Tyrosinase inhibiting peptide and use thereof
By preparing novel tyrosinase inhibitory peptides, the problems of poor solubility and stability of existing tyrosinase inhibitors have been solved, achieving highly effective anti-aging effects and enabling their application in cosmetics and pharmaceuticals.
Patent Information
- Application Number
- CN202510643364.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-26
- Publication Date
- 2026-01-02
- Estimated Expiration
- 2044-06-26
AI Technical Summary
Existing tyrosinase inhibitors have poor solubility, stability, and safety, which limits their application in the field of anti-aging.
A novel tyrosinase inhibitory peptide was developed, with amino acid sequences 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). It was prepared by conventional synthetic methods and used in the preparation of anti-aging cosmetics and pharmaceuticals.
The active peptides exhibit good anti-aging activity, especially SEQ.ID.NO.1 and SEQ.ID.NO.2, which have high tyrosinase inhibition rates and can effectively inhibit tyrosinase activity. They can be used in cosmetics and pharmaceuticals to delay aging.
Smart Images
Figure CN120665146B_ABST
Abstract
Description
[0001] This application is a divisional application of patent application No. 202410834916.0, filed on June 26, 2024, with the title of "Active Peptide with Anti-aging Effect and Application Thereof". TECHNICAL FIELD
[0002] The present application belongs to the field of bioactive peptides, and specifically relates to a tyrosinase inhibiting peptide and application thereof. BACKGROUND
[0003] With the aggravation of population aging, various diseases related to aging, such as chronic diseases such as cardiovascular and cerebrovascular diseases, neurodegenerative diseases, and cancer, have a high incidence, bringing a series of health, social, and medical problems. "Healthy aging" has become an urgent demand of the public, and how to delay aging and develop functional foods with anti-aging effect has become a research hotspot in the field of life sciences. It has been found that some polysaccharides (such as fungal polysaccharides, gynostemma pentaphyllum polysaccharides, and algal polysaccharides), unsaturated fatty acids (such as DHA and alpha-linolenic acid), and polypeptides (such as sea cucumber polypeptides, tilapia polypeptides, and glutathione) have good anti-aging effect. Among them, polypeptides are concerned due to their good solubility, easy absorption, low allergenicity, and multiple physiological functions. SUMMARY
[0004] The purpose of the present application is to provide a tyrosinase inhibiting peptide and application thereof.
[0005] The purpose of the present application is achieved by the following technical solutions.
[0006] A tyrosinase inhibiting peptide, the amino acid sequences of which 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), respectively.
[0007] The tyrosinase inhibiting peptide is prepared by a conventional synthesis method.
[0008] The tyrosinase inhibiting peptide has anti-aging activity and can be used for preparing cosmetics and drugs with anti-aging activity.
[0009] The present application has the following advantages and effects compared with the prior art.
[0010] The present application provides an active peptide with a novel amino acid composition. Research shows that the active peptide has good anti-aging activity. Therefore, it has important application value to use the active peptide as an active ingredient for preparing cosmetics or drugs with anti-aging activity. BRIEF DESCRIPTION OF DRAWINGS
[0011] Figure 1 HPLC purity profile of the active peptide of the amino acid sequence as depicted in SEQ ID NO. 1 prepared according to the method described in Example 1.
[0012] Figure 2 Mass spectrum of the active peptide of the amino acid sequence as depicted in SEQ ID NO. 1 prepared according to the method described in Example 1.
[0013] Figure 3 HPLC purity profile of the active peptide of the amino acid sequence as depicted in SEQ ID NO. 2 prepared according to the method described in Example 2.
[0014] Figure 4 Mass spectrum of the active peptide of the amino acid sequence as depicted in SEQ ID NO. 2 prepared according to the method described in Example 2.
[0015] Figure 5 HPLC purity profile of the active peptide of the amino acid sequence as depicted in SEQ ID NO. 3 prepared according to the method described in Example 3.
[0016] Figure 6 Mass spectrum of the active peptide of the amino acid sequence as depicted in SEQ ID NO. 3 prepared according to the method described in Example 3.
[0017] Figure 7 HPLC purity profile of the active peptide of the amino acid sequence as depicted in SEQ ID NO. 4 prepared according to the method described in Example 4.
[0018] Figure 8 Mass spectrum of the active peptide of the amino acid sequence as depicted in SEQ ID NO. 4 prepared according to the method described in Example 4.
[0019] Figure 9 HPLC purity profile of the active peptide of the amino acid sequence as depicted in SEQ ID NO. 5 prepared according to the method described in Example 5.
[0020] Figure 10 Mass spectrum of the active peptide of the amino acid sequence as depicted in SEQ ID NO. 5 prepared according to the method described in Example 5. DETAILED DESCRIPTION
[0021] The application will be further described with reference to the following examples and drawings, but the embodiments of the application are not limited thereto.
[0022] Example 1: Synthesis of active peptides SEQ ID NO. 1-5
[0023] The embodiment adopts the conventional solid-phase polypeptide synthesis method to synthesize the active peptide of the amino acid sequence (YFPFH) shown in SEQ ID NO. 1-5.
[0024] 1. The solid-phase polypeptide synthesis operation steps are as follows:
[0025] I. Resin swelling
[0026] 3g of 2-Chlorotrityl Chloride Resin resin with a substitution degree of 0.84 mmol / g is weighed, the resin is placed in a reaction tube, DCM (15 mL / g) is added, and oscillation is performed for 30 min.
[0027] II. Adding the first amino acid
[0028] The solvent is filtered out through a sand core, 1.5 times the molar excess of Fmoc-L-His (Trt)-OH amino acid is added, 10 times the molar excess of DIEA is added, a small amount of DMF is finally added for dissolution, oscillation is performed for 1 h, and washing is performed with DMF and DCM alternately for 6 times.
[0029] III. Capping
[0030] A certain amount of methanol is added to the reaction solution. The excess reaction sites are closed to avoid affecting the subsequent reaction.
[0031] IV. Deprotection
[0032] 15 mL of 20% piperidine DMF solution (15 mL / g) is added, 5 min, and 15 ml of 20% piperidine DMF solution (15 mL / g) is added again, 15 min.
[0033] V. Detection
[0034] The piperidine solution is filtered out, a dozen resin particles are taken, washed with ethanol for three times, one drop of ninhydrin, KCN, and phenol solution is added, heating is performed at 105-110°C for 5 min, and dark blue color is taken as a positive reaction.
[0035] VI. Washing
[0036] DMF (10 mL / g) twice, methanol (10 mL / g) twice, and DMF (10 mL / g) twice
[0037] VII. Condensation
[0038] The protected amino acid (Fmoc-L-Trp (Boc)-OH) is three times excess, HBTU is three times excess, both are dissolved in as little DMF as possible, added to the reaction tube, and NMM is added immediately ten times excess. Reaction is performed for 30 min.
[0039] VIII. Washing
[0040] DMF (10 mL / g) once, methanol (10 mL / g) twice, DMF (10 mL / g) twice
[0041] IX. Repeat steps IV to VIII, connecting the amino acids in the sequence from right to left.
[0042] X. After the last amino acid is coupled, deprotect the resin and wash the resin according to the following procedure.
[0043] DMF (10 mL / g) twice, methanol (10 mL / g) twice, DMF (10 mL / g) twice, DCM (10 mL / g) twice, 10 min under vacuum.
[0044] XI. Cleavage of the polypeptide from the resin
[0045] Prepare the cleavage solution (10 mL / g) TFA 94.5%; water 2.5%; EDT 2.5%; TIS 1%
[0046] Load the resin into a flask or centrifuge tube, resin to cleavage solution ratio 10 mL / g, constant temperature shaking, time: 120 min
[0047] XII. Blow dry the wash
[0048] Blow the cleavage solution dry with nitrogen, chromatograph the cleavage solution with ether, then wash with ether six times, then dry at room temperature. The crude peptide sequence is obtained.
[0049] XIII. Purify the polypeptide using HPLC
[0050] Specific operation steps:
[0051] (1) Take 200 mg of the crude peptide and dissolve it in a beaker with 2-5 mL of 50% aqueous acetonitrile solution. It can be slightly ultrasonicated for 2 min.
[0052] (2) Filter the dissolved solution with a 0.45 um filter membrane.
[0053] (3) Analysis: Take 3 uL of the crude product and analyze it using analytical HPLC. The mobile phase is water and acetonitrile, time 30 min, gradient elution, first equilibrate the HPLC with the starting gradient for 5 min, then inject the sample, starting gradient water 95%, acetonitrile 5%, ending gradient water 5%, acetonitrile 95% (Note: High Performance Liquid Chromatography, Beijing Qingbohua P1300)
[0054] (4) Preparation: Prepare the sample for injection. Equilibrate the preparative HPLC for 10 min, starting gradient water 95%, acetonitrile 5%, ending gradient water 25%, acetonitrile 75%, gradient time 40 min. Collect the sample from the detector. (Note: High Performance Liquid Chromatography, Beijing Qingbohua P1300)
[0055] (5) Identification: The collected sample was taken for purity and MS identification. (Note: waters ZQ2000)
[0056] XIV. Finally, the purified solution was lyophilized to obtain the finished product.
[0057] XV. Analysis of polypeptide purity. (Note: waters 2695 liquid chromatograph)
[0058] (1) Polypeptide in white powder form, 1 mg, appropriate amount of H2O was dissolved, if water solubility is poor, appropriate amount of organic solvent can be used for dissolution.
[0059] (2) According to the length of the sequence, select appropriate acetonitrile gradient analysis.
[0060] (3) Analysis qualified, sealed packaging, -20 degrees storage.
[0061] 2, structure identification method:
[0062] 2.1 Instrument parameters
[0063] 1) Instrument: Ultimate U3000 nano-Lumos three-in-one liquid chromatograph-mass spectrometer
[0064] 2) Column: filler is 1.9 μm, pre-column: 2 cm x 100 μm; analysis column: 15 cm x 100 μm
[0065] 3) Mobile phase A: 0.1% formic acid 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: 300 nL / min
[0068] 5) Mass spectrometry parameters:
[0069] Spray voltage: 2.2 kV
[0070] Capillary temperature: 320℃
[0071] Primary scan: resolution 60000, scan 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 into.mgf format file by MSConvert software, and then data processing was performed using pNovo software. The main parameters were as follows:
[0075] 1) Fragmentation mode: HCD
[0076] 2) Enzyme: non-specific
[0077] 3) Mass error: 20ppm for both primary and secondary spectra
[0078] 4) Modification: Oxidation (Met) as variable modification.
[0079] Figures 1-10 The HPLC purity analysis graph and mass spectrum graph of the polypeptides shown in SEQ ID NO. 1-5, respectively.
[0080] Experimental Example 2: Analysis of the anti-aging activity of the active peptide in vitro
[0081] Tyrosinase is a rate-limiting enzyme that regulates melanin production and can participate in catalyzing the generation of L-dopa and its product, dopaquinone. Appropriate melanin can protect the skin from external ultraviolet light, but excessive accumulation can lead to pigmentation, such as the occurrence of liver spots, freckles, age spots, vitiligo, Alzheimer's disease, and Parkinson's disease. Since the solubility, stability, safety, and effectiveness of current tyrosinase inhibitors (such as arbutin, azelaic acid, and kojic acid) are poor, their clinical application is limited. Therefore, it is necessary to further develop new, safe, and efficient tyrosinase inhibitors. The tyrosinase inhibition rate is used to evaluate the anti-aging activity of the pigeon blood active peptide.
[0082] (1) Sample preparation and incubation of pigeon blood active peptide
[0083] 100 μL of pigeon blood active peptide sample with a concentration of 5 mg / mL was mixed with an equal volume of 125 U / mL tyrosinase (dissolved in 0.05 mol / L PBS, pH 6.8), and incubated 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 content of dopa pigment in the reaction mixture was determined by detecting the absorbance at 475 nm. The formula for calculating the tyrosinase inhibition activity is as follows:
[0088]
[0089] In the formula: 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 not containing tyrosinase.
[0090] The sample to be tested is an active peptide of the amino acid sequence 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 rate test results of the active peptide of the present application
[0092] Active peptide of pigeon blood 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 peptide of the amino acid sequence shown in SEQ ID NO. 1 and SEQ ID NO. 2 has a higher tyrosinase inhibition rate, while the tyrosinase inhibition rate of SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5 is relatively lower than the former two, but the activity can be improved by increasing the concentration; the above results show that the active peptide of the present application has good anti-aging activity.
[0094] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.
Claims
1. A tyrosinase-inhibiting peptide, characterized in that The amino acid sequence is shown as SEQ ID NO.
2.
2. Use of the tyrosinase-inhibiting peptide of claim 1 in the preparation of cosmetics and pharmaceuticals for combating skin aging.