CN114163500B - Oyster protein source anti-skin photoaging active peptide and preparation method and application thereof - Google Patents
Oyster protein source anti-skin photoaging active peptide and preparation method and application thereof Download PDFInfo
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- CN114163500B CN114163500B CN202111339694.8A CN202111339694A CN114163500B CN 114163500 B CN114163500 B CN 114163500B CN 202111339694 A CN202111339694 A CN 202111339694A CN 114163500 B CN114163500 B CN 114163500B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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Abstract
The invention belongs to the technical field of proteins, and discloses an oyster protein source anti-skin photoaging active peptide, a preparation method and application thereof. The oyster protein source skin photoaging resisting active peptide has the amino acid sequence as follows: a) WNLNP (SEQ ID NO. 1); or b) an amino acid sequence which is functionally identical or similar after substitution or deletion of one or more amino acids of the amino acid sequence shown in SEQ ID NO. 1. The active peptide has antioxidant activity, can promote the secretion of type I collagen precursor, inhibit MMP-1 expression, has protective effect on keratinocyte, can be used for preparing anti-skin photoaging or anti-aging products, preventing or repairing keratinocyte photodamage caused by UVB, and preventing or improving skin damage caused by UVB radiation.
Description
Technical Field
The invention belongs to the technical field of proteins, and particularly relates to an oyster protein source anti-skin photoaging active peptide, and a preparation method and application thereof.
Background
In recent years, more ultraviolet (Ultraviolet light, UV) is radiated to the earth's surface through the ozone layer due to the destruction of the ozone layer, so that patients suffering from skin diseases are increased year by year, seriously endangering the physical and psychological health of the masses. Photoaging of skin is a phenomenon of premature skin aging caused by the accumulation of excessive ultraviolet radiation. Ultraviolet rays radiated to the ground can be classified into three types according to their wavelengths: UVA (320-420 nm); UVB (275-320 nm) and UVC (200-275 nm), wherein UVB is able to penetrate the epidermis layer up to the interface between the epidermis layer and the dermis layer, are the main causes of photo-aging of the skin. Photoaging of the skin not only impairs cosmetic appearance but is also closely linked to the occurrence of many diseases. How to prevent and treat skin photoaging has become a hotspot in the field of skin and cosmetic research.
Excessive UVB radiation of the skin not only causes damage to biological macromolecules (proteins and DNA) within the cell, but also reduces intracellular antioxidant enzymes, resulting in increased levels of intracellular Reactive Oxygen Species (ROS). Excessive ROS production can promote expression of Matrix Metalloproteinases (MMPs) through the MAPK signaling pathway. MMPs are a group consisting of Zn 2+ The dependent endopeptidase family can accelerate the degradation of collagen fibers in extracellular matrix, thereby leading to skin collagen loss and promoting the formation of wrinkles. Collagen precursors play a vital role in collagen formation, thus inhibiting ROS activity, and inhibiting collagen degradation, provide new strategies for preventing and treating photoaging.
The hong Kong oyster (Crassostrea hongkongensis) is an important cultured shellfish distributed in the coastal area of the south of China. According to the materia medica outline record written in the Ming dynasty of plum time law: oyster meat is very beautiful to eat, and has fine skin and beautiful color. At present, research reports of anti-UVB-induced skin photoaging of the hong Kong oyster and related products thereof are not seen.
Disclosure of Invention
The object of the first aspect of the invention is to provide an oyster protein source anti-skin photoaging active peptide.
The object of the second aspect of the present invention is to provide a nucleic acid molecule encoding the oyster protein source anti-skin photoaging active peptide of the first aspect of the present invention.
The object of the third aspect of the invention is an expression cassette, a recombinant vector or a transgenic cell comprising the nucleic acid molecule of the second aspect of the invention.
The fourth aspect of the present invention is to provide a method for preparing the oyster protein source anti-skin photoaging active peptide according to the first aspect of the present invention.
The fifth aspect of the invention aims to provide a chemically modified oyster protein source anti-skin photoaging active peptide.
The sixth aspect of the present invention is to provide the use of the oyster protein source anti-skin photo-aging active peptide of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression cassette, recombinant vector or transgenic cell of the third aspect of the present invention, and the chemically modified oyster protein source anti-skin photo-aging active peptide of the fifth aspect of the present invention.
A seventh aspect of the invention is directed to a product.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided an oyster protein source anti-skin photo-aging active peptide, the amino acid sequence of which is:
a) WNLNP (SEQ ID NO. 1); or (b)
b) The amino acid sequence shown in SEQ ID NO.1 has the same or similar function after one or more amino acid substitutions or deletions.
In a second aspect of the invention there is provided a nucleic acid molecule encoding an oyster protein source anti-skin photoaging active peptide of the first aspect of the invention.
In a third aspect of the invention there is provided an expression cassette, recombinant vector or transgenic cell comprising the nucleic acid molecule of the second aspect of the invention.
Preferably, the transgenic cell does not comprise propagation material.
In a fourth aspect, the present invention provides a method for producing the oyster protein-derived anti-skin photoaging active peptide according to the first aspect, wherein the method comprises any one of (a) to (c):
(a) Extracting oyster as raw material;
(b) Synthesizing by adopting a liquid phase or solid phase synthesis method;
(c) Culturing the transgenic cell of the third aspect of the invention.
In a fifth aspect of the present invention, there is provided a chemically modified oyster protein-derived anti-skin photo-aging active peptide modified at the N-terminus and/or the C-terminus of the oyster protein-derived anti-skin photo-aging active peptide of the first aspect of the present invention.
Preferably, the chemically modified oyster protein-derived anti-skin photo-aging active peptide has the same or similar function as the oyster protein-derived anti-skin photo-aging active peptide.
Preferably, the chemically modified oyster protein-derived anti-skin photo-aging active peptide is subjected to acetylation modification or palmitoylation modification at the N-terminus of the oyster protein-derived anti-skin photo-aging active peptide of the first aspect of the present invention.
Preferably, the amino acid sequence of the chemically modified oyster protein source anti-skin photo-aging active peptide is shown as SEQ ID NO.4 or SEQ ID NO. 5.
Preferably, the chemically modified oyster protein source anti-skin photo-aging active peptide is amidated modified at the C-terminal end of the oyster protein source anti-skin photo-aging active peptide of the first aspect of the present invention.
Preferably, the amino acid sequence of the chemically modified oyster protein source anti-skin photo-aging active peptide is shown as SEQ ID NO. 6.
In a sixth aspect, the invention provides the use of the oyster protein source anti-skin photo-aging active peptide of the first aspect, the nucleic acid molecule of the second aspect, the expression cassette, recombinant vector or transgenic cell of the third aspect, and the chemically modified oyster protein source anti-skin photo-aging active peptide of the fifth aspect.
(d) The use of any one of (h) to (n) in any one of (h) to (g);
(d) The oyster protein source anti-skin photoaging active peptide of the first aspect of the invention;
(e) The nucleic acid molecule of the second aspect of the invention;
(f) An expression cassette, recombinant vector or transgenic cell of the third aspect of the invention;
(g) The chemically modified oyster protein source anti-skin photoaging active peptide of the fifth aspect of the present invention;
(h) Preparing an antioxidant product;
(i) Preparing a product for promoting secretion of type I collagen precursors;
(j) Preparing a product for inhibiting MMP-1 expression;
(k) Preparing an anti-skin photoaging product;
(l) Preparing an anti-aging product;
(m) preparing a product for preventing or repairing photodamage of keratinocytes due to UVB;
(n) preparing a product for preventing or improving skin damage caused by UVB radiation.
Preferably, the product is at least one of a cosmetic, a pharmaceutical and an agent.
In a seventh aspect of the invention, there is provided a product comprising: at least one of (d) to (g);
(d) The oyster protein source anti-skin photoaging active peptide of the first aspect of the invention;
(e) The nucleic acid molecule of the second aspect of the invention;
(f) An expression cassette, recombinant vector or transgenic cell of the third aspect of the invention;
(g) The chemically modified oyster protein source anti-skin photoaging active peptide of the fifth aspect of the present invention.
Preferably, the product is at least one of a cosmetic, a pharmaceutical and an agent.
Preferably, the product has any one of functions (o) - (u):
(o) antioxidant;
(p) promoting secretion of type I collagen precursors;
(q) inhibiting MMP-1 expression;
(r) anti-skin photoaging;
(s) anti-aging;
(t) preventing or repairing photodamage to keratinocytes due to UVB;
(u) preventing or ameliorating skin damage due to UVB radiation.
The beneficial effects of the invention are as follows:
the invention discloses an oyster protein source anti-skin photoaging active peptide for the first time, which has antioxidant activity, can promote the secretion of type I collagen precursors, can inhibit the expression of MMP-1, has a protective effect on keratinocytes, can be used for preparing anti-skin photoaging or anti-aging products, and can be used for preventing or repairing the photodamage of the keratinocytes caused by UVB and preventing or improving the skin damage caused by UVB radiation.
The invention also provides a chemically modified oyster protein source anti-skin photo-aging active peptide, which is subjected to acetylation modification or palmitoylation modification at the N end of the oyster protein source anti-skin photo-aging active peptide, and has a protective effect on keratinocytes as the oyster protein source anti-skin photo-aging active peptide, and can inhibit the expression of MMP-1: wherein N-terminal palmitoylation can increase the protective effect of oyster protein source anti-skin photoaging active peptide on keratinocytes and the inhibiting effect on MMP-1 expression.
Drawings
FIG. 1 is a technical roadmap of an embodiment of the invention.
FIG. 2 is a graph of the effect of UVB at different irradiation intensities on HaCaT cell viability; where, P <0.01 compared to the CT group (control group, the same applies below).
FIG. 3 is a graph of the effect of OPEH and various ultrafiltration components on HaCaT cell viability after UVB irradiation: wherein P <0.01 compared to CT group; # denotes p <0.01 compared to UVB group; # indicates p <0.05 compared to UVB group.
FIG. 4 is a graph of the effect of OPEH and various ultrafiltration components on HaCaT cell morphology after UVB irradiation.
FIG. 5 is a graph of the effect of OPEH and various ultrafiltration components on the ROS levels in HaCaT cells following UVB irradiation.
FIG. 6 is a graph of the effect of OPEH and various ultrafiltration components on the production of ROS in HaCaT cells following UVB irradiation, and secretion of extracellular type I collagen precursors: wherein A is a graph of the effect of OPEH and the respective ultrafiltration components on the relative production of ROS in HaCaT cells after UVB irradiation; b is an influence diagram of OPEH and each ultrafiltration component on secretion of the extracellular type I collagen of HaCaT after UVB irradiation; * P <0.01 compared to CT group; # denotes p <0.01 compared to UVB group.
FIG. 7 is a graph of the G25 sephadex elution profile of OPEH-1.
FIG. 8 is a graph of the anti-skin photoaging activity of each elution peak (P1 to P6) of the G25 gel chromatography of OPEH-1: wherein A is a HaCaT cell activity influence diagram of P1-P6 on UVB irradiation; b is a Western Blotting result graph of the influence of P1-P6 on MMP-1 expression in UVB-irradiated HaCaT cells; c is a statistical result diagram of the effect of P1-P6 on MMP-1 expression in UVB irradiated HaCaT cells; different letters represent differences (p < 0.05).
FIG. 9 is an elution profile of RP-HPLC of P4.
FIG. 10 is a graph of the anti-skin photoaging activity of each elution peak (P4-1 to P4-6) of RP-HPLC of P4: wherein A is a HaCaT cell activity influence diagram of P4-1 to P4-6 on UVB irradiation; b is a Western Blotting result graph of the influence of P4-1 to P4-6 on MMP-1 expression in UVB-irradiated HaCaT cells; c is a statistical result graph of the effect of P4-1 to P4-6 on MMP-1 expression in UVB-irradiated HaCaT cells; different letters represent differences (p < 0.05).
Fig. 11 is a primary and secondary mass spectrum of YTVTF: wherein A is a primary mass spectrum and B is a secondary mass spectrum.
Fig. 12 is a primary and secondary mass spectrum of RKNEVLGK: wherein A is a primary mass spectrum and B is a secondary mass spectrum.
Fig. 13 is a primary and secondary mass spectrum of wnnp: wherein A is a primary mass spectrum and B is a secondary mass spectrum.
Fig. 14 is a primary and secondary mass spectrum of VTY: wherein A is a primary mass spectrum and B is a secondary mass spectrum.
FIG. 15 is a RP-HPLC purity identification spectrum of YTTTF.
FIG. 16 is an ESI-MS spectrum of YTTTF.
FIG. 17 is a RP-HPLC purity identification spectrum of RKNEVLGK.
FIG. 18 is an ESI-MS spectrum of RKNEVLGK.
FIG. 19 is a RP-HPLC purity identifying chart of WNLNP.
FIG. 20 is an ESI-MS spectrum of WNLNP.
FIG. 21 is a RP-HPLC purity identification spectrum of VTY.
FIG. 22 is an ESI-MS spectrum of VTY.
FIG. 23 is a graph of the anti-skin photoaging activity of four synthetic polypeptides: wherein A is a HaCaT cell viability influence diagram of four synthetic polypeptides on UVB irradiation; b is a Western Blotting result graph of the influence of four synthetic polypeptides on MMP-1 expression in UVB-irradiated HaCaT cells; c is a graph of statistical results of the effect of four synthetic polypeptides on MMP-1 expression in UVB-irradiated HaCaT cells; different letters represent differences (p < 0.05); in the figure, pep1 represents the polypeptide YTVTF; pep2 represents the polypeptide rkevlgk; pep3 represents the polypeptide WNLNP; pep4 represents the polypeptide VTY.
FIG. 24 is a graph showing the result of the protection effect of WNLNP on HaCaT cells after UVB irradiation: wherein A is an visual result diagram of DCF fluorescence influence of WNLNP on HaCaT cells after UVB irradiation; b is a graph of the effect of WNLNP on survival of HaCaT cells after UVB irradiation; c is a statistical result graph of the influence of WNLNP on DCF relative fluorescence intensity of HaCaT cells after UVB irradiation; d is a graph of statistical results of the effect of WNLNP on the secretion of extracellular type I procollagen of HaCaT after UVB irradiation; * P <0.01 compared to CT group; # denotes p <0.01 compared to UVB group.
FIG. 25 is a RP-HPLC purity identifying chart of AC-WNLNP.
FIG. 26 is an ESI/MS mass spectrum of AC-WNLNP.
FIG. 27 is WNLNP-NH 2 RP-HPLC purity identification profile of (C).
FIG. 28 is WNLNP-NH 2 Is an ESI/MS mass spectrum of (C).
FIG. 29 is a RP-HPLC purity identifying chart of Palm-WNLNP.
FIG. 30 is an ESI/MS mass spectrum of Palm-WNLNP.
FIG. 31 is a graph of the anti-skin photoaging activity of chemically modified polypeptides: wherein A is a HaCaT cell viability influence diagram of the chemically modified polypeptide on UVB irradiation; b is an SDS-PAGE polyacrylamide gel electrophoresis result diagram of the influence of the chemical modification polypeptide on MMP-1 expression in UVB irradiated HaCaT cells; c is a graph of statistical results of the effect of chemically modified polypeptides on MMP-1 expression in UVB-irradiated HaCaT cells; different letters represent differences (p < 0.05).
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The materials, reagents and the like used in this example are commercially available ones unless otherwise specified.
The materials and reagents used in this example were as follows: fresh hong Kong oyster meat is purchased from the Dongfeng aquatic products market in Zhanjiang city of Guangdong province; ultrafiltration membranes (8, 5, 3 kDa) and sterile filtration membranes (0.22. Mu.M) were purchased from Miybbo, germany; naOH (500 g/bottle), hydrochloric acid from Guangzhou chemical reagent plant; neutral protease (30000U/g) purchased from Guangxi nan Ning Pang Bo Bio-engineering Co., ltd; ABTS total antioxidant assay kit and ROS reactive oxygen species assay kit were purchased from bi yun-tian biotechnology limited; haCaT cells were purchased from north na alliance biotechnology limited, su; CCK-8 kit was purchased from Japan Tonic chemical institute; DEME (Dulbecco's modified eagle medium) high sugar medium and fetal bovine serum (Fetal Bovine Serum, FBS) were purchased from Gibco;0.25% pancreatin was purchased from Hyclone company; 96-well, 24-well, 6-well cell culture plates, 25T cell culture flasks and cell cryopreservation tubes were purchased from Corning corporation; cell cryopreservation solution was purchased from rich biotechnology limited; sterile syringes were purchased from Dongguan Kang Run instrument and device limited; serum-free cell cryopreservation solution was purchased from Fuheng biotechnology limited, and sterile syringe was purchased from Dongguan Kang Run instrument equipment limited; reagents such as cell lysate, PMSF, BCA protein concentration determination kit, SDS-PAGE polyacrylamide preformed gel (10%), electrophoresis buffer, transfer buffer, WB sealing solution, primary anti-dilution solution, secondary anti-dilution solution, WB washing solution, loading buffer, 10-170kDa color pre-dyeing Marker and the like are purchased from Biyunsian biotechnology Co., ltd; MMP-1, an Actin murine IgG monoclonal antibody, and a rabbit anti-mouse IgG secondary antibody were purchased from Santa Cruz; chemiluminescent fluids were purchased from brillouin biotechnology limited.
The instrumentation used in this example is as follows: wall breaking machine (Jiuyang home electric appliance company); t18 high speed disperser ULTRA-TURRAX (IKA company, germany); -an 80 degree ultra low temperature refrigerator (siemens appliance, germany); freeze dryer (tokyo chemical company, japan); UVB ultraviolet lamp (9 w) (philips lighting limited); varioskan Flash full-automatic microplate reader (sammer, feier, usa); carbon dioxide incubator (sammer, feier, usa); ultra clean bench (su zhou purification equipment limited); FD-551 large vertical freeze dryer (Tokyo physical and chemical instruments Co., ltd.); DM2000 LED fluorescence inverted microscope (Leica company, germany); BSA224S-CW model ten-thousandth electronic balance (cerdelisco instruments); protein purification System (AKTA Co., USA); ultimate3000 UPLC-MS (ESI)/MS mass spectrometer (Simer Feier Co., USA); vertical electrophoresis tank (Shanghai Techno Co., ltd.); electrophoresis apparatus (Beijing Liuyi instrumentation); chemiluminescent instrument (Shanghai Technical Co., ltd.).
The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The materials, reagents and the like used in this example are commercially available ones unless otherwise specified.
The technical scheme of the embodiment is shown in fig. 1.
EXAMPLE 1 preparation of oyster protein source anti-skin photoaging active peptide
1. Preparation of oyster protein hydrolysate (Oyster protein enzymatic hydrolysate, OPEH) and ultrafiltration fractions
Extraction of oyster protein: fresh hong Kong oyster meat, cleaning and weighing, preliminary pulping, adding distilled water according to a material ratio of 1:3 (100 g oyster meat: 300mL distilled water), regulating pH to 12.0 by 0.2mol/L NaOH, homogenizing for 5min by a high-speed homogenizer (ice bath), stirring for 3h by a chromatography cabinet at 4 ℃, centrifuging (10000 r/min,4 ℃ for 20 min), collecting supernatant, regulating pH of the supernatant by 0.2mol/L HCl to 4.8, centrifuging at 4 ℃ for 20min (10000 r/min,4 ℃ for 20 min), collecting precipitate, and freeze-drying to obtain oyster protein.
Enzymolysis of oyster protein: weighing oyster protein, adding distilled water according to the material ratio of 1:3 (100 g oyster protein: 300mL distilled water), adjusting the pH of the sample solution to 7.0 by 0.2mol/L NaOH, adding neutral protease for enzymolysis (the mass ratio of enzyme to substrate is 2%,50 ℃ C., 3 h), inactivating enzyme in a water bath at 95 ℃ for 10min, cooling, centrifuging (10000 r/min,4 ℃ C., 20 min), and taking supernatant for standby (OPEH).
Preparation of the ultrafiltration fraction of OPEH: under ice bath condition, ultrafiltering OPEH with ultrafilter membrane of 8kDa, 5kDa and 3kDa to obtain ultrafilter components of <3kDa (OPEH-1), 3-5 kDa (OPEH-2), 5-8 kDa (OPEH-3), and >8kDa (OPEH-4), concentrating by rotary evaporation, freeze drying, and storing in refrigerator at-80deg.C.
The total antioxidant capacity of OPEH and each ultrafiltration fraction was determined by the ABTS method (see product description for specific procedures), and the results are shown in Table 1: OPEH-1, OPEH-2 and OPEH all showed good ABTS + Radical scavenging ability, ABTS + The half-purge concentrations (IC 50) were 156.083. Mu.g/mL, 273.505. Mu.g/mL, 243.133. Mu.g/mL, respectively. ABTS of OPEH-1 + The lowest IC50 of the free radical (compared with the positive control Vitamin C (VC)) indicates that OPEH-1 has the best total antioxidant capacity in vitro.
TABLE 1 OPEH in vitro Total antioxidant Capacity of the respective Ultrafiltration Components
Measurement of HaCaT cell Activity (CCK-8 method) and cell morphology observations: will HaCaT cells were seeded into 96-well plates (about 1 x 10 per well) 4 Cell), after 12h of cells are completely adhered, the culture medium is sucked, the culture medium is washed 3 times by PBS, a little liquid is reserved at the bottom of the pore plate, a series of irradiation doses are carried out on HaCaT cells by adopting a UVB ultraviolet irradiation instrument, and the optimal irradiation dose of UVB is determined to be 35mJ/cm 2 Is a radiation intensity of (2); after irradiation of HaCaT cells by UVB irradiation, 100. Mu.L of serum-free medium is added, then samples with different concentrations are added for treating the cells for 24 hours, and then the morphological change of the cells is photographed under an inverted microscope, or 10. Mu.L of CCK-8 reagent is added to a 96-well plate, and after 2 hours, the OD value is detected at a wavelength of 450 nm.
Keratinocytes are the major cell type of the epidermis of skin tissue, playing a key role in skin defense, and during their continued cell differentiation and renewal, the morphology, size and arrangement of cells change regularly, eventually forming keratin-rich keratinocytes. Excessive aging and apoptosis of keratinocytes are a typical pathological feature of photoaging of skin. As can be seen from FIG. 2, 35mJ/cm compared with the control group 2 After irradiation of HaCaT cells with UVB at irradiation intensity, cell viability was significantly decreased (p<0.01 Only about 61% of the control group. The research of other previous subject groups shows that when the cell activity is about 60%, the cells are not damaged and overweight due to UVB irradiation, and meanwhile, the anti-photoaging related proteins in the cells are expressed, so that the photoaging cell model modeling is considered to be successful under the irradiation dose.
After the HaCaT cells after UVB irradiation are treated for 24 hours by adding OPEH with different concentrations and ultrafiltration components thereof, OPEH-1, OPEH-2 and OPEH have protective effects on the cell viability of photo-aged HaCaT cells after UVB irradiation and show a better dose-dependent relationship: OPEH-1 significantly improved HaCaT cell viability (p < 0.05) after UVB irradiation at concentrations >50 μg/mL; OPEH-2 and OPEH also significantly improved the viability of HaCaT cells (p <0.05, respectively) after UVB irradiation at concentrations >100 μg/mL (CT is blank, UVB is model, UVB+OPEH-1 is OPEH-1 treated group, as shown in FIG. 3); from the experimental results of CCK-8, OPEH-1 has the best protection effect on the cell viability of HaCaT cells after UVB irradiation on a HaCaT photoaging cell model induced by UVB irradiation.
The results of the effects of OPEH and the various ultrafiltration components on HaCaT cell morphology are shown in FIG. 4: the normal HaCaT cells are closely arranged, the cell morphology is consistent, the cell is elliptical, the boundary is clear, and the cell light transmittance is good; in the model group, after UVB irradiates HaCaT cells, cell gaps become large and shrink, morphology changes irregularly, cell edges become blurred, and cell debris is generated; after 100 mug/mL of OPEH and various ultrafiltration components are added to HaCaT cells after UVB irradiation for treating the cells for 24 hours, the results are consistent with the cell viability test results, and OPEH-1, OPEH-2 and OPEH have certain improvement effects on the change of the morphology of the HaCaT cells; wherein OPEH-1 has the best effect of improving the morphological change of HaCaT cells; the HaCaT cells after UVB irradiation treated by OPEH-1, although slightly broken, basically recovered to the level of the blank control group, and the improvement effect of the positive control drug Vitamin C (Vitamin C, VC) of 5 mug/mL was nearly consistent.
The extracellular content of the type I collagen precursor in the HaCaT cells is measured by adopting an enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA) kit, the generation of ROS in the HaCaT cells is detected by adopting an active oxygen detection kit (specific operation steps refer to product specifications), and the ultraviolet irradiation and administration treatment refer to the measurement of the activity of the HaCaT cells, and the results are shown in figures 5 and 6: after the HaCaT of the CT group is incubated for 20min by using the DCFH-DA fluorescent probe, only weak green fluorescence appears in cells; after UVB irradiates HaCaT cells, ROS are accumulated in the cells of the UVB group, and a large amount of green fluorescence is visible after the cells are dyed by a fluorescent probe; OPEH, ultrafiltration components and positive control drug VC can obviously weaken the intensity of green fluorescence in cells and inhibit the generation of ROS at the administration concentration of 100 mug/m L; meanwhile, the inhibition effect of OPEH-1, OPEH-2 and OPEH on intracellular ROS is consistent with that of a positive control medicament VC of 5 mug/mL (figure 5); the UVB irradiation leads to the large-scale generation of ROS in HaCaT cells (p < 0.01), and OPEH and each ultrafiltration component and positive control drug VC of 5 mug/mL can extremely remarkably inhibit the generation of ROS (p < 0.01), wherein the inhibition effect of OPEH-1 and OPEH-2 on the ROS in cells is higher than that of other components (p < 0.01) (A in fig. 6); the level of type I collagen precursor secreted to the extracellular by HaCaT cells after UVB irradiation was significantly reduced (p < 0.01); of OPEH and each ultrafiltration fraction, only OPEH-1 significantly increased the secretion of type I collagen precursor (p < 0.01) (B in FIG. 6).
Therefore, OPEH-1 has the best in vitro antioxidant capacity, ABTS + The IC50 of the free radical is 156.083 mug/mL; on the UVB-induced HaCaT photoaging cell model, at drug administration concentrations>The activity of HaCaT cells after UVB irradiation can be remarkably improved after 50 mug/mL, and a certain dose dependency relationship is presented; the HaCaT cell gap enlargement and shrinkage caused by UVB irradiation can be improved, and the morphology is changed irregularly; inhibit intracellular ROS production, and promote secretion of extracellular type I collagen.
2. Performing next separation and purification on OPEH-1 by using Sephadex G-25 gel chromatographic column
(1) Packing of Sephadex G-25 gel chromatography gel column:
g25 particle pretreatment: firstly, G-25 dry glue particles are soaked in distilled water with the volume of 5-10 times of that of the dry glue for 24 hours, and then heated in a boiling water bath for 0.5 hour to expand; after removing impurities from the supernatant, the supernatant was re-purified with distilled water according to 3:1 by volume.
(2) Isolating polypeptides
1) Sample pretreatment: dissolving a sample (OPEH-1) in ultrapure water to prepare a solution of 35mg/mL, removing impurities by using a syringe through a 0.22 mu m filter membrane, and discharging redundant bubbles by using ultrasonic vibration;
2) Loading: injecting a sample (2 mL) into a G25 sephadex column from a sample inlet, eluting with ultrapure water, controlling the flow rate to be 1.5mL/min, monitoring the eluent under the absorbance of 280nm, collecting a tube of effluent every 3min, and collecting 6 components according to an elution curve on a protein purifier;
3) The collected components are directly frozen and dried and stored in a refrigerator at the temperature of-80 ℃ for standby.
Under the conditions of separation and purification, after OPEH-1 was subjected to G25 sephadex chromatography, the elution profile shown in FIG. 7 was obtained: OPEH-1 is subjected to G25 sephadex chromatography, and 6 components, namely P1, P2, P3, P4, P5 and P6 are collected under the detection wavelength of 280 nm; cytotoxicity assays were performed on HaCaT cells for these six components; the six components do not affect the cell viability of the HaCaT cells in the concentration range of 0-100 mug/mL.
The final concentrations of the samples (six components) were set at 50 μg/mL and their protective effect on the viability of UVB aged HaCaT cells and their inhibitory effect on MMP-1 expression were compared as follows:
HaCaT cells were seeded into 96-well plates (about 1 x 10 per well) 4 Individual cells), after 12h of complete adherence, the medium was blotted, washed 3 times with PBS and left with a little liquid at the bottom of the well plate; firstly, carrying out a series of irradiation doses on HaCaT cells by adopting a UVB ultraviolet irradiation instrument to irradiate the cells, and determining that the optimal irradiation dose of UVB is 35mJ/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the After HaCaT cells are irradiated, 100 mu L of serum-free culture medium is added, then samples with different concentrations are added for treating the cells for 24 hours, 10 mu L of CCK-8 reagent is added into a 96-well plate, and after 2 hours, OD value is detected at the wavelength of 450 nm; the results are shown in FIG. 8: when the final concentration of each separated component sample is 50 mug/mL, only P4 and P5 have a certain protection effect on the activity of the HaCaT cells after UVB irradiation, and the cell activity of the HaCaT cells after UVB irradiation is remarkably improved: wherein the P4 component improves the cell viability of the HaCaT cells after UVB irradiation from 60.80% of the normal control group to 84.31%; the P5 component improves the cell viability of the HaCaT cells after UVB irradiation from 60.80% of the normal control group to 70.52%; the P4 fraction was found to be better protective for UVB aged HaCaT cells than the P5 fraction.
HaCaT cells were seeded in 6-well plates (about 1 x 10 per well) 4 Cell,) after 12h of complete adherence, sucking up the culture medium, washing 3 times with PBS, leaving a little liquid at the bottom of the pore plate, after HaCaT cell irradiation, adding 100 mu L of serum-free culture medium, and then adding sample to treat cells; after 24h, the cells were washed twice with pre-chilled PBS, the PBS was aspirated off, pre-chilled cell lysates (with 100. Mu.M protease inhibitor PMSF added) were added and lysed in ice water for 10min, after sufficient lysis, 10000-14000g was centrifuged for 5min and the supernatant was used for the subsequent Western Blotting experiments.
Prior to the Western Blotting experiment, the protein concentration of the cell extract was determined according to the instructions of the BCA protein assay kit. Then adding protein loading buffer solution into protein extraction supernatant according to a certain proportion, and water-bathing at 95 ℃ for 5 min. The sample was added to SDS-PAGE polyacrylamide gel, and the loading per well was 20. Mu.g. The inhibition of MMP-1 expression in UVB-aged HaCaT cells was measured by the conventional Western Blotting method and the results are shown in FIG. 8: in normal HaCaT cells, the expression level of MMP-1 is very low, and after the HaCaT cells are irradiated by UVB, the expression level of MMP-1 is remarkably increased (p < 0.01), and the expression level is increased to about 2.55 times of that of a blank control; of the 6 fractions (P1 to P6) of OPEH-1 separated by G25 gel chromatography, about four fractions (P3, P4, P5, P6) have inhibitory effect on the high expression of MMP-1 by UVB irradiation; of these, the two components with the best inhibition effect are the P4 and P6 components.
The protection of UVB-aged HaCaT cells and the inhibition of MMP-1 by 6 separate fractions were compared together, among which P4 had the best anti-photoaging activity. Notably, while P4 is the most optimal anti-photoaging component, the P3 and P5 components adjacent thereto also exhibit inhibition of MMP-1 overexpression. To a certain extent, the separation effect of G25 sephadex chromatography is not ideal, and further separation of P4 component is needed.
(3) C18 reverse phase high performance liquid chromatography
The next step was performed for separation and purification of component F4 with optimal anti-photoaging activity after G25 sephadex chromatography by a Waters reversed-phase high performance liquid chromatography (RP-HPLC) instrument using a C18 (Xterra MS 5. Mu.M, 4.6X250 mm) RP-HPLC column. The sample loading concentration was 5mg/mL, and the loading amount was 100. Mu.L each time. The liquid phase system has two channels, wherein the mobile phase A is deionized water, and the mobile phase B is trifluoroacetic acid. The elution gradient was as follows: the initial proportion of the liquid phase B is 5v/v%, the proportion of the mobile phase B is 5% to 30% in 0-15 min, the mobile phase B is kept at 30% to 16min, and the proportion of the mobile phase B is reduced from 30% to 5% in 17min to 19 min. Maintaining the proportion of mobile phase B5% until 20min is reached; the elution flow rate is 1mL/min; the detection wavelength was set at 214nm, and each peak sample was collected according to the elution profile. After freeze drying, the mixture was stored in a refrigerator at-80℃for use.
The P4 fraction was purified by RP-HPLC to give about 6 peaks (FIG. 9), and samples of these 6 peaks (P4-1-6) were examined for cytotoxicity against HaCaT cells, as well as the concentration of these 6 samples was found to be from 1. Mu.g/mL to 100. Mu.g/mL without cytotoxicity and without adversely affecting the growth of HaCaT cells.
The activity of HaCaT cells was measured by CCK-8 (the method is identical to P1-P6 in protecting the activity of UVB aged HaCaT cells, except that the final concentration of the sample is 25. Mu.g/mL), and the results are shown in FIG. 10: after UVB irradiation, the activity of the HaCaT cells is obviously reduced, and three samples in 6 samples separated and purified by P4 component RP-HPLC can obviously improve the activity of the HaCaT cells after UVB irradiation (P4-3, P4-4 and P4-5 respectively). Of these, P4-4 was the best protective for UVB aged HaCaT cells, increasing their cell viability from 59.06% to 90.43% in the CT group.
The inhibition of MMP-1 expression in UVB aged HaCaT cells was determined using Western Blotting (the method is similar to P1-P6 in terms of inhibition of MMP-1 expression in HaCaT cells, except that the sample concentration was 25. Mu.g/mL), and the results are shown in FIG. 10: MMP-1 is expressed in large amounts after UVB irradiation of HaCaT cells; 3 samples can obviously inhibit the expression of MMP-1 in UVB aged HaCaT cells, namely P4-4, P4-5 and P4-6; wherein, the P4-4 and the P4-6 have the most obvious inhibition effect on the over-expression of the MMP-1, so that the expression level of the MMP-1 is almost restored to the normal level; notably, the P4-3 component, while significantly enhancing cell viability of HaCaT cells after UVB aging, failed to inhibit MMP-1 overexpression after UVB aging; in contrast, the P4-6 component, while here capable of significantly inhibiting MMP-1 overexpression after UVB aging, failed to provide significant protection for the cell viability of HaCaT cells after UVB irradiation; thus, it can be seen that MMP-1 protein may only participate in degradation of extracellular type I collagen within HaCaT cells, and that aging and other proteins and signaling pathways have no obvious effect on apoptosis of HaCaT cells.
In conclusion, the P4-component RP-HPLC (high Performance liquid chromatography) separated and purified P4-4 sample has the optimal protection activity on the activity of HaCaT cells after UVB irradiation, and can also remarkably inhibit the over-expression of MMP-1 after UVB aging. It is shown that it may have the best anti-photoaging effect and that the peak is a single sharp peak, so we choose this component for further isolation, purification and identification.
(4) UPLC-MS/MS mass spectrometry
1) Alkylation treatment of sample:
adding 100 mu L dithiothreitol with the concentration of 10mmol/L into a sample tube, reducing for 1h in a water bath at 56 ℃, sucking out and discarding; then adding 100 mu L of acetonitrile with the concentration of 55mmol/L, reacting for 1h in dark at room temperature, sucking out and discarding; then desalted using a self-packed desalting column, and the dry solvent was evaporated in a vacuum centrifugal concentrator at 45 ℃. The alkylated samples were then subjected to mass spectrometry.
2) Capillary ultra high performance liquid chromatography (UPLC) conditions
Chromatographic column information: 75 μm i.d. times. 150mm,packed with Acclaim PepMap RPLC C18,3 μm,
mobile phase a:0.1% formic acid, 2% acetonitrile; mobile phase B:0.1% formic acid, 80% acetonitrile; flow rate: 300nL/min; sample analysis time 78min; the sample loading was 10. Mu.L.
Liquid chromatography gradient: phase B is 0-8 min 6-9%, 8-24 min 9-14%, 24-60 min 14-30%, 60-75 min 30-40%, 75-78 min and 40-95%.
3) Mass spectral parameters
The separated peptide segment enters an electrospray-combined ion trap Orbitrap mass spectrometer Q exact TM Hybrid Quadrupole-Orbitrap TM Mass Spectrometer, the specific parameters are as follows:
primary mass spectrometry parameters: resolution:70,000agc target:3e6 maximm IT:40ms Scan range:350to1800m/z;
secondary mass spectrometry parameters: resolution:75,000agc target:1e5MaximumIT:60ms TopN:20NCE/stepped NCE:27.
4) Database retrieval
The mass spectrum original file was searched for uniprot database using maxquat (1.6.2.10) with the following search parameters:
fixation modification (Fixed modifications): carbamidomethyl (C);
variable modification (Variable modifications): oxidation (M);
enzyme (Enzyme): unsectical;
missing cleavage site (Maximum Missed Cleavages): 2 first order mass spectrometry error (Peptide Mass Tolerance): 20ppm secondary mass spectrometry error (Fragment Mass Tolerance): 0.6Da;
mass number of peptide fragment ion (Mass value): monoisotopic significance threshold (Significance threshold): 0.01.
after the P4-4 sample is subjected to ESI/MS mass spectrum, the four peak response values with the mass-to-charge ratios of the primary mass spectrum of 381.52, 944.46, 322.16 and 315.66 are higher; after ESI/MS/MS secondary mass spectrometry, combining PEAKS software to carry out spectrum decomposition on the original data, identifying the amino acid sequence of the polypeptide, and finally comparing the amino acid sequence with known protein data Unipro to judge the source of the polypeptide; four polypeptides were identified by ESI/MS/MS, the sequences of which are (1) pentapeptides Tyr-Thr-Val-Thr-Phe (abbreviated as YTTTF (SEQ ID NO. 2), the theoretical relative molecular mass is 629.30), and are fragments of a protein (ID K1R 801) derived from oyster; (2) The octapeptide Arg-Lys-Asn-Glu-Val-Leu-Gly-Lys (abbreviated as RKNEVLGK (SEQ ID NO. 3), the theoretical relative molecular mass is 942.56), is a fragment of the dystrobrevin-binding protein 1 protein (ID K1R1Y 2) derived from oyster muscle cells; (3) Pentapeptide Trp-Asn-Leu-Asn-Pro (abbreviated as WNLNP (SEQ ID NO. 1), theoretical relative molecular mass: 642.31); is a fragment of dimethylaniline monooxygenase (ID K1PRN 4) from oyster mitochondria; (4) Tripeptide Val-Thr-Tyr (abbreviated as VTY, theoretical relative molecular mass: 381.19) is a fragment of the Lectoxin-Lio3 protein (ID K1Q1W 6) from oyster (primary and secondary mass spectra of the polypeptide are shown in FIGS. 11, 12, 13, and 14).
3. Synthesis and purity characterization of polypeptides
(1) YTTTF synthesis and purity identification
The method comprises the steps of synthesizing polypeptide by a solid phase method (assisted by Jiangsu blaze biotechnology Co., ltd.) and according to the amino acid sequence of YTTTF, tyr-Thr-Val-Thr-Phe, firstly, selecting a macromolecular resin material, connecting the carboxyl of Phe with a resin in a covalent bond mode, then carrying out the amino glycidyl reaction on the carboxyl of Thr and the Phe, sequentially adding amino acids from right to left, and cutting off the connection between the carboxyl of the first amino acid Phe and the resin after the reaction between the carboxyl of the last amino acid and the amino of the last amino acid is completed, thus obtaining YTTTF. The purity of the synthetic peptides was then identified using RP-HPLC in combination with a Kromasil C18 column (100-5C 18,4.6mm x 250mm,5 mcron, column temperature, 30 ℃). The RP-HPLC liquid phase system has two channels, wherein the solution A of channel A is acetonitrile containing 1% trifluoroacetic acid, and the solution B of channel B is deionized water containing 1% trifluoroacetic acid. The elution conditions were as follows: the initial proportion of A is 25%, the proportion of A rises to 50% in 20min, the proportion of A rises to 100% in 20min to 20.1min, 100% is kept running for 25min to stop, the detection wavelength is 220nm, and the flow rate is 1mL/min. RP-HPLC purity identifications are shown in FIG. 15. The main peak with the peak time of 5.04s is very large in percentage, and only a very small number of impurity peaks (5.40 s) appear in the chromatogram, and the main peak accounts for 99.36% of the total peak area by calculating the area of each peak, which means that the purity of the synthesized polypeptide YTTTF is 99.36%.
The main peak solution was collected, and after liquid nitrogen flash freeze-dried, the components were identified by ESI-MS mass spectrometry, as shown in FIG. 16. Major ion peak of synthetic peptide [ M+H ]] + 630.53. Step 2 identification of the first-order mass spectrum ion peak of YTTTF by mass spectrum [ M+H ]] 2+ For 315.66, the synthetic peptide was the same as YTTTF identified in step 2.
(2) Synthesis and purity characterization of RKNEVLGK
The polypeptide is synthesized by a solid phase method (assisted by Jiangsu blaze biotechnology Co., ltd.) according to the amino acid sequence of RKNEVLGK, which is Arg-Lys-Asn-Glu-Val-Leu-Gly-Lys, firstly preparing a polymer resin, then connecting carboxyl of Lys with the resin in a covalent bond form, then performing amino glycidyl reaction on carboxyl of Gly and Lys, sequentially adding amino acid from right to left until the last amino acid is added, and then removing the connection between the carboxyl of Lys and the resin to obtain RKNEVLGK. Finally, RP-HPLC was used in combination with Kromasil C18 column (100-5C 18,4.6 mm. Times.250 mm,5 mini-column, column temperature, 30 ℃ C.) for purity identification, and the main peak solution was collected and used for sample identification by ESI/MS. The RP-HPLC liquid phase system is provided with two channels, and the solution A of the A channel is acetonitrile containing 1% trifluoroacetic acid; solution B of channel B was deionized water of 1% trifluoroacetic acid. The elution conditions were as follows: the initial proportion of A is 10%, the proportion of A rises to 35% in 20min, the proportion of A rises to 100% in 20min to 20.1min, 100% is kept running for 25min to stop, the detection wavelength is 220nm, and the flow rate is 1mL/min. RP-HPLC purity was identified as shown in FIG. 17. The main peak (6.793 s) was found to be a very large percentage, and only a few impurity peaks (6.640 s,7.317 s) were present in the chromatogram, and the purity of the main peak was found to be 99.05% by comparing the peak areas.
The main peak solution was collected, flash cooled with liquid nitrogen, and after lyophilization, the structure was identified by ESI-MS as shown in fig. 18. From the figure, it can be seen that the synthetic peptide ion peak [ M+H ]] + 472.67, is consistent with the ion peak of RKNEVLGK identified by mass spectrometry in step 2.
(3) Synthesis and purity identification of WNLNP
The method comprises the steps of adopting a solid phase method to synthesize polypeptide (which is assisted by Jiangsu blaze biotechnology Co., ltd.) and preparing a polymer resin molecule according to the amino acid sequence Trp-Asn-Leu-Asn-Pro of WNLNP, connecting the carboxyl of Pro with a resin in a covalent bond form according to the amino acid sequence, then carrying out an amino glycidyl reaction on the carboxyl of Asn and the amino of Pro, sequentially adding amino acid from right to left until the last Trp amino acid reacts, and then connecting the carboxyl of Pro with the resin to obtain the WNLNP. The purity was checked using RP-HPLC on a Kromasil C18 column (100-5C 18,4.6mm x 250mm,5 mcron, column temperature, 30 ℃). The liquid phase system has two channels, the solution of the A channel is acetonitrile containing 1% trifluoroacetic acid, and the solution of the B channel is deionized water containing 1% trifluoroacetic acid. The elution conditions were as follows: the initial proportion of A is 20%, the proportion of A rises to 45% in 20min, the proportion of A rises to 100% in 20min to 20.1min, 100% is kept running for 25min to stop, the detection wavelength is 220nm, and the flow rate is 1mL/min. RP-HPLC purity was identified as shown in FIG. 19 below. The main peak (9.156 s) was extremely large in percentage, and only a few impurity peaks appeared in the chromatogram, and the purity of the main peak was 98.34% as seen by comparing the peak areas.
The main peak solution was collected, flash cooled with liquid nitrogen, and after lyophilization, the structure was identified by ESI-MS as shown in fig. 20. From the figure, it can be seen that the ion peak [ M+H ] of the synthetic peptide] + 643.66, is consistent with the WNLNP ion peak identified by mass spectrometry in step 2.
(4) Synthesis and purity characterization of VTY
The polypeptide is synthesized by a solid phase method (which is assisted by Jiangsu blaze biotechnology Co., ltd.) according to the amino acid sequence Val-Thr-Tyr of VTY, firstly, carboxyl of Try is connected with a resin in a covalent bond form, then amino of Thr and carboxyl of Try are subjected to shrinkage reaction, finally amino of VaL and carboxyl of Thr are subjected to reaction, and the connection is removed from the resin, so that the VTY is obtained. RP-HPLC was used in conjunction with a Kromasil C18 column (100-5C 18,4.6 mm. Times.250 mm,5micron, column temperature, 30 ℃). The solution of the A channel of the liquid phase system is acetonitrile containing 1% of trifluoroacetic acid, and the solution of the B channel is deionized water containing 1% of trifluoroacetic acid. The elution conditions were as follows: the initial proportion of A is 12%, the proportion of A rises to 37% in 20min, the proportion of A rises to 100% in 20min to 20.1min, 100% is kept running for 25min to stop, the detection wavelength is 220nm, and the flow rate is 1mL/min. RP-HPLC purity was identified as shown in FIG. 21. The main peak is extremely large in percentage, only a few impurity peaks appear in the chromatogram, and the purity of the main peak is 99.13% as can be seen by comparing the areas of the peaks.
The main peak solution was collected, flash cooled with liquid nitrogen, and after lyophilization, the structure was identified by ESI-MS as shown in fig. 22. From the figure, it can be seen that the synthetic peptide ion peak [ M+H ]] + 382.60, is consistent with the VTY ion peak identified by mass spectrometry in step 2.
EXAMPLE 2 verification of anti-photoaging Activity of synthetic Polypeptides
The CCK-8 method was used to determine HaCaT cell viability (the method is the same as P1-P6 of example 1 for protection of UVB aged HaCaT cell viability, except that the final sample concentration was 20 μm), and the results are shown in fig. 23: after UVB irradiation, the cell viability of HaCaT cells is reduced to 55.61% of CT group, and the synthesized polypeptide RKNEVLGK and WNLNP can remarkably improve the cell viability of UVB aged HaCaT cells at a final concentration of 20 mu M, so that the cell viability of HaCaT cells is respectively improved to 77.86% and 84.08% of CT group.
The inhibition of MMP-1 expression in UVB aged HaCaT cells was determined by Western Blotting (the inhibition of MMP-1 expression in HaCaT cells by P1-P6 in example 1 was only distinguished by the final concentration of the sample of 20. Mu.M), and the results are shown in FIG. 23: only the polypeptide RKNEVLGK and the WNLNP extremely remarkably inhibit the overexpression of MMP-1 protein caused by UVB irradiation; among them, WNLNP has the best anti-photoaging activity.
EXAMPLE 3 WNLNP protection against UVB aged HaCaT
The activity of HaCaT cells was determined by CCK-8 method, the extracellular content of type I collagen precursor was determined by enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA) kit, the ROS production in HaCaT cells was detected by reactive oxygen species detection kit (see product description for specific procedures), the inhibition of MMP-1 expression in UVB-aged HaCaT cells by the sample was determined by Western Blotting method (the protection of UVB-aged HaCaT cells by P1-P6 in example 1 was the same except that the final concentration of sample was 10, 20, 50 μm), the results were shown in fig. 24: after UVB irradiation of HaCaT cells, cell viability decreased significantly (p < 0.01); after the WNLNP administration treatment, the cell viability is obviously improved and has a certain dose-dependent relationship; meanwhile, in the HaCaT cells of the normal group (CT group), only a small amount of ROS are formed, and DCF fluorescence in the cells is weak; after UVB irradiates HaCaT cells, a large amount of ROS are formed in the cells, and DCF in the cells emits macroscopic green fluorescence under a fluorescence inversion microscope; indicating successful establishment of UVB-aged HaCaT cells; after the WNLNP treated cells with different concentration gradients are added, the generation of ROS is obviously inhibited, and a certain dose-dependent relationship is formed; the DCF fluorescence signal is collected by adopting a fluorescence microplate reader, as shown in fig. 24, after UVB radiation, a large amount of ROS in HaCat cells is generated (p < 0.01), and the generation of ROS can be remarkably inhibited by high-concentration WNLNP treatment (p < 0.01); meanwhile, the level of the type I collagen precursor secreted to the outside of cells by HaCaT cells after UVB irradiation is obviously reduced (p is less than 0.01); the medium and high concentration wnnp treatment significantly increased the secretion of type I collagen precursors and was dose dependent.
EXAMPLE 4 chemical modification of WNLNP
1. Prediction of skin penetration ability of polypeptides
There are many mathematical models available today to predict the penetration of compounds into human skin. The most common models among these are the boz and bivalve equations:
logK p ＝0.71logP (octanol/water) -0.0061MW-2.74；
wherein: kp is the skin penetration capacity of the compound expressed as (cm/h), P (octanol/water) is the octanol/water partition coefficient of the compound, MW is the relative molecular mass of the compound. A large number of software can predict log P (octanol/water) values. The log P (octanol/water) value online prediction software adopted in the embodiment is provided by Shanghai organic chemistry research institute, and the specific website is http: the ratio of/(www.siocccbg.ac.cn)/software/xlogp 3.WNLNP, N-terminally palmitoylated WNLNP (Palm-WNLNP, SEQ ID No. 4), N-terminally acetylated WNLNP (AC-WNLNP, SEQ ID No. 5), C-terminally amidated WNLNP (WNLNP-NH) 2 The skin penetration capacity of SEQ ID NO. 6) is shown in Table 2: although N-terminal acetylation modification can raise the Logp (octanol/water) of WNLNP from-0.73 to-0.29, the acetylation modification increases the relative molecular mass, so that the passive permeability coefficient Kp coefficient of skin is 6.62×10 -s cm/h down to 7.5 x 10 -8 cm/h; the C-terminal amidation modification, although having no influence on the relative molecular mass, greatly reduces the Log p (octanol/water) coefficient of the polypeptide WNLNP, so that the skin passive permeability coefficient Kp coefficient is 6.62×10 -8 cm/h down to 7.63 x 10 - 10 cm/h, reduced by about 2 orders of magnitude; n-terminal palmitoylation modification will slightly increase the relative molecular mass of the polypeptide, but palmitoylation modification willThe Log p (octanol/water) of WNLNP was increased from-0.75 to 6.40, and the skin passive permeability coefficient Kp coefficient was increased from 6.62×10 -8 cm/h increased to 2.68 x 10 -4 cm/h, the skin penetration capacity is improved by more than about 4 orders of magnitude.
Table 2 skin penetration Capacity of polypeptides
2. Synthesis and purity identification of N-terminally acetylated WNLNP (AC-WNLNP)
The synthesis and chemical modification of the polypeptide are completed with assistance of Jiangsu blaze biotechnology limited company, the WNLNP is synthesized according to the amino acid sequence solid-phase synthesis step of AC-Trp-Asn-Leu-Asn-Pro, and then acetic anhydride reacts with the amino group of Trp at the N end of a peptide chain under the catalysis of piperidine to obtain N-end acetylated WNLNP (AC-WNLNP). Purifying by RP-HPLC, and freeze drying to obtain the final product. Purity was identified using RP-HPLC on a Kromasil C18 column (100-5C 18,4.6mm x 250mm,5 mcron, column temperature, 30 ℃). The liquid phase system has two channels: the solution of channel a was acetonitrile containing 1% trifluoroacetic acid. Solution B of channel B was deionized water of 1% trifluoroacetic acid. The elution conditions were as follows: the initial proportion of A is 27%, the proportion of A rises to 53% in 20min, the proportion of A rises to 100% in 20min to 20.1min, 100% is kept running for 25min to stop, the detection wavelength is 220nm, and the flow rate is 1mL/min. RP-HPLC purity was identified as shown in FIG. 25. Only a few impurity peaks appear in the chromatogram, the main peak occupies a very large percentage, and the purity of the main peak is 97.91% as can be seen by comparing the areas of the peaks. The main peak solution was collected, rapidly cooled in liquid nitrogen, lyophilized, and then identified by ESI-MS. As shown in FIG. 26, the peak of the AC-Trp-Asn-Leu-Asn-Pro ion is 683.7, which is quite close to its theoretical relative molecular mass 684.72.
3. C-terminal amidated WNLNP (WNLNP-NH) 2 ) Synthesis and purity identification of (C)
The synthesis and chemical modification of the polypeptide are assisted by Jiangsu blaze Biotechnology Co., ltd, according to Trp-Asn-Leu-Asn-Pro-NH 2 Is characterized in that Pro is first carboxylatedThe radical is connected with resin with activated amino group in covalent bond form, then the subsequent amino acid is added according to solid phase synthesis method, so as to obtain the target WNLNP-NH 2 . Purifying by RP-HPLC, and freeze drying to obtain the final product. Purity was performed using RP-HPLC on Kromasil C18 column (100-5C 18,4.6mm x 250mm,5 mcron, column temperature, 30 ℃). The A channel of the liquid phase system is acetonitrile containing 1% of trifluoroacetic acid. The B channel is deionized water with 1% trifluoroacetic acid. The elution conditions were as follows: the initial proportion of A is 20%, the proportion of A rises to 50% in 20min, the proportion of A rises to 100% in 20min to 20.1min, 100% is kept running for 25min to stop, the detection wavelength is 220nm, and the flow rate is 1mL/min. The RP-HPLC purity determination result is shown in FIG. 27. Only a few impurity peaks appear in the chromatogram, the main peak occupies a very large percentage, and the purity of the main peak is 98.67% as can be seen by comparing the areas of the peaks. The main peak solution was collected, rapidly cooled with liquid nitrogen, and lyophilized, and then the structure was identified by ESI-MS. As shown in FIG. 28, WNLNP-NH 2 The ion peak is 642.8, which is close to its theoretical relative molecular mass 641.74.
4. N-terminal palmitoylated WNLNP (Palm-WNLNP) synthesis and purity identification
The synthesis and chemical modification of the polypeptide are completed with the assistance of Jiangsu blaze biotechnology limited company, WNLNP is synthesized according to the amino acid sequence solid-phase synthesis step of Palm-Trp-Asn-Leu-Asn-Pro, then activated palmitic acid is used as amino acid, and the activated palmitic acid reacts with the amino group of Trp at the N end of a peptide chain under the catalysis of HOBT (1-hydroxybenzotriazole) +DIC (diisopropylcarbodiimide), so that the target polypeptide Palm-WNLNP can be obtained. Purifying by RP-HPLC, and freeze drying to obtain the final product. Purity was identified using RP-HPLC on a Kromasil C18 column (100-5C 18,4.6mm x 250mm,5 mcron, column temperature, 30 ℃). The liquid phase system has two channels, wherein solution A of channel A is acetonitrile containing 1% trifluoroacetic acid. The B channel is deionized water with 1% trifluoroacetic acid as solution B. The elution conditions were as follows: the initial proportion of A is 75%, the proportion of A rises to 95% within 20min, the proportion of A rises to 100% within 20min to 20.1min, 100% operation is kept for 25min, the detection wavelength is 220nm, and the flow rate is 1mL/min. RP-HPLC purity was identified as shown in FIG. 29. In the chromatogram, the main peak was a very large percentage, and only a few impurity peaks appeared in the chromatogram, and the purity of the main peak was 96.01% as seen by comparing the peak areas. The main peak solution was collected, rapidly cooled with liquid nitrogen, and lyophilized, and then the structure was identified by ESI-MS. As shown in FIG. 30, the Palm-WNLNP ion peak was 880.1, which is quite close to its theoretical relative molecular mass 881.14.
5. Protection of HaCaT cells by three chemical modifications of WNLNP
The CCK-8 method was used to determine HaCaT cell viability (the method is the same as P1-P6 of example 1 for protection of UVB aged HaCaT cell viability, except that the final sample concentration was 20 μm), and the results are shown in fig. 31: the synthesized polypeptides AC-WNLN, palm-WNLNP and WNLNP can significantly improve the activity of UVB aged HaCaT cells at the concentration of 20 mu M (p <0.01 respectively): wherein Palm-WNLNP has stronger protection effect on UVB aged HaCaT cell viability than WNLNP (p < 0.05), indicating that N-terminal palmitoylation can increase the protection effect of WNLNP on UVB aged HaCaT cell viability.
The inhibition of MMP-1 expression in UVB aged HaCaT cells was determined by Western Blotting (the method is identical to P1-P6 in terms of inhibition of MMP-1 expression in HaCaT cells, except that the sample concentration was 20. Mu.M), and the results are shown in FIG. 31: WNLNP-NH 2 Palm-WNLNP, WNLNP significantly inhibited MMP-1 protein overexpression caused by UVB irradiation (p<0.01 A) is provided; wherein, N-terminal palmitoylation modification can improve the inhibition of the MMP-1 expression by WNLNP (p<0.05)。
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
SEQUENCE LISTING
<110> university of Guangdong ocean
<120> an oyster protein source peptide with skin photoaging resisting effect, and its preparation method and application
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 5
<212> PRT
<213> Crassostrea hongkongensis
<400> 1
Trp Asn Leu Asn Pro
1 5
<210> 2
<211> 5
<212> PRT
<213> Crassostrea hongkongensis
<400> 2
Tyr Thr Val Thr Phe
1 5
<210> 3
<211> 8
<212> PRT
<213> Crassostrea hongkongensis
<400> 3
Arg Lys Asn Glu Val Leu Gly Lys
1 5
<210> 4
<211> 5
<212> PRT
<213> artificial sequence
<220>
<221> MOD-RES
<222> (1)..(1)
<223> Xaa(1)=Palm-Trp
<400> 4
Xaa Asn Leu Asn Pro
1 5
<210> 5
<211> 5
<212> PRT
<213> artificial sequence
<220>
<221> MOD-RES
<222> (1)..(1)
<223> Xaa(1)=AC-Trp
<400> 5
Xaa Asn Leu Asn Pro
1 5
<210> 6
<211> 5
<212> PRT
<213> artificial sequence
<220>
<221> MOD-RES
<222> (5)..(5)
<223> Xaa(5)=Pro-NH2
<400> 6
Trp Asn Leu Asn Xaa
1 5
Claims (9)
1. An active peptide, wherein the amino acid sequence of the active peptide is WNLNP.
2. A nucleic acid molecule encoding the active peptide of claim 1.
3. An expression cassette, recombinant vector or transgenic cell comprising the nucleic acid molecule of claim 2; the transgenic cells do not comprise propagation material.
4. The method for producing an active peptide according to claim 1, wherein the method comprises any one of (a) to (c):
(a) Extracting oyster as raw material;
(b) Synthesizing by adopting a liquid phase or solid phase synthesis method;
(c) A transgenic cell according to claim 3.
5. A chemically modified active peptide modified at the N-terminus and/or C-terminus of the active peptide of claim 1.
6. The chemically modified active peptide of claim 5 wherein:
an N-terminal of the active peptide according to claim 1, which is subjected to an acetylation modification or a palmitoylation modification.
7. The chemically modified active peptide of claim 5 wherein:
amidation modification is performed at the C-terminal end of the active peptide according to claim 1.
8. (d) The application of any one of (h) - (j) to any one of (g);
(d) The active peptide of claim 1;
(e) The nucleic acid molecule of claim 2;
(f) The expression cassette, recombinant vector or transgenic cell of claim 3;
(g) A chemically modified active peptide according to any one of claims 5 to 7;
(h) Preparing an anti-skin photoaging product;
(i) Preparing an anti-aging product;
(j) Preparing a product for preventing or improving skin damage caused by UVB radiation;
the product is a cosmetic, pharmaceutical or reagent.
9. A product, comprising: at least one of (d) to (g);
(d) The active peptide of claim 1;
(e) The nucleic acid molecule of claim 2;
(f) The expression cassette, recombinant vector or transgenic cell of claim 3;
(g) A chemically modified active peptide according to any one of claims 5 to 7;
the product is a cosmetic, pharmaceutical or reagent.
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| CN202111339694.8A CN114163500B (en) | 2021-11-12 | 2021-11-12 | Oyster protein source anti-skin photoaging active peptide and preparation method and application thereof |
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| CN202111339694.8A CN114163500B (en) | 2021-11-12 | 2021-11-12 | Oyster protein source anti-skin photoaging active peptide and preparation method and application thereof |
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| CN114163500B true CN114163500B (en) | 2024-02-09 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010101331A1 (en) * | 2009-03-03 | 2010-09-10 | 경상대학교산학협력단 | External use skin composition which suppresses skin aging |
| KR20160147293A (en) * | 2015-06-15 | 2016-12-23 | 이정복 | Skin external application composition and cosmetic composition comprising peptide derived silk cocoon or its derivatives with an anti-aging, antioxidant or anti-inflammatory effect |
| CN109400678A (en) * | 2018-10-18 | 2019-03-01 | 大连深蓝肽科技研发有限公司 | A kind of anti-oxidant and DPP-IV inhibitory activity peptide in stichopus japonicus source |
-
2021
- 2021-11-12 CN CN202111339694.8A patent/CN114163500B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010101331A1 (en) * | 2009-03-03 | 2010-09-10 | 경상대학교산학협력단 | External use skin composition which suppresses skin aging |
| KR20160147293A (en) * | 2015-06-15 | 2016-12-23 | 이정복 | Skin external application composition and cosmetic composition comprising peptide derived silk cocoon or its derivatives with an anti-aging, antioxidant or anti-inflammatory effect |
| CN109400678A (en) * | 2018-10-18 | 2019-03-01 | 大连深蓝肽科技研发有限公司 | A kind of anti-oxidant and DPP-IV inhibitory activity peptide in stichopus japonicus source |
Non-Patent Citations (3)
| Title |
|---|
| Purification and Identification of Peptides from Oyster (Crassostrea hongkongensis) Protein Enzymatic Hydrolysates and Their Anti-Skin Photoaging Effects on UVB-Irradiated HaCaT Cells;Zhilan Peng，等;Marine drugs;第20卷(第12期);第1-18页 * |
| 牡蛎蛋白源抗皮肤光老化活性肽的分离纯化、鉴定及其作用机理研究;彭志兰;中国博士学位论文全文数据库（电子期刊）工程科技辑Ⅰ辑(第2期);第B024-51页 * |
| 牡蛎蛋白酶解产物及其超滤组分抗皮肤光老化活性研究;彭志兰，等;食品与发酵工业;第47卷(第13期);第66-71页 * |
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| CN114163500A (en) | 2022-03-11 |
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