Anti-wrinkle polypeptide, polypeptide-containing composition and application thereof

Abstract

The present invention relates to an anti-wrinkle polypeptide, a composition containing the polypeptide and an application thereof. The present invention obtains a nonapeptide-III active peptide having significant elastase inhibition activity and antioxidant activity by fermenting agrimony with Lactobacillus rhamnosus and Streptococcus thermophilus, filtering with Sephadex G-15 and separating and purifying with reversed-phase high-performance liquid chromatography. In addition to having good antioxidant and elastase inhibition activities, the composition containing the polypeptide also has the effects of increasing the improvement rate of wrinkles and reducing the wrinkle area ratio, thereby being able to achieve a good skin anti-wrinkle effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetic peptides, and in particular relates to an anti-wrinkle polypeptide, a composition containing the polypeptide and applications thereof. Background Art

[0002] The skin is the largest organ of the human body, which is directly exposed to the external environment and protects the internal organs through the barrier function of the epidermis. As the first line of defense, the skin must adapt to the consequences caused by solar radiation, atmospheric pollution, physical wounds and biological hazards - oxidative stress, cytotoxicity, inflammation, etc. Otherwise, it may develop accelerated aging, pigmentation, atopic dermatitis, psoriasis and skin cancer.

[0003] The medicinal use of crane herb has a long history. It has the effects of tonifying deficiency, astringing and stopping bleeding, stopping malaria and stopping dysentery, detoxifying and killing insects, and relieving pain. It is widely used in the treatment of respiratory, circulatory, digestive, endocrine, blood, tumor, gynecological and other related diseases. There are many chemical components in crane herb, such as flavonoids, triterpenes, tannins, phenols, volatile oils, esters, glycosides, organic acids, trace elements, etc. Among them, flavonoids and phenolic active substances are the most important pharmacological components of crane herb. Modern pharmacological studies have shown that crane herb has analgesic and anti-inflammatory, antioxidant, hemostatic, hypoglycemic, anti-tumor, platelet aggregation inhibition, insecticidal, anti-malarial, anti-arrhythmic, and blood pressure lowering effects.

[0004] Macromolecular proteins such as collagen and elastin are widely used in anti-aging cosmetics as raw materials for anti-wrinkle and firming, but these macromolecular proteins have poor transdermal absorption and are difficult to penetrate the stratum corneum of the skin for absorption. Small molecule peptides (mainly dipeptides and tripeptides) are easily absorbed transdermally. Peptides regulate cell proliferation and migration, inflammation, angiogenesis and protein synthesis in the skin. Current research mainly focuses on the antioxidant and anti-aging effects of macromolecular proteins such as collagen and elastin. The efficacy evaluation methods are mostly concentrated on biochemistry and cell biology experiments. There are few studies on the extraction of small molecule active peptides from the fermentation of Agrimony for antioxidant and anti-wrinkle effects. Summary of the invention

[0005] In order to solve the above problems, the present invention firstly provides an active peptide extracted from Agrimony foetus fermentation which can effectively inhibit the activity of elastase and has antioxidant activity, and an anti-wrinkle composition with significant anti-wrinkle effect.

[0006] The invention provides an anti-wrinkle polypeptide, the amino acid sequence of the polypeptide is AGSRLYCVI.

[0007] The invention also provides an anti-wrinkle composition, comprising active peptides and active substances extracted from fermented agrimony.

[0008] In certain embodiments, the active substance is selected from at least one of hyaluronic acid, ergothioneine and γ-aminobutyric acid.

[0009] The present invention also provides an application of the anti-wrinkle composition in the preparation of skin care products.

[0010] The invention also discloses a skin care product, comprising the anti-wrinkle composition or anti-wrinkle polypeptide.

[0011] Compared with the prior art, the present invention has at least the following beneficial effects:

[0012] The present invention obtains anti-wrinkle polypeptides from Agrimoniae fermentation for the first time, which have significant elastase inhibition activity and antioxidant activity;

[0013] The present invention provides an anti-wrinkle polypeptide composition for the first time, which has better anti-wrinkle effect than other anti-wrinkle polypeptide compositions in the prior art, mainly reflected in the improvement rate of wrinkles and the reduction of wrinkle area ratio. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Sephadex G-15 gel chromatography profile;

[0015] Figure 2 DPPH free radical scavenging test and elastase inhibition test of each component of A1, A2, A3 and A4;

[0016] Figure 3 Separation and Purification of Component A1 by Reversed-Phase High Performance Liquid Chromatography

[0017] Figure 4 DPPH free radical scavenging test and elastase inhibition rate test for groups I, II, and III;

[0018] Figure 5 LC-ESI-MS / MS was used to identify group III active peptides;

[0019] Figure 6 Analysis of the improvement rate of anti-wrinkle composition aqueous solution on wrinkles;

[0020] Figure 7 Analysis of the percentage of wrinkle area after using the anti-wrinkle composition aqueous solution. DETAILED DESCRIPTION

[0021] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0022] Example 1 Preparation and Identification of Active Peptides Extracted from Agrimonia Fermentation

[0023] Step 1 Preparation of starter culture: centrifuge Lactobacillus rhamnosus and Streptococcus thermophilus activated to logarithmic growth phase (8000r / min, 10min) and retain the bacteria, add an equal volume of 0.9% sterile saline to rinse three times, count by plate count method, and make the bacterial suspension concentration 10 7 CFU / mL.

[0024] Step 2 Preparation of fermented liquid: Weigh 40g of Agrimony, add 600mL of distilled water, soak overnight at 4°C, beat, liquefy for 40min at 90°C with α-amylase, and after pasteurization (80°C, 20min), cool to room temperature. According to the inoculum amount of 5% by mass fraction, draw the fermentation agent bacterial suspension (Lactobacillus rhamnosus: Streptococcus thermophilus = 1:2) into the Agrimony liquid with a pH value of 6.5, ferment in a 37°C biochemical incubator for 4d, centrifuge the fermented liquid at 8000r / min for 5min, and take the supernatant.

[0025] Step 3 Separation and purification of Sephadex G-15: 2 mL of the fermentation broth in step 2 was filtered through a filter membrane (0.45 μm) before loading, and separated and purified using a gel chromatography column Sephadex G-15 (1.6 cm × 60 cm), eluted with ultrapure water at a flow rate of 1.6 mL / min, and a Sephadex G-15 gel chromatography spectrum was obtained, see Figure 1 .

[0026] Figure 1 The results showed that four peaks were obtained after Sephadex G-15 separation of the fermentation supernatant of Agrimoniae truncatula, which were named A1, A2, A3 and A4.

[0027] Components A1, A2, A3, and A4 were freeze-dried respectively, and 100 mg / mL active peptide solution was prepared with PBS solution for DPPH free radical scavenging test and elastase inhibition rate test.

[0028] DPPH free radical scavenging test: 100 μL of 100 mg / mL active peptide sample solution of each component of A1, A2, A3, and A4 and 100 μL of DPPH solution (0.1 mmol / L) were mixed evenly, and reacted in a light-shielded water bath at 25°C for 30 min, and the absorbance at 517 nm was measured.

[0029] DPPH free radical scavenging rate = (1-(Ai-Aj) / A0) × 100%

[0030] Wherein: Ai is the absorbance of the sample solution and DPPH solution; Aj is the absorbance of the sample solution and PBS dilution; A0 is the absorbance of the PBS dilution and DPPH solution.

[0031] Elastase inhibition test: Take 15 μL of 100 mg / mL active peptide sample solution of each component of A1, A2, A3, and A4 and 85 μL of Tris-HCl buffer solution (50 mmol / L) with a pH of 8.0, mix thoroughly, then add 25 μL of elastase solution (60 mU / mL), mix well, and react for 15 min (25°C). Then add 25 μL of elastase substrate AAA-pNA solution (1.015 mmol / L), mix well, react for 15 min (25°C), and measure the absorbance at 410 nm.

[0032] Elastase inhibition rate = (1-A sample / A blank) × 100%

[0033] Wherein: A sample is the absorbance of sample solution, PBS substrate solution, Tris-HCl buffer and enzyme mixture; A blank is the absorbance of PBS substrate solution, Tris-HCl buffer and enzyme mixture.

[0034] Depend on Figure 2 It can be seen that A1 has a significant DPPH free radical scavenging rate and elastase inhibition rate compared with other components, which shows that the A1 sample can effectively inhibit the activity of elastase and has antioxidant activity, which can reduce the formation of wrinkles and delay aging.

[0035] Step 4: Reverse-phase high performance liquid chromatography separation and purification:

[0036] Fraction A1 was collected for further separation and loaded onto RP-HPLC (BioBasic-18, 250 mm × 4.6 mm, 5μm), mobile phase A was 0.1% trifluoroacetic acid-water solution, mobile phase B was 0.1% trifluoroacetic acid-acetonitrile solution, gradient elution program: 0-5min, 90% A; 5-20min, 90%-70% A; 21-30min, 50%-10% A; 35-40min, 10%-90% A. Injection 50μL, flow rate 0.8mL / min, column temperature 25℃, detection wavelength: 220nm, see Figure 3 .

[0037] Figure 3 The results showed that after RP-HPLC separation, three peaks were obtained for component A1, namely group I, group II and group III.

[0038] The components of group I, group II and group III were freeze-dried, and 100 mg / mL active peptide solution was prepared with PBS solution for DPPH free radical scavenging test and elastase inhibition rate test.

[0039] DPPH free radical scavenging test: 100 μL of 100 mg / mL active peptide sample solution of each component of group I, group II, and group III and 100 μL of DPPH solution (0.1 mmol / L) were mixed evenly, reacted in a light-shielded water bath at 25°C for 30 min, and the absorbance value at 517 nm was measured.

[0040] DPPH free radical scavenging rate = (1-(Ai-Aj) / A0) × 100%

[0041] Wherein: Ai is the absorbance of the sample solution and DPPH solution; Aj is the absorbance of the sample solution and PBS dilution; A0 is the absorbance of the PBS dilution and DPPH solution.

[0042] Elastase inhibition test: Take 15 μL of 100 mg / mL active peptide sample solution of each component of group I, group II, and group III and mix them thoroughly with 85 μL of Tris-HCl buffer solution (50 mmol / L) at pH 8.0, then add 25 μL of elastase solution (60 mU / mL), mix well, and react for 15 min (25°C). Then add 25 μL of elastase substrate AAA-pNA solution (1.015 mmol / L), mix well, react for 15 min (25°C), and measure the absorbance value at 410 nm.

[0043] Elastase inhibition rate = (1-A sample / A blank) × 100%

[0044] Wherein: A sample is the absorbance of sample solution, PBS substrate solution, Tris-HCl buffer and enzyme mixture; A blank is the absorbance of PBS substrate solution, Tris-HCl buffer and enzyme mixture.

[0045] Depend on Figure 4 It can be seen that group III has a significant DPPH free radical scavenging rate and elastase inhibition rate compared with other components, which shows that group III can effectively inhibit elastase activity and has antioxidant activity, and can further play a role in reducing wrinkles and delaying aging.

[0046] Step 5: LC-ESI-MS / MS was used to identify the active peptides of group III, and the secondary mass spectra and mass-to-charge ratio (m / z) of the active peptides were determined. The identification conditions were: mass scanning range 1200 m / z, drying gas flow rate 5 L / min, and drying gas temperature 150 °C.

[0047] Depend on Figure 5 It can be seen that the amino acid sequence of the active peptide of group III is AGSRLYCVI, labeled as nonapeptide-III.

[0048] Example 2 Study on the anti-wrinkle efficacy of the composition containing nonapeptide-III active peptide

[0049] Anti-wrinkle composition 1: acetyl tetrapeptide-2, thiolated hyaluronic acid, ergothioneine and gamma-aminobutyric acid, in a mass ratio of 1:1:1;

[0050] Anti-wrinkle composition 2: acetyl hexapeptide-8, thiolated hyaluronic acid, ergothioneine and gamma-aminobutyric acid, in a mass ratio of 1:1:1;

[0051] Anti-wrinkle composition 3: nonapeptide-III active peptide, thiolated hyaluronic acid, ergothioneine and gamma-aminobutyric acid, in a mass ratio of 1:1:1;

[0052] The anti-wrinkle composition 1-3, p-hydroxyacetophenone and 1,2-hexanediol are respectively added to deionized water and stirred evenly to prepare the corresponding anti-wrinkle composition aqueous solution 1-3; in the anti-wrinkle composition aqueous solution 1-3, the concentration of acetyl tetrapeptide-2 or modified acetyl tetrapeptide-2 is 0.1wt%, the concentration of p-hydroxyacetophenone is 0.5wt%, and the concentration of 1,2-hexanediol is 0.5wt%. 30 volunteers aged 40-60 are selected as testers to sign a written informed consent form; the anti-wrinkle composition aqueous solution 1-3 is applied to one side of the face of the tester, twice a day, once in the morning and once in the evening, for a total of 35 days, and the wrinkle depth (D0) of the tester before and after use and the wrinkle depth (D30) after 35 days of use are recorded, and then the improvement rate of wrinkles is calculated, and the calculation formula of the improvement rate of wrinkles is: improvement rate (%) = (D0-D30) / D0×100%.

[0053] Figure 6 It can be seen that after 30 days of use, the anti-wrinkle composition aqueous solutions 1-3 have an improvement effect on wrinkles, and the improvement rate is higher than 8%; the anti-wrinkle composition aqueous solution 3 containing the nonapeptide-III active peptide has a higher improvement rate on wrinkles than the anti-wrinkle composition aqueous solution 1 and the anti-wrinkle composition aqueous solution 2.

[0054] The wrinkle condition of the test subjects was analyzed by using Image-Pro Plus software to analyze the wrinkle area ratio of the Standard 2 light source facial images taken by VISIA-CR. The wrinkle area ratio represents the ratio of the wrinkle area to the analysis area, expressed as a percentage. The smaller the wrinkle area ratio, the less obvious the wrinkles in the image, and the better the anti-wrinkle effect of the sample. The wrinkle area ratio analysis results after using the anti-wrinkle composition aqueous agent 1-3 are shown in Figure 7 .

[0055] Figure 7 It can be seen that the wrinkle area ratio of the image in the fifth week after the use of the anti-wrinkle composition aqueous solution 3 was significantly reduced by 2.68% compared with the initial value, and the effect of reducing wrinkles was significantly better than that of the anti-wrinkle composition aqueous solution 1-2.

[0056] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An anti-wrinkle polypeptide, characterized in that The amino acid sequence of the polypeptide is AGSRLYCVI.

2. A composition containing the anti-wrinkle polypeptide according to claim 1, characterized in that: The composition further comprises an active substance, and the active substance is selected from at least one of hyaluronic acid substances, ergothioneine and gamma-aminobutyric acid.

3. Use of the anti-wrinkle polypeptide according to claim 1 in the preparation of anti-wrinkle skin care products.

4. Use of the composition according to claim 2 in the preparation of anti-wrinkle skin care products.

5. An anti-wrinkle skin care product, comprising the anti-wrinkle polypeptide according to claim 1 or the composition according to claim 2.

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