Active peptides with anti-aging effects and their applications

Abstract

The present application discloses an active peptide with anti-aging effect and its application. The amino acid sequences are 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) respectively. The said active peptide has anti-aging activity and can be used for preparing cosmetics and drugs with anti-aging activity. The present invention provides an active peptide with a brand-new amino acid composition. Research shows that the active peptide of the present invention has good anti-aging activity. Therefore, further using it as an active ingredient for preparing cosmetics or drugs with anti-aging activity has important application value.

Description

Technical Field

[0001] The present invention belongs to the field of bioactive peptides, and particularly relates to active peptides with anti-aging effects and their applications. Background Art

[0002] With the aggravation of population aging, various chronic diseases related to aging, such as cardiovascular and cerebrovascular diseases, neurodegenerative diseases, cancers, etc., have a high incidence, bringing a series of health, social and medical problems. "Healthy aging" has become an urgent need of the public. It has been found that some substances such as polysaccharides (such as fungal polysaccharides, wolfberry polysaccharides, algal polysaccharides), unsaturated fatty acids (such as DHA, α-linolenic acid, etc.) and polypeptides (such as sea cucumber polypeptides, tilapia polypeptides, glutathione, etc.) have good anti-aging effects. Among them, polypeptides have attracted much attention due to their good solubility, easy absorption, low allergenicity and various physiological functions. Summary of the Invention

[0003] The purpose of the present invention is to provide an active peptide with anti-aging effects and its application.

[0004] The purpose of the present invention is achieved by the following technical solutions:

[0005] An active peptide with anti-aging effects, whose amino acid sequences 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).

[0006] The said active peptide is prepared by a conventional synthesis method.

[0007] The said active peptide has anti-aging activity and can be used to prepare cosmetics and drugs with anti-aging activity.

[0008] The present invention has the following advantages and effects compared with the prior art:

[0009] The present invention provides an active peptide with a completely new amino acid composition. Research shows that the active peptide described in the present invention has good anti-aging activity; therefore, further using it as an active ingredient to prepare cosmetics or drugs with anti-aging activity has important application value. Brief Description of the Drawings

[0010] Figure 1 It is an HPLC purity analysis chart of the active peptide with the amino acid sequence shown as SEQ ID NO.1 prepared by the method described in Example 1.

[0011] Figure 2Mass spectrometry diagram of the bioactive peptide with the amino acid sequence shown in SEQ ID NO.1 prepared by the method described in Example 1.

[0012] Figure 3 HPLC purity analysis diagram of the bioactive peptide with the amino acid sequence shown in SEQ ID NO.2 prepared by the method described in Example 2.

[0013] Figure 4 Mass spectrometry diagram of the bioactive peptide with the amino acid sequence shown in SEQ ID NO.2 prepared by the method described in Example 2.

[0014] Figure 5 HPLC purity analysis diagram of the bioactive peptide with the amino acid sequence shown in SEQ ID NO.3 prepared by the method described in Example 3.

[0015] Figure 6 Mass spectrometry diagram of the bioactive peptide with the amino acid sequence shown in SEQ ID NO.3 prepared by the method described in Example 3.

[0016] Figure 7 HPLC purity analysis diagram of the bioactive peptide with the amino acid sequence shown in SEQ ID NO.4 prepared by the method described in Example 4.

[0017] Figure 8 Mass spectrometry diagram of the bioactive peptide with the amino acid sequence shown in SEQ ID NO.4 prepared by the method described in Example 4.

[0018] Figure 9 HPLC purity analysis diagram of the bioactive peptide with the amino acid sequence shown in SEQ ID NO.5 prepared by the method described in Example 5.

[0019] Figure 10 Mass spectrometry diagram of the bioactive peptide with the amino acid sequence shown in SEQ ID NO.5 prepared by the method described in Example 5. Detailed implementation manners

[0020] The present invention will be further described in detail below in conjunction with examples and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0021] Example 1: Synthesis of bioactive peptides SEQ ID NO.1 - 5

[0022] In this example, conventional solid-phase peptide synthesis method was used to synthesize the bioactive peptides with the amino acid sequences (YFPFH) shown in SEQ ID NO.1 - 5.

[0023] 1. Operating steps of solid-phase peptide synthesis:

[0024] I. Swelling of resin

[0025] Weigh 3 g of 2-Chlorotrityl Chloride Resin with a substitution degree of 0.84 mmol / g. Put the resin into a reaction tube, add DCM (15 mL / g), and shake for 30 min.

[0026] II. Attach the first amino acid

[0027] Filter off the solvent through a sintered glass funnel. Add 1.5-fold molar excess of Fmoc-L-His(Trt)-OH amino acid, then add 10-fold molar excess of DIEA, and finally add a small amount of DMF to dissolve. Shake for 1 h. Wash alternately with DMF and DCM for 6 times.

[0028] III. Capping

[0029] Add a certain amount of methanol to the reaction solution to block the excess reaction sites to avoid affecting subsequent reactions.

[0030] IV. Deprotection

[0031] Add 15 mL of 20% piperidine in DMF solution (15 mL / g), for 5 min, then remove and add another 15 mL of 20% piperidine in DMF solution (15 mL / g) for 15 min.

[0032] V. Detection

[0033] Filter off the piperidine solution. Take a dozen resin particles, wash them three times with ethanol, add one drop each of ninhydrin, KCN, and phenol solution, and heat at 105 °C - 110 °C for 5 min. A dark blue color change indicates a positive reaction.

[0034] VI. Washing

[0035] Wash twice with DMF (10 mL / g), twice with methanol (10 mL / g), and twice with DMF (10 mL / g)

[0036] VII. Condensation

[0037] Use three-fold excess of protected amino acid (Fmoc-L-Trp(Boc)-OH) and three-fold excess of HBTU. Dissolve them in as little DMF as possible, add them to the reaction tube, and immediately add ten-fold excess of NMM. React for 30 min.

[0038] VIII. Washing

[0039] Wash once with DMF (10 mL / g), twice with methanol (10 mL / g), and twice with DMF (10 mL / g)

[0040] IX. Repeat steps IV to VIII to sequentially attach the amino acids in the sequence from right to left.

[0041] X. After the last amino acid is coupled, deprotect and wash the resin as follows.

[0042] DMF (10 mL / g) twice, methanol (10 mL / g) twice, DMF (10 mL / g) twice, DCM (10 mL / g) twice, and drain for 10 min.

[0043] XI. Cleave the polypeptide from the resin

[0044] Prepare the cleavage solution (10 mL / g): 94.5% TFA; 2.5% water; 2.5% EDT; 1% TIS

[0045] Load the resin into a flask or centrifuge tube. The ratio of resin to cleavage solution is 10 mL / g. Shake at a constant temperature for 120 min

[0046] XII. Blow-dry and wash

[0047] Blow-dry the cleavage solution as much as possible with nitrogen, chromatograph with ether, wash with ether six times, and then evaporate to dryness at room temperature. The crude peptide sequence is obtained.

[0048] XIII. Purify the polypeptide by HPLC

[0049] Specific operating steps:

[0050] (1) Take 200 mg of the crude peptide and put it into a container. Dissolve it with 2 - 5 mL of 50% aqueous acetonitrile solution. Slightly sonicate for 2 min.

[0051] (2) Filter the dissolved solution through a 0.45 µm filter membrane.

[0052] (3) Analysis: Take 3 µL and analyze the crude product by analytical HPLC. The mobile phase is water and acetonitrile. The time is 30 min, with gradient elution. First, equilibrate the HPLC with the starting gradient for 5 min and then inject the sample. The starting gradient is 95% water and 5% acetonitrile, and the ending ratio is 5% water and 95% acetonitrile (Note: High-performance liquid chromatography, Innovent LC3000)

[0053] (4) Preparation: Prepare the dissolved sample for injection. Equilibrate the preparative HPLC for 10 min. The starting gradient is 95% water and 5% acetonitrile, and the ending gradient is 25% water and 75% acetonitrile. The gradient time is 40 min. Collect the sample coming out of the detector. (Note: High-performance liquid chromatography, Beijing Qingbohua P1300)

[0054] (5) Identification: Take a sample of the collected sample for purity and MS identification. (Note: Waters ZQ2000)

[0055] XIV. Finally, lyophilize the purified solution to obtain the finished product.

[0056] XV. Analyze the purity of the polypeptide. (Note: Waters 2695 liquid phase analyzer)

[0057] (1) Take 1 mg of the white powdery polypeptide and dissolve it in an appropriate amount of H2O. If the water solubility is poor, an appropriate amount of organic solvent can be used to assist in dissolution.

[0058] (2) Select an appropriate acetonitrile gradient for analysis according to the sequence length.

[0059] (3) If the analysis is qualified, seal and package it, and store it at -20 °C.

[0060] 2. Structure identification method:

[0061] 2.1 Instrument parameters

[0062] 1) Instrument: Ultimate U3000 nano-Lumos triple quadrupole liquid chromatography-mass spectrometry

[0063] 2) Chromatographic column: The packing material is 1.9 μm for both. Pre-column: 2 cm x 100 μm; Analytical column: 15 cm x 100 μm

[0064] 3) Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid, 80% acetonitrile

[0065] 4) Chromatographic gradient: 0 - 8 min 2% B; 8 - 9 min 2 - 10% B; 9 - 63 min 10 - 44% B; 63 - 68 min

[0066] 44 - 99% B. Flow rate: 300 nL / min

[0067] 5) Mass spectrometry parameters:

[0068] Spray voltage: 2.2 kV

[0069] Capillary temperature: 320 °C

[0070] Primary scan: Resolution 60000, scan range 350 - 1600 m / z

[0071] Secondary scan: Resolution 15000, HCD collision energy: 30%

[0072] 2.2 Data processing

[0073] Convert the raw data in.raw format to.mgf format file through MSConvert software, and then use pNovo software for data processing. The main parameters are:

[0074] 1) Fragmentation method: HCD

[0075] 2) Enzyme digestion: non-specific

[0076] 3) Mass error: 20 ppm for both the first-order spectrum and the second-order spectrum

[0077] 4) Modification: Oxidation(Met) is a variable modification.

[0078] Figures 1-10 They are respectively the HPLC purity analysis charts and mass spectrometry charts of the polypeptides shown in SEQ ID NO.1 - 5.

[0079] Experimental Example 2: Analysis of the anti - aging activity of active peptides in vitro

[0080] Tyrosinase is the rate - limiting enzyme that regulates melanin production and can participate in catalyzing tyrosine to form L - dopa and its product dopaquinone. Appropriate melanin can protect the skin from external ultraviolet damage, but excessive accumulation can lead to pigmentation disorders such as liver spots, freckles, senile plaques, vitiligo, Alzheimer's disease, and Parkinson's disease. Since the current tyrosinase inhibitors (such as arbutin, azelaic acid, and kojic acid) have poor solubility, stability, safety, and effectiveness, their clinical applications are limited. Therefore, it is necessary to further develop new, safe, and highly efficient tyrosinase inhibitors. In this invention, the tyrosinase inhibition rate is used to evaluate the anti - aging activity of pigeon blood active peptides.

[0081] (1) Sample preparation and incubation of pigeon blood active peptides

[0082] Mix 100 μL of pigeon blood active peptide sample with a concentration of 5 mg / mL with an equal volume of 125 U / mL tyrosinase (dissolved in 0.05 mol / L PBS, pH 6.8) and incubate at 25 °C for 5 min.

[0083] (2) Color reaction

[0084] After incubation, add an equal volume of 10 mmol / L L - DOPA (dissolved in 0.05 mol / L PBS, pH 6.8), and incubate the resulting reaction mixture at 25 °C for another 5 min.

[0085] (3) Determination of tyrosinase inhibition rate

[0086] The content of dopachrome in the reaction mixture is determined by detecting the absorbance at 475 nm. The calculation formula for tyrosinase inhibition activity is as follows:

[0087]

[0088] In the formula: A is the tyrosinase mixture without the sample; B is the mixture without the sample and tyrosinase; C is the mixture of the sample and tyrosinase; D is the mixture containing the sample but without tyrosinase.

[0089] The sample to be tested is an active peptide with the 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.

[0090] Table 1. Test results of tyrosinase inhibition rate of the active peptide of the present invention

[0091] Pigeon blood active peptide Tyrosinase inhibition rate Active peptide with the amino acid sequence shown in SEQ ID NO.1 98.77％ Active peptide with the amino acid sequence shown in SEQ ID NO.2 98.32％ Active peptide with the amino acid sequence shown in SEQ ID NO.3 30.41％ Active peptide with the amino acid sequence shown in SEQ ID NO.4 30.15％ Active peptide with the amino acid sequence shown in SEQ ID NO.5 37.64％

[0092] As can be seen from the experimental results in Table 1, the active peptides with the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2 have a relatively high tyrosinase inhibition rate, while the tyrosinase inhibition rates of SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 are relatively lower than the former 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.

[0093] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An application of an active peptide in the preparation of cosmetics and medicines for resisting skin aging, characterized in that: The amino acid sequence of the active peptide is shown in SEQ.ID.NO.1.

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