 5 are selected as test samples to carry out anti-inflammatory activity evaluation, anti-malassezia experiment, mouse hair follicle regeneration experiment, stability test, and skin irritation test, and the results are compared, as follows: Test 1: Anti-inflammatory activity evaluation Test scheme: inoculate mouse mononuclear macrophages (RAW264.7) in a 96-well plate at a density of 1×10 4Cell, lipopolysaccharide (LPS) (1 μg / mL) was added to stimulate 24 hours, induced inflammatory response; respectively, the concentration of 50 μg / mL, 24 hours of culture; ELISA method was used to determine the concentration of IL-6 and TNF-α in cell supernatant, and the inhibition rate was calculated.

[0024] Test results: see Table 1.

[0025] Table 1 Anti-inflammatory activity evaluation results Sample IL-6 inhibition rate (%) TNF-α inhibition rate (%) Example 3 78.5 ± 2.1 82.3 ± 1.8 Example 4 55.2 ± 3.0 58.7 ± 2.5 Example 5 36.4 ± 4.2 40.1 ± 3.7 Table 1 shows that the IL-6 and TNF-α inhibition rates of Example 3 are significantly higher than those of Example 4 and Example 5 (p<0.05), indicating that its anti-inflammatory effect is the best, which benefits from the synergistic effect of ozonated Hirudo peptides liposomes and anti-inflammatory ingredients.

[0026] Test 2: Mouse hair follicle regeneration experiment Test scheme: C57BL / 6 mice were depilated on the back (diameter 2 cm), and test samples (Example 3, 4, 5, prepared with physiological saline to a concentration of 0.3%) and blank control (physiological saline) were applied daily. After 14 days, hematoxylin-eosin (HE) staining was performed, and the hair follicle density and regeneration cycle were analyzed by histological section.

[0027] Test results: see Table 2.

[0028] Table 2 Results of mouse hair follicle regeneration experiment Sample Hair follicle density (pieces / mm2) Regeneration cycle (days) Example 3 41.2 ± 3.8 18.5 ± 1.2 Example 4 38.7 ± 4.1 20.3 ± 1.5 Example 5 29.6 ± 2.5 28.7 ± 2.1 Blank control 26.4 ± 2.1 30.2 ± 2.3 Table 2 shows that the hair follicle density of Example 3 is the highest (increased by 56.1% compared with the blank control), and the regeneration cycle is shortened by 38.7%, which is significantly better than Example 5 (shortened by only 4.9%), indicating that the combination of ozonated Hirudo peptides liposomes and panthenol can effectively promote the proliferation of hair papilla cells.

[0029] Test 3: Anti-malassezia experiment Test scheme: Malassezia furfur was inoculated in Sabouraud liquid medium containing olive oil and cultured at 37°C for 48 hours. Microdilution method was used to gradiently dilute the samples (Example 3, 4, 5) (0.1%-5%), inoculate the bacterial suspension (1×10 6 CFU / mL), and observe the minimum inhibitory concentration for 48 hours.

[0030] Test results: see Table 3.

[0031] Table 3 Results of anti-malassezia experiment Sample MIC (%) Example 3 0.15 ± 0.02 Example 4 0.48 ± 0.05 Example 5 0.75 ± 0.10 Table 3 shows that the MIC value (0.15%) of Example 3 (with liposome) is significantly lower than that of Example 4 (without liposome, 0.48%) and Example 5 (free polypeptide, 0.75%), indicating that the liposome encapsulation greatly enhances the antibacterial activity of climbazole.

[0032] Test 4: Stability test Test plan: The samples (Examples 3, 4, 5) were stored at 25°C / 60% RH and 40°C / 75% RH for 3 months, and the liposome encapsulation rate, pH value, microbial limit and appearance change were detected every month.

[0033] Test results: See Table 4.

[0034]

[0035] Table 4 shows that the encapsulation rate of Example 3 remains 78% under high temperature and high humidity conditions, the pH is stable, the microbial limit is qualified, and the appearance has no change, which is significantly better than the other groups.

[0036] Test 5: Skin irritation evaluation Experimental animals: 3 healthy adult New Zealand rabbits (male and female, body weight 2.0-2.5 kg) were selected, the back skin was shaved (area about 6 cm²) before the experiment, and adaptive feeding for 7 days.

[0037] Test article treatment: Take 0.5 mL of Examples 3, 4, 5 samples, evenly apply to the shaved area, cover with semi-closed dressing (4 hours of contact time).

[0038] Observation time: 1 hour, 24 hours, 48 hours and 72 hours after removing the dressing, record the erythema and edema reaction (0-4 points) according to the scoring standard of "Cosmetic Safety Technology Standard".

[0039] Scoring criteria: (1) Erythema: 0 points (none), 1 point (mild), 2 points (obvious), 3 points (moderate to severe), 4 points (purpura / necrosis); (2) Edema: 0 points (none), 1 point (mild), 2 points (obvious), 3 points (moderate to severe).

[0040] Data analysis: Calculate the total irritation score (erythema + edema) of each animal, take the average value, and the data is expressed as mean ± standard deviation (SD).

[0041] Test results: See Table 5.

[0042]

[0043] Table 5 shows that the total irritation score of Example 3 at all time points is less than 0.5 points, indicating that its mildness is significantly better than that of Example 4 and Example 5.

[0044] Conclusion In summary, the cosmetic of the present application is compounded by ozone water leech polypeptide liposome and functional ingredients, which can significantly inhibit inflammation, regulate sebum secretion, shorten the hair follicle regeneration cycle, has excellent stability, is suitable for long-term care of patients with seborrheic alopecia, and has high efficiency, safety and industrial application value.

Claims

1. Ozonated Hirudo Polypeptide Liposomal Cosmetics for Hair Loss, characterized by: The cosmetic is composed of 5-15% ozonated Hirudo nipponia polypeptide liposome, 0.5-2% zinc pyrithione, 0.1-0.5% climbazole, 1-3% panthenol, 1-3% hydrogenated lecithin, the balance being purified water and cosmetically acceptable adjuvants by weight percentage; wherein the ozonated Hirudo nipponia polypeptide liposome is prepared by the following steps: S1: a co-solvent system: the mass ratio of the water frog polypeptide with a molecular weight of 1000-3000 Da prepared from artificially bred water frogs or leeches and oil with a purity of 98% or more is 1:3, oil is weighed and dissolved in ethanol with a mass-volume ratio of 1:25, the water frog polypeptide is dissolved in purified water with a mass-volume ratio of 1:25, ultrasonic treatment is performed at 100 W for 5 minutes, 0.5% polysorbate 80 is added, and stirring is maintained to uniformly disperse, and the pH is adjusted to 7.0 with a phosphate buffer; S2: ozonation: ozone is introduced into the co-solvent system at a concentration of 15 mg / L and a flow rate of 1.0 L / min, and synchronous ultraviolet light irradiation is performed at 254 nm with a power of 30 W at 10°C for 60 minutes, and 0.1% sodium thiosulfate is added to quench ozone; S3: lipidization: soybean lecithin and cholesterol are dissolved in ethanol at a mass ratio of 4:1, a lipid membrane is prepared by rotary evaporation, the ozonation mixture is hydrated at 45°C for 2 hours, ultrasonic treatment is performed at a power of 300 W for 5 minutes, 0.22 μm filter membrane is used for filtration, 5% mannitol freeze-drying protectant is added, and freeze-drying is performed to obtain a liposome powder.

2. The cosmetic product according to claim 1, characterized in that: The addition amount of the ozonated Hirudo nipponia polypeptide liposome in the cosmetic is 10%, the addition amount of zinc pyrithione is 1%, the addition amount of climbazole is 0.3%, the addition amount of panthenol is 2%, and the addition amount of hydrogenated lecithin is 2%.

3. The cosmetic according to claim 1 or 2, characterized in that: The cosmetic is prepared by the following steps: St1: hydrogenated lecithin is dispersed in purified water at 60°C, ozonated Hirudo nipponia polypeptide liposome is added, homogenization is performed at 5000 rpm for 10 minutes; St2: zinc pyrithione and climbazole are dissolved in propylene glycol at 5%, the mixture of step St1 is slowly added, and stirring and dispersion are performed; St3: panthenol and preservatives are added, preferably: 0.5% phenoxyethanol, citric acid is added to adjust the pH to 5.5-6.0, homogenization is performed at 8000 rpm for 5 minutes, and cooling and filling are performed.

4. The cosmetic product of claim 3, wherein: The cosmetic is used for inhibiting IL-6 and TNF-α, regulating scalp microecology, and promoting hair follicle regeneration.

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