Mink peptide compound with acne-removing skin care effect and separation and preparation thereof

Abstract

The application discloses a mink peptide compound with acne-removing and skin-protecting effects and a separation and preparation method thereof. The mink peptide raw material is successfully separated and prepared into a cyclic peptide compound with acne-removing and skin-protecting effects, and the structure is as follows.

Description

Technical Field

[0001] This invention relates to a mink peptide compound and its isolation, preparation, and application. Background Technology

[0002] Cephalosporins and defensins are among the most widely studied antimicrobial peptides, exhibiting strong inhibitory activity against Propionibacterium acnes. However, their use in acne treatment still requires consideration of safety and drug resistance issues. Animal peptides, through multiple synergistic mechanisms including direct antibacterial action (against Propionibacterium acnes), potent anti-inflammatory effects, regulation of sebum secretion, and repair of the skin barrier, intervene in multiple pathogenic stages of acne. They are not only bactericides but also inflammatory signal modulators and microbial ecosystem managers, providing a novel solution for acne treatment, especially suitable for skin that is unresponsive to traditional therapies or is sensitive and intolerant.

[0003] The greatest advantage of animal peptides is their ability to alleviate inflammation that causes redness, swelling, and pain. For example, palmitoyl tripeptide-8, by mimicking the body's own anti-inflammatory signals, can effectively inhibit TLR2 / 1-mediated inflammatory responses, directly reducing the inflammatory response of acne from the upstream, and is particularly effective in relieving red and swollen inflammatory pimples. Another example is copper peptides, which not only promote collagen production and wound healing, but also have anti-inflammatory and antioxidant capabilities, showing outstanding performance in the repair of late-stage acne and the prevention of acne scars and pits.

[0004] Therefore, there is an urgent need to accurately isolate the corresponding active ingredients from various animal peptides, such as mink peptides, to efficiently prepare products such as acne-removing skin care products. Summary of the Invention

[0005] The purpose of this invention is to provide a novel mink peptide compound with acne-removing and skin-care effects.

[0006] According to one aspect of the present invention, a cyclic peptide compound is provided, having the following structure:

[0007]

[0008] According to another aspect of the present invention, an acne-reducing skincare composition is also provided, comprising the above-mentioned cyclic peptide compound and a suitable carrier. The dosage form of the composition includes, but is not limited to, transdermal absorption formulations.

[0009] According to another aspect of the present invention, the application of the above-mentioned cyclic peptide compound in the preparation of acne-removing skin care products is also provided.

[0010] According to another aspect of the present invention, a method for preparing the above-mentioned cyclic peptide compound is also provided, comprising:

[0011] Provides pure mink meat;

[0012] Degreased mink meat protein was obtained by placing pure mink meat in ethanol for static defatting treatment.

[0013] The obtained defatted mink meat protein was extracted by reflux with water and then filtered to obtain the filtrate.

[0014] The obtained filtrate was concentrated and dried to obtain mink peptide raw material;

[0015] The obtained mink peptide raw material was dissolved in water and then enzymatically hydrolyzed with protease. After enzyme inactivation, the supernatant was collected by centrifugation.

[0016] The supernatant obtained by fractional filtration was collected and the components were gathered.

[0017] The collected components were separated and purified to obtain the cyclic peptide compound.

[0018] The weight-to-volume ratio of pure mink meat to ethanol can be (8~12):1. The weight-to-volume ratio of defatted mink meat protein to water can be 1:(8~12).

[0019] The protease may include pepsin and / or trypsin. Enzymatic hydrolysis conditions can be: pH 2–9, temperature 22°C–40°C, and time 4–7 hours. The amount of enzyme added (by weight) can be 0.5%–2% of the mink peptide raw material.

[0020] The reflux extraction temperature can be 85℃~95℃, and the time can be 1~3 hours.

[0021] The fractional filtration may include: sequentially filtering the resulting supernatant through ultrafiltration membranes with molecular weight cutoffs of 10KD, 5KD and 3KD, and collecting the 0~3KD fraction.

[0022] Separation and purification may include:

[0023] The collected fractions were subjected to chromatography using a Sephadex LH-20 column, eluted sequentially with methanol / water at concentrations of 5%, 10%, 15%, 20%, 25%, 35%, 55%, 75%, 90%, and 100%, and the corresponding eluted fractions were collected.

[0024] The elution fraction corresponding to the methanol / water mixture with a methanol volume concentration of 35% was selected for separation and preparation by reversed-phase high-performance liquid chromatography. The chromatographic conditions were: C18 column, isocratic elution with methanol / water mixture with a methanol volume concentration of 35%, and detection wavelength of 220 nm. The cyclic peptide compound was obtained after collecting the target peak.

[0025] Concentrating and drying the obtained filtrate to obtain mink peptide raw material may include: concentrating the obtained filtrate under reduced pressure until a gel-like solution is obtained; and freeze-drying the obtained gel-like solution at -90°C to -70°C for 30 to 60 hours to obtain mink peptide raw material.

[0026] This invention is the first to isolate and prepare a cyclic peptide compound or short peptide FFVRP with significant acne-removing activity from mink peptides. Experiments show that it can significantly inhibit Propionibacterium acnes and the activity of hyaluronidase in the skin, and has a significant acne-removing effect. Attached Figure Description

[0027] Figure 1 The proton NMR spectrum of the mink peptide FFVRP isolated and prepared according to the present invention.

[0028] Figure 2 The carbon NMR spectrum of the mink peptide FFVRP isolated and prepared according to the present invention. Detailed Implementation

[0029] Preparation of mink peptide raw materials

[0030] 1) Take mink meat (with fur and internal organs removed) and mix it with ethanol at a weight / volume ratio of 10:1 and let it stand. Filter the residue, concentrate and dry it to obtain defatted mink meat protein.

[0031] 2) Mix the obtained defatted mink meat protein with pure water at a weight / volume ratio of 1:10, heat to 90℃ and reflux for 2 hours, then filter to obtain the filtrate.

[0032] 3) Concentrate the obtained filtrate under reduced pressure until a gel-like solution is obtained.

[0033] 4) The obtained gel solution was dried in a freeze dryer at -80℃ for 48 hours to obtain mink peptide raw material.

[0034] Preparation of mink peptide supernatant by enzymatic hydrolysis

[0035] Weigh 100 g of the above mink peptide raw material and dissolve it in 100 mL of pure water to prepare a 1000 g / L solution. Add pepsin for enzymatic hydrolysis: the hydrolysis conditions are pH 3 (the pH is adjusted using 0.1 mol / L NaOH or HCl solution during the hydrolysis process to maintain a stable pH value), temperature 25℃, hydrolysis time 6 hours, and the amount of enzyme added is 1% of the weight of the mink peptide raw material. After hydrolysis, boil for 10 minutes to inactivate the enzyme, cool to room temperature, and centrifuge at 4℃ and 10000 r / min for 20 minutes, collecting the supernatant.

[0036] Weigh 100 g of the above mink peptide raw material and dissolve it in 100 mL of ultrapure water to prepare a 1000 g / L solution. Add trypsin for enzymatic hydrolysis: the hydrolysis conditions are pH 8 (the pH is adjusted using 0.1 mol / L NaOH or HCl solution during the hydrolysis process to maintain a stable pH value), temperature 37℃, hydrolysis time 5 hours, and enzyme dosage is 1% of the weight of the mink peptide raw material. After hydrolysis, boil for 10 minutes to inactivate the enzyme, cool to room temperature, and centrifuge at 4℃ and 10000 r / min for 20 minutes, collecting the supernatant 2.

[0037] Preparation of mink peptide FFVRP

[0038] The supernatants 1 and 2 were combined and filtered sequentially through ultrafiltration membranes with molecular weight cutoffs of 10 KD, 5 KD, and 3 KD, collecting the 0-3 KD fraction. This fraction was then subjected to Sephadex LH-20 column chromatography, using a gradient elution of methanol / water at concentrations of 5%, 10%, 15%, 20%, 25%, 35%, 55%, 75%, 90%, and 100% to obtain 10 fractions, Fr.1–10. The Fr.6 fraction (corresponding to 35% methanol / water) was selected and separated using reversed-phase high-performance liquid chromatography (RP-HPLC; using an LC3000 semi-preparative liquid chromatography system from Beijing Innovation Tongheng Co., Ltd.). Chromatographic conditions: C18 column (4.6 × 250 mm, 5 μm), isocratic elution with 35% methanol / water (containing 0.1% trifluoroacetic acid), flow rate 1.0 mL / min, detection wavelength 220 nm, injection volume 50 μL / injection, and collection retention time 8.4 min. The target peak was collected and freeze-dried to obtain white mink peptide FFVRP powder.

[0039] Compound structure identification

[0040] Figure 1 The 1H NMR spectrum of white mink peptide FFVRP is shown. Figure 2 The carbon NMR spectrum is used to identify compounds with the following structures.

[0041]

[0042] 1 H NMR (700 MHz, DMSO- d 6) δ 8.69 - 8.58 (m, 1H), 8.32 (d, J = 7.8 Hz, 1H), 8.22 (d, J = 7.1 Hz, 1H), 8.07 (d, J = 7.8 Hz, 1H), 7.98 - 7.86 (m, 7H), 7.28 (m, 4H), 4.44 - 4.38 (m, 2H), 4.23 (td, J = 8.2, 5.2 Hz, 1H), 4.18 (q, J = 7.3 Hz, 1H), 3.62 (m, 2H), 3.11 (q, J = 6.6 Hz, 2H), 2.81 (dd,J = 13.5, 4.6 Hz, 1H), 2.74 (tt, J = 8.4, 5.5 Hz, 3H), 1.70 - 1.65 (m, 2H), 1.53 (m,6H), 1.44 - 1.31 (m, 2H), 1.27 (d, J = 7.3 Hz, 3H).

[0043] 13 C NMR (176 MHz, DMSO) δ 174.46, 171.57, 171.52, 169.86, 166.37,159.04, 158.86, 157.41, 157.37, 118.48, 116.78, 55.67, 52.77, 52.73, 48.02,40.84, 40.60, 39.12, 31.74, 29.82, 27.10, 26.75, 25.32, 22.51, 17.58.

[0044] The obtained mink peptide FFVRP showed inhibitory activity against Propionibacterium acnes.

[0045] The standard strain was inoculated onto a Clostridium enrichment slant containing agar powder and anaerobically cultured at 37°C for 50 h. Propionibacterium acnes was eluted with 5 mL of Clostridium enrichment broth, and the eluent was collected in a 10 mL centrifuge tube. The bacterial concentration was adjusted to 10⁻⁶ using a turbidimetric method. 5 ~10 6 CFU / mL, viable bacteria were counted using the plate method; 19 mL of Clostridium enrichment agar was poured into disposable petri dishes, with a pour thickness of approximately 5 mm. After the plates solidified, 100 μL of Propionibacterium acnes solution was placed on the surface of the culture medium and quickly spread evenly with a sterile spreader; sterile Oxford cups (6 mm inner diameter, 8 mm outer diameter, 10 mm height) were placed on the spread culture medium, gently pressed to ensure contact without gaps, with each Oxford cup at least 14 mm from the edge of the plate and at least 30 mm between the centers of any two Oxford cups. Approximately 200 μL of the test sample was added to the Oxford cup, ensuring no overflow. Each test sample was prepared in triplicate, with erythromycin as a positive control. The samples were anaerobically incubated at 37°C for 50 h, and the results were observed. The clear ring inhibition zone was observed, and its diameter was measured using calipers. The average value of the three sets of data was recorded. The test results are shown in Table 1.

[0046]

[0047] The inhibitory effect of the obtained mink peptide FFVRP on hyaluronidase activity

[0048] Prepare acetate buffer, hyaluronidase solution, sodium hyaluronate solution, and cetyltrimethylammonium bromide (CTAB) solution separately. Then, prepare 0.2 g / ml solutions for each sample. Divide each sample solution into two groups, A and B. Add 0.1 ml of hyaluronidase solution to solution A, incubate at 37°C for 10 min, then add 0.1 ml of 0.04% sodium hyaluronate solution, incubate at 37°C for 45 min, then add 0.2 ml of CTAB solution, incubate at 37°C for 5 min, and collect 200 μL of the reaction solution in a 96-well plate. Measure the absorbance at 400 nm to obtain the result for A. Add 0.1 ml of acetate buffer to solution B, incubate at 37°C for 10 min, then add 0.1 ml of 0.04% sodium hyaluronate solution, incubate at 37°C for 45 min, then add 0.2 ml of CTAB solution, incubate at 37°C for 5 min, and collect 200 μL of the reaction solution in a 96-well plate. In a 96-well plate, the absorbance at 400 nm was measured to obtain result B. Weigh 0.1 ml of deionized water, add 0.1 ml of hyaluronidase solution, incubate at 37°C for 10 min, then add 0.1 ml of 0.04% sodium hyaluronate solution, incubate at 37°C for 45 min, then add 0.2 ml of CTAB solution, precipitate at 37°C for 5 min, and pipette 200 μL of the reaction solution into a 96-well plate, measuring the absorbance at 400 nm to obtain result C. Weigh 0.1 ml of [unspecified ingredient], add 0.1 ml of [unspecified ingredient] to deionized water, add 0.1 ml of acetate buffer, incubate at 37°C for 10 min, then add 0.1 ml of 0.04% sodium hyaluronate solution, incubate at 37°C for 45 min, then add 0.2 ml of [unspecified ingredient]... The CTAB solution was precipitated at 37℃ for 5 min. 200 μL of the resulting solution was then transferred to a 96-well plate, and the absorbance at 400 nm was measured to obtain result D. The hyaluronidase inhibition rate was calculated as {(Result D - Result C) - (Result B - Result A)} / (Result D - Result C) × 100%. The presence of a soothing effect was determined based on the positive correlation between sample concentration and hyaluronidase inhibition rate. The test results are shown in Table 2.

[0049]

[0050] As can be seen, mink peptide FFVRP has significant skin acne-removing effects both in vitro and in vivo.

[0051] In summary, this invention, through a specific sorting process, for the first time separates and prepares a novel short peptide—basic pentapeptide FFVRP—from mink peptide raw materials, and has been verified to have excellent acne-removing and skin-care effects as described above.

Claims

1. A short peptide FFVRP, the structure of which is as follows: 。 2. The application of the short peptide FFVRP according to claim 1 in the preparation of acne-removing skin care products.

3. The method for preparing the short peptide FFVRP according to claim 1, comprising: Provides pure mink meat; Degreased mink meat protein was obtained by placing pure mink meat in ethanol for static defatting treatment. The obtained defatted mink meat protein was refluxed with water and then filtered to obtain the filtrate. The reflux extraction temperature was 85℃~95℃ and the time was 1~3 hours. The obtained filtrate was concentrated and dried to obtain mink peptide raw material; The obtained mink peptide raw material was dissolved in water and then enzymatically hydrolyzed with protease. After enzyme inactivation, the supernatant was collected by centrifugation. The protease included pepsin and / or trypsin. The hydrolysis conditions were pH 2-9, temperature 22℃-40℃, time 4-7 hours, and the amount of enzyme added was 0.5%-2% of the weight of the mink peptide raw material. The resulting supernatant was sequentially filtered through ultrafiltration membranes with molecular weight cutoffs of 10KD, 5KD and 3KD, and the fractions of 0-3KD were collected. The collected components were separated and purified to obtain the short peptide FFVRP. The separation and purification process includes: The collected fractions were subjected to chromatography using a Sephadex LH-20 column, eluted sequentially with methanol / water at concentrations of 5%, 10%, 15%, 20%, 25%, 35%, 55%, 75%, 90%, and 100%, and the corresponding eluted fractions were collected. The elution fraction corresponding to the above-mentioned methanol / water mixture with a methanol volume concentration of 35% was selected for reversed-phase high-performance liquid chromatography (RP-HPLC) separation and preparation. Chromatographic conditions: C18 column, isocratic elution with methanol / water mixture with a methanol volume concentration of 35%, detection wavelength of 220 nm; the short peptide FFVRP was obtained after collecting the target peak.

4. According to the preparation method of claim 3, the mink peptide raw material is obtained by concentrating and drying the obtained filtrate, comprising: The resulting filtrate was concentrated under reduced pressure until a gel-like solution was obtained; The resulting gel solution was freeze-dried at -90°C to -70°C for 30 to 60 hours to obtain mink peptide raw material.

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