An anti-aging composition containing collagen and its application in treating premature aging

Abstract

The present invention relates to an anti-aging composition containing collagen and its application in treating premature aging. The present invention separates, screens and identifies a polypeptide with anti-oxidation and anti-aging effects from forest frog skin. The polypeptide has good antioxidant properties and can promote the secretion of collagen in cells. The polypeptide can effectively treat premature skin aging when used alone or in combination with collagen, and has broad application prospects.

Description

Technical Field

[0001] The present application relates to the biological field, and in particular to an anti-aging composition comprising collagen and its application in treating premature aging. Background Art

[0002] The skin is the largest and most widely distributed organ in the human body, and is responsible for physiological functions such as temperature regulation, secretion, excretion, and immunity. As we age, the skin undergoes degenerative changes in structure and function, causing skin structural atrophy and functional decline, which in turn leads to skin aging and accelerates the development of many skin diseases. Skin aging is the most intuitive manifestation of body aging, and its essence and basis is the aging of skin cells. Intervening in the skin aging process and improving skin condition and structure are effective treatments for preventing premature skin aging.

[0003] Premature skin aging is a complex biological process, including aging caused by various intrinsic factors such as DNA damage and epigenome, free radicals, skin-related proteins, mitochondrial damage, telomere shortening, etc. Reactive oxygen species (ROS) are representatives of free radicals. The accumulation of ROS and the resulting oxidative stress can induce and accelerate the aging process, including skin aging. Reactive oxygen species (ROS) are byproducts of the electron transport chain of mitochondrial aerobic metabolism. The accumulation of ROS can cause oxidative stress that leads to damage to lipids, proteins, nucleic acids and organelles, thereby leading to the occurrence of cell aging. In addition to genetic factors. ROS is the main cause of endogenous aging. In the skin, about 1.5%-5% of oxygen consumption is converted into ROS through intrinsic processes. Among the various factors of skin aging, most of them cause skin aging through reactive oxygen species, such as photoaging. ROS accumulation can directly damage DNA, proteins and lipids, triggering the downregulation of collagen production, leading to dermal aging. The accumulation of ROS is mainly caused by the inactivation of protein tyrosine phosphatases, which leads to the activation of receptor tyrosine kinases, thereby RTK phosphorylation, and downstream signaling pathways such as the MAPK (mitogen-activated protein kinase) family are activated. Corresponding ROS can induce the expression of matrix metalloproteinases (MMPs) by stimulating mitogen-activated protein kinases and activating heterodimer activator protein 1. MMPs are one of the main components of the senescence-associated secretory phenotype (SASP). SASP is expressed by senescent cells and in turn destroys the microenvironment, damages tissue structure and function, thereby promoting local and systemic aging-related pathologies and promoting cancer. At the same time, SASP inhibits the clearance of senescent cells in the dermis by macrophages, leading to the accumulation of senescent cells and aggravating the aging of tissue cells. MMPs can also mediate skin aging by specifically targeting and decomposing collagen and elastin fibers. ROS and activated mitogen-activated protein kinase signaling pathways can activate NF-κB, thereby affecting the expression of multiple cytokines that mediate inflammation. Activated NF-κB reactively promotes the further generation of ROS, aggravating skin aging. In summary, ROS promotes the occurrence and development of aging, including skin, through various mechanisms of interaction.

[0004] For a long time, sunscreens have been considered an effective method to prevent UV damage to the skin and treat premature skin aging. However, due to the improper use of sunscreens, the use of narrow-spectrum sunscreens, and the toxicity of the sunscreens themselves, their role in the actual application of preventing skin photoaging is limited, and other more effective methods for preventing and treating skin photoaging must be found. Given the important role of ROS in the occurrence of skin photoaging and premature aging, the use of antioxidants to remove ROS may be a reasonable and effective strategy for preventing and treating photoaging and premature skin aging. Traditional antioxidants such as vitamin C, vitamin E, β-carotene, zinc, and selenium have certain UV protection effects. Recent studies have shown that several plant antioxidants such as Ectoin (a plant found in Egyptian waters), tea extracts, Polypodium vulgare extracts, lycopene, N-acetylcysteine, and genistein have certain effects in preventing premature skin aging. With the in-depth study of the molecular mechanism of premature skin aging in recent years, the specific pathways and methods of ROS in the process of premature skin aging have become increasingly clear. This new knowledge helps to screen targeted antioxidants for the prevention of premature skin aging.

[0005] However, at present, it is a consensus in the field that small molecule peptide drugs can be better absorbed and utilized by the skin, but the development of peptide drugs with particularly good antioxidant effects is still insufficient, and further research is needed, especially the development of peptide compositions for the treatment of premature skin aging. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides an anti-aging composition comprising collagen and application thereof in treating premature aging.

[0007] Specifically, the anti-aging composition of the present invention contains antioxidant and anti-aging polypeptides specifically isolated and identified from the skin of Rana sylvatica.

[0008] Specifically, the amino acid sequence of the antioxidant and anti-aging polypeptide is shown in SEQ ID NO: 1.

[0009] Furthermore, the polypeptide may be modified.

[0010] Optionally, when determining the degree of amino acid similarity, the skilled person may also consider so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains are glycine, alanine, valine, leucine and isoleucine; a group of amino acids with aliphatic hydroxyl side chains are serine and threonine; a group of amino acids with amide-containing side chains are asparagine and glutamine; a group of amino acids with aromatic side chains are phenylalanine, tyrosine and tryptophan; a group of amino acids with basic side chains are lysine, arginine and histidine; and a group of amino acids with sulfur-containing side chains are cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine. Substitution variants of the amino acid sequences disclosed herein are those in which at least one residue in the disclosed sequence has been removed and a different residue has been inserted in its place. Preferably, the amino acid changes are conservative. Preferred conservative substitutions for each naturally occurring amino acid are as follows: Ala to ser; Arg to lys; Asp to glu; Cys to ser or ala; Glu to asp; Gly to pro; Leu to ile or val; Lys to arg; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and Val to ile or leu.

[0011] Specifically, in the present invention, the term "pharmaceutically acceptable" refers to an amount sufficient to produce a therapeutic effect without causing side effects, and refers to the type of disease, the patient's age, weight, health status, gender and the patient's sensitivity to the drug. , Those skilled in the art can easily determine based on factors well known in the art, such as route of administration, method of administration, frequency of administration, treatment cycle, combination or simultaneous use of drugs. The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable excipient. Excipients are not particularly limited, but for oral administration, adhesives, lubricants, disintegrants, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavoring agents, etc. can be used. For injections, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used.

[0012] The preparation of the composition of the present invention can be prepared in various ways by mixing it with a pharmaceutically acceptable excipient as described above. For example, for oral administration, it can be manufactured in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be manufactured in the form of unit dose ampoules or multiple dosage forms. In addition, it can be formulated into solutions, suspensions, tablets, pills, capsules, sustained-release preparations, etc.

[0013] Meanwhile, the examples of carriers, excipients and diluents suitable for preparations include glucose, fructose, galactose, lactose, maltose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, amino acid (such as methionine), gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, sodium acetate, cellulose, methylcellulose, microcrystalline cellulose, polysorbate, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum, magnesium stearate or mineral oil. In addition, fillers, anticoagulants, lubricants, wetting agents, spices, preservatives etc. can be included in addition.

[0014] In addition, the pharmaceutical composition of the present invention may have any dosage form selected from tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, lyophilized preparations and suppositories.

[0015] In addition, according to conventional methods in the pharmaceutical field, the composition is formulated into a unit dosage form suitable for administration to a patient, specifically, formulated into a form of a preparation that can be used to administer a protein drug, and can be administered orally or parenterally using conventional administration methods in the art, including but not limited to skin, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal or rectal routes. Most preferably, for the skin.

[0016] Furthermore, the present invention provides an antioxidant and anti-aging polypeptide for use in preparing a drug for treating premature skin aging, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.

[0017] More specifically, the present invention also provides use of a combination of an antioxidant and anti-aging polypeptide and collagen in the preparation of a drug for treating premature skin aging, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.

[0018] Beneficial Effects

[0019] The present invention isolates, screens and identifies polypeptides with anti-oxidation and anti-aging effects from forest frog skin. The polypeptides have good anti-oxidation properties and can promote the secretion of collagen in cells. The polypeptides can effectively treat premature skin aging when used alone or in combination with collagen, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Effects of peptides on HaCaT cell viability

[0021] Figure 2 Effects of peptides on the expression of extracellular type I collagen precursor

[0022] Figure 3 Effects of peptides on intracellular ROS expression

[0023] Figure 4 Effects of each treatment group on the expression level of MMP-1 in mouse skin tissue DETAILED DESCRIPTION

[0024] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, if not specifically stated, are all conventional methods, equipment, and materials in the art, and can be purchased from the market.

[0025] Example 1 Screening and Identification of Rana sylvatica Antioxidant and Anti-aging Peptides

[0026] The skin of artificially reared forest frogs was taken, and after being frozen and crushed in liquid nitrogen, 3500U / g of neutral protease was added under the optimized enzymatic hydrolysis conditions, i.e., pH 6.5, material-liquid ratio of 1:1.8, and temperature of 46℃. After enzymatic hydrolysis for 2.3h, the enzyme was inactivated at 90℃ and centrifuged to obtain the enzymatic hydrolyzate. The supernatant was obtained by centrifugation (10000r / min, 20min) to obtain the supernatant of the enzymatic hydrolyzate. Then the supernatant was passed through 8k, 5k, and 3kDa molecular sieve ultrafiltration membranes step by step, and a total of 4 ultrafiltration fractions of <3kDa (LW-1), 3k-5kDa (LW-2), 5k-8kDa (OPEH-3) and >8kDa (LW-4) were retained. The total antioxidant capacity of each ultrafiltration fraction was determined by the ABTS method. The specific operation steps refer to the product manual (total antioxidant capacity (ABTS method) test kit product brand: mlBio / enzyme-linked biology). The results are shown in Table 1.

[0027] Table 1 Comparison of ABTS cation radical inhibition mass concentrations of each component

[0028] sample Half inhibitory concentration (IC50 / (μg / mL)) LW-1 137.14±8.64 LW-2 375.81±10.83 LW-3 783.72±28.76 LW-4 483.69±15.36 Vc 113.51±5.37

[0029] As shown in Table 1, LW-1 exhibited the best ABTS cation radical scavenging ability, indicating that LW-1 had the best total antioxidant capacity in vitro.

[0030] The LW-1 component was further separated by DEAE-52 cellulose chromatography. Specifically, a 16mm×250mm glass chromatography column was selected, and the cellulose column was equilibrated with 50mmol·L-1Tris-HCl (pH7.8) buffer, and gradient elution was performed with 0-0.7mol / L buffer, with an elution rate of 6min per tube. The absorbance value was detected at a wavelength of 280nm, and 4 corresponding components were collected and named LW-1-A, LW-1-B, LW-1-C, LW-1-, LW-1-A, respectively. The protein content was determined and freeze-dried. Subsequently, a solution with a mass concentration of 1mg / mL was prepared, and its scavenging rate for DPPH, ABTS and ·OH free radicals was determined. The highest one was the LW-1-C component, with scavenging rates of 92.5±2.8, 93.5±1.9, and 88.9±2.0, respectively. The LW-1-C component was purified by Sephadex G-25 gel chromatography. A 10 mm × 600 mm glass chromatography column was selected, and deionized water was used for continuous elution. The sample volume was 5 mL, the flow rate of the constant flow pump was 1.5 mL / min, and the elution rate was 5 min per tube. The absorbance value was detected at 280 nm, and a total of 3 components were collected, which were named LW-1-CI, LW-1-C-II, and LW-1-C-III, respectively. The protein content was determined, and the powder was freeze-dried and prepared into a solution with a mass concentration of 1 mg / mL. The scavenging rate of DPPH, ABTS and ·OH free radicals was determined. The LW-1-C-II component with a higher free radical scavenging rate (the scavenging rates were 98.2±2.5, 97.3±1.7, and 94.1±1.5, respectively) was selected for mass spectrometry identification, and the amino acid sequence obtained by identification was shown in SEQ ID NO: 1. The amino acid sequence is rich in glycine. As a semi-essential amino acid, glycine intake plays an important role in collagen renewal and glutathione stability.

[0031] Example 2 Identification of the Antioxidant and Anti-aging Functions of LW-1-C-II Polypeptide

[0032] When HaCaT cells grew to 70%-80% in T25 cell culture flasks, they were digested with 0.25% trypsin to prepare a cell suspension. Cells (1×10 4After the cells are completely attached to the wall, the medium is aspirated and the cells are washed three times with PBS solution, leaving a little liquid at the bottom. The 96-well plate is placed under a radiation meter consisting of 8 UVB lamps arranged side by side (9W, emission spectrum between 285-350nm, peak at 310-315nm, lamp tubes 16cm away from the well plate). 2 The cells were irradiated with a radiation intensity of 100 μL, and then 100 μL of DMEM basal medium with a final concentration of 10 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL of the sample to be tested was added to the cells. After 24 hours, the cell viability of the CCK-8 kit was measured. The cell viability of the blank cell without UVB treatment was set as 1. The results are as follows: Figure 1 shown.

[0033] Depend on Figure 1 It can be seen that compared with the control group, the cell viability of human horny layer HaCaT cells decreased significantly after UVB irradiation (P<0.01). After adding different concentrations of antioxidant and anti-aging peptides to the UVB-irradiated HaCaT cells for 24 hours, different concentrations of peptides can protect the cell viability of photoaged HaCaT cells after UVB irradiation, and show a good dose dependence. At a mass concentration of 200μg / mL, the relative cell viability increased by nearly 1 times compared with the UVB group without added peptides, and the effect was significant.

[0034] Determination of extracellular type I collagen precursor expression: Enzyme-linked immunosorbent assay (ELISA) kit was used to determine the extracellular content of type I collagen precursor in HaCaT cells. The culture medium of each group after culture was collected and centrifuged (2500r / min, 4°C, 20min), the supernatant was collected, and the concentration was determined according to the ELISA reagent manual. The results are shown in Figure 2 shown.

[0035] like Figure 2 As shown in the figure, the ELISA results showed that after UVB radiation, the level of type I collagen precursor secreted into the extracellular space by HaCaT cells decreased significantly compared with the blank control group CT (P<0.01). After UVB radiation and simultaneous addition of peptide treatment, the concentration of type I collagen precursor secreted into the extracellular space also gradually increased with the increase of peptide concentration, indicating that the peptide has a good effect in promoting the secretion of type I collagen precursor into the extracellular space.

[0036] Determination of intracellular ROS expression: The generation of ROS in HaCaT cells was detected using a reactive oxygen species detection kit. HaCaT cells were inoculated in a 96-well fluorescent ELISA plate (inoculation concentration was 1×10 5 / well), UV radiation and drug treatment were the same as before. After 12 hours, the culture medium was aspirated, and fresh serum-free culture medium containing 10 μmol / L DCFH-DA was added, and incubated at 37°C in the dark for 20 minutes. Subsequently, the cells were washed three times with PBS solution. The intensity of DCF fluorescence in the 96-well fluorescent enzyme-labeled plate was detected on a fluorescent microplate reader, and the amount of CT was used as the relative basic amount. The results are shown in Figure 3 shown.

[0037] from Figure 3 It can be seen that after UVB radiation, a large amount of ROS was produced in HaCaT cells (P<0.01), and different concentrations of polypeptides could significantly inhibit the production of ROS, especially when the mass concentration was 200 μg / mL, the relative content of ROS in the UVB group cells was reduced by nearly 6 times compared with that in the UVB group without adding polypeptides (P<0.01).

[0038] Example 3 Zoological Experiments on LW-1-C-II Polypeptide and Its Composition

[0039] In an environment of about 25°C and relative humidity of 60%, the experimental mice were free to drink water and eat. After one week of adaptive feeding, they were divided into a control group, a model group, and a treatment group by a random number table method, with 10 mice in each group. All mice were depilated once every 5 days using a 4% sodium sulfide solution to remove the surface hair on the back of the mice, exposing skin of about 4cmX4cm in size. Each mouse in the model group was subcutaneously injected with 5% D-galactose 10mg / kg on the back of the neck; and a UVB lamp was placed 40cm vertically above the mouse, and the radiation intensity was measured before irradiation. The radiation dose was 120mJ / cm 2 , lasting 40 days. The control group was injected with 0.9% sodium chloride solution (i.e., saline) 10 mg / kg subcutaneously at the back of the neck every day, but was kept under normal light. The 40W UVB lamp (311-312nm, TL40W / 01-RS) was a product of Philips, Germany. The experimental group was based on the model group, and was gavaged with LW-1-C-II polypeptide every day (10 mg / kg in the low concentration group and 100 mg / kg in the high concentration group), control collagen group (collagen type I, CAS:9007-34-5, Sigma, gavage 100 mg / kg every day); combined treatment group (gavage LW-1-C-II polypeptide 10 mg / kg and collagen type I, CAS:9007-34-5, Sigma, 100 mg / kg every day), positive control group (gavage Vc 100 mg / kg every day); normal group and model group were gavaged with saline. The administration time was also 40 days.

[0040] On the 42nd day of the experiment, all mice were killed by cervical dislocation after intraperitoneal anesthesia, and the depilated skin of 2cmX2cm on the back of the neck was cut, and the excess subcutaneous fat was removed. After being cut with ophthalmic scissors, 0.9% sodium chloride solution was added according to the ratio of tissue mass (g): reagent volume (mL) of 1:9, and the mixture was placed in an ice water bath for homogenization and centrifuged at 2000xg for 10 minutes. The supernatant was taken and diluted with 0.9% sodium chloride solution to 1% skin tissue slurry, and then the SOD and GSH-PX activities were determined according to the instructions of the kit. The results are shown in Table 2.

[0041] Table 2 Results of SOD and GSH-PX activities in each group

[0042]

[0043]

[0044] From the results in Table 2, it can be seen that compared with the control group, the SOD and GSH-PX activities of the model group mice skin were significantly reduced, and the differences were statistically significant (all P values ​​<0.01). The SOD and GSH-PX activities of the model group mice skin were significantly improved, especially the combined treatment group, which was significantly improved compared with the model group SOD and GSH-PX activities, and the differences were statistically significant (all P values ​​<0.01). This shows that the combination of the LW-1-C-II polypeptide of the present invention and collagen can better improve the immune activity of mice and improve the antioxidant capacity of mice.

[0045] Furthermore, the expression of MMP-1 in skin tissue was detected by western blotting. The back skin of the mice in the above groups was cut into pieces with ophthalmic scissors and RIPA lysis buffer was added. After centrifugation at 4°C and 12000xg for 5 minutes, the supernatant was collected. The protein concentration in the supernatant was determined by the BCA method, and SDS-PAGE was performed. Then, the bands separated by electrophoresis were transferred to a PVDF membrane. After blocking with 1% BSA for 1 hour, a 1:2000 diluted primary antibody (rabbit anti-mouse MMP-1 monoclonal antibody) was added and incubated at 4°C overnight. Then, a 1:10000 diluted secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG) was added and incubated at room temperature for 50 minutes. Then, ECL luminescent solution was used for color development, and Image J software was used for color band analysis. The results are as follows: Figure 4 shown.

[0046] from Figure 4It can be seen that the expression level of MMP-1 in the mouse skin tissue of the model group was significantly increased compared with that of the control group, and the differences were statistically significant (P<0.01). After treatment, the expression level of MMP-1 was significantly reduced in the polypeptide group, the combination treatment group and the positive control group. This also shows that polypeptides and polypeptides combined with collagen have a good synergistic therapeutic effect and can effectively inhibit the expression of MMP-1. It is well known in the art that MMP-1 can degrade type III fibrous collagen, causing the fragmentation of collagen fibers, and then causing premature aging of the skin. Therefore, the polypeptides and polypeptides combined with collagen of the present invention have a good skin repair effect and a good effect in treating premature skin aging.

[0047] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit it. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in this field should understand that they can still modify the technical solutions recorded in the above embodiments, or replace part or all of the technical features therein with equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antioxidant and anti-aging polypeptide LW-1-C-II, characterized in that The polypeptide amino acid sequence is shown in SEQ ID NO:

1.

2. Use of the antioxidant and anti-aging polypeptide LW-1-C-II in the preparation of a drug for treating skin antioxidant effects, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

3. Use of the antioxidant and anti-aging polypeptide LW-1-C-II in the preparation of a drug for treating premature skin aging, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1, and the premature skin aging is caused by oxidation.

4. Use of the antioxidant and anti-aging polypeptide LW-1-C-II and collagen in the preparation of a pharmaceutical composition for treating premature skin aging, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1, and the premature skin aging is caused by oxidation.

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