CN112812150A - Active cyclic peptide, active cyclic peptide composition and application of active cyclic peptide composition in preparation of products with antioxidant or anti-inflammatory effects - Google Patents
Active cyclic peptide, active cyclic peptide composition and application of active cyclic peptide composition in preparation of products with antioxidant or anti-inflammatory effects Download PDFInfo
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- CN112812150A CN112812150A CN202110124497.8A CN202110124497A CN112812150A CN 112812150 A CN112812150 A CN 112812150A CN 202110124497 A CN202110124497 A CN 202110124497A CN 112812150 A CN112812150 A CN 112812150A
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- bifidobacterium longum
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/12—Cyclic peptides with only normal peptide bonds in the ring
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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- Nutrition Science (AREA)
- Mycology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to the technical field of biomedicine, and particularly discloses an active cyclic peptide and application thereof in preparing a product with an antioxidant or anti-inflammatory effect. The active cyclic peptide has a structure shown in a formula I or a formula II. The experimental results show that: the bifidobacterium longum cyclopeptide-1 and the bifidobacterium longum cyclopeptide-2 have obvious antioxidant and anti-inflammatory effects, and particularly have excellent effects of resisting oxidative damage and inflammatory damage of cells caused by UVB. Can be used for preparing cosmetics, skin care products, foods, health products or medicines with antioxidant or antiinflammatory effects.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to an active cyclic peptide, an active cyclic peptide composition and application thereof in preparing a product with antioxidant or anti-inflammatory effects.
Background
External damage stimuli have been considered by studies to be a significant cause of skin aging. Among them, ultraviolet B (UVB, wavelength 280-315nm) irradiation is one of the important causes of skin photoaging, which causes facial wrinkles. UVB has been reported to affect fibroblast physiological functions, and UVB irradiation significantly promotes apoptosis in human keratinocytes. Most skin diseases, such as cancer, photoaging, sunburn and pigmentation, are reported to be closely related to UVB exposure. Studies with human epidermoid carcinoma a431 cells indicate that UVB radiation induces autophagy of a431 cells.
The incidence of skin cancer is greatly increased in skin that is over-exposed to uv radiation and is damaged by uv radiation. Ultraviolet radiation is one of the major external factors that cause oxidative stress in skin cells. Reactive Oxygen Species (ROS) have been shown to be associated with a number of pathologies, including skin aging caused by alterations in energy metabolism resulting from uv irradiation. ROS trigger a series of cascade reactions simultaneously, leading to overexpression of MMPs and ultimately photoaging. Furthermore, increased MMPs expression is the most prominent feature in ultraviolet b (uvb) -induced photoaged skin.
ROS have also been shown to be involved in many pathological processes, including aging due to altered energy metabolism. The tricarboxylic acid cycle (TCA cycle) is the basic metabolic pathway in which various active enzymes of the cell participate. UVB radiation causes a decrease in dihydrolipoyl dehydrogenase (DLD) expression in the TCA cycle. UVB targets DLD in the skin with antioxidant activity, which means that DLD can protect skin cells from uv-induced oxidative stress through the endogenous cellular defense system. In addition, the nuclear factor E2 release factor 2(Nrf2) signal pathway of the main defense system of the skin plays an important role in resisting oxidative damage caused by UVB. In the absence of stimulation, Nrf2 was captured by Kelch-like ECH-associated proteins and degraded by proteasomes. Cytoplasmic Nrf2 is ready to translocate to the nucleus where it binds to a small Maf protein and to the Antioxidant Response Element (ARE) of the upstream promoter region, driving the expression of cytoprotective enzymes and AREs, such as nad (p) H: quinone oxidoreductase 1(NQO-1) and heme oxygenase 1 (HO-1). The cellular antioxidant system is considered to be the most important endogenous defense system against oxidative stress. Nrf2 and ARE reportedly key regulators of oxidative stress, and various natural products can promote the transfer of Nrf2 to the nucleus.
Oxidative stress caused by ultraviolet light directly or indirectly exacerbates the risk of skin damage. The increased incidence of human skin cancer with harmful stimuli such as high quarterly uv radiation has placed a growing need for new preventive strategies.
However, there are many factors that cause skin oxidation and inflammatory damage, and the existing antioxidant and anti-inflammatory drugs are not necessarily capable of treating skin cell oxidation and inflammatory damage caused by ultraviolet b (uvb). Therefore, the development of a compound having the effects of resisting skin cell oxidation and inflammatory injury caused by UV has important application value for preventing skin diseases.
Disclosure of Invention
In view of the above, the invention firstly provides an active cyclic peptide with a novel structure, and further research shows that the active cyclic peptide has the effects of resisting skin cell oxidation and inflammatory injury caused by UV.
The detailed technical scheme of the invention is as follows:
in a first aspect, the present invention provides an active cyclic peptide having a structure represented by formula i or formula ii:
preferably, the active cyclic peptide shown in the formula I is formed by head-to-tail connection and cyclization of a linear peptide shown in SEQ ID No.1 (His-Phe-Leu-Val);
the active cyclic peptide shown in the formula II is formed by head-to-tail connection and cyclization of a linear peptide shown in SEQ ID No.2 (Thr-His-Ala-Trp).
Preferably, the active cyclic peptide shown in the formula I is formed by connecting an amino group of His and a carboxyl group of Val through an amide bond condensation reaction to form a ring;
the active cyclopeptide shown in the formula II is formed by connecting an amino group of Thr and a carboxyl group of Trp through an amido bond condensation reaction.
The active cyclic peptide with the structure shown in the formula I is named as bifidobacterium longum cyclic peptide-1 (abbreviated as CP-1). The amino acid sequence of the bifidobacterium longum cyclopeptide-1 is as follows: cycle- [ His-Phe-Leu-Val ]; the acyclic linear amino acid sequence can be the amino acid sequence shown as SEQ ID No.1 in a sequence table.
The active cyclic peptide with the structure shown in the formula II is named as bifidobacterium longum cyclic peptide-2 (abbreviated as CP-2). The amino acid sequence of the bifidobacterium longum cyclopeptide-2 is as follows: cycle- [ Thr-His-Ala-Trp ]; the acyclic linear amino acid sequence can be the amino acid sequence shown as SEQ ID No.2 in the sequence table.
The bifidobacterium longum cyclopeptide-1 and the bifidobacterium longum cyclopeptide-2 can be obtained by separating from bifidobacterium; can also be prepared according to the methods in the examples.
In a second aspect of the invention, an active cyclic peptide composition is provided, which comprises an active cyclic peptide having a structure shown in formula I and an active cyclic peptide having a structure shown in formula II.
Preferably, the molar ratio of the active cyclic peptide with the structure shown in the formula I to the active cyclic peptide with the structure shown in the formula II is 1-6: 1-6.
Further preferably, the molar ratio of the active cyclic peptide with the structure shown in the formula I to the active cyclic peptide with the structure shown in the formula II is 1-4: 1-4.
Most preferably, the molar ratio of the active cyclic peptide having the structure shown in formula I to the active cyclic peptide having the structure shown in formula II is 1: 1.
In a third aspect of the invention, there is provided a use of the above active cyclic peptide in the preparation of a product having antioxidant and/or anti-inflammatory effects.
Preferably, the antioxidant and/or anti-inflammatory effect is specifically an anti-UV-induced skin cell oxidation and/or inflammatory damage effect.
In a fourth aspect of the invention, there is provided the use of an active cyclic peptide as described above in the manufacture of a product having anti-photoaging, anti-inflammatory, anti-wrinkle, sunscreen or anti-pigmentation properties, or in the manufacture of a product having skin cancer treatment or prevention properties.
Preferably, the product is a cosmetic, a skin care product, a food, a health product or a medicament.
Further preferably, the cosmetic or skin care product comprises an emulsion, a cream, a gel, a water, an oil, a powder or a mask.
Further preferably, the food, health product or pharmaceutical is in the form of a tablet, capsule, powder, granule, pill, syrup, solution, suspension or aerosol.
Has the advantages that: the invention provides an active cyclic peptide with a brand-new structure; test results show that the bifidobacterium longum cyclopeptide-1 and the bifidobacterium longum cyclopeptide-2 can obviously improve the cell activity after UVB irradiates HaCaT cells and can obviously improve the cell activity after BaP stimulates the HaCaT cells; the results show that the bifidobacterium longum cyclopeptide-1 and the bifidobacterium longum cyclopeptide-2 with the brand-new structures have excellent antioxidant and anti-inflammatory effects, and particularly have excellent effects of resisting oxidative damage and inflammatory damage of cells caused by UVB. The activity of the bifidobacterium longum cyclopeptide-1 is better than that of the bifidobacterium longum cyclopeptide-2; therefore, we further provide an active cyclic peptide composition consisting of bifidobacterium longum cyclic peptide-1 and bifidobacterium longum cyclic peptide-2, which helps to improve the activity of bifidobacterium longum cyclic peptide-2. Further research shows that the bifidobacterium longum cyclopeptide-1 and the bifidobacterium longum cyclopeptide-2 have obvious effect of improving the cell activity after UVB irradiates HaCaT cells. Further research shows that bifidobacterium longum cyclopeptide-1 and bifidobacterium longum cyclopeptide-2 can inhibit apoptosis caused by UVB irradiation by reducing excessive ROS production induced by UVB in cells and less excessive secretion of inflammatory factors, thereby achieving the purposes of resisting oxidative damage and inflammatory damage. Further research shows that the bifidobacterium longum cyclopeptide-1 and the bifidobacterium longum cyclopeptide-2 can play the activities of resisting oxidative damage and inflammatory damage caused by UVB irradiation by regulating DLD/Nrf2/ARE and NF-kB signal channels. Skin diseases such as cancer, photoaging, sunburn and pigmentation are closely related to UVB-induced skin cell oxidation and inflammatory injury activity; therefore, the bifidobacterium longum cyclopeptide-1, the bifidobacterium longum cyclopeptide-2 and the combination thereof can be further used for preparing cosmetics, skin care products, foods, health products or medicines with the effects of preventing cancers, preventing sun, preventing pigmentation or resisting photoaging. In addition, the preparation process of the active cyclic peptide is simple, the operation is convenient, the purity of the prepared antioxidant and anti-inflammatory phenol amide micromolecule compound is high, and the production cost can be reduced when the compound is popularized and applied.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only drawings of some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a graph showing the results of high performance liquid chromatography measurement of Bifidobacterium longum cyclic peptide-1.
FIG. 2 is a graph showing the results of high performance liquid chromatography measurement of Bifidobacterium longum cyclopeptide-2.
FIG. 3 is a mass spectrum of Bifidobacterium longum cyclopeptide-1.
FIG. 4 is a mass spectrum of Bifidobacterium longum cyclopeptide-2.
FIG. 5 is a graph showing the result of anti-UVB oxidative damage to HaCaT cells when Bifidobacterium longum cyclic peptide-1 is short peptide, Bifidobacterium longum cyclic peptide-2 and the composition thereof. Wherein, FIG. 5A is a graph showing the effect on the viability of HaCaT cells after UVB irradiation; FIG. 5B is a graph showing the effect on ROS content in HaCaT cells after UVB irradiation; FIG. 5C is a graph showing the effect on mitochondrial membrane potential of HaCaT cells after UVB irradiation; FIG. 5D is a graph showing the effect on mitochondrial cytochrome C in HaCaT cells after UVB irradiation.
FIG. 6 is a graph showing the results of experiments on the inhibition of apoptosis induced by UVB-irradiated HaCaT cells by using Bifidobacterium longum cyclopeptide-1 as short peptide, Bifidobacterium longum cyclopeptide-2 and compositions thereof.
FIG. 7 is a graph showing the results of experiments on the inhibition of the over-expression of MMPs in cells induced by UVB irradiation on HaCaT cells by using Bifidobacterium longum cyclopeptide-1 as short peptide and Bifidobacterium longum cyclopeptide-2 and compositions thereof.
FIG. 8 is a graph of the results of experiments in which Bifidobacterium longum cyclopeptide-1 is short peptide, Bifidobacterium longum cyclopeptide-2 and combinations thereof inhibit nuclear transfer of HaCaT cells Nrf2 caused by UVB irradiation.
FIG. 9 is a graph showing the results of experiments in which Bifidobacterium longum cyclic peptide-1 is short peptide, Bifidobacterium longum cyclic peptide-2 and combinations thereof inhibit activation of DLD/HO-1/NQO-1 signaling pathway in HaCaT cells caused by UVB irradiation.
FIG. 10 is a graph showing the results of experiments on the inhibition of the activation of NF- κ B signaling pathway in HaCaT cells by BaP stimulation with Bifidobacterium longum cyclopeptide-1 as short peptide, Bifidobacterium longum cyclopeptide-2 and combinations thereof.
Detailed Description
The technical solution of the present invention will be clearly and completely described with reference to the following examples. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 isolation of Cyclic peptide-1 of Bifidobacterium longum and Cyclic peptide-2 of Bifidobacterium longum
Mixing 50g of bifidobacterium longum with 500mL of ethyl acetate and 2000mL of water uniformly, standing, extracting to obtain an aqueous phase layer, repeating the extraction process for multiple times, carrying out vacuum low-temperature freeze-drying on the aqueous phase layer, and carrying out preparative separation by preparative HPLC to obtain the active cyclic peptide bifidobacterium longum cyclic peptide-1 (CP-1).
Mixing 50g of bifidobacterium longum with 500mL of ethyl acetate and 2000mL of water uniformly, standing, extracting to obtain an aqueous phase layer, repeating the extraction process for multiple times, carrying out vacuum low-temperature freeze-drying on the aqueous phase layer, and carrying out preparative separation by using preparative HPLC to obtain the cyclic peptide-2 (CP-2) of bifidobacterium longum.
The preparation conditions of the preparative HPLC were as follows: taking 0.1% trifluoroacetic acid water solution as a mobile phase A, taking 0.1% trifluoroacetic acid acetonitrile solution as the mobile phase A, wherein the ratio of the mobile phase A: the mobile phase B is 60:40, and the measuring wavelength is 310 μm. From FIGS. 1 and 2, it can be seen that the peak-off times of Bifidobacterium longum cyclic peptide-1 (CP-1) and Bifidobacterium longum cyclic peptide-2 (CP-2) were 8.058min and 10.905min, respectively.
EXAMPLE 2 chemical Synthesis of Bifidobacterium longum cyclopeptide-1 and Bifidobacterium longum cyclopeptide-2
(1) Placing 100mg Fmoc-His Wang Resin in a solid phase synthesis tube, adding N, N-Dimethylformamide (DMF), standing to fully swell the Resin, filtering to remove the solvent, adding piperidine DMF solution, oscillating, and filtering to remove the solvent. Dissolving Fmoc-Phe-OH, 1-hydroxy benzotriazole and O-benzotriazole-tetramethylurea hexafluorophosphate in DMF, adding N, N-diisopropylethylamine, mixing uniformly and keeping out of the sun, activating, adding into resin, stirring for 2 hours at 25 ℃ under the action of nitrogen blowing, carrying out suction filtration, washing with DMF and dichloromethane in sequence, and drying the solvent. Repeating the steps, sequentially adding activated Fmoc-Leu-OH and Fmoc-Val-OH into resin, stirring for 2 hours under the action of nitrogen blowing at 25 ℃, washing the resin after complete reaction, evaporating the obtained filtrate under reduced pressure to remove the solvent to obtain a precipitate Z-1, cutting the resin from Z-1 by using a TFA solution to obtain a free-OH end, evaporating the solid obtained by removing the TFA under reduced pressure at 40 ℃ in a water bath, adding the solid into ethyl acetate, mixing the solid with equimolar p-nitrophenol, preparing the p-nitrophenol active ester Z-2 by using DCC as a condensing agent, removing the protecting group Fmoc from Z-2 by using a piperidine DMF solution, dissociating an N end, removing the redundant solvent to obtain a crude peptide solid, adding alkali Na2CO310 is prepared from solvent dioxane-3-10-4Diluting the solution, reacting at 25 deg.C for 5 hr, removing solvent by rotary evaporation at 40 deg.C in organic phase water bath, and freeze drying to obtain Bifidobacterium longum cyclic peptide-1 (CP-1) with amino acid sequence of Cycle- [ His-Phe-Leu-Val]。
(2) 100mg of Fmoc-Thr Wang Resin is put into a solid phase synthesis tube, N-Dimethylformamide (DMF) is added, and the mixture is stood to fully swell the ResinThe solvent was filtered off, followed by addition of piperidine DMF solution and shaking. Dissolving Fmoc-His-OH, 1-hydroxybenzotriazole and O-benzotriazol-tetramethylurea hexafluorophosphate in DMF, adding N, N-diisopropylethylamine, uniformly mixing, keeping out of the sun, activating, adding into resin, stirring for 2 hours at 25 ℃ under the action of nitrogen blowing, carrying out suction filtration, washing with DMF and dichloromethane in sequence, and drying the solvent. Repeating the steps, sequentially adding activated Fmoc-Ala-OH and Fmoc-Trp-OH into resin, stirring for 2 hours under the action of nitrogen blowing at 25 ℃, washing the resin after complete reaction, carrying out reduced pressure evaporation on the obtained filtrate to remove the solvent to obtain a precipitate Y-1, cutting off the resin by using a TFA solution to obtain a free-OH end by using Y-1, adding a solid obtained by reduced pressure evaporation of TFA in water bath at 40 ℃ into ethyl acetate, mixing the solid with equimolar p-nitrophenol, preparing p-nitrophenol active ester Y-2 by using DCC as a condensing agent, removing Fmoc from Y-2 by using a piperidine DMF solution, dissociating an N end, removing the redundant solvent to obtain a crude peptide solid, adding alkali Na2CO310 is prepared from solvent dioxane-3-10-4Diluting the solution, reacting at 25 deg.C for 5 hr, removing solvent by rotary evaporation in organic phase water bath at 40 deg.C, and freeze drying to obtain Bifidobacterium longum cyclic peptide-2 (CP-2) with amino acid sequence of Cycle- [ Thr-His-Ala-Trp]。
Measuring the cyclic peptide-1 of the bifidobacterium longum and the cyclic peptide-2 of the bifidobacterium longum by using a mass spectrum, wherein the mass spectrum measuring conditions comprise ESI positive ion mode capillary voltage of 3kV, taper hole voltage of 50V, extraction voltage of 5V, desolvation temperature of 350 ℃ and atomized air flow of 350L/h; the high performance liquid chromatography measurement conditions were that a Boston Green ODS-AQ chromatographic column (250 × 4.6mm) was used, a 0.1% aqueous trifluoroacetic acid solution was used as a mobile phase a, a 0.1% acetonitrile solution of trifluoroacetic acid was used as a mobile phase a, and the mobile phase a: the mobile phase B was 60:40, the flow rate was 1mL/min, the detection wavelength was 310 μm, and the sample size was 10 μ L.
497.2843[ M + H ] from the mass spectral data (see FIG. 3)]+,469.2897[M-CO+H]+,398.2164[M-Val+H2O+H]+,384.2012[M-Leu+H2O+H]+,380.2053[M-Val+H]+,350.2168[M-Phe+H2O+H]+,285.1325[His-Phe-2H2O+H]+,261.1582[Leu-Phe-2H2O+H]+,237.1332[His-Val-2H2O+H]+,212.1169[Val-Leu-2H2O+H]+,138.0656[His-H2O+H]+,110.0708[His-COOH+H]+,86.0975[Leu-COOH+H]+. Finally, the amino acid sequence of the active cyclic peptide bifidobacterium longum cyclic peptide-1 (CP-1) is determined to be Cycle- [ His-Phe-Leu-Val]I.e. the chemical structure shown in formula I;
496.7325[ M + H ] from the mass spectral data (see FIG. 4)]+,478.2213[M-H2O+H]+,460.7127[M-2H2O+H]+,425.1942[M-Ala+H2O+H]+,258.1244[Trp-Ala-2H2O+H]+,239.1140[His-Thr-2H2O+H]+,209.1035[Ala-His-2H2O+H]+,159.0920[Trp-COOH+H]+,110.0721[His-COOH+H]+. Finally, the amino acid sequence of the bifidobacterium longum cyclopeptide-2 (CP-2) is determined to be Cycle- [ Thr-His-Ala-Trp](ii) a Namely the chemical structure shown in formula II.
Examples of the experiments
To evaluate the biological activities of bifidobacterium longum cyclopeptide-1 and bifidobacterium longum cyclopeptide-2 and combinations thereof of the present invention, the following effect examples were performed.
Culturing HaCaT cells of immortalized keratinocyte cell line in a cell culture box at 37 ℃ and 5% CO2(DMEM medium). The irradiation intensity of UVB is 80mJ/cm2The irradiation light source was spaced 15cm from the cells. During irradiation, the culture cell culture solution of each group is sucked off, washed for 2 times by PBS, and then a small amount of solution is added to cover the bottom surface to avoid drying. The plates were irradiated in a room temperature water bath to avoid overheating after irradiation. After irradiation, PBS is discarded, and DMEM medium or the medium containing bifidobacterium cyclopeptide (20 mu M) is added again for further culture for 24 hours. The CCK8 method is used for detecting cell viability and collecting cells and culture solution.
In the test of the effect of compounds on the viability of HaCaT cells, the experiments were divided into 4 groups: normal Control group (Control); a UVB irradiation group; CP-1 group (UVB irradiation + CP-120. mu.M); CP-2 group (UVB irradiation + CP-220. mu.M). Each treatment condition was replicated 3 times in 3 wells and the experiment was repeated 3 times. The normal control group did not receive UVB irradiation, and the UVB irradiation group, CP-1 group and CP-2 group received UVB irradiation respectively.
The experiments were divided into 4 groups: normal Control group (Control); a UVB irradiation group; CP-1 group (UVB irradiation + CP-120. mu.M); CP-2 group (UVB irradiation + CP-220. mu.M). Each treatment condition was replicated 3 times in 3 wells and the experiment was repeated 3 times. The normal control group did not receive UVB irradiation, and the UVB irradiation group, CP-1 group and CP-2 group received UVB irradiation respectively. After the HaCaT cells are treated according to the experimental design, the cells are collected; detecting the secretion of ROS by referring to an ELISA kit operating instruction; detecting the change condition of the cell mitochondrial membrane potential by adopting flow cytometry; and (5) detecting the content change condition of the MMPs by referring to the operation instruction of the ELISA kit.
After the HaCaT cells were processed as designed, the cells were collected and centrifuged at 2000g for 3min at room temperature. Cells were suspended in pre-cooled 1 × PBS, centrifuged at 2000g for 3min, and the cells washed. Annexin V-FITC/PI double staining experiments were performed according to the manufacturer's instructions. Apoptosis was detected by flow cytometry and all experiments were repeated at least 3 times.
After the HaCaT cells were treated according to the experimental design, the cells were collected, washed 2 times with precooled PBS, added with 50 μ L of cell lysate, and left to stand at 4 ℃ for 30 min. Centrifuging at 10000r/min for 15min, taking supernatant to extract total protein, and performing protein quantification by using a BCA method. Total proteins were separated by SDS-PAGE and transferred to PVDF membrane. Blocking with 5% skimmed milk powder at room temperature for 2 h. The primary antibody was then added, shaken gently overnight at 4 ℃, washed 3 times with TBST, the corresponding secondary antibody was added, incubated 1h at room temperature, and rinsed 3 times.
The results are as follows:
as can be seen from the data in FIG. 5A, the active cyclic peptides Bifidobacterium longum cyclic peptide-1 (CP-1) and Bifidobacterium longum cyclic peptide-2 (CP-2) have significantly improved HaCaT cell viability after UVB irradiation; and the active cyclic peptide bifidobacterium longum cyclic peptide-1 (CP-1) is improved more obviously than the bifidobacterium longum cyclic peptide-2 (CP-2). This shows that the cyclic peptide-1 (CP-1) and cyclic peptide-2 (CP-2) of Bifidobacterium longum can improve the activity of cells against UVB damage and reduce the damage of UVB irradiation to cells; and the activity of the bifidobacterium longum cyclic peptide-1 (CP-1) is greater than that of the bifidobacterium longum cyclic peptide-2 (CP-2). As shown in FIG. 5B, UVB irradiation increased the ROS content in HaCaT cells compared to Control, while CP-1 and CP-2 reduced UVB irradiation induced ROS, and the CP-1 reduction was more pronounced; as shown in fig. 5C, compared to the Control group, UVB irradiation significantly decreased the mitochondrial membrane potential of HaCaT cells, whereas the CP-1 group and CP-2 group inhibited UVB irradiation-induced decrease in mitochondrial membrane potential of cells, and the inhibition effect of CP-1 group was more significant; as shown in FIG. 5D, UVB irradiation significantly leaked mitochondrial cytochrome C from HaCaT cells to cytoplasm, whereas CP-1 and CP-2 suppressed the UVB irradiation-induced leakage of mitochondrial cytochrome C, and the inhibition of CP-1 was more significant, as compared to Control. The results of the experiments in FIG. 5 show that CP-1 and CP-2 are resistant to UVB-induced oxidative damage of cells; and the effect of CP-1 is stronger than that of CP-2.
As shown in FIG. 6, UVB irradiation resulted in increased apoptosis, while HaCaT cell apoptosis rate was significantly decreased by CP-1 and CP-2 groups. We speculate that CP-1 and CP-2 can reduce the damage to the skin by reducing UVB radiation-induced HaCaT cell apoptosis; UVB activates the secretion of MMPs, which is one of the signs of skin damage and aging. To investigate the effect of CP-1 and CP-2 on UVB-induced MMPs expression, we examined the effect of CP-1 and CP-2 on UVB-induced MMPs secretion. The result shows that UVB irradiation can obviously increase MMP-1 and MMP-3 expression of HaCaT cells, and CP-1 and CP-2 can inhibit UVB-induced MMP-1 and MMP-3 expression. ELISA results were similar to Western blotting results, with increased secretion of MMP-1 and-3 following UVB irradiation of HaCaT cells. Whereas CP-1 and CP-2 reduced MMP-1 and-3 secretion from HaCaT cells induced by UVB irradiation (see FIG. 7). It can also be seen from FIG. 7 that CP-1 has a stronger activity than CP-2.
As shown in fig. 8, UVB irradiation resulted in a significant increase in nuclear transfer of ncat 2 in HaCaT cells, whereas CP-1 and CP-2 inhibited UVB-induced nuclear transfer of Nrf 2; further CP-1 and CP-2 stabilized the changes in expression of DLD, HO-1 and NQO-1 caused by UVB irradiation (see FIG. 9).
In the research, UVB irradiation can remarkably up-regulate the expression of p65, I kappa B alpha and p50, and CP-1 and CP-2 remarkably inhibit UVB irradiation expression caused by UVB irradiation, and the inhibition effect of CP-1 is greater than that of CP-2; this suggests that CP-1 and CP-2 regulate the inflammatory injury of cells caused by UVB irradiation by inhibiting the activation of NF- κ B signaling pathway (see FIG. 10); and CP-1 has a greater activity than CP-2.
In conclusion, CP-1 and CP-2 can inhibit oxidative damage and inflammatory damage of cells caused by UVB irradiation; CP-1 and CP-2 can be used for preparing products for resisting skin cell oxidation and/or inflammatory injury caused by UV.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Sequence listing
<110> Shenzhen sea-invasive Biotech Limited
<120> an active cyclic peptide, an active cyclic peptide composition and the application thereof in the preparation of products with antioxidant or anti-inflammatory effects
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
His Phe Leu Val
1
<210> 2
<211> 4
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Thr His Ala Trp
1
Claims (8)
1. An active cyclic peptide having a structure represented by formula i or formula ii:
2. an active cyclic peptide composition, which is characterized by comprising an active cyclic peptide with a structure shown in a formula I and an active cyclic peptide with a structure shown in a formula II.
3. The active cyclic peptide composition of claim 2, wherein the molar ratio of the active cyclic peptide having the structure shown in formula I to the active cyclic peptide having the structure shown in formula II is 1-6: 1-6.
4. The active cyclic peptide composition of claim 2, wherein the molar ratio of the active cyclic peptide with the structure shown in formula I to the active cyclic peptide with the structure shown in formula II is 1-4: 1-4; most preferably, the molar ratio of the active cyclic peptide having the structure shown in formula I to the active cyclic peptide having the structure shown in formula II is 1: 1.
5. Use of an active cyclic peptide or an active cyclic peptide composition according to any one of claims 1 to 4 for the preparation of a product having antioxidant and/or anti-inflammatory activity.
6. Use according to claim 5, characterized in that the antioxidant and/or anti-inflammatory action is in particular an action against UV-induced skin cell oxidation and/or inflammatory damage.
7. Use of an active cyclic peptide or an active cyclic peptide according to any one of claims 1 to 4 for the preparation of a product having anti-photoaging, anti-inflammatory, anti-wrinkle, sunscreen or anti-pigmentation effects; or in the preparation of products for treating or preventing skin cancer.
8. The use according to any one of claims 5 to 7, wherein the product is a cosmetic, skin care product, food, health product or pharmaceutical.
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| CN112920075A (en) * | 2021-02-01 | 2021-06-08 | 深圳海创生物科技有限公司 | Amide compound, composition and application of amide compound and composition in preparation of products with antioxidant or anti-inflammatory effects |
| CN113832061A (en) * | 2021-09-26 | 2021-12-24 | 广东一元兰欣生物科技有限公司 | Bifidobacterium longum NX-8 and application thereof in preparation of anti-aging drugs |
| CN114621319A (en) * | 2021-12-22 | 2022-06-14 | 深圳科兴药业有限公司 | Probiotic active peptide, active peptide composition and application thereof in preparation of products with anti-inflammatory and/or antioxidant effects |
| CN114790228A (en) * | 2022-05-19 | 2022-07-26 | 浙江湃肽生物股份有限公司 | Anti-skin-aging cyclic hexapeptide compound and preparation method thereof |
| CN117462440A (en) * | 2023-12-26 | 2024-01-30 | 杭州湃肽生化科技有限公司 | Functional cyclic peptide and preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112920075A (en) * | 2021-02-01 | 2021-06-08 | 深圳海创生物科技有限公司 | Amide compound, composition and application of amide compound and composition in preparation of products with antioxidant or anti-inflammatory effects |
| CN113832061A (en) * | 2021-09-26 | 2021-12-24 | 广东一元兰欣生物科技有限公司 | Bifidobacterium longum NX-8 and application thereof in preparation of anti-aging drugs |
| CN114621319A (en) * | 2021-12-22 | 2022-06-14 | 深圳科兴药业有限公司 | Probiotic active peptide, active peptide composition and application thereof in preparation of products with anti-inflammatory and/or antioxidant effects |
| CN114790228A (en) * | 2022-05-19 | 2022-07-26 | 浙江湃肽生物股份有限公司 | Anti-skin-aging cyclic hexapeptide compound and preparation method thereof |
| CN117462440A (en) * | 2023-12-26 | 2024-01-30 | 杭州湃肽生化科技有限公司 | Functional cyclic peptide and preparation method and application thereof |
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