Anti-aging and anti-oxidation cysteine-rich polypeptide and its application

Abstract

The present invention discloses an anti-aging and anti-oxidation cysteine-rich polypeptide and its application, belonging to the field of biomedicine technology, the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO: 1-9, the amino acids at sites 3, 7, 15, 20, 22 and 32 of the polypeptide are all cysteine, wherein site 3 and site 20, site 7 and site 22, and site 15 and site 32 form three pairs of disulfide bonds. The polypeptide provided by the present invention has the effects of clearing cell ROS, resisting reactive oxygen-induced cell apoptosis, promoting the proliferation of mesenchymal stem cells and delaying the life span of Caenorhabditis elegans under natural conditions, and has the effects of anti-oxidation and anti-aging; the cysteine-rich polypeptide provided by the present invention can be used to prepare anti-oxidation and anti-aging food, medicine and cosmetics, and has important promotion and application value.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an anti-aging and anti-oxidation cysteine-rich polypeptide and application thereof. Background Art

[0002] Aging refers to the comprehensive manifestation of the gradual decline of body functions with age. When the body enters aging, it is easy to induce a variety of chronic diseases, such as Alzheimer's disease, cancer, diabetes and cardiovascular disease. The body will continuously produce reactive oxygen molecules during metabolism, which have strong oxidative effects and accelerate cell aging and death. Energy metabolism in aging cells is prone to disorder, and mitochondria are the "energy factory" of cells. After metabolic disorders, oxygen consumption increases, and more free radicals are produced to damage cells. Eliminating free radicals or reducing the level of reactive oxygen can delay cell apoptosis and anti-aging.

[0003] There is a hydrolase called β-galactosidase (SA-β-Gal) in the cell lysosome, which is only expressed in senescent cells to catalyze the hydrolysis of β-galactoside. Lipofuscin is the "garbage" left over after the lysosome cannot digest it. As the cells age, lipofuscin slowly deposits. Common age spots are caused by lipofuscin. Therefore, senescent cells can be identified by detecting the activity of β-galactosidase. In senescent cells, β-glucosidase is often highly expressed and is a marker molecule indicating cell aging. As a classic model animal for studying aging, Caenorhabditis elegans has a gene homology of 60% to 80% with humans. It has the advantages of a short life cycle and simple experimental operation, and is often used as a tool to explore anti-aging under natural conditions.

[0004] Beans are not only one of the main sources of protein, dietary fiber, minerals and vitamins for humans, but also contain rich functional nutrients. Studies have shown that bean seeds are rich in antioxidant active substances such as peptides, vitamin E, and flavonoids, and long-term consumption can help delay aging. In recent years, many new substances in legumes have been continuously discovered and applied to the research of tumors, diabetes, cardiovascular diseases, Alzheimer's disease, antioxidants and anti-aging. However, the efficacy of natural oral peptides has rarely been reported. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-aging and anti-oxidation cysteine-rich polypeptide and its application to solve the problems existing in the above-mentioned prior art. The cysteine-rich polypeptide provided by the present invention has antioxidant and anti-aging effects, can be used to prepare antioxidant and anti-aging medicines and cosmetics, and has important promotion and application value.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an anti-aging and anti-oxidation polypeptide, the amino acid sequence of the polypeptide is shown in any one of SEQ ID NOs: 1-9.

[0008] Furthermore, the amino acids at positions 3, 7, 15, 20, 22 and 32 of the polypeptides shown in SEQ ID NOs: 1-9 are all cysteine, wherein position 3 and position 20, position 7 and position 22, and position 15 and position 32 form three pairs of disulfide bonds.

[0009] The present invention also provides an application of the polypeptide in preparing an antioxidant product.

[0010] The present invention also provides an application of the polypeptide in preparing anti-aging products.

[0011] Furthermore, the polypeptide resists aging through one or more of the following pathways:

[0012] S1. Remove cellular reactive oxygen species;

[0013] S2. Promote cell proliferation;

[0014] S3. Inhibit cell apoptosis and senescence.

[0015] Furthermore, the product comprises an effective dose of the polypeptide and excipients.

[0016] Furthermore, the auxiliary materials include one or more of diluents, binders, disintegrants, lubricants and flavoring agents.

[0017] The present invention discloses the following technical effects:

[0018] The present invention provides 9 cysteine-rich polypeptides, which are characterized by containing 37 amino acids and 6 cysteines in the molecule, all of which are located at sites 3, 7, 15, 20, 22 and 32, wherein sites 3 and 20, sites 7 and 22, and sites 15 and 32 form three pairs of disulfide bonds, and these three pairs of disulfide bonds form a cysteine ​​domain. The polypeptides provided by the present invention have the effects of clearing cell ROS, resisting reactive oxygen-induced cell apoptosis, promoting the proliferation of mesenchymal stem cells and delaying the life span of Caenorhabditis elegans under natural conditions, and have antioxidant and anti-aging effects. The cysteine-rich polypeptides provided by the present invention can be used to prepare antioxidant and anti-aging drugs and cosmetics, and have important promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 are sequences of cysteine-rich polypeptides derived from different legumes;

[0021] Figure 2 To evaluate the scavenging effects of cysteine-rich peptides from different legumes on reactive oxygen species;

[0022] Figure 3 The cysteine-rich polypeptide Vg derived from soybean promotes the proliferation of mesenchymal stem cells in vitro;

[0023] Figure 4 Vg reverses hydrogen peroxide-induced apoptosis of mesenchymal stem cells;

[0024] Figure 5 Vg inhibits hydrogen peroxide-induced mesenchymal stem cell senescence;

[0025] Figure 6 Vg delays the lifespan of Caenorhabditis elegans in culture. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1

[0032] Extraction and identification of cysteine-rich peptides from different legumes

[0033] (1) Raw material cleaning: Taking 500 g of soybeans as an example, weigh 500 g of soybeans into a container and add water to clean three times.

[0034] (2) Soaking: Add 4-5 times the amount of deionized water to the container containing the washed soybeans, cover the mouth of the container with gauze to prevent dust from entering. The soaking time varies with the temperature (the soaking time is 5-7 hours when the temperature is 30-35°C). When the temperature is low, the soaking time should be appropriately extended until the soybeans absorb water and expand to their maximum volume.

[0035] (3) Grinding extraction: Grind the soybeans with 4-5 times deionized water, add 25 ml of 1% acetic acid solution and stir to mix thoroughly for extraction. Stir after a certain period of time to ensure sufficient extraction. The extraction time is about 8 hours at an extraction temperature of 10°C.

[0036] (4) Slurry separation: The extracted material obtained in step (3) is subjected to slurry separation using a filter bag. Freeze drying: The collected filtrate is freeze-dried per plate volume for 48 hours to obtain a crude extract after drying.

[0037] The content of a portion of the crude product obtained in the experiment was determined by high performance liquid chromatography (HPLC).

[0038] Chromatographic conditions: chromatographic column-Zorbax C18 4.6×150mm, 5um particles, column temperature-30℃, mobile phase A-0.1% trifluoroacetic acid aqueous solution, mobile phase B-0.1% trifluoroacetic acid acetonitrile solution, flow rate-1ml / min, detector-UV214nm, injection volume-20μl, gradient: 50 minutes B liquid from 10% to 60%, mobile phase A and B liquid need to be ultrasonically degassed before use. Then use 100% B liquid to elute for 30min to completely wash off the residual substances on the column, and then use the corresponding concentration of A liquid to balance the column to form a complete chromatographic process. The single peak obtained by gradient elution is subjected to mass spectrometry to determine the molecular weight. Combined with the species database comparison, the amino acid sequence of the isolate is determined.

[0039] Referring to the above method, in this example, 9 cysteine-rich polypeptides with different sequences were separated and identified from adzuki bean, mung bean, pea, soybean and astragalus, respectively. The sequences were azuki bean leguinsulin ADCNGACSPFEMPPCGSTDCRCVPIALVGGFCIHPTG (SEQ ID NO: 1), mung bean leguinsulin ADCNGACSPFQMPPCGSTDCLCIPAGLLFGYCTYPSG (SEQ ID NO: 2), pea leguinsulin ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRHPSG (SEQ ID NO: 3), soybean leguinsulin ADCNGACSPFEVPPCRSRDCRCVPIGLFVGFCIHPTG (SEQ ID NO: 4), soybean aglycin ASCNGVCGPFEMPPCSSSACGCIPVRLVVGYCRHPSG (SEQ ID NO: 5), soybean iglycin ISCNGVCGPFDIPPCSTPLCGCIPARLFVGKCRHPYG (SEQ ID NO: 6), soybean vglycin 1 VSCNGVCGPFDIPPCSTPLCGCIPYRLFVGNCRHPYG (SEQ ID NO: 7), soybean vglycin 2 VSCNGVCGPFEMPPCSSSACGCIPYRLVVGNCRHPSG (SEQ ID NO: 8), Astragalus aM1VDCSGACSPFEVPPCGSRDCRCIPIGLVVGFCIYPTG (SEQ ID NO: 9).

[0040] The nine sequences obtained all contain 37 amino acids and 6 cysteines in the molecule, located at positions 3, 7, 15, 20, 22 and 32, of which three pairs of disulfide bonds are formed between positions 3 and 20, between positions 7 and 22, and between positions 15 and 32. These three pairs of disulfide bonds form a cysteine-rich domain (see Figure 1 ).

[0041] Example 2

[0042] Evaluation of the scavenging effects of cysteine-rich peptides from different legumes on reactive oxygen species

[0043] Cells: Human umbilical cord blood mesenchymal stem cells were purchased from Qilu Stem Cell Engineering Co., Ltd., and DCFHDA fluorescent probes were purchased from Bio-Tech Biotechnology Co., Ltd.

[0044] The fifth generation MSC cells in logarithmic phase were selected and digested with trypsin to prepare cell suspension. 5 The cells were inoculated into 6-well plates per well and placed in a cell culture incubator for culture. After 24 hours of culture, the old culture medium was removed, washed twice with PBS, and cell culture medium containing 200 μM cysteine-rich peptides was added to stimulate the cells, and the cells were incubated for another 24 hours. The positive control group was treated with NAC. After the incubation, the old culture medium was discarded, washed twice with PBS, and digested with 0.05% trypsin, and the cells were collected by centrifugation at 1200r for 3min. The DCFHDA fluorescent probe was diluted to a working concentration (10μM) using preheated serum-free DMEM. The cells collected in the previous step were resuspended in 1ml / well of the prepared DCFHDA working solution, and incubated in a 37°C, 5% CO2 incubator in the dark for 30min, and mixed by inverting every 3 to 5min. After the incubation, the cells were centrifuged at 1200r for 3min to precipitate the cells, the staining working solution was discarded, the cells were washed with preheated serum-free DMEM, centrifuged at 1200r for 3min, and the washing was repeated 3 times to obtain a cell pellet. Resuspend the cell pellet in PBS and detect it on a flow cytometer. The test results are analyzed using FlowJo.

[0045] In this example, it was found that the above polypeptides all have the activity of reducing the reactive oxygen species of mesenchymal stem cells (MSC). Compared with the control group, the cysteine-rich polypeptides from different sources can remove the reactive oxygen levels of MSC cells, and the removal rate is above 50%. Among them, the antioxidant activity of vglycin1 from soybean is close to that of the reactive oxygen scavenger acetylcysteine ​​NAC ( Figure 2 ), this type of polypeptide is also resistant to acid, alkali, gastrointestinal digestive enzymes, and high temperature, so it can be taken orally. The above results show that these cysteine-rich polypeptides have the potential to develop antioxidant and anti-aging drugs.

[0046] Example 3

[0047] Soybean-derived cysteine-rich peptide Vg promotes the proliferation of mesenchymal stem cells in vitro

[0048] In order to further verify the biological activity of cysteine-rich peptides, vglycin1 (Vg) was selected for subsequent experiments.

[0049] The MTT experiment was used to detect the effect of Vg on cell viability. The MTT method is a method used to detect cell survival and growth. The detection principle is that there is a substance called succinate dehydrogenase in the mitochondria of living cells, which can reduce exogenous MTT to water-insoluble blue-purple crystals. This blue-purple crystal is formazan, so formazan is deposited in the cells. Only living cells can produce formazan, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve formazan, and its light absorption value is measured at a wavelength of 490nm using an enzyme marker, which can indirectly reflect the number of living cells. Within a certain range of cell numbers, the amount of MTT crystals formed is proportional to the number of cells.

[0050] Take the 5th generation mesenchymal stem cells (MSC) in the logarithmic growth phase, discard the old culture medium, add 1ml PBS buffer to rinse the cells, aspirate the PBS, use 0.05% trypsin to digest the cells for 2-3 minutes, place them under a microscope, and add cell culture medium to terminate the digestion after visually observing that the cells shrink, become round, and have a tendency to slide. Immediately transfer the digested cells to a 1.5ml EP tube, centrifuge at 1200r for 3min, discard the supernatant, resuspend in PBS 1-2 times, add 1ml cell culture medium to the EP tube and gently pipette to make a cell suspension. Use a cell counter to measure the cell concentration, and use 5×10 3 cells / well, 100 μL per well was inoculated in a 96-well plate. After the well plate was placed in a cell culture incubator for 24 hours, 200 μM and two-fold gradient dilutions of Vg were added, and 3 replicates were set up for each group. The blank group was added with an equal amount of normal cell culture fluid. After 72 hours, the supernatant was discarded, and the cells were washed 3 times with PBS buffer. 30 μL of pre-configured MTT working solution was added to each well at a concentration of 5 mg / mL, and the cells were placed in a cell culture incubator for further incubation for 4 hours. After the incubation, the MTT solution was carefully discarded, and 50 μL of DMSO was added to each well to dissolve the precipitate. Before the test, observe whether the precipitate is completely dissolved. If there is any precipitate that has not dissolved, it can be placed on a shaker at room temperature and gently shaken to dissolve it. The absorbance of each well was detected at OD490 nm, and the relative cell viability was calculated.

[0051] The dysfunction and decrease in number of stem cells in tissues may be the decisive factors affecting human aging. Under in vitro culture conditions, Vg has the function of promoting the proliferation of mesenchymal stem cells (MSC) in vitro, and has a concentration-dependent effect ( Figure 3 ).

[0052] Example 4

[0053] Vg reverses hydrogen peroxide-induced apoptosis of mesenchymal stem cells

[0054] The fifth generation MSC cells in logarithmic phase were selected and digested with trypsin to prepare cell suspension. 4 / dish was inoculated in a 6cm small dish and placed in a 5% CO2, 37℃ incubator for continued culture. After 24h of culture, the old culture medium was discarded and washed 2-3 times with PBS. It was divided into 4 groups, namely control group, Vg treatment group, hydrogen peroxide (H2O2) group and Vg intervention group (H2O2+Vg). The H2O2 group and Vg intervention group were treated with 200μM hydrogen peroxide for 30 minutes, and fresh culture medium was replaced and PBS or 50μM Vg was added, and the cells were placed in a cell culture incubator for continued culture for 24h. After the apoptosis induction was completed, the old culture medium was collected and transferred to a 5ml EP tube, washed 2-3 times with PBS, and the cells were digested with EDTA-free trypsin. Pay attention to the digestion time to avoid over-digestion. Use the old culture medium to terminate the digestion, transfer the cell suspension to the EP tube, and centrifuge at 1200r for 5min together with the old culture medium collected in the previous step. Get the cell pellet, discard the supernatant, resuspend the cells with pre-cooled PBS, centrifuge at 900r for 3min, and wash with PBS 2-3 times. Aspirate and discard the PBS, resuspend the cells with 400μL Binding buffer, and divide into 4 tubes, 100μL each, marked as blank tube, AnnexinV-FITC single staining tube, PI single staining tube and AnnexinV-FITC / PI double staining tube. Add 5μL AnnexinV-FITC to the FITC single staining tube and stain at room temperature in the dark for 5min. Add 10μLPI to the PI single staining tube and stain at room temperature in the dark for 15min. Add 5μL AnnexinV-FITC to the FITC / PI double staining tube, place at room temperature for 15min, and then add 10μL PI. Add 400μL Binding buffer to each tube to make up to 500μL, mix well, place on ice, and detect within 1h. Use flow cytometry for apoptosis detection, and use FlowJo to analyze data, plot and calculate apoptosis rate.

[0055] Hydrogen peroxide-induced mesenchymal stem cell senescence is one of the classic cell senescence models. Figure 4As shown, about 23.7% (Q2+Q3) of mesenchymal stem cells showed apoptosis when cultured to the ninth generation, and the apoptosis rate was reduced to 16.3% after treatment with 20μg / mL Vg. After treatment with hydrogen peroxide, the apoptosis rate of mesenchymal stem cells was as high as 83.8%, while co-culture with Vg after hydrogen peroxide treatment reversed the apoptosis of mesenchymal stem cells, and the apoptosis rate was reduced to 22.7%. This shows that the cysteine-rich polypeptide vglycin1 (Vg) has strong antioxidant activity and reverses hydrogen peroxide-induced apoptosis of mesenchymal stem cells.

[0056] Example 5

[0057] Vg inhibits hydrogen peroxide-induced senescence of mesenchymal stem cells

[0058] The fifth generation MSC cells in logarithmic phase were selected and digested with trypsin to prepare cell suspension. 4 / dish was inoculated in a 6 cm small dish and placed in a 5% CO2, 37°C incubator for continued culture. After 24 hours of culture, the old culture medium was discarded and washed 2 to 3 times with PBS. It was divided into 4 groups, namely the control group, Vg treatment group, hydrogen peroxide (H2O2) group and Vg intervention group (H2O2+Vg). The H2O2 group and the Vg intervention group were treated with 200μM hydrogen peroxide for 30 minutes, and fresh culture medium was replaced and PBS or 50μM Vg was added, and the cells were placed in a cell culture incubator for continued culture for 24 hours. β-galactosidase staining was performed. Staining steps: Carefully remove the culture medium in the 24-well plate; add 500μl of washing solution (liquid A) to each well to wash the cell surface; after removing the washing solution in the well, add 500μl of fixative solution (liquid B) to each well and incubate for 5min; remove the fixative solution, add 500μl of acid solution (liquid C) to each well, repeat once, remove liquid C, add 400μl of staining solution to each well, put in a 37℃ incubator, and incubate for 3 to 16h. Observe and count under an optical microscope. Cells expressing senescence-specific β-galactosidase are positive cells and appear blue.

[0059] To further prove that Vg can reverse cell senescence caused by oxidative stress, β-galactosidase staining was used to find that the positive rate of senescent cells in the tenth generation of mesenchymal stem cells was 11.2%. Vg treatment slightly reduced the positive rate of senescent cells to 9.6%. The positive rate of senescent cells after hydrogen peroxide treatment was as high as 92.7%. After hydrogen peroxide treatment, adding Vg to co-culture greatly delayed cell senescence, and the positive rate was reduced to 34.2% ( Figure 5 ).

[0060] Example 6

[0061] Vg delays lifespan of Caenorhabditis elegans in culture

[0062] Because of its short lifespan (2-3 weeks under natural conditions) and multiple core metabolic pathways and key factors that are very similar to those of humans, C. elegans is often the best biological model for investigating the aging of the body. When the L1 nematodes develop to the L4 stage, 12μg / ml 5-FUDR is used to treat the C. elegans to inhibit its egg laying; when the nematodes develop into adults, Vg treatment begins, and they are divided into control group, 5μg / ml, 20μg / ml and 50μg / ml groups, and the drug is continued every 24 hours, and the number of nematodes that survive is observed and recorded every day until the last nematode dies. The criterion for judging death is: the C. elegans does not respond to strong light or knocking on the plate, the pharyngeal muscles do not move under a high-power microscope, and finally the head of the nematode is knocked with a worm needle. If there is still no response, it can be judged as dead. The dead nematodes need to be picked out of the culture dish, and E. coli OP50 is added in time when it is exhausted. The survival time of each group of nematodes was statistically analyzed, and the data results are presented as Mean±SE.

[0063] The results are as follows Figure 6 As shown, when 20 μg / mL and 50 μg / mL of Vg were added to the culture medium, the lifespan of Caenorhabditis elegans was extended from 17 days under natural conditions to about 26 days, confirming that the cysteine-rich polypeptide vglycin1 (Vg) can delay the lifespan of Caenorhabditis elegans in culture.

[0064] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a polypeptide in the preparation of an antioxidant product, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO: 7; as shown in SEQ ID NO: 7, the amino acids at positions 3, 7, 15, 20, 22 and 32 of the polypeptide are all cysteine, wherein three pairs of disulfide bonds are formed between position 3 and position 20, between position 7 and position 22, and between position 15 and position 32; and the product is a medicine or a cosmetic.

2. Use of a polypeptide in the preparation of an anti-aging product, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO: 7; as shown in SEQ ID NO: 7, the amino acids at positions 3, 7, 15, 20, 22 and 32 of the polypeptide are all cysteine, wherein three pairs of disulfide bonds are formed between position 3 and position 20, between position 7 and position 22, and between position 15 and position 32; and the product is a medicine or a cosmetic.

3. The use according to claim 1 or 2, characterized in that: The product comprises an effective dose of the polypeptide and excipients.

4. The use according to claim 3, characterized in that: The auxiliary materials include one or more of diluents, binders, disintegrants, lubricants and flavoring agents.

Images