Preparation of andiroba collagen tripeptide with skin anti-aging function and application thereof

Abstract

The application discloses and application of a giant salamander collagen tripeptide with skin anti-aging function, and belongs to the technical field of biological medicine. The giant salamander bone collagen hydrolysate is prepared by enzymolysis of giant salamander bones, the filtrate is collected by filtering through a 1 kDa ultrafiltration membrane to obtain the giant salamander bone collagen hydrolysate with a molecular weight of less than 1 kDa, and then the peptide segments are identified by LC-MS / MS and the collagen tripeptides FGI, PFI and VLF are obtained by combining virtual screening. The tripeptides FGI, PFI and VLF can improve the degradation of type I collagen and elastin caused by UVB irradiation, FGI can further reduce the inflammatory response by inhibiting the activation of the NF-kappa B signaling pathway, thereby reducing the skin photoaging caused by UVB, delaying the skin aging process, improving the skin elasticity, reducing wrinkles and improving the skin repair capacity. The application provides a theoretical basis for skin anti-aging or skin anti-photoaging.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the preparation and application of a giant salamander collagen tripeptide with anti-skin aging function. Background Technology

[0002] Skin aging is a complex process caused by the interaction of various internal and external factors, among which ultraviolet (UV) radiation is one of the main external factors leading to photoaging. Long-term UV exposure can activate the NF-κB signaling pathway, causing the degradation of structural proteins such as collagen and elastin in the dermis, thereby accelerating the skin aging process. With age, the dermis's ability to synthesize collagen declines, and elastin degradation accelerates, leading to reduced skin elasticity, increased wrinkles, and impaired skin barrier function, resulting in a series of aging phenomena such as sagging and dryness. Current anti-aging research mainly focuses on improving skin aging problems by supplementing with collagen peptides. Collagen peptides have received widespread attention due to their ability to promote skin repair, improve skin elasticity, and delay aging. Traditional collagen peptides are mainly derived from mammals such as pigs and cattle, which not only pose potential risks of zoonotic diseases but are also restricted by religious taboos. Therefore, developing pure collagen peptides derived from non-mammals not only helps avoid the risks of zoonotic diseases but also promises greater consistency in efficacy and product quality, and may exhibit superior bioactivity. Current research indicates that collagen peptides can exert anti-aging effects by inhibiting the NF-κB signaling pathway, thereby slowing down UV-induced collagen degradation and inflammatory responses. However, existing research largely focuses on mixed systems of common collagen hydrolysates, with insufficient systematic studies on specific small-molecule collagen peptides, particularly lacking in-depth exploration of specific tripeptides with anti-photoaging activity. Therefore, screening and developing novel collagen tripeptides with unique peptide compositions that can efficiently regulate key pathways of skin aging has become an important research direction in the field of collagen anti-aging. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing and applying giant salamander collagen tripeptides with anti-skin aging functions, thereby addressing the problems existing in the prior art. Through enzymatic hydrolysis, ultrafiltration, virtual screening, and artificial synthesis, collagen tripeptides FGI, PFI, and VLF are obtained. These tripeptides can improve the degradation of type I collagen and elastin caused by UVB irradiation. FGI can also reduce inflammatory responses by inhibiting the activation of the NF-κB signaling pathway, thus mitigating the UVB-induced photoaging process of the skin. This invention provides a theoretical basis for combating skin photoaging.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] This invention provides the application of giant salamander collagen tripeptide in any of the following:

[0006] (1) Application in the preparation of anti-skin photoaging products;

[0007] (2) Application in the preparation of anti-aging products;

[0008] The amino acid sequence of the giant salamander collagen tripeptide includes FGI, PFI, or VLF.

[0009] This invention also provides the application of giant salamander collagen tripeptide in the preparation of dietary supplements, characterized in that the amino acid sequence of the giant salamander collagen tripeptide includes FGI, PFI, or VLF.

[0010] Preferably, the preparation method of the giant salamander collagen peptide includes the following steps:

[0011] The dried giant salamander bone powder was mixed with water, and a compound enzyme was added for enzymatic hydrolysis. The supernatant was collected after centrifugation of the hydrolysate, dried, and freeze-dried to obtain giant salamander bone collagen hydrolysate.

[0012] The giant salamander bone collagen hydrolysate was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, the filtrate was collected and freeze-dried to obtain a low molecular weight collagen hydrolysate with a molecular weight cutoff of <1 kDa.

[0013] The low molecular weight collagen hydrolysate with a molecular weight of <1 kDa was separated and identified using LC-MS / MS technology to obtain giant salamander bone collagen peptides.

[0014] Preferably, the mass-to-volume ratio of the giant salamander bone powder to water is 1:2.

[0015] Preferably, based on the mass of crude protein in the giant salamander bone meal, 6000U of a complex enzyme is added per gram of crude protein in the giant salamander bone meal, said complex enzyme being Novozym. ® 11039, Protamex ® It is a mixture of 1.6X and neutral protease in an enzyme activity ratio of 1:1:1.

[0016] Preferably, the enzymatic hydrolysis conditions are: continuous stirring at 55°C for 2 hours.

[0017] Preferably, the conditions for separation and identification using the LC-MS / MS technology are as follows:

[0018] The analytical column used for mass spectrometry detection is C10. 18The column flow rate was controlled at 300 nL / min; the column temperature was 40℃; the electrospray voltage was 2kV; mobile phase A was ultrapure water containing 0.1% formic acid, and mobile phase B was 0.1% formic acid, 80% acetonitrile, and 19.9% ​​ultrapure water; the detection gradient started from 2.2% of phase B, increased to 90% nonlinearly in 54.5 minutes, increased to 99% within 0.5 minutes, and was maintained for 5 minutes.

[0019] The mass spectrometer operates in data-dependent acquisition mode and automatically switches between MS and MS / MS acquisition. The mass spectrometry parameters are set as follows: (1) MS: Scan range (m / z): 200-1500; Resolution: 60000; Normalized AGC target: 300%; Maximum injection time: 25 ms; (2) HCD-MS / MS: Resolution: 15000; Normalized AGC target: 50%; Maximum injection time: 22 ms; Collision energy: 30%; Dynamic exclusion time: 5 s;

[0020] Peptide card value -10lgP≥20.

[0021] Preferably, the product includes pharmaceuticals and cosmetics. The cosmetics include face creams, serums, lotions, and repair serums.

[0022] The present invention also provides a drug or cosmetic for anti-photoaging of the skin, containing giant salamander collagen tripeptide, wherein the amino acid sequence of the giant salamander collagen tripeptide includes FGI, PFI or VLF.

[0023] The present invention also provides a dietary supplement containing giant salamander collagen tripeptide, wherein the amino acid sequence of the giant salamander collagen tripeptide includes FGI, PFI or VLF.

[0024] The present invention discloses the following technical effects:

[0025] This invention prepares giant salamander bone collagen hydrolysate using a compound enzyme hydrolysis technique and obtains low molecular weight collagen hydrolysate through <1 kDa ultrafiltration membrane filtration. This method can effectively obtain collagen peptides with a molecular weight of less than 1 kDa, significantly improving their bioactivity and absorption efficiency. Based on virtual screening technology, combined with predictions of peptide activity, toxicity, sensitization, and gastrointestinal absorbability, a non-toxic, non-sensitizing collagen tripeptide FGI (Phe-Gly-Ile), PFI (Pro-Phe-Ile), and VFL (Val-Phe-Leu) with high gastrointestinal absorbability were successfully identified. In human skin fibroblast experiments, FGI, PFI, and VFL all promoted the expression of type I collagen and elastin. Among them, FGI can also repair UV-induced photoaging of the skin by inhibiting the activation of the NF-κB signaling pathway caused by UVB irradiation. Compared with traditional photodynamic therapy, laser therapy, and antioxidant therapy, this invention provides a deep and lasting repair effect, offering a new technical approach to solving the problem of photoaging of the skin. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The molecular weight distribution of the collagen hydrolysate from the bones of the giant salamander;

[0028] Figure 2 The effects of collagen tripeptides FGI (A), PFI (B), and VLF (C) on the vitality of human skin fibroblasts;

[0029] Figure 3 The effects of collagen tripeptides FGI, PFI, and VLF on the expression of type I collagen and elastin in human fibroblasts were investigated. (A) Western blot results after FGI intervention; (B) Type I collagen expression level after FGI intervention; (C) Elastin expression level after FGI intervention; (D) Western blot results after PFI and VLF intervention; (E) Type I collagen expression level after PFI and VLF intervention; (F) Elastin expression level after PFI and VLF intervention.

[0030] Figure 4The effect of collagen tripeptide FGI on the expression levels of NF-κB pathway proteins; (A) Western blot results; (B) expression levels of p-p65 / t-p65; (C) expression levels of p-IKBα / t-IKBα. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Example 1

[0037] 1. Preparation of giant salamander collagen peptides

[0038] The farmed giant salamander bones were thoroughly cleaned, and any remaining muscle, tendons, and cartilage were removed. They were then dried in an oven at 60°C. The dried bones were then pulverized using a high-speed grinder to obtain bone meal. The crude protein content of the bone meal was determined to be 90% using the Kjeldahl method.

[0039] After mixing bone meal and deionized water at a ratio of 1:2 (w / v), a complex enzyme (prepared by mixing Novozym® 11039, Protamex® 1.6X, and neutral protease at an enzyme activity ratio of 1:1:1) was added based on the crude protein content of the giant salamander bone meal. The mixture was stirred continuously at 55°C for 2 hours. The resulting enzymatic hydrolysate was then heated at 95°C for 15 minutes to inactivate the enzyme. After centrifugation (4°C, 10000 rpm, 30 min), the supernatant was collected. The supernatant was then rotary evaporated (50°C, 80 rpm) for 1 hour to remove excess water and then freeze-dried to obtain the giant salamander bone collagen hydrolysate.

[0040] 2. Molecular weight determination of collagen hydrolysate from giant salamander bones

[0041] Collagen hydrolysate was dissolved in a mobile phase to prepare a collagen solution with a concentration of 1 mg / mL. The mobile phase consisted of 54.9% ultrapure water, 45% acetonitrile, and 0.1% trifluoroacetic acid (v / v). The collagen solution was then filtered through a 0.22 μm filter to remove potential impurities and particles. Subsequently, molecular weight distribution analysis was performed using LC-16 high-performance liquid chromatography (HPLC) at a flow rate of 0.5 mL / min and a monitoring wavelength of 220 nm. The molecular weight distribution of the collagen hydrolysate was calculated from the chromatographic data.

[0042] 3. Peptide identification of collagen hydrolysates

[0043] Collagen hydrolysate was filtered through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The filtrate was collected and lyophilized to obtain low molecular weight collagen hydrolysate with a molecular weight cutoff of <1 kDa. Subsequently, LC-MS / MS was used to identify the peptides in the hydrolysate and analyze its peptide composition. The LC-MS / MS identification conditions were as follows:

[0044] Pretreatment: Take an appropriate amount of sample and desalinate it using a C18 desalting column.

[0045] Instrument parameters: Samples were analyzed by LC-MS / MS equipped with an online nano-spray ion source. The entire system was an OrbitrapExploris 480 mass spectrometer (Thermo Fisher Scientific, MA, USA) with EASY-nanoLC 1200 in series. A total of 2 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm × 25 cm), and the sample was separated by a gradient over 60 min. The column flow rate was controlled at 300 nL / min, the column temperature at 40°C, and the electrospray voltage at 2 kV. Mobile phase A was ultrapure water containing 0.1% formic acid (v / v), and mobile phase B was 0.1% formic acid + 80% acetonitrile + 19.9% ​​ultrapure water (v / v). The gradient started from 2.2% of phase B, increased to 90% nonlinearly in 54.5 min, increased to 99% within 0.5 min, and maintained for 5 min.

[0046] The mass spectrometer operates in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters are set as follows: (1) MS: Scan range (m / z): 200-1500; Resolution: 60000; Normalized AGC target: 300%; Maximum injection time: 25 ms; (2) HCD-MS / MS: Resolution: 15000; Normalized AGC target: 50%; Maximum injection time: 22 ms; Collision energy: 30%; Dynamic exclusion time: 5 s.

[0047] Peptide card value -10lgP≥20.

[0048] 4. Virtual screening of peptides

[0049] The identified peptides will undergo bioactivity and related property prediction using multiple databases. First, the PeptideRanker database (http: / / distilldeep.ucd.ie / PeptideRanker / ) will be used to predict the bioactivity of the peptides; peptides with a score greater than 0.5 will be considered bioactive. Next, the ToxinPred database (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) will be used to assess the toxicity of the peptides, and the AllerTOP database (https: / / www.ddg-pharmfac.net / AllerTOP / feedback.py) will be used to predict the sensitization potential. Furthermore, the Swiss ADME database (http: / / www.swissadme.ch / index.php) will be used to predict the gastrointestinal absorption of the peptides. Finally, the PlifePred database (https: / / webs.iiitd.edu.in / raghava / plifepred / batch.php) will be used to assess the plasma half-life of the peptides.

[0050] 5. Results and Analysis

[0051] like Figure 1 As shown, this is the molecular weight distribution of the collagen hydrolysate from the giant salamander bone. The results show that the proportion of the collagen hydrolysate with a molecular weight of <1kDa is 86.27%.

[0052] As shown in Table 1, the tripeptides FGI, PFI, and VLF obtained through virtual screening exhibited superior physicochemical and safety evaluation results. Their PeptideRanker scores were 0.92, 0.92, and 0.73, respectively, indicating that all three collagen tripeptides possess strong biological activity. Furthermore, the prediction results showed that the three collagen tripeptides were non-toxic, non-allergenic, and had high gastrointestinal absorption capacity. The plasma half-life of all three collagen tripeptides reached 834.81 s, indicating that these peptides have relatively long-lasting biological activity potential in vivo.

[0053] Table 1. Analysis of the results of virtual screening of giant salamander bone collagen hydrolysate.

[0054]

[0055] Example 2

[0056] 1. Construction of a cellular senescence model and intervention with collagen tripeptide FGI

[0057] Human skin fibroblasts (HSF) were used at a rate of 1×10 4Cells were seeded at a density of [insert density here] / well in 96-well plates and cultured for 24 hours in high-glucose DMEM medium containing 15% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin. Next, the medium was removed, and 100 μL of sterile PBS was added to each well. The plates were then placed under UVB irradiation at a dose of 200 mJ / cm². 2 After irradiation, PBS was removed, and complete culture medium containing different concentrations of collagen tripeptides FGI, PFI, and VLF was added, followed by incubation for 24 hours. The blank control group did not receive UVB irradiation, while the model group received UVB irradiation followed by incubation in normal culture medium. Finally, cell viability was determined using the CCK-8 assay.

[0058] 2. Effects of collagen tripeptides FGI, PFI, and VLF on type I collagen, elastin, and NF-κB pathway proteins

[0059] Human skin fibroblasts (HSF) were used at a rate of 1×10 6 Cells were seeded at a density of [insert density here] / well in 6-well plates, and UVB modeling and collagen tripeptide intervention were performed according to the above steps. After 24 hours of treatment, cells were collected using a cell scraper and transferred to centrifuge tubes. 500 μL of RIPA lysis buffer was added to each tube, and lysis was performed for 30 minutes. The lysis buffer was then centrifuged at 13000 rpm for 20 minutes (4°C), and the supernatant was collected. The protein concentration in the supernatant was determined using a BCA protein assay kit. The supernatant was then mixed with SDS-PAGE protein loading buffer (5×) at a ratio of 4:1 (v / v) and heated at 100°C for 10 minutes to denature the protein. Finally, the expression levels of type I collagen, elastin, and NF-κB pathway-related proteins were detected by Western blot.

[0060] 3. Results and Analysis

[0061] like Figure 2 As shown, cell viability significantly decreased to approximately 50% after UVB irradiation, indicating the successful establishment of the photoaging model. Based on this, interventions with 4, 8, and 16 μM FGI, PFI, and VLF, respectively, resulted in dose-dependent changes in cell viability: compared to the model group, all concentrations of FGI and PFI significantly increased cell viability. Although VLF did not promote an increase in cell viability, the difference compared to the model group was not significant, indicating its lack of cytotoxicity. These results suggest that within an appropriate concentration range, the three collagen tripeptides can effectively improve UVB-induced photoaging damage and possess potential anti-skin aging activity.

[0062] like Figure 3As shown, UVB irradiation significantly reduced the expression levels of type I collagen and elastin in human skin fibroblasts, indicating the successful establishment of the cellular photoaging model. After intervention with collagen tripeptides FGI, PFI, and VLF, the expression levels of both type I collagen and elastin significantly increased compared to the model group. Specifically, 4 μM and 16 μM FGI significantly promoted type I collagen expression, while all concentrations (4-16 μM) of FGI significantly increased elastin expression. PFI and VLF promoted type I collagen synthesis at all concentrations (4-16 μM); PFI at 8-16 μM promoted elastin synthesis, while VLF at 4-8 μM promoted elastin synthesis. These results indicate that FGI, PFI, and VLF can effectively alleviate UVB-induced skin photoaging damage, demonstrating good anti-aging potential.

[0063] like Figure 4 As shown, UVB irradiation significantly increased the expression levels of p-p65 and p-IKBα, indicating activation of the NF-κB signaling pathway, leading to an inflammatory response and further degradation of type I collagen and elastin. This also demonstrates the successful construction of the cellular photoaging model. After intervention with collagen tripeptide FGI, the expression levels of p-p65 and p-IKBα were significantly decreased compared to the model group. Specifically, 4 μM, 8 μM, and 16 μM FGI significantly reduced the expression level of p-p65, while 8 μM FGI significantly reduced the expression level of p-IKBα. These results indicate that collagen tripeptide FGI can inhibit UVB irradiation-induced activation of the NF-κB signaling pathway, thereby suppressing the inflammatory response and exhibiting a significant anti-skin aging effect.

[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of giant salamander collagen tripeptide in the preparation of drugs or cosmetics for anti-photoaging of the skin, characterized in that, The amino acid sequence of the giant salamander collagen tripeptide is FGI.

2. The application of giant salamander collagen tripeptide in the preparation of dietary supplements, characterized in that, The amino acid sequence of the giant salamander collagen tripeptide is FGI.

3. The application as described in claim 1 or 2, characterized in that, The preparation method of the giant salamander collagen tripeptide includes the following steps: The dried giant salamander bone powder was mixed with water, and a compound enzyme was added for enzymatic hydrolysis. The supernatant was collected after centrifugation of the hydrolysate and freeze-dried to obtain giant salamander bone collagen hydrolysate. The giant salamander bone collagen hydrolysate was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, the filtrate was collected and freeze-dried to obtain a low molecular weight collagen hydrolysate with a molecular weight cutoff of <1 kDa. The low molecular weight collagen hydrolysate with a molecular weight of <1 kDa was separated and identified using LC-MS / MS technology to obtain the giant salamander bone collagen tripeptide.

4. The application as described in claim 3, characterized in that, The mass-to-volume ratio of the giant salamander bone powder to water is 1:

2.

5. The application as described in claim 3, characterized in that, Based on the mass of crude protein in giant salamander bone meal, 6000U of a complex enzyme is added per gram of crude protein in giant salamander bone meal. This complex enzyme is produced by Novozym. ® 11039, Protamex ® It is a mixture of 1.6X and neutral protease in an enzyme activity ratio of 1:1:

1.

6. The application as described in claim 3, characterized in that, The enzymatic hydrolysis conditions were: continuous stirring at 55°C for 2 hours.

7. The application as described in claim 3, characterized in that, The conditions for separation and identification using the LC-MS / MS technology are as follows: The analytical column used for mass spectrometry detection is C10. 18 The column flow rate was controlled at 300 nL / min; the column temperature was 40℃; the electrospray voltage was 2 kV; mobile phase A was ultrapure water containing 0.1% formic acid, and mobile phase B was 0.1% formic acid, 80% acetonitrile, and 19.9% ​​ultrapure water; the detection gradient started from 2.2% of phase B, increased to 90% nonlinearly in 54.5 minutes, increased to 99% within 0.5 minutes, and was maintained for 5 minutes. The mass spectrometer operates in data-dependent acquisition mode and automatically switches between MS and MS / MS acquisition; the mass spectrometry parameters are set as follows: (1) MS: scan range (m / z): 200-1500; Resolution: 60000; Normalized AGC target: 300%; Maximum injection time: 25 ms; (2) HCD-MS / MS: Resolution: 15000; Normalized AGC target: 50%; Maximum injection time: 22 ms; Collision energy: 30%; Dynamic exclusion time: 5 s; Peptide card value -10lgP≥20.

8. A drug or cosmetic for combating skin photoaging, characterized in that, It contains giant salamander collagen tripeptide, the amino acid sequence of which is FGI.

9. A dietary supplement, characterized in that, It contains giant salamander collagen tripeptide, the amino acid sequence of which is FGI.

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