Mink peptide compound with whitening and skincare effects and separation and preparation of mink peptide compound

Abstract

The invention discloses a mink peptide compound with whitening and skin care effects and separation and preparation thereof. The invention successfully separates and prepares a small peptide with whitening activity from a mink peptide raw material, and the structure of the small peptide is shown in the specification.

Description

Technical Field

[0001] This invention relates generally to the field of skin whitening and skincare, and specifically to a mink peptide compound and its separation, preparation, and application. Background Technology

[0002] Animal peptides have shown great potential in the field of skin whitening due to their high activity, high specificity, relative safety, and good skin permeability. They primarily achieve skin whitening and other effects through the synergistic effects of multiple pathways, including inhibiting tyrosinase activity, blocking melanocyte activation signals, promoting stratum corneum renewal, and anti-inflammatory and antioxidant properties. Currently, various active peptides derived from marine fish (such as tilapia and cod skin and scales), silk, and milk have been successfully developed and applied in high-end skincare products. Studies have shown that they not only assist in whitening through antioxidant and anti-inflammatory effects, but their hydrolysates can also provide nutrients to the skin, improve overall skin texture, and strengthen the skin barrier, thus making the whitening effect more lasting and healthy. For example, sericin peptides derived from the hydrolysis of silk sericin can significantly inhibit tyrosinase activity and melanocyte proliferation, and also have excellent moisturizing properties, achieving a dual effect of whitening and moisturizing.

[0003] Therefore, it is still necessary to accurately isolate the corresponding active ingredients from various animal peptides, such as mink peptides, in order to prepare more effective whitening skin care products. Summary of the Invention

[0004] The purpose of this invention is to provide a novel mink peptide compound with skin-whitening and skin-care effects.

[0005] According to one aspect of the present invention, a small peptide is provided having the following structure:

[0006] According to another aspect of the present invention, a skin-whitening composition is also provided, comprising the above-mentioned small peptides and a suitable carrier. The dosage form of the composition includes, but is not limited to, transdermal absorption formulations.

[0007] According to another aspect of the present invention, the application of the above-mentioned small peptides in the preparation of skin care and whitening products is also provided.

[0008] According to another aspect of the present invention, a method for preparing the above-mentioned small peptide is also provided, 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 extracted by reflux with water and then filtered to obtain the filtrate. 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 supernatant obtained by fractional filtration was collected and the components were gathered. The collected components were separated and purified to obtain the small peptide.

[0009] 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).

[0010] 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.

[0011] The reflux extraction temperature can be 85℃~95℃, and the time can be 1~3 hours.

[0012] 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.

[0013] Separation and purification may include: 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 90% was selected for separation and preparation by reversed-phase high-performance liquid chromatography. Chromatographic conditions: C18 column, isocratic elution with methanol / water mixture with a methanol volume concentration of 14%, detection wavelength of 220 nm; the small peptide was obtained after collecting the target peak.

[0014] 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.

[0015] This invention is the first to isolate and prepare GRCKA small peptides or short peptides with significant whitening activity from mink peptides. Experiments show that they can significantly inhibit tyrosinase activity, have a significant inhibitory effect on melanoma cells and intracellular tyrosinase, and have a significant whitening effect on guinea pigs with skin pigmentation induced by UVB irradiation. Attached Figure Description

[0016] Figure 1 The proton NMR spectrum of the mink peptide GRCKA isolated and prepared according to the present invention.

[0017] Figure 2The carbon NMR spectrum of the mink peptide GRCKA isolated and prepared according to the present invention. Detailed Implementation

[0018] Preparation of mink peptide raw materials 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.

[0019] 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.

[0020] 3) Concentrate the obtained filtrate under reduced pressure until a gel-like solution is obtained.

[0021] 4) The obtained gel solution was dried in a freeze dryer at -80℃ for 48 hours to obtain mink peptide raw material.

[0022] Preparation of mink peptide supernatant by enzymatic hydrolysis 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.

[0023] 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.

[0024] Preparation of mink peptide GRCKA 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 Fr9 fraction (corresponding to 90% methanol / water) was selected for separation and preparation by reversed-phase high-performance liquid chromatography (RP-HPLC; using the LC3000 semi-preparative liquid chromatography system of Beijing Innovation Tongheng Co., Ltd.). Chromatographic conditions: C18 column (4.6×250 mm, 5 μm), isocratic elution with 14% 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 GRCKA powder.

[0025] Compound structure identification Figure 1 The 1H NMR spectrum of white powdered mink peptide GRCKA. Figure 2 The carbon NMR spectrum is used to identify compounds with the following structures.

[0026]

[0027] 1 H NMR (700 MHz, DMSO- d 6) δ 8.83 (d, J = 8.1 Hz, 1H), 8.24 (d, J = 8.8Hz, 1H), 8.17 (d, J = 7.3 Hz, 1H), 8.06 (s, 3H), 7.79 - 7.78 (m, 1H), 7.33(d, J = 7.6 Hz, 2H), 7.27 (q, J = 7.0, 6.4 Hz, 6H), 7.21 (t, J = 7.3 Hz, 1H), 4.73 (d, J = 3.3 Hz, 1H), 4.47 (d, J = 5.9 Hz, 1H), 4.27 - 4.25 (m, 1H), 4.22(dd, J= 8.6, 4.9 Hz, 1H), 4.02 (s, 1H), 3.74 (d, J = 9.4 Hz, 1H), 3.57 (dd, J = 6.4, 2.5 Hz, 1H), 3.13 (ddd, J = 26.5, 13.8, 5.5 Hz, 3H), 3.04 (dd, J =14.1, 3.8 Hz, 1H), 2.94 (dd, J = 14.2, 7.9 Hz, 1H), 2.84 (dd, J = 14.0, 9.9Hz, 1H), 2.14 (dd, J = 12.2, 7.9 Hz, 1H), 2.00 - 1.98 (m, 1H), 1.92 (dd, J =13.2, 6.4 Hz, 1H), 1.88 (dd, J = 12.8, 6.5 Hz, 1H), 1.84 (dq, J = 11.8, 6.4,5.8 Hz, 2H), 1.74 - 1.72 (m, 1H), 1.59 - 1.57 (m, 2H), 0.87 (dd, J = 13.6,6.8 Hz, 6H). 13 C NMR (176 MHz, DMSO) δ 173.64, 171.15, 171.12, 170.07, 168.42,157.35, 138.00, 135.08, 130.19, 129.80, 129.74, 128.91, 128.57, 127.59,126.85, 58.96, 57.94, 54.52, 53.44, 50.55, 46.98, 41.02, 37.95, 37.31, 31.21,29.11, 28.56, 25.12, 25.07, 19.66, 18.60. The inhibitory effect of the obtained mink peptide GRCKA on the activity of tyrosinase and diphenolase Using levodopa as a substrate, the mixture consisted of 320 μL phosphate-buffered saline (PBS, pH 6.8), 150 μL levodopa solution (2.5 mM), and 50 μL of mink peptide GRCKA sample solutions of different concentrations. After incubation at 30°C for 20 min, 80 μL tyrosinase solution (250 U / mL) was added, and the absorbance at 475 nm was immediately measured. Three replicates were performed, and the average value was taken. Nicotinamide was used as a positive control. The test results are shown in Table 1.

[0028]

[0029] The inhibitory effect of the obtained mink peptide GRCKA on tyrosinase monophenolase activity Using L-tyrosine as a substrate, the system consisted of 300 μL phosphate-buffered saline (PBS, pH 6.8), 120 μL L-tyrosine solution (1 mM), and 50 μL of 100 μg / mL mink peptide GRCKA sample solution. After incubation at 30°C for 20 min, 80 μL tyrosinase solution (250 U / mL) was added, and the absorbance at 475 nm was immediately measured. Nicotinamide was used as a positive control. Three replicates were set up, and the average value was taken. The test results are shown in Table 2.

[0030]

[0031] The obtained mink peptide GRCKA inhibited the activity of tyrosinase in melanoma cells. B-16 melanoma cell line was cultured in IMDM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were passaged using 0.25% trypsin. Cells from the same passage were used for each experiment, and after digestion, they were seeded evenly in 6-well plates with 10% IMDM medium, with three replicates for each concentration. Cells were divided into six groups: a normal control group and five dose groups of mink peptide GRCKA (low to high). 24 hours after seeding, the culture medium was aspirated, and serum-free culture medium was added. The next day, different concentrations of the test drug were added, and cells and culture supernatant were collected at 24, 36, and 48 hours. The same amount of cell freeze-thawed solution with the same protein content was mixed with 0.5 ml of L-dopa solution at a concentration of 1.214 mg / ml, and the volume was adjusted to 1 ml with PBS. The initial OD value and the OD value after 10 min in a 37℃ water bath were measured at 475 nm with a light path of 0.5 cm. The inhibition rate of enzyme activity was calculated as: OD value of experimental group - OD value of blank group / OD value of normal group - OD value of blank group. The test results are shown in Table 3.

[0032]

[0033] The whitening effect of the obtained mink peptide GRCKA on UVB-induced skin pigmentation in guinea pigs Seven male guinea pigs (weighing 500-700 grams) were selected and placed separately. The fur on their backs was shaved, and six 1.5 cm diameter slots were installed on their exposed skin. Two slots were placed in each group, resulting in three groups: the first group served as the blank control, the second as the matrix control, and the third as the experimental group (containing 100 ug / mL of GRCKA peptide solution). The exposed skin was subjected to ultraviolet (UV) irradiation at an energy of 1 J / cm², every other day for two weeks. A one-week waiting period was then allowed for the UV-induced pigmentation to stabilize. In the blank control group, the pigmented skin areas received no treatment. In the experimental group, 0.1 mL of the sample solution was applied evenly twice daily to the pigmented skin areas. In the matrix control group, 0.1 mL of the solution was applied evenly twice daily to the pigmented skin areas; the solution composition was identical to the experimental sample except that it did not contain GRCKA peptide. The skin photometric intensity (L*) of each area was measured using a colorimeter before treatment and on days 7, 14, 21, and 28 after treatment. The change in the luminance index L* before and after medication is represented by ΔL*. The test results are shown in Table 4.

[0034]

[0035] As can be seen, the mink peptide GRCKA has significant skin whitening effects both in vitro and in vivo.

[0036] In summary, this invention, through a specific sorting process, for the first time separates and prepares a novel small peptide or short peptide—the basic pentapeptide GRCKA—from mink peptide raw materials, and has been verified to have excellent skin whitening effects as described above.

Claims

1. A small peptide with the following structure: 。 2. The application of the small peptide according to claim 1 in the preparation of skin care and whitening products.

3. The method for preparing the small peptide 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 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 small peptide, wherein the separation and purification included: 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 methanol / water mixture with a methanol volume concentration of 90% 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 14%, and detection wavelength of 220 nm. The small peptide 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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