Stable and efficient ozonized leech polypeptide liposome as well as preparation method and application thereof

Abstract

The invention discloses stable and efficient ozonized leech polypeptide lipidosome as well as a preparation method and application thereof. The method comprises the following steps: preparing leech polypeptide with the molecular weight of 100-5000 Da by taking artificially bred leeches or poecilobdella manillensis as a raw material, dissolving the leech polypeptide and high-purity oleic acid (greater than or equal to 98%) in an ethanol-water cosolvent (in a volume ratio of 3: 1) according to a mass ratio of 1: 3, and further realizing sulfonation of the leech polypeptide and efficient conversion of oleic acid into azelaic acid through combination of ozone oxidation (10-20 mg / L) and ultraviolet irradiation (254 nm, 30-50 W). The sulfated leech polypeptide is directly hydrated with a lipid membrane material (soybean lecithin: cholesterol = 4: 1) to form liposome, and the liposome with high entrapment efficiency (sulfated leech polypeptide is greater than or equal to 75% and azelaic acid is greater than or equal to 85%), strong transdermal absorption and synergistic anti-inflammatory and hair-growing effects is obtained after freeze-drying, and the liposome is suitable for alopecia care and scalp repair products.

Description

TECHNICAL FIELD

[0001] The present application relates to the preparation technology of bioactive ingredients, in particular to a stable and efficient ozonated hirudin polypeptide liposome and its preparation method and application, which are used in the fields of anti-inflammatory, hair follicle regeneration and hair growth promotion. BACKGROUND

[0002] In the prior art, the application of hirudin polypeptide in cosmetics includes promoting blood circulation and improving microcirculation, anti-inflammatory and repair, antioxidant and free radical scavenging, etc. Chinese patent CN118047829A discloses a hirudin polypeptide extraction method, but does not involve ozonated modification and liposome delivery. The application of azelaic acid (moutan flower acid) in cosmetics includes acne and anti-acne, whitening and lightening of color spots, oil control and skin conditioning, anti-inflammatory and sensitive soothing, etc. However, it mostly relies on chemical synthesis, especially the preparation of azelaic acid by ozonated oxidation of oleic acid, which needs separate purification, complex process and high cost. Moreover, separate use has problems such as high irritation and poor transdermal absorption. Chinese patent CN115590777A proposes azelaic acid compounding technology, but does not involve polypeptide and acid synergistic delivery technology.

[0003] In the existing literature, there is no research on the synergistic oxidation of hirudin polypeptide and oleic acid, sulfonated modification and azelaic acid conversion, physicochemical properties and functional synergy of liposome delivery. Traditional process needs to handle hirudin polypeptide and oleic acid separately, which is complicated and has serious activity loss. The ozonated oxidation products need to be separated and purified, resulting in high cost. The liposome encapsulation rate is low, and the physicochemical properties and functional synergy cannot be realized.

[0004] The present application first proposes a one-step preparation technology of ozonated hirudin polypeptide-oleic acid liposome without separation, which directly uses the ozonated mixed system to complete the preparation of liposome through in-situ ozonation-lipidation integrated process, significantly simplifies the process, and significantly improves the physicochemical properties and functional synergy of active ingredients, filling the technical gap. SUMMARY

[0005] The first object of the present application is to provide a stable and efficient ozonated hirudin polypeptide liposome and its preparation method and application. The second object is to provide the use of a stable and efficient ozonated hirudin polypeptide liposome.

[0006] The first object of the present application is achieved by the following technical scheme: 1. Technical route design A specific type and proportion of hirudin polypeptide and oleic acid are compounded, the solvent system is optimized, the ozonation reaction conditions and parameters are controlled, so that the mixed solution after ozonation does not need to be separated and purified, and directly combines with the optimized and screened lipid membrane material to form a stable and efficient functional liposome.

[0007] 2. Complete process steps and parameters (1) Construction of integrated ozone oxidation-lipidization system ① Raw material ratio: Leech polypeptide: 1 part by mass (molecular weight 100-5000 Da, preferably 1000-3000 Da); Oleic acid: 3 parts by mass (purity ≥98%); Ozone concentration: 10-20 mg / L, preferably 15 mg / L; Lipid material: soy lecithin and cholesterol mixed in a mass ratio of 4:1.

[0008] ② Solvent system: Ethanol-water cosolvent system (volume ratio 3:1): add 0.5%-1% polysorbate 80 (preferably 0.5%) and 0.1% sodium thiosulfate (ozone quencher); pH adjuster: phosphate buffer.

[0009] (2) One-step ozone oxidation-lipidization process Step 1: Cosolvent system construction Leech polypeptide was weighed in mass parts and dissolved in purified water (mass-volume ratio g:mL 1:25), and oleic acid was dissolved in ethanol (mass-volume ratio g:mL 1:25), and then ultrasonically mixed (100 W, 5 minutes); polysorbate 80 was added, and stirring was maintained to disperse uniformly, and the pH was adjusted to 6.5-7.0 (preferably pH 7.0) with phosphate buffer to form a uniform and stable emulsion.

[0010] Step 2: Ozone oxidation and simultaneous lipid membrane fusion Ozone control: ozone was introduced into the mixed emulsion (flow rate 1.0 L / min), and the ice water bath was controlled at 5-10°C (preferably 10°C), and the reaction was carried out for 45-90 minutes (preferably 60 minutes).

[0011] Ultraviolet light assistance: 254 nm ultraviolet light (power 30-50 W, preferably 30 W) was used vertically to accelerate the sulfonation of leech polypeptide and ozone cracking of oleic acid.

[0012] Reaction termination: 0.1% sodium thiosulfate was added to quench residual ozone.

[0013] Key innovation: Soy lecithin and cholesterol were dissolved in ethanol in a mass ratio of 4:1, and a lipid membrane was formed by rotary evaporation; the ozone oxidation mixture (containing sulfonated leech polypeptide and azelaic acid) was directly added to the lipid membrane, and the water was hydrated at 45°C for 2 hours, without the need for separation and purification.

[0014] Step 3: In situ self-assembly of liposomes Ultrasonic treatment: probe ultrasonication (300 W) for 5 minutes, and then pass through a 0.22 μm filter membrane to obtain a liposome suspension.

[0015] Lyophilization protection: 5% mannitol was added, and the lyophilized liposome powder was obtained by freeze-drying.

[0016] 3. Key process parameter verification (1) Oxidation conversion and liposome performance Parameters Indicators Detection methods Conversion rate of oleic acid→azelaic acid ≥80% HPLC (C18 column, acetonitrile / 0.1% phosphoric acid) Sulfonated rate of leech polypeptide ≥ 60% (mass spectrometry - SO3H modification) MALDI-TOF MS Liposome encapsulation rate Sulfonated leech polypeptide ≥75% azelaic acid ≥85% Ultrafiltration-HPLC combination Liposome particle size and distribution uniformity (PDI) 100±15 nm, PDI <0.2 Dynamic light scattering (DLS) (2) Process compatibility optimization Solvent residue control: ethanol residue <0.5% (GC-MS detection), in line with cosmetic safety standards; Oxidation by-product inhibition: polysorbate 80 combined with sodium thiosulfate, free radical clearance rate > 95% (ESR detection).

[0017] 4. Technical advantages (1) Raw material optimization and process innovation Leech polypeptide molecular weight optimization: 100-5000 Da, preferably 1000-3000 Da. Ensure sulfonation, lipidation and maximize transdermal absorption.

[0018] Oleic acid purity optimization: ≥98.0%. Ensure oxidation conversion efficiency.

[0019] Leech polypeptide-oleic acid mass ratio optimization: 1:3. Ensure ozone oxidation, lipidation and biological activity of both.

[0020] Co-solvent system design: water-ethanol volume ratio 3:1, containing 0.5%-1% polysorbate 80 (preferably 0.5%), phosphate buffer to adjust pH 6.5-7.0 (preferably pH 7.0), to solve the contradiction between oleic acid dispersion and leech polypeptide solubility.

[0021] Ozone-ultraviolet synergistic oxidation: ozone concentration 10-20 mg / L (preferably 15 mg / L), 254 nm ultraviolet light (power 30-50 W, preferably 30 W) catalysis, one-step realization of polypeptide sulfonation (-SH→-SO3H) and oleic acid→azelaic acid conversion. Among them: polypeptide sulfonation rate ≥60.0%, oleic acid→azelaic acid conversion rate ≥80%.

[0022] Free separation lipidation: oxidation mixture is directly hydrated with lipid membrane (soy lecithin: cholesterol = 4:1), ultrasonic liposome is formed, and the encapsulation efficiency is increased by 18%-22%; ozone oxidation and liposome preparation are completed in one step, reducing the purification steps and shortening the production cycle by more than 50%.

[0023] In-situ synergism: ozone oxidation of leech polypeptide mixed with oleic acid, sulfonated rate of leech polypeptide increased by 10%-13%, conversion rate of oleic acid-azelaic acid increased by 17%-21%; active oxygen (ROS) generated in the oxidation process promotes the self-assembly of lipid membrane, and the encapsulation rate increases by 18-22%.

[0024] (2) Breakthrough of functional synergy Anti-inflammatory-hair growth synergy: The sulfonic group of ozonated leech polypeptide and azelaic acid jointly inhibit the NF-κB pathway, and the inhibition rate of IL-6 and TNF-α induced by LPS is significantly improved, showing obvious synergistic effect.

[0025] Liposome targets hair follicle, mouse model shows that the hair regeneration cycle is shortened by 35% (tissue section analysis).

[0026] Stability enhancement: Liposome isolates ozone residues, 40°C accelerated test for 14 days, degradation rate of ozonated leech polypeptide and azelaic acid <10%, while degradation rate of control free state >30%, stability is significantly enhanced.

[0027] (3) Cost and environmental protection advantages Solvent recovery: ethanol recovery rate ≥90%, reducing raw material consumption.

[0028] Energy consumption optimization: ultraviolet-ozone synergistically reduces reaction time, energy consumption is reduced by 40%.

[0029] 5. Summary The present application successfully realizes the preparation of functional liposomes with separation-free and high efficiency and synergy through in-situ integrated technology of ozone oxidation-lipidization, which has the advantages of process simplification, efficacy enhancement and cost advantage, and provides a new demonstration for the development of anti-inflammatory and hair growth cosmetics.

[0030] The second object of the present application is achieved by the following technical scheme: Application of the ozonated leech polypeptide liposome in the preparation of anti-inflammatory and hair growth cosmetics. Specific embodiments

[0031] The present application will be further described below with reference to specific embodiments. Those skilled in the art can understand that these embodiments are only used to illustrate the present application, and do not limit the scope of the present application in any way, and any changes or substitutions made based on the teaching of the present application are within the scope of the present application.

[0032] Example 1 Preparation of leech polypeptide After homogenization of Hirudo nipponica, enzymatic hydrolysis (trypsin, pH 8.0, 37℃, 4 hours), ultrafiltration, the filtrate was collected and purified by Sephadex G-25 column, and freeze-dried to obtain polypeptide with molecular weight of 1000-3000 Da.

[0033] Example 2 Preparation of oleic acid The commercially available standard oleic acid was purchased and tested to have a purity of 98.5%.

[0034] Example 3 One-step method for liposome preparation (1) 10 g of leech polypeptide prepared in Example 1 was dissolved in 250 mL of purified water; (2) 30 g of oleic acid prepared in Example 2 was dissolved in 750 mL of ethanol; (3) The mixture was ultrasonicated (100 W) for 5 minutes, 0.5% polysorbate 80 was added, and the pH was adjusted to 7.0 with a phosphate buffer; (4) Ozone (concentration 15 mg / L, flow rate 1.0 L / min) was introduced, and 254 nm ultraviolet light (30 W) was irradiated synchronously for 60 minutes at 10°C; (5) 0.1% sodium thiosulfate was added to quench the ozone; (6) Soybean lecithin and cholesterol (4:1) were dissolved in ethanol, and a lipid film was formed by rotary evaporation, and the oxidation mixture was hydrated at 45°C for 2 hours; (7) The mixture was ultrasonicated (300 W) for 5 minutes, filtered through a 0.22 μm filter, and freeze-dried with the addition of 5% mannitol to obtain a liposome powder.

[0035] Example 4 Two-step method for control liposome preparation The leech polypeptide and oleic acid were separately oxidized and purified according to steps (1)-(5) of Example 3, and the purified sulfonated leech polypeptide solution was mixed with azelaic acid conversion solution, and liposomes were prepared according to steps (6)-(7) of Example 3.

[0036] Example 5 Preparation of free ozone-oxidized leech polypeptide The oxidation mixture was prepared according to steps (1)-(5) of Example 3, and was directly freeze-dried without lipidization.

[0037] Example 6 Preparation of ozone-oxidized leech polypeptide liposomes The leech polypeptide was ozone-oxidized and lipidized according to steps (1), (4), (5), (6), and (7) of Example 3 to obtain ozone-oxidized leech polypeptide liposomes.

[0038] Example 7 Preparation of ozone-oxidized oleic acid liposomes The oleic acid was ozone-oxidized and lipidized according to steps (2), (4), (5), (6), and (7) of Example 3 to obtain ozone-oxidized oleic acid liposomes.

[0039] To further illustrate the technical effects of the present application, the liposome of Example 3 (A), the control liposome of Example 4 (B), the free ozonated leech polypeptide of Example 5 (C), the ozonated liposome of leech polypeptide of Example 6 (D), and the ozonated liposome of oleic acid of Example 7 (E) are selected as test samples to carry out sulfonation / conversion rate and encapsulation rate detection, anti-inflammatory activity evaluation, mouse hair follicle regeneration experiment, and stability test, and the results are compared, as follows: Test 1: Polypeptide sulfonation / oleic acid-sebacic acid conversion rate and encapsulation rate detection Test samples: A, B, C, D, and E Test method: Mass spectrometry for sulfonation rate; HPLC for oleic acid-sebacic acid conversion rate; ultrafiltration-HPLC for encapsulation rate.

[0040] Test results: See Table 1.

[0041] Table 1: Polypeptide sulfonation / sebacic acid conversion rate and encapsulation rate detection results Sample Sulfonated rate of polypeptide (%) Conversion rate of azelaic acid (%) OHP encapsulation rate (%) Azelaic acid encapsulation rate (%) A (one-step method) 62.5 82.5 78.3 87.6 B (two-step method) 55.3 68.3 65.4 72.1 C (free) 60.7 80.1 —— —— D (only polypeptide) 55.3 —— 66.3 —— E (only oleic acid) —— 68.3 —— 73.5 Note: OHP represents ozonated leech polypeptide (the same below).

[0042] Table 1 shows: (1) The liposome prepared by the one-step method of the present application (A) has significantly better polypeptide sulfonation rate, sebacic acid conversion rate, OHP encapsulation rate, and sebacic acid encapsulation rate than other control samples; (2) Compared with the ozonation of leech polypeptide and oleic acid separately, the ozonation of their mixture has a 9.8%-13.0% increase in polypeptide sulfonation rate and a 17.3%-20.8% increase in sebacic acid conversion rate.

[0043] (3) Compared with the two-step method (B) and the ozonation and liposome formation of the two separately, the liposome prepared by the one-step method of the present application (A) has an 18.1%-19.7% increase in OHP encapsulation rate and a 19.2%-21.5% increase in sebacic acid encapsulation rate.

[0044] Test 2: Anti-inflammatory activity evaluation Test samples: A, B, C, D, and E Test method: Mouse monocyte macrophages (RAW264.7) were inoculated in a 96-well plate, lipopolysaccharide (LPS) (1 μg / mL) was used to induce inflammation, and 50 μg / mL of each of samples A, B, C, D, and E was added, and enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) levels, and the inhibition rates of each test sample on IL-6 and TNF-a were calculated.

[0045] Test results: See Table 2.

[0046] Table 2 Evaluation results of anti-inflammatory activity Sample IL-6 inhibition rate (%) TNF-α inhibition rate (%) A (one-step method) 78.5 71.2 B (two-step method) 62.3 54.8 C (free) 58.7 49.6 D (only polypeptide) 52.1 43.7 E (only oleic acid) 45.2 38.1 Table 2 shows that the liposome (A) prepared by the one-step method of the application significantly improves the inhibition rate of IL-6 and TNF-α induced by LPS, and shows obvious synergistic effect, because the sulfonic acid group of ozonated leech polypeptide and azelaic acid jointly inhibit the NF-κB pathway.

[0047] Test 3: Mouse hair follicle regeneration experiment Test sample: A, B, C, D, E Test method: C57BL / 6 mice were depilated on the back (diameter 2 cm), and samples A, B, C, D, and E were applied daily (prepared with physiological saline to a concentration of 0.3%), and after 14 days, hematoxylin-eosin (HE) staining was performed, and the hair follicle density and regeneration cycle were analyzed by histological section.

[0048] Test results: See Table 3.

[0049] Table 3 Mouse hair follicle density and regeneration cycle Sample Hair follicle density (pieces / mm²) Regeneration cycle (days) A (one-step method) 42.3 12.5 B (two-step method) 34.1 16.8 C (free) 30.5 18.2 D (only polypeptide) 28.3 19.1 E (only oleic acid) 26.8 20.5 Table 3 shows that the one-step liposome (A) of the application targets the mouse hair follicle, significantly increases the hair follicle density, and shortens the hair regeneration cycle by more than 35%.

[0050] Test 4: Stability test Test sample: A, B, C, D, E Test method: The samples were subjected to accelerated testing at 40°C for 14 days, and the residual rates of OHP and azelaic acid were detected by HPLC.

[0051] Results: See Table 4.

[0052] Table 4 Residual rate of stability test Sample OHP residual rate (%) Azelaic acid residual rate (%) A (one-step method) 92.1 90.4 B (two-step method) 78.5 73.2 C (free) 65.3 58.7 D (only polypeptide) 80.7 —— E (only oleic acid) —— 75.8 Table 4 shows that the liposome isolates ozone residues, and after 14 days of accelerated testing at 40°C, the degradation rate of ozonated leech polypeptide and azelaic acid is <10%, while the degradation rate of the control free state is >30%, and the stability is significantly improved.

[0053] In summary: The one-step ozonation-lipidation integrated process of the application realizes the efficient synergy of sulfonation of leech polypeptide and conversion of oleic acid, and the liposome encapsulation rate, anti-inflammatory effect, and hair growth effect are significantly better than those of the traditional method, and the application has outstanding creativity and industrial application value.

Claims

1. A stable and efficient ozonated Hirudo polypeptide liposome, characterized in that: The ozonated leech polypeptide liposome is prepared by co-dissolving, ozonating and lipidizing leech polypeptide and oleic acid with a mass ratio of 1:3; the leech polypeptide is derived from artificially bred leeches or Philander, and is obtained by enzymatic hydrolysis and ultrafiltration purification, and has a molecular weight of 100-5000 Da; the oleic acid has a purity of 98%; and the ozonated leech polypeptide liposome is prepared according to the following steps: S1 co-dissolving system: oleic acid is weighed with a mass ratio and dissolved in ethanol with a mass-volume ratio of 1:25; leech polypeptide is dissolved in purified water with a mass-volume ratio of 1:25; ultrasonic treatment is performed for 5 minutes at 100 W; 0.5%-1% polysorbate 80 is added; stirring is performed to maintain uniform dispersion; and the pH is adjusted to 6.5-7.0 by using a phosphate buffer; S2 ozonation: ozone is introduced into the co-dissolving system at a concentration of 10-20 mg / L and a flow rate of 1.0 L / min; ultraviolet light irradiation is performed at 254 nm at a power of 30-50 W; the reaction is performed at 5-10℃ for 45-90 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, and a lipid film is prepared by rotary evaporation; the ozonated mixture is hydrated at 45℃ for 2 hours; ultrasonic treatment is performed at a power of 300 W for 5 minutes; the mixture is filtered through a 0.22 μm filter; 5% mannitol is added as a freeze-drying protective agent; and the mixture is freeze-dried to obtain a liposome powder.

2. The ozonated limbus polypeptide liposome of claim 1, wherein: The leech polypeptide has a molecular weight of 1000-3000 Da; the co-dissolving system contains 0.5% polysorbate 80, and the pH is adjusted to 7.0; the ozonation is performed at an ozone concentration of 15 mg / L, an ultraviolet light power of 30 W, a temperature of 10℃ and a reaction time of 60 minutes.

3. The ozonated limbus polypeptide liposome according to any one of claims 1-2, characterized in that: The ozonated leech polypeptide liposome is used for preparing anti-inflammatory and hair growth cosmetics.