Tyrosinase inhibitory peptide and application thereof

Abstract

The invention discloses a tyrosinase inhibitory peptide and application thereof, the amino acid sequences of the tyrosinase inhibitory peptide are respectively shown as SEQ ID NO.1 (NFKLL, SEQ ID NO.2 (PFGLFP), SEQ ID NO.3 (YFPDHF), SEQ ID NO.4 (YFPFH) and SEQ ID NO.5 (QGRTLYGFGG), and the active peptide has anti-aging activity and can be used for preparing cosmetics and medicines with anti-aging activity. The invention provides an active peptide composed of brand new amino acids, and research shows that the active peptide has good anti-aging activity; therefore, further application value in preparing cosmetics or medicines with anti-aging activity by using the compound as an active ingredient is important.

Description

[0001] This application is a divisional application of the patent application with application number 202410834916.0, application date 2024.06.26, and invention name “Active peptides with anti-aging effects and their applications”. Technical Field

[0002] The present invention belongs to the field of bioactive peptides, and in particular relates to a tyrosinase inhibitory peptide and an application thereof. Background Art

[0003] With the increasing aging of the population, various age-related diseases such as cardiovascular and cerebrovascular diseases, neurodegenerative diseases, cancer and other chronic diseases are becoming more prevalent, bringing about a series of health, social and medical problems. "Healthy aging" has become an urgent public demand, and how to delay aging and develop functional foods with anti-aging effects has become a research hotspot in the life sciences. Some polysaccharides (such as fungal polysaccharides, wolfberry polysaccharides, and algae polysaccharides), unsaturated fatty acids (such as DHA and α-linolenic acid), and peptides (such as sea cucumber peptides, tilapia peptides, and glutathione) have been found to have excellent anti-aging effects. Among them, peptides have attracted much attention due to their good solubility, easy absorption, low allergenicity, and multiple physiological functions. Summary of the Invention

[0004] The purpose of the present invention is to provide a tyrosinase inhibitory peptide and its application.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A tyrosinase inhibitory peptide, whose amino acid sequences are shown in SEQ.ID.NO.1 (NFKLL), SEQ.ID.NO.2 (PFGLFP), SEQ.ID.NO.3 (YFPDHF), SEQ.ID.NO.4 (YFPFH) and SEQ.ID.NO.5 (QGRTLYGFGG).

[0007] The tyrosinase inhibitory peptide is prepared by a conventional synthesis method.

[0008] The tyrosinase inhibitory peptide has anti-aging activity and can be used to prepare cosmetics and medicines with anti-aging activity.

[0009] The present invention has the following advantages and effects compared to the prior art:

[0010] The present invention provides an active peptide composed of a new amino acid composition. Studies have shown that the active peptide of the present invention has good anti-aging activity; therefore, further using it as an active ingredient in the preparation of cosmetics or medicines with anti-aging activity has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a HPLC purity analysis chart of the active peptide with the amino acid sequence shown in SEQ ID NO.1 prepared by the method described in Example 1.

[0012] Figure 2 This is the mass spectrum of the active peptide with the amino acid sequence shown in SEQ ID NO.1 prepared by the method described in Example 1.

[0013] Figure 3 This is a HPLC purity analysis chart of the active peptide with the amino acid sequence shown in SEQ ID NO. 2 prepared by the method described in Example 2.

[0014] Figure 4 This is the mass spectrum of the active peptide with the amino acid sequence shown in SEQ ID NO. 2 prepared by the method described in Example 2.

[0015] Figure 5 This is a HPLC purity analysis chart of the active peptide with the amino acid sequence shown in SEQ ID NO. 3 prepared by the method described in Example 3.

[0016] Figure 6 This is the mass spectrum of the active peptide with the amino acid sequence shown in SEQ ID NO.3 prepared by the method described in Example 3.

[0017] Figure 7 This is a HPLC purity analysis chart of the active peptide with the amino acid sequence shown in SEQ ID NO. 4 prepared by the method described in Example 4.

[0018] Figure 8 This is the mass spectrum of the active peptide with the amino acid sequence shown in SEQ ID NO.4 prepared by the method described in Example 4.

[0019] Figure 9 This is a HPLC purity analysis chart of the active peptide with the amino acid sequence shown in SEQ ID NO.5 prepared by the method described in Example 5.

[0020] Figure 10 The mass spectrum of the active peptide with the amino acid sequence shown in SEQ ID NO.5 prepared by the method described in Example 5. DETAILED DESCRIPTION

[0021] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0022] Example 1: Synthesis of active peptides SEQ ID NO. 1 to 5

[0023] In this example, conventional solid-phase peptide synthesis was used to synthesize active peptides of the amino acid sequences (YFPFH) shown in SEQ ID NOs. 1 to 5.

[0024] 1. Solid phase peptide synthesis operation steps:

[0025] 1. Resin swelling

[0026] Weigh 3 g of 2-Chlorotrityl Chloride Resin (DN) with a degree of substitution of 0.84 mmol / g, place the resin in a reaction tube, add DCM (15 mL / g), and shake for 30 min.

[0027] 2. Connect the first amino acid

[0028] The solvent was removed by filtration through a sand core, and a 1.5-fold molar excess of Fmoc-L-His(Trt)-OH amino acid was added, followed by a 10-fold molar excess of DIEA. Finally, a small amount of DMF was added to dissolve the mixture, and the mixture was shaken for 1 hour. The mixture was then washed alternately with DMF and DCM 6 times.

[0029] 3. End Capping

[0030] Add a certain amount of methanol to the reaction solution to block the excess reaction sites to avoid affecting subsequent reactions.

[0031] 4. Deprotection

[0032] Add 15 mL of 20% piperidine DMF solution (15 mL / g) for 5 min, remove and add 15 mL of 20% piperidine DMF solution (15 mL / g) for 15 min.

[0033] 5. Testing

[0034] Drain the piperidine solution, take a dozen resin pellets, wash them three times with ethanol, add one drop each of ninhydrin, KCN, and phenol solution, heat at 105℃-110℃ for 5 minutes, and turn dark blue for a positive reaction.

[0035] 6. Wash

[0036] DMF (10 mL / g) twice, methanol (10 mL / g) twice, DMF (10 mL / g) twice

[0037] 7. Condensation

[0038] A three-fold excess of the protected amino acid (Fmoc-L-Trp(Boc)-OH) and a three-fold excess of HBTU, both dissolved in minimal DMF, were added to the reaction tube. Immediately, a ten-fold excess of NMM was added. The reaction was allowed to proceed for 30 min.

[0039] 8. Wash

[0040] DMF (10 mL / g) once, methanol (10 mL / g) twice, DMF (10 mL / g) twice

[0041] 9. Repeat steps 4 to 8, connecting the amino acids in the sequence from right to left.

[0042] 10. After the last amino acid is connected, deprotect it and wash the resin according to the following method.

[0043] DMF (10 mL / g) twice, methanol (10 mL / g) twice, DMF (10 mL / g) twice, DCM (10 mL / g) twice, and pump to dryness for 10 min.

[0044] 11. Cleavage of the Peptide from the Resin

[0045] Prepare cutting solution (10 mL / g): TFA 94.5%; water 2.5%; EDT 2.5%; TIS 1%

[0046] Place the resin in a flask or centrifuge tube with a resin to cutting fluid ratio of 10 mL / g and shake at a constant temperature for 120 minutes.

[0047] 12. Blow dry and wash

[0048] The lysate was blown dry with nitrogen as much as possible, separated by ether chromatography, washed with ether six times, and then evaporated to dryness at room temperature to obtain the crude peptide sequence.

[0049] 13. Purification of peptides by HPLC

[0050] Specific steps:

[0051] (1) Place 200 mg of the crude peptide in a container. Dissolve it in 2-5 mL of 50% acetonitrile solution. Sonicate gently for 2 minutes.

[0052] (2) Filter the solution using a 0.45 μm filter membrane.

[0053] (3) Analysis: Take 3 μL of the crude product and analyze it using analytical grade HPLC. The mobile phase is water and acetonitrile, the time is 30 minutes, and the gradient elution is performed. First, the HPLC is equilibrated with a starting gradient for 5 minutes before injection. The starting gradient is 95% water, 5% acetonitrile, and the ending ratio is 5% water, 95% acetonitrile (Note: HPLC: Chuangxin Tongheng LC3000)

[0054] (4) Preparation: Prepare the dissolved sample for injection. Equilibrate the preparative HPLC for 10 minutes, with a starting gradient of 95% water, 5% acetonitrile, and an ending gradient of 25% water, 75% acetonitrile over a 40-minute gradient. Collect the sample from the detector. (Note: HPLC: Beijing Qingbohua P1300)

[0055] (5) Identification: Take samples from the collected samples for purity and MS identification. (Note: watersZQ2000)

[0056] 14. Finally, the purified solution is freeze-dried to obtain the finished product.

[0057] 15. Analyze peptide purity. (Note: Waters 2695 liquid chromatography analyzer)

[0058] (1) Take 1 mg of white powdered polypeptide and dissolve it in an appropriate amount of H2O. If it is poorly water-soluble, it can be dissolved with the help of an appropriate amount of organic solvent.

[0059] (2) According to the length of the sequence, select the appropriate acetonitrile gradient analysis.

[0060] (3) If qualified, seal and store at -20℃.

[0061] 2. Structural identification method:

[0062] 2.1 Instrument parameters

[0063] 1) Instrument: Ultimate U3000 nano-Lumos three-in-one liquid chromatography-mass spectrometry instrument

[0064] 2) Chromatographic columns: Fillers are all 1.9 μm, pre-column: 2 cm x 100 μm; analytical column: 15 cm x 100 μm

[0065] 3) Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid, 80% acetonitrile

[0066] 4) Chromatographic gradient: 0-8 min 2% B; 8-9 min 2-10% B; 9-63 min 10-44% B; 63-68 min

[0067] 44-99% B. Flow rate: 300nL / min

[0068] 5) Mass spectrometry parameters:

[0069] Spray voltage: 2.2kV

[0070] Capillary temperature: 320℃

[0071] Level 1 scan: resolution 60,000, scanning range 350-1600 m / z

[0072] Secondary scan: resolution 15000, HCD collision energy: 30%

[0073] 2.2 Data Processing

[0074] The raw data in .raw format was converted to .mgf format using MSConvert software, and then the data was processed using pNovo software. The main parameters are:

[0075] 1) Fragmentation method: HCD

[0076] 2) Enzyme digestion: non-specific

[0077] 3) Mass error: 20 ppm for both primary and secondary spectra

[0078] 4) Modification: Oxidation (Met) is a variable modification.

[0079] Figures 1 to 10 They are HPLC purity analysis chart and mass spectrum chart of the polypeptides shown in SEQ ID NO.1 to 5 respectively.

[0080] Experimental Example 2: Analysis of the anti-aging activity of active peptides in vitro

[0081] Tyrosinase is the rate-limiting enzyme that regulates melanin production and can participate in the catalysis of tyrosine to generate L-DOPA and its product, dopaquinone. Appropriate melanin can protect the skin from the harm of external ultraviolet rays, but excessive accumulation can cause pigmentation, such as the generation of diseases such as liver spots, freckles, age spots, vitiligo, Alzheimer's disease and Parkinson's disease. Because current tyrosinase inhibitors (such as arbutin, azelaic acid and kojic acid) have poor solubility, stability, safety and effectiveness, clinical application is restricted. Therefore, it is necessary to further develop novel, safe and efficient tyrosinase inhibitors. The present invention adopts tyrosinase inhibition rate to evaluate the anti-aging activity of pigeon blood active peptide.

[0082] (1) Sample preparation and incubation of pigeon blood active peptides

[0083] 100 μL of 5 mg / mL pigeon blood active peptide sample was incubated with an equal volume of 125 U / mL tyrosine kinase (dissolved in 0.05 mol / L PBS, pH 6.8) at 25° C. for 5 min.

[0084] (2) Color reaction

[0085] After incubation, an equal volume of 10 mmol / L L-DOPA (dissolved in 0.05 mol / L PBS, pH 6.8) was added, and the resulting reaction mixture was incubated at 25°C for another 5 min.

[0086] (3) Determination of tyrosinase inhibition rate

[0087] The dopachrome content in the reaction mixture was determined by measuring the absorbance at 475 nm. The tyrosine kinase inhibitory activity was calculated as follows:

[0088]

[0089] Wherein: A is a tyrosinase mixture without sample; B is a mixture without sample and tyrosinase; C is a mixture of sample and tyrosinase; D is a mixture containing sample but without tyrosinase.

[0090] The samples to be tested are active peptides with amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5; the test results are shown in Table 1.

[0091] Table 1. Tyrosinase inhibition test results of the active peptides of the present invention

[0092] Pigeon blood active peptide Tyrosinase inhibition rate Active peptide of the amino acid sequence shown in SEQ ID NO.1 98.77％ Active peptide of the amino acid sequence shown in SEQ ID NO.2 98.32％ Active peptide of the amino acid sequence shown in SEQ ID NO.3 30.41％ Active peptide of the amino acid sequence shown in SEQ ID NO.4 30.15％ Active peptide of the amino acid sequence shown in SEQ ID NO.5 37.64％

[0093] As can be seen from the experimental results in Table 1, the active peptides with amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2 have higher tyrosinase inhibition rates, while the tyrosinase inhibition rates of SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 are relatively low compared to the first two, but the activity can be improved by increasing the concentration; the above results indicate that the active peptides described in the present invention all have good anti-aging activity.

[0094] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A tyrosinase inhibitory peptide, characterized in that The amino acid sequences are shown in SEQ.ID.NO.2 respectively.

2. Use of the tyrosinase inhibitory peptide according to claim 1 in the preparation of cosmetics and medicines.

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