Polypeptides, compositions, and uses having anti-photoaging activity

Abstract

The application discloses a kind of polypeptides with anti-photoaging activity, composition and application, belong to polypeptide technical field.The amino acid sequence of polypeptide with anti-photoaging activity of the present application is at least one of AVSAANDPNGI, GNTELQAA.The present application is separated and purified from phycocyanin by enzymolysis, ultrafiltration, gel chromatography, liquid chromatography separation technology, and two polypeptides with anti-photoaging activity are obtained, these polypeptides and complex peptide segment all have good tyrosinase inhibitory activity, and the tyrosinase inhibitory activity of complex peptide segment is significantly better than single peptide segment.The polypeptide obtained in the present application has good application prospect in the preparation of products with anti-photoaging effect, and lays a foundation for high value-added utilization of phycocyanin and promotes the research and application of functional active peptides.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of polypeptides, and particularly relates to a polypeptide with anti-photoaging activity, a composition and application. BACKGROUND

[0002] Photoaging is a common type of skin aging caused by the sun, especially long-term UV radiation, mainly manifested in epidermal hyperplasia, skin laxity and darkening, and coarse wrinkles. Tyrosinase is an important rate-limiting enzyme in the melanin production reaction and is widely distributed in the human body. The accumulation of melanin in the human body can cause pigmented spots and malignant melanoma and other diseases, and reducing the activity of tyrosinase can achieve the effect of preventing skin diseases. However, the safety and practicality of many new tyrosinase inhibitors reported in recent years are poor, so relatively safe natural extracts have become the research direction for developing new tyrosinase inhibitors.

[0003] Phycocyanin is a functional protein in spirulina, which has the effects of anti-tumor, anti-inflammatory and anti-oxidation. However, as a biological macromolecule, phycocyanin has poor stability and is easy to be inactivated, and is not easy to be digested and absorbed by the human body, which limits its further application in the medical field.

[0004] Polypeptides are specific protein fragments that have a positive impact on the health of the body. Compared with complete proteins, small molecule peptide fragments have multiple advantages: they are more easily absorbed and utilized by the human body, have high activity, can play a physiological role even at low concentrations, have small molecular weights, are easy to modify and transform, and can be obtained through artificial chemical synthesis. Compared with single amino acids, small molecule peptides not only have special physiological activity, but also have unique advantages in absorption channels and speed.

[0005] Based on phycocyanin, small molecule polypeptides with anti-photoaging activity are developed, which lays a foundation for high-value utilization of phycocyanin and promotes the research and application of anti-photoaging activity peptide products. SUMMARY

[0006] In view of the problems in the prior art, the purpose of the present application is to provide a polypeptide with anti-photoaging activity, a composition and application.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] The polypeptide with anti-photoaging activity has at least one of the amino acid sequences AVSAANDPNGI and GNTELQAA.

[0009] The polypeptide composition with anti-photoaging activity has an effective component of a polypeptide with the amino acid sequence AVSAANDPNGI and GNTELQAA.

[0010] On the basis of the above scheme, the mass ratio of the AVSAANDPNGI and the GNTELQAA is 1:1.

[0011] The polypeptide composition with the anti-photoaging activity is applied to the preparation of a medicine with the anti-photoaging effect.

[0012] On the basis of the above scheme, the medicine further comprises a pharmaceutically acceptable excipient.

[0013] The advantages of the technical scheme of the present application are as follows:

[0014] The present application separates and purifies two polypeptides with the anti-photoaging activity from phycocyanin through enzymolysis, ultrafiltration, gel chromatography and liquid chromatography separation technology, and preliminarily identifies the components and sequences of the polypeptides through liquid chromatography-mass spectrometry technology. The polypeptide segments are chemically synthesized according to the identified peptide segment sequences, and the tyrosinase inhibitory activity of the single polypeptide segment and the complex polypeptide segment is detected. The polypeptides and the complex polypeptide segment all have good tyrosinase inhibitory activity, and the tyrosinase inhibitory activity of the complex polypeptide segment is significantly better than that of the single polypeptide segment. The polypeptides obtained by the present application have good application prospect in the preparation of products with the anti-photoaging effect, and lay a foundation for the high-value utilization of phycocyanin and the research and application of functional polypeptides. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Hydrolysis degree of different phycocyanin enzymolysis products;

[0016] Figure 2 Determination results of tyrosinase inhibitory rates of different phycocyanin enzymolysis products;

[0017] Figure 3 Electrophoresis analysis diagram of phycocyanin (lane 1) and products obtained by trypsin enzymolysis of phycocyanin (lane 2) (protein marker: molecular weight is 3.3-20.1 KDa);

[0018] Figure 4 Determination results of tyrosinase inhibitory rates of different molecular weight products obtained by trypsin enzymolysis of phycocyanin;

[0019] Figure 5 Superdex™ Increase 10 / 300 GL gel chromatogram of the component with a molecular weight less than 3 KDa;

[0020] Figure 6 Determination results of tyrosinase inhibitory rates of each component collected from the Superdex™ Increase 10 / 300 GL filtration;

[0021] Figure 7RP-HPLC chromatogram of the active ingredient of Superdex™ Increase 10 / 300 GL loaded on a C18 column;

[0022] Figure 8 Results of the determination of tyrosinase inhibition rate of each component collected by RP-HPLC;

[0023] Figure 9 Mass spectrometry analysis results of phycocyanin peptide AVSAANDPNGI;

[0024] Figure 10 Mass spectrometry analysis results of phycocyanin polypeptide GNTELQAA;

[0025] Figure 11 Molecular weight diagram of peptide AVSAANDPNGI mass spectrometry;

[0026] Figure 12 Molecular weight diagram of the GNTELQAA peptide by mass spectrometry;

[0027] Figure 13 Results of tyrosinase inhibition rate determination for chemically synthesized peptides AVSAANDPNGI, GNTELQAA, and the complex peptide (AVSAANDPNGI+GNTELQAA). Detailed Implementation

[0028] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and are not intended to limit the scope of the invention in any way.

[0029] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the experimental materials, reagents, and chemicals used in the following embodiments can be obtained through general channels.

[0030] Determination of the degree of hydrolysis of phycocyanin

[0031] The hydrolysis of phycocyanin was determined using the o-phthalaldehyde (OPA) method. The specific procedure is as follows: 80 mg of OPA was dissolved in 2 mL of β-mercaptoethanol, 5 mL of 10% SDS (w / v), and 92.8 mL of 0.1 M sodium tetraborate to prepare a 100 mL OPA reagent solution. Four mL of phycocyanin hydrolysates from different proteases were mixed with 4 mL of OPA reagent, incubated at room temperature for 2 min, and the absorbance was measured at 340 nm.

[0032] The phycocyanin was placed in 6M HC1 and reacted at 4°C for 24h. The amount of free amino acids was determined using a standard curve of serine as the amount of free amino acids produced by complete hydrolysis of phycocyanin.

[0033] The degree of hydrolysis of phycocyanin was calculated according to the following formula:

[0034] Degree of hydrolysis (%) = [(NH2) t - (NH2) t0 )] / (NH2) T

[0035] In the formula: (NH2) t represents the amount of free amino acids in the enzymatic product at t; (NH2) t0 represents the amount of free amino acids without enzymatic hydrolysis; (NH2) T represents the amount of free amino acids after complete hydrolysis.

[0036] The determination of tyrosinase inhibitory activity was carried out by the following steps:

[0037] First, mark the positions of A1, A2, A3 and A4 in the 96-well plate, and add 40 μL of sample solution (1 mg / mL) to A1 and A2, 40 μL of phosphate buffer to A3 and A4, 40 μL of tyrosinase (2 mg / mL) to A1 and A3, and 40 μL of phosphate buffer to A2 and A4, and mix in the wells of the 96-well plate, and shake gently for 30 s. After incubation at 37°C for 5 min, add 50 μL of L-tyrosine (0.2 mg / mL) to A1 and A3, and 50 μL of phosphate buffer to A2 and A4. To produce the maximum coloration of the reaction mixture, react for 25 min at the same temperature. Then measure the absorbance of the sample at 290 nm. Each sample was in triplicate. The inhibition of tyrosinase was determined by the residual activity of the negative control. The test sample was replaced with buffer solution, and the enzyme solution was replaced with blank buffer solution.

[0038] The anti-photoaging activity was calculated using the following formula:

[0039] Tyrosinase inhibitory activity (%) = [(A1-A2)-(A3-A4)] / (A1-A2)

[0040] Where A1 is the absorbance of the sample group containing tyrosinase, A2 is the absorbance of the sample group without tyrosinase, A3 is the PBS buffer containing tyrosinase, and A4 is the PBS buffer.

[0041] The phycocyanin used in the following examples was purchased from Zhejiang Binmei Biotechnology Co., Ltd.

[0042] Example 1

[0043] A polypeptide having anti-photoaging activity, an amino acid sequence of the polypeptide being shown in at least one of SEQ ID NO: 1~SEQ ID NO: 2.

[0044] SEQ ID NO: 1 AVSAANDPNGI

[0045] SEQ ID NO: 2 GNTELQAA

[0046] Example 2

[0047] A method for preparing a polypeptide having anti-photoaging activity can be directly synthesized by using an existing method such as a chemical synthesis method, or can be extracted and prepared by using the following method, and the specific steps are as follows:

[0048] (1) 5 g of phycocyanin powder was prepared into a protein solution with a concentration of 5% (w / v) using ultrapure water,

[0049] Trypsin was added to the prepared phycocyanin aqueous solution at an enzyme to substrate ratio of 1:100 (w / w), and the enzyme was hydrolyzed at 37°C for 6 h. During the process, 0.05 mol / L NaOH and HCl were used to adjust the pH value of the reaction system. After the enzyme hydrolysis reaction was completed, the enzyme hydrolysate was boiled at 100°C for 10 min to terminate the reaction. The enzyme hydrolysate was centrifuged at 6000 r / min at 4°C for 15 min, and the supernatant was reserved.

[0050] Effects of different proteases on the degree of hydrolysis of phycocyanin enzyme hydrolysate and the inhibition of tyrosinase activity related to anti-photoaging activity

[0051] Alkaline protease, papain, trypsin, pepsin, neutral protease and acid protease were used to hydrolyze phycocyanin, respectively. The temperature and pH of the enzyme hydrolysis were at the optimum temperature and pH of each protease (Table 1), and the other conditions were the same as those of trypsin. After the enzyme hydrolysis was completed, the effects of different proteases on the degree of hydrolysis (DH, %) of phycocyanin enzyme hydrolysate and the inhibition of tyrosinase activity related to anti-photoaging activity were determined. The results are shown in Table 2. Figure 1 The results show that the hydrolysis ability of trypsin is better Figure 1 , and the anti-photoaging efficacy of the hydrolysate is significant Figure 2 .

[0052] Table 1 Optimum temperature and pH of different proteases

[0053] Protease name Enzyme activity (U / g) Optimum temperature / °C Optimum pH Alkaline protease 2000000 50 10.5 Papain 100000 50 7.0 Trypsin 250000 37 7.5 Pepsin 250000 37 3.0 Neutral protease 50000 50 10.5 Acid protease 50000 50 3.0

[0054] (2) The supernatant obtained by trypsin digestion was fractionated into different molecular weights using ultrafiltration centrifuge tubes with a 10 kDa and 3 kDa molecular weight cutoff. Fractions were collected as follows: >10 kDa, peptides retained and did not pass through the 10 kDa membrane; 3-10 kDa, peptides permeated through the 10 kDa membrane but not the 3 kDa membrane; <3 kDa, peptides permeated through the 3 kDa membrane. Thus, proteolytic solutions with molecular weight ranges of <3 kDa, 3-10 kDa, and >10 kDa were obtained. Figure 3 ).

[0055] The different molecular weight components were assayed for their ability to inhibit tyrosinase activity associated with anti-photoaging activity. The results showed that the <3 kDa protein peptides had better effects on the inhibition of tyrosinase activity. Figure 4 .

[0056] (4) The <3 kDa proteolytic solution was further purified using a Superdex™ Increase 10 / 300 GL gel permeation column (10 x 300 mm). The column was eluted with deionized water (pH 7) and fractions were collected at a flow rate of 1 mL / min. Fractions were detected at 280 nm.

[0057] Three polypeptide components were collected in the order of the time of elution and were designated F1, F2, and F3. Figure 5 The three polypeptide components were assayed for their ability to inhibit tyrosinase activity. The results showed that the three components had the ability to inhibit tyrosinase activity. Figure 6 Among them, component F2 had the most significant effect.

[0058] (5) The component F2 obtained from the gel permeation chromatography was further purified using reverse phase HPLC on a C 18 column. A linear gradient of acetonitrile (0-40%) with 0.1% trifluoroacetic acid (TFA) was used at a flow rate of 1 mL / min. Elution peaks were detected at 215 nm and active peaks were concentrated using a rotary evaporator. The potent peaks were collected and lyophilized. The amino acid sequence of the final purified peptide was analyzed.

[0059] Five polypeptide components were collected in the order of the time of elution and were designated F2-I, F2-II, F2-III, F2-IV, and F2-V. Figure 7 The active peaks were concentrated using a rotary evaporator and were assayed for their ability to inhibit tyrosinase activity. The results showed that F2-I had a better effect on the inhibition of tyrosinase activity. Figure 8 .

[0060] (6) The amino acid sequence of F2-I was determined.

[0061] The polypeptide sample obtained in step (5) was dried by centrifugation, then redissolved in Nano-LC mobile phase A (0.1% formic acid / water) and bottled for loading, and subjected to online LCMS analysis. The liquid phase was an Easy nLC 1200 nanoliter liquid phase system, and the dissolved sample was loaded onto a nanoViper C18 pre-column (3 μm, 100 A) in a suitable volume, and then 20 μl was used for desalting. After desalting on the pre-column, the sample was separated by an analytical column, and the analytical column was a C18 reversed-phase column (Acclaim PepMap RSLC, 75 μm x 25 cm C18-2 μm 100 A). The gradient used in the experiment was that the mobile phase B (80% acetonitrile, 0.1% formic acid) was increased from 5% to 38% in 30 min. The mass spectrometer was a ThermoFisher Q Exactive system (ThermoFisher, USA) combined with a nanospray Nano Flex ion source (ThermoFisher, USA), with a spray voltage of 1.9 kV and an ion transmission tube heating temperature of 275°C. The mass spectrometry scanning mode was a data-dependent acquisition mode (DDA, Data Dependent Analysis), with a first mass spectrometry scanning resolution of 70000 and a scanning range of 350-2000 m / z, and a maximum injection time of 100 ms. A maximum of 20 secondary spectra with a charge of 2+ to 5+ were collected in each DDA cycle, and the maximum injection time of the secondary mass spectrometry ions was 50 ms. The collision chamber energy (high-energy collision-induced dissociation, HCD) was set to 28 eV, suitable for all precursor ions, and the dynamic exclusion setting was 25 s.

[0062] Two peptide segments were obtained (Table 2), and the secondary mass spectrometry analysis was as follows: a peptide with a molecular weight of 1028.4774, the sequence of which was AVSAANDPNGI Figure 9 ). Figure 10 .

[0063] Table 2 List of amino acid sequences determined by liquid chromatography-mass spectrometry for component F2-I

[0064] Peptide z m / z Mass Length AVSAANDPNGI 1 515.2480 1028.4774 11 GNTELQAA 1 402.2005 802.3831 8

[0065] The sequences AVSAANDPNGI and GNTELQAA in Table 2 above were chemically synthesized, and the mass spectrometry molecular weight graphs of the two obtained peptide segments are shown in Figure 11 and Figure 12 .

[0066] Example 3

[0067] A polypeptide composition with anti-photoaging activity, the effective component of the composition is AVSAANDPNGI peptide segment and GNTELQAA peptide segment, wherein the mass ratio of AVSAANDPNGI peptide segment and GNTELQAA peptide segment is 1:1.

[0068] The tyrosinase inhibiting ability of single peptide segment AVSAANDPNGI, GNTELQAA and complex peptide segment AVSAANDPNGI+GNTELQAA is determined.

[0069] The results are shown in Table 1, wherein the tyrosinase inhibiting effect of single peptide segment AVSAANDPNGI and GNTELQAA is 33.19% and 52.35% respectively. Figure 13 The tyrosinase inhibiting effect of complex peptide segment AVSAANDPNGI+GNTELQAA is 88.24%. It can be seen that the tyrosinase inhibiting effect of complex peptide segment is significantly better than that of single peptide segment, and has better anti-photoaging effect.

[0070] The above is only the preferred embodiment of the present application, and does not limit the other forms of the present application. Any skilled person in the art can use the disclosed technical content to make changes or modifications into equivalent embodiments. However, any simple modification, equivalent change and modification made on the above embodiments without departing from the technical solution of the present application, and according to the technical essence of the present application, still belongs to the protection scope of the technical solution of the present application.

Claims

1. A polypeptide with anti-photoaging activity, characterized in that, The amino acid sequence of the polypeptide with anti-photoaging activity is AVSAANDPNGI or GNTELQAA.

2. A polypeptide composition with anti-photoaging activity, characterized in that, The active ingredient in the polypeptide composition with anti-photoaging activity is a polypeptide with the amino acid sequences AVSAANDPNGI and GNTELQAA.

3. The polypeptide composition with anti-photoaging activity according to claim 2, characterized in that, The mass ratio of AVSAANDPNGI to GNTELQAA is 1:

1.

4. The use of the polypeptide composition with anti-photoaging activity according to any one of claims 2-3 in the preparation of a medicament with anti-photoaging activity.

5. The application according to claim 4, characterized in that, The drug also contains pharmaceutically acceptable excipients.

Images