Anti-aging earthworm polypeptide as well as preparation method and application thereof

Abstract

The invention relates to the field of polypeptides, in particular to an anti-aging earthworm polypeptide as well as a preparation method and application thereof. The earthworm polypeptide disclosed by the invention comprises at least one of sequences shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, has a remarkable effect in the aspects of resisting oxidation and promoting collagen generation, has a remarkable anti-aging effect, and can be widely applied to preparation of anti-aging cosmetics, skin care products, health care products and medicines.

Description

Technical Field

[0001] The present invention relates to the field of polypeptides, and in particular to an anti-aging earthworm polypeptide and a preparation method and application thereof. Background Art

[0002] Earthworms, also known as earth dragons, are rich in functional ingredients such as protein, amino acids, trace elements, enzymes, hormones, etc. The protein content of dried earthworms reaches 70%. They are the raw material for preparing medicines, cosmetics, health foods, etc. and have great economic value.

[0003] Earthworm polypeptide is a bioactive substance with multiple physiological effects and functions. It is widely used in the fields of medicine, health products and agriculture, and shows significant therapeutic effects and promotion. In medicine, earthworm polypeptide can enhance human immunity, promote the production and activation of white blood cells, and increase the resistance of immune cells. In addition, earthworm polypeptide also has antibacterial, antiviral and anti-tumor properties. It can inhibit the growth of bacteria and viruses, reduce the occurrence of infection and disease, and is currently often used in the preparation of cardiovascular and anti-tumor drugs.

[0004] Earthworm peptides also have a unique role in beauty and health care. Earthworm peptides can promote the production and repair of collagen, increase the elasticity and firmness of the skin. They can also reduce wrinkles, spots and acne scars and other skin problems, making the skin younger and healthier. In addition, earthworm peptides also have moisturizing and anti-oxidation effects, effectively preventing skin dryness and aging, making the skin more tender and shiny.

[0005] However, earthworm polypeptide is a general term for a class of peptides, and there are few reports on which specific polypeptides in earthworm polypeptide play a role. Summary of the invention

[0006] In view of the shortcomings and deficiencies of the prior art, the present invention provides an anti-aging earthworm polypeptide and a preparation method and application thereof. The specific scheme is as follows:

[0007] The first aspect of the present invention provides an anti-aging earthworm polypeptide, the polypeptide comprising at least one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. The present invention also provides a nucleotide encoding an earthworm polypeptide having an amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, the nucleotide sequence comprising a nucleotide sequence that has been disclosed and a nucleotide sequence optimized according to actual needs.

[0008] The present invention further provides a biomaterial, wherein the biomaterial is selected from any one of the following 1) to 2):

[0009] 1) A vector having a nucleotide encoding an earthworm polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4;

[0010] 2) A recombinant cell capable of producing one or more of the earthworm polypeptides with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, and / or a recombinant cell containing nucleotides encoding the earthworm polypeptides with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0011] The second aspect of the present invention provides the use of the polypeptide or the nucleotide or the biomaterial in anti-oxidation and / or promoting collagen production. Preferably, the collagen is type I collagen.

[0012] The present invention also provides the use of the polypeptide or the nucleotide or the biological material in the preparation of anti-aging cosmetics, skin care products, health products and medicines.

[0013] The third aspect of the present invention provides a composition, characterized in that the composition comprises the earthworm polypeptide.

[0014] Furthermore, the present invention also provides the use of the composition in anti-oxidation and / or promoting collagen production. Preferably, the collagen is type I collagen.

[0015] Furthermore, the present invention also provides the use of the composition in the preparation of anti-aging cosmetics, skin care products, health care products, and medicines.

[0016] The last aspect of the present invention provides a method for preparing the earthworm polypeptide, the method comprising the following steps:

[0017] (1) extracting earthworms and pure water at a ratio of 1:12 to 1:24 at 30 to 40° C. for 14 to 20 hours to obtain a solution containing earthworm extracts. In a specific embodiment of the present invention, extracting earthworms and pure water at a ratio of 1:20 at 35° C. for 16 hours to obtain a solution containing earthworm extracts;

[0018] (2) filtering the solution containing the earthworm extract obtained in step (1) through an ultrafiltration membrane with a molecular weight cutoff of less than or equal to 3000 Daltons, and then freeze-drying to obtain earthworm polypeptides.

[0019] Further, the earthworm is selected from at least one of Eisenia fetida, Pheretima leucoderma, Pheretima williamica, Pheretima philadelphica, and Pheretima zhiliensis. Preferably, the earthworm is Eisenia fetida.

[0020] The beneficial effects of the present invention include but are not limited to:

[0021] The earthworm polypeptide prepared by the present invention has a simple preparation method, is highly safe, has obvious anti-oxidation and / or collagen production promotion and anti-oxidation effects, can effectively promote the growth and proliferation of human skin fibroblasts and promote the expression of type I collagen. It has obvious anti-aging effects and can be widely used in the preparation of anti-aging cosmetics, skin care products, foods, and medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0023] Figure 1 is the mass spectrum of QY-1 in Example 2 of the present invention;

[0024] Figure 2 is the mass spectrum of QY-2 in Example 2 of the present invention;

[0025] Figure 3 is the mass spectrum of QY-3 in Example 2 of the present invention;

[0026] Figure 4 It is the mass spectrum of QY-4 in Example 2 of the present invention. DETAILED DESCRIPTION

[0027] The present invention is described in detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present invention are purchased through commercial channels.

[0028] Example 1 Isolation and Extraction of Earthworm Polypeptides

[0029] (1) washing fresh earthworms, extracting the earthworms and pure water at a ratio of 1:20 at 35° C. for 16 hours to obtain a solution containing earthworm extracts, wherein the earthworms are selected from at least one of Eisenia fetida, Pheretima leucoderma, Pheretima wilmii, Pheretima genus, and Pheretima zhiliensis;

[0030] (2) The solution containing the earthworm extract obtained in step (1) is filtered through an ultrafiltration membrane with a molecular weight cutoff of less than or equal to 2500 Da, and then freeze-dried to obtain earthworm polypeptides.

[0031] Example 2 Identification of earthworm polypeptides

[0032] (1) UPLC analysis

[0033] UPLC conditions were as follows:

[0034] Instrument: Waters ACQUITY UPLC ultra-high performance liquid chromatography-electrospray-quadrupole-time-of-flight mass spectrometer; chromatographic column specifications: BEH C18 chromatographic column; flow rate: 0.5 mL / min; temperature: 48°C; UV detection wavelength: 210 nm; injection volume: 2.5 μL.

[0035] Gradient conditions: Solution A: water containing 0.1% formic acid (v / v), Solution B: acetonitrile containing 0.1% formic acid (v / v). The specific liquid phase gradient is shown in Table 1.

[0036] Table 1

[0037] Time(min) ％A ％B 0 90.0 10.0 1.50 75.0 25.0 3.50 60.0 40.0 5.00 40.0 60.0 7.50 20.0 80.0 8.50 0.0 100 10.00 0.0 100 10.50 90.0 10.0 12.00 90.0 10.0

[0038] 2) Mass spectrometry analysis

[0039] The mass spectrometry conditions were as follows:

[0040] Ion mode: ES+; mass range (m / z): 100-1000; capillary voltage (kV): 3.0; sampling cone (V): 35.0; ion source temperature (℃): 115; desolvation temperature (℃): 350; desolvation gas flow (L / hr): 700.0; collision energy (eV): 4.0; scan time (sec): 0.25; internal scan time (sec): 0.02.

[0041] According to the above analysis method, the earthworm polypeptide obtained in Example 1 was subjected to chromatography and mass spectrometry analysis using ultra-high performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight mass spectrometry.

[0042] 3) Results

[0043] The mass spectrum of earthworm peptides is Figure 1-Figure 4 As shown, according to the breakage of az and by, the sequence of the earthworm polypeptide was obtained through analysis and calculation with Mascot software as shown in Table 2.

[0044] Table 2

[0045] serial number Sequence ID M / Z Molecular weight sequence QY-1 SEQ ID NO: 1 1925.0344 1925.0344 SPVLP SDGPR LCAEL TRL QY-2 SEQ ID NO: 2 1513.7931 1513.7931 LVMVL PIGSE EQAQ QY-3 SEQ ID NO: 3 1799.8366 1799.8366 YESYQ VLSNH FVDSL QY-4 SEQ ID NO: 4 1382.7162 1382.7162 PENVV ANDII LGE

[0046] Example 3 Antioxidant Experiment of Earthworm Peptide

[0047] 1) Experimental reagents and instruments:

[0048] Reagents: Experimental animals: ICR mice (male, 5 weeks old), Shanghai Experimental Animal Center; D-gal, Sinopharm Chemical Reagent Co., Ltd.; paraformaldehyde, Sinopharm Chemical Reagent Co., Ltd.; sodium chloride, Sinopharm Chemical Reagent Co., Ltd.; earthworm polypeptide obtained in Example 2, chemically synthesized by Gill Biochemical (Shanghai) Co., Ltd.; BCA protein kit, Nanjing Keygen Biotechnology Co., Ltd.; MDA lipid peroxide kit, Nanjing Keygen Biotechnology Co., Ltd.; SOD superoxide dismutase kit, Nanjing Jiancheng Biotechnology Co., Ltd.; T-AOC total antioxidant capacity kit, Nanjing Jiancheng Biotechnology Co., Ltd.

[0049] Instruments and equipment: CM-230 Moore ultrapure water, Shanghai Moore Scientific Instrument Co., Ltd.; MilliporeMILLEX GP0.22μm filter membrane, Millipore Corporation, USA; GL-22M high-speed refrigerated centrifuge, Shanghai Lu Xiangyi Centrifuge Instrument Co., Ltd.

[0050] 2) Experimental methods

[0051] (1) Animal aging model

[0052] After one week of adaptive feeding, ICR mice were divided into 4 groups, 6 mice in each group. Group 1 was a low-dose gavage group, in which mice were subcutaneously injected with D-gal at a dose of 500 mg / kg in the neck and back every day, and earthworm polypeptides QY-1, QY-2, QY-3, and QY-4 were gavaged at a dose of 1 mg / mouse / day; Group 2 was a high-dose gavage group, in which mice were subcutaneously injected with D-gal at a dose of 500 mg / kg in the neck and back every day, and earthworm polypeptides QY-1, QY-2, QY-3, and QY-4 were gavaged at a dose of 3 mg / mouse / day; Group 3 was a blank group, and mice grew normally; Group 4 was an animal model group, in which mice were subcutaneously injected with D-gal at a dose of 500 mg / kg in the neck and back every day, and were gavaged with 0.9% saline; the injection cycle of D-gal and the gavage cycle of polypeptides were both 42 days. The litter was replaced every 3 days, and the supply of feed and distilled water was guaranteed. The mice were weighed every five days, and D-gal injection solution was prepared according to the weight of the mice. The D-gal injection solution was filtered through a 0.22 μm syringe filter membrane to ensure sterility.

[0053] (2) Obtaining animal organs

[0054] After the experimental cycle is completed, the blood of the mouse is obtained by removing the eyeball and bleeding. After obtaining the blood, the mouse is killed by dislocating the neck, and then the body of the mouse is placed on a low-temperature ice box, and the brain, spleen, liver and kidney of the mouse are quickly removed. The organs obtained are placed in a pre-sterilized 1.5mL centrifuge tube, and all organ samples are stored in a -80℃ refrigerator for inspection. The removed mouse spleen is directly immersed in a pre-prepared 4% paraformaldehyde solution to fix its morphology. Paraformaldehyde powder is difficult to dissolve, and a trace amount of sodium bicarbonate can be added to adjust the pH value to alkaline to aid dissolution. The preparation of the paraformaldehyde solution needs to be completed in a fume hood.

[0055] (3) Sample testing

[0056] All organs to be tested were ground under low temperature and diluted into 10% tissue homogenate with 4°C sterile PBS solution. After centrifugation at 4000g at 4°C, the supernatant was aspirated and the precipitate was discarded. The test was performed according to the instructions of the kit or placed in a -80°C refrigerator for testing.

[0057] 3) Experimental results and analysis: The experimental results are shown in Tables 3 to 5.

[0058] Table 3

[0059]

[0060]

[0061] As shown in Table 3, compared with the mice in the animal model group, the SOD content in the liver and kidney of the mice in the polypeptide gavage group (Group 1 and Group 2) showed a significant increase. This means that although the mice in the polypeptide gavage group were stimulated by long-term large doses of D-gal, even excessive injection of D-gal did not completely destroy the SOD enzyme system in the mice, indicating that during the injection period, the experimental animals were continuously stimulated by aging factors, which would lead to a decrease in the SOD content in different organs, but at the same time, the intake of a certain amount of earthworm polypeptide had a certain protective effect on oxidative damage in mice.

[0062] Table 4

[0063]

[0064] As shown in Table 4, the MDA content in the liver of the mice in the animal model group was 27.45±5.93nmol / L. Compared with the animal model group, the MDA content in the liver of the two groups of mice gavaged with polypeptides showed significant differences. Since MDA can be used to estimate the accumulation of lipid peroxides in animals, it can be seen that in the process of forming the aging model of the mice in the animal model group, due to the long-term injection of excessive D-gal, the sugar metabolism pathway of the mice was disordered, a large number of free radicals were produced, and oxidative damage was caused. A large number of lipid peroxides appeared in their liver tissues. As a lipid peroxide, the increase in the content of MDA in the animal body can reflect the decrease in the activity of the antioxidant enzyme system in the mice from the side. The significant decrease in the MDA content in the liver of the mice in the polypeptide gavage group shows that the intake of earthworm polypeptides can effectively protect important tissues and organs from the stimulation of adverse factors to produce a large number of lipid peroxides.

[0065] Table 5

[0066]

[0067] As shown in Table 5, the liver T-AOC value of the mice in the animal model group was 0.59±0.16U / mgprot. Compared with the model group, the mice in the high-dose and low-dose gavage groups of the peptide showed significant differences; the kidney T-AOC content of the mice in the blank group was 0.56±0.23U / mgprot. Compared with the mice in the animal model group, the low-dose gavage group showed significant differences, and the high-dose gavage group also showed significant differences. This result shows that during the entire experimental period, due to the continuous stimulation of aging factors by the experimental animals, the liver and kidney tissues of the mice in the animal model group were damaged, resulting in a decrease in their total antioxidant capacity. Compared with the animal model group and the blank group, the total antioxidant capacity of the main organs of the mice in the peptide gavage group was always maintained at a higher level during the stimulation of aging factors, indicating that the intake of earthworm peptides enables the animal body and its main organs to have a higher self-protection function.

[0068] Example 4 Experiment on promoting collagen production by earthworm polypeptide

[0069] (1) Cell seeding: 8×10 4 Cells were seeded into 24-well plates at a density of 1 / well and incubated in an incubator (37°C, 5% CO2).

[0070] Incubate overnight.

[0071] (2) Experimental grouping: The experiment set up a blank control group, a negative control group and an earthworm polypeptide group, and a concentration gradient was set for the earthworm polypeptide group.

[0072] (3) Solution preparation: Prepare working solutions of the test substance of different concentrations according to Table 6.

[0073] Table 6

[0074]

[0075]

[0076] (4) Radiation: When the cell plating rate in the 24-well plate reached 40% to 60%, irradiation was performed. The total dose received by the negative control group and the experimental group was 9 J / cm 2 At the same time, the blank control group was placed in the same environment but did not receive UVA radiation.

[0077] (5) Administration: Administration was carried out after the irradiation was completed. 1 mL of cell culture medium was added to each well of the blank control group and the negative control group; 1 mL of culture medium containing 100 μg / mL VC and 7 μg / mL VE was added to each well of the positive control group; and 1 mL of culture medium containing the test substance at the corresponding concentration was added to each well of the sample group.

[0078] (6) Collecting cells: After incubation for 24 h, add 0.5 mL of lysis buffer to each well and leave at room temperature for 5 min to allow complete lysis. Transfer the cells to a 1.5 mL RNase-free Eppendorf tube and extract RNA according to the operating procedures.

[0079] (7) Reverse transcription: Synthesize cDNA according to the instructions of the RNA reverse transcription kit.

[0080] (8) qRT-PCR detection of CollagenⅠ gene.

[0081] (9) Statistical analysis of results: The inter-group comparison was performed using t-test statistical analysis. The results are shown in Table 7.

[0082] Table 7

[0083]

[0084]

[0085] Based on the UVA-stimulated human fibroblast model, the experimental groups containing earthworm peptides showed a significant improvement in the gene expression of type I collagen.

[0086] The above description is only an embodiment of the present invention. The protection scope of the present invention is not limited by these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent substitution, improvement, etc. made within the technical ideas and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-aging earthworm polypeptide, characterized in that: The polypeptide comprises at least one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

2. A nucleotide encoding the earthworm polypeptide as claimed in claim 1.

3. A biomaterial, characterized in that: The biological material is selected from any one of the following 1) to 2): 1) A vector having one or more nucleotides as claimed in claim 2; 2) A recombinant cell capable of producing one or more of the earthworm polypeptides as described in claim 1, and / or containing one or more of the nucleotides as described in claim 2.

4. Use of the polypeptide according to claim 1, the nucleotide according to claim 2, or the biomaterial according to claim 3 in anti-oxidation and / or promoting collagen production, preferably, the collagen is type I collagen.

5. Use of the polypeptide according to claim 1, the nucleotide according to claim 2, or the biomaterial according to claim 3 in the preparation of anti-aging cosmetics, skin care products, health products, and medicines.

6. A composition, characterized in that The composition comprises the earthworm polypeptide of claim 1.

7. Use of the composition according to claim 6 in anti-oxidation and / or promotion of collagen production, preferably, the collagen is type I collagen.

8. Use of the composition as claimed in claim 6 in the preparation of anti-aging cosmetics, skin care products, health products and medicines.

9. A method for preparing the earthworm polypeptide according to claim 1, characterized in that: The method comprises the following steps: (1) extracting earthworms and pure water at a ratio of 1:12 to 1:24 at 30 to 40° C. for 14 to 20 hours to obtain a solution containing an earthworm extract; (2) filtering the solution containing the earthworm extract obtained in step (1) through an ultrafiltration membrane with a molecular weight cutoff of less than or equal to 2500 Daltons, and then freeze-drying to obtain earthworm polypeptides.

10. The method according to claim 9, characterized in that The earthworm is selected from at least one of Eisenia fetida, Pheretima leucoderma, Pheretima william, Pheretima vulgaris and Pheretima zhiliensis.

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