Anti-skin aging yak skin peptide-anthocyanin complex and its preparation method and application

Abstract

The present invention proposes a yak skin peptide-anthocyanin complex for resisting skin aging and its preparation method and application, which belongs to the technical field of functional polypeptides. The preparation method comprises the following steps: (1) after dissolving blueberry anthocyanin in water to obtain an anthocyanin solution, filtering through an aqueous microporous membrane to obtain an anthocyanin stock solution; (2) after adding yak skin peptide to the above-mentioned anthocyanin stock solution, adjusting the pH value to 4-6, reacting for 1-2 h, and obtaining a reaction solution; (3) adding anhydrous ethanol to the above-mentioned reaction solution, after centrifugation, collecting the precipitate, freeze-drying, and obtaining a yak skin peptide-anthocyanin complex. The obtained complex is mainly used to prepare food and medicine for improving skin aging. By adjusting the mass ratio of yak skin peptide to anthocyanin, reaction pH, reaction time and yak skin peptide concentration, the binding ability of the two is optimized, and better stability is shown, its bioavailability is improved, and the effect of improving skin aging is better played.

Description

Technical Field

[0001] The invention belongs to the technical field of functional polypeptides, and specifically relates to a yak hide peptide-anthocyanin complex for resisting skin aging, and a preparation method and application thereof. Background Art

[0002] Skin aging is the most intuitive result of aging, affecting the epidermis, dermis and subcutaneous layers. The dermis contains collagen fibers, elastic fibers and other extracellular matrix (ECM), and is the layer of the skin that changes the most due to aging. Human dermal fibroblasts (HDFs) are the main cell type in the dermis, responsible for the synthesis of collagen and elastic fibers. With age, the number and proliferation rate of HDFs decrease, and HDFs lose the ability to produce collagen and release senescence-associated secretory proteins (SASPs) and matrix-degrading metalloproteinases (MMPs), leading to ECM degradation, which leads to increased skin wrinkles and decreased skin elasticity and mechanical resistance.

[0003] Yak skin collagen peptide, also known as yak skin peptide, can promote endogenous collagen synthesis and inhibit collagen degradation. At the same time, it has physiological activities such as moisturizing, moisture absorption, tyrosinase inhibition, and anti-oxidation, and has attracted attention from the cosmetics industry in recent years.

[0004] There is growing interest in the use of anthocyanins, natural water-soluble pigments with antioxidant and biological properties that can combat diabetes, cardiovascular disease, inflammation and aging, but their use as active ingredients is limited due to their instability and low bioavailability. Summary of the invention

[0005] The present invention provides a yak hide peptide-anthocyanin complex for resisting skin aging and a preparation method and application thereof, so as to solve the problems of instability and low bioavailability of anthocyanin.

[0006] The present invention provides a method for preparing a yak hide peptide-anthocyanin complex for resisting skin aging, comprising the following steps:

[0007] (1) dissolving blueberry anthocyanins in water to obtain an anthocyanin solution, and filtering the solution through an aqueous microporous membrane to obtain an anthocyanin stock solution;

[0008] (2) After adding yak skin peptide to the above anthocyanin stock solution, the pH value is adjusted to 4-6, and the reaction is carried out for 1-2 hours to obtain a reaction solution; wherein the mass ratio of yak skin peptide to anthocyanin is 5-6:1, and the mass concentration of yak skin peptide is 1%-2%;

[0009] (3) adding anhydrous ethanol to the above reaction solution to precipitate the yak skin peptide-anthocyanin complex; collecting the precipitate after centrifugation and freeze-drying to obtain the yak skin peptide-anthocyanin complex.

[0010] Furthermore, in step (1), the concentration of the anthocyanin solution is 1-10 mg / ml.

[0011] Furthermore, in step (1), the pore size of the aqueous phase microporous membrane is 220 μm.

[0012] Furthermore, in step (2), the yak skin peptide is selected from yak skin collagen oligopeptide; the molecular weight of the yak skin collagen oligopeptide is less than 2 kDa.

[0013] Furthermore, in step (3), the centrifugal speed is 6000-10000 r / min, and the centrifugal time is 10-20 min.

[0014] Furthermore, in step (3), the freeze-drying temperature is -40 to -60°C; and the freeze-drying vacuum degree is 10 to 30Pa.

[0015] The present invention also provides a yak skin peptide-anthocyanin complex for resisting skin aging prepared by any of the above-mentioned preparation methods.

[0016] The present invention also proposes the use of the yak skin peptide-anthocyanin complex in the preparation of medicines for improving skin aging.

[0017] The present invention has the following advantages:

[0018] The method for preparing the anti-skin aging yak skin peptide-anthocyanin complex proposed in the present invention combines yak skin peptide with blueberry anthocyanin, and optimizes the binding ability of the two by adjusting the mass ratio of yak skin peptide to anthocyanin, reaction pH, reaction time and yak skin peptide concentration. The obtained complex can show better stability at suitable temperature, pH and simulated gastrointestinal tract, effectively reduce the degradation of anthocyanin in the body, thereby improving its bioavailability and better playing the role of improving skin aging. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0020] Figure 1 is the UV-visible spectrum of the yak hide peptide-anthocyanin complex in Test Example 2 of the present invention;

[0021] Figure 2is the Fourier transform infrared spectrum of the yak skin peptide-anthocyanin complex in Test Example 2 of the present invention;

[0022] Figure 3 is an anthocyanin stability diagram of the yak hide peptide-anthocyanin complex in Test Example 3 of the present invention; wherein 3a represents the thermal stability of anthocyanins, 3b represents the pH stability of anthocyanins, and 3c represents the simulated gastrointestinal stability of anthocyanins;

[0023] Figure 4 This is a macroscopic appearance of the skin of mice in each drug-administered group in Test Example 4 of the present invention;

[0024] Figure 5 is a graph showing the degree of skin relaxation of mice in each drug administration group in Test Example 4 of the present invention;

[0025] Figure 6 This is a graph showing the skin moisture content of mice in each drug administration group in Test Example 4 of the present invention;

[0026] Figure 7 It is HE and Masson staining pictures of mouse skin in each drug-administered group in Test Example 4 of the present invention;

[0027] Figure 8 It is a graph of the hydroxyproline (a) and hyaluronic acid (b) contents in the skin of mice in each administration group in Test Example 4 of the present invention;

[0028] Fig. 9 Graph showing the content of MDA (a), SOD (b), and GSH-Px (c) in the skin of mice in each drug-administered group in Test Example 4 of the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. In the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0030] Yak skin collagen peptide, also known as yak skin peptide, is an easily absorbed small molecule peptide obtained by deep processing of yak skin and has antioxidant properties. The inventors of the present application have found that combining low molecular weight yak skin peptide and anthocyanin by a specific method can effectively solve the problem of anthocyanin's instability and low bioavailability.

[0031] On the one hand, the present invention provides a method for preparing a yak skin peptide-anthocyanin complex for anti-skin aging, comprising the following steps:

[0032] (1) dissolving blueberry anthocyanins in water to obtain an anthocyanin solution, and filtering the solution through an aqueous microporous membrane to obtain an anthocyanin stock solution;

[0033] (2) After adding yak skin peptide to the above anthocyanin stock solution, the pH value is adjusted to 4-6, and the reaction is carried out for 1-2 hours to obtain a reaction solution; wherein the mass ratio of yak skin peptide to anthocyanin is 5-6:1, and the mass concentration of yak skin peptide is 1%-2%;

[0034] (3) adding anhydrous ethanol to the above reaction solution to precipitate the yak skin peptide-anthocyanin complex; collecting the precipitate after centrifugation and freeze-drying to obtain the yak skin peptide-anthocyanin complex.

[0035] The method for preparing the anti-skin aging yak skin peptide-anthocyanin complex proposed in the embodiment of the present invention combines yak skin peptide with blueberry anthocyanin, and optimizes the binding ability of the two by adjusting the mass ratio of yak skin peptide to anthocyanin, reaction pH, reaction time and yak skin peptide concentration. The complex obtained in the embodiment of the present invention can show better stability at suitable temperature, pH and simulated gastrointestinal tract, effectively reduce the degradation of anthocyanin in the body, thereby improving its bioavailability and better playing the role of improving skin aging.

[0036] In the embodiment of the present invention, the mass ratio of yak skin peptide to anthocyanin is 5-6:1. When the mass ratio of yak skin peptide to anthocyanin is too high or too low, the combination of the two will be affected.

[0037] In the embodiment of the present invention, the mass concentration of yak skin peptide is 1%-2%. Appropriate peptide concentration is conducive to the binding of anthocyanins thereto. Too low a concentration may result in insufficient binding of the peptide and anthocyanins, while too high a concentration may be detrimental to the dispersion of the solute, resulting in a decrease in the binding rate.

[0038] In the embodiment of the present invention, the pH value is 4-6. Under acidic conditions, when the pH increases, the hydrogen ions on -COOH are more easily ionized, thereby having more opportunities to bind with anthocyanins. When the pH is greater than 7, anthocyanins are easily degraded in the environment, resulting in a decrease in the binding rate.

[0039] In the embodiment of the present invention, the reaction time is 1-2 h. Too short a reaction time may lead to incomplete reaction, while too long a reaction time may destroy the interaction between the complexes due to too long stirring, resulting in a decrease in the binding rate.

[0040] In one embodiment of the present invention, in step (1), the concentration of the anthocyanin solution is 1-10 mg / ml. Preferably, the concentration of the anthocyanin solution is 5 mg / ml.

[0041] In one embodiment of the present invention, in step (1), the pore size of the aqueous microporous membrane is 220 μm. In the embodiment of the present invention, filtering through a 220 μm aqueous microporous membrane is mainly for removing insoluble impurities.

[0042] Furthermore, in step (1), the water is preferably distilled water.

[0043] In one embodiment of the present invention, in step (2), the yak skin peptide is selected from yak skin collagen oligopeptide. Preferably, the molecular weight of yak skin collagen oligopeptide is less than 2kDa. In the embodiment of the present invention, small molecular weight peptides are selected to be more easily combined with anthocyanins, and small molecular weight peptides are easily absorbed.

[0044] Furthermore, in step (2), the pH value is adjusted using a 1 mol / L NaOH solution or a 1 mol / L HCl solution.

[0045] In one embodiment of the present invention, in step (3), the volume ratio of anhydrous ethanol to the reaction liquid is 3 to 5: 1. Preferably, in step (3), the volume ratio of anhydrous ethanol to the reaction liquid is 4: 1.

[0046] In one embodiment of the present invention, in step (3), the centrifugal speed is 6000-10000 r / min, and the centrifugal time is 10-20 min. Preferably, the centrifugal speed is 8000 r / min, and the centrifugal time is 15 min.

[0047] In one embodiment of the present invention, in step (3), the freeze-drying temperature is -40 to -60°C; and the freeze-drying vacuum is 10 to 30 Pa. Preferably, in step (3), the freeze-drying temperature is -56°C; and the freeze-drying vacuum is 20 Pa.

[0048] On the other hand, the present invention also provides a yak skin peptide-anthocyanin complex for skin aging prepared by the above preparation method. The yak skin peptide-anthocyanin complex obtained by the present invention effectively improves the stability of anthocyanin and increases its bioavailability.

[0049] On the other hand, the embodiments of the present invention also propose the use of the above-mentioned yak skin peptide-anthocyanin complex in the preparation of medicines for improving skin aging. The present invention is of great significance for the development of the application of yak skin collagen peptides and blueberry anthocyanins in medicines for improving skin aging, lays an experimental foundation for the deep processing industrialization and high-value utilization of yak skin and blueberries, and fills the gap in the prior art of yak skin peptides for improving skin aging.

[0050] The present invention will be described in detail below with reference to embodiments and drawings.

[0051] Example 1 A method for preparing a yak hide peptide-anthocyanin complex for anti-skin aging comprises the following steps:

[0052] (1) dissolving blueberry anthocyanins in distilled water to prepare a 5 mg / ml anthocyanin solution;

[0053] (2) passing the anthocyanin solution obtained in step (1) through a 220 μm aqueous microporous membrane to remove insoluble impurities, thereby obtaining an anthocyanin stock solution for standby use;

[0054] (3) adding yak skin peptide (the molecular weight of yak skin collagen oligopeptide is <2 kDa) to the anthocyanin stock solution obtained in step (2) at a mass ratio of 5:1, and maintaining the mass concentration of yak skin peptide at 1.5%, to obtain a mixed solution;

[0055] (4) adjusting the pH value of the mixed solution obtained in step (3) to 5 using 1 mol / L HCl, reacting for 2 h to obtain a reaction solution;

[0056] (5) Add 4 times the volume of anhydrous ethanol to the reaction solution obtained in step (4), let it stand for half an hour until precipitation occurs, centrifuge at 8000 r / min for 15 min, collect the precipitate, and freeze-dry (the freeze-drying temperature is -56°C; the freeze-drying vacuum is 20 Pa) to obtain a yak skin peptide-anthocyanin complex.

[0057] Example 2 A method for preparing a yak hide peptide-anthocyanin complex for anti-skin aging comprises the following steps:

[0058] (1) dissolving blueberry anthocyanins in distilled water to prepare a 5 mg / ml anthocyanin solution;

[0059] (2) passing the anthocyanin solution obtained in step (1) through a 220 μm aqueous microporous membrane to remove insoluble impurities, thereby obtaining an anthocyanin stock solution for standby use;

[0060] (3) adding yak skin peptide (the molecular weight of yak skin collagen oligopeptide is <2 kDa) to the anthocyanin stock solution obtained in step (2) at a mass ratio of 6:1, and maintaining the mass concentration of yak skin peptide at 1.5%, to obtain a mixed solution;

[0061] (4) adjusting the pH value of the mixed solution obtained in step (3) to 5 using 1 mol / L HCl solution, reacting for 2 h to obtain a reaction solution;

[0062] (5) Add 4 times the volume of anhydrous ethanol to the reaction solution obtained in step (4), let it stand for half an hour until precipitation occurs, centrifuge at 8000 r / min for 15 min, collect the precipitate, and freeze-dry (the freeze-drying temperature is -56°C; the freeze-drying vacuum is 20 Pa) to obtain a yak skin peptide-anthocyanin complex.

[0063] Example 3 A method for preparing a yak hide peptide-anthocyanin complex for anti-skin aging comprises the following steps:

[0064] (1) dissolving blueberry anthocyanins in distilled water to prepare a 5 mg / ml anthocyanin solution;

[0065] (2) passing the anthocyanin solution obtained in step (1) through a 220 μm aqueous microporous membrane to remove insoluble impurities, thereby obtaining an anthocyanin stock solution for standby use;

[0066] (3) adding yak skin peptide (the molecular weight of yak skin collagen oligopeptide is <2 kDa) to the anthocyanin stock solution obtained in step (2) at a mass ratio of 6:1, and maintaining the mass concentration of yak skin peptide at 1.5%, to obtain a mixed solution;

[0067] (4) adjusting the pH value of the mixed solution obtained in step (3) to 6 using 1 mol / L HCl, reacting for 2 h to obtain a reaction solution;

[0068] (5) Add 4 times the volume of anhydrous ethanol to the reaction solution obtained in step (4), let it stand for half an hour until precipitation occurs, centrifuge at 8000 r / min for 15 min, collect the precipitate, and freeze-dry (the freeze-drying temperature is -56°C; the freeze-drying vacuum is 20 Pa) to obtain a yak skin peptide-anthocyanin complex.

[0069] Example 4 A method for preparing a yak hide peptide-anthocyanin complex for anti-skin aging comprises the following steps:

[0070] (1) dissolving blueberry anthocyanins in distilled water to prepare a 5 mg / ml anthocyanin solution;

[0071] (2) passing the anthocyanin solution obtained in step (1) through a 220 μm aqueous microporous membrane to remove insoluble impurities, thereby obtaining an anthocyanin stock solution for standby use;

[0072] (3) adding yak skin peptide (the molecular weight of yak skin collagen oligopeptide is <2 kDa) to the anthocyanin stock solution obtained in step (2) at a mass ratio of 6:1, and maintaining the mass concentration of yak skin peptide at 1.5%, to obtain a mixed solution;

[0073] (4) adjusting the pH value of the mixed solution obtained in step (3) to 5 using 1 mol / L HCl, reacting for 1 h to obtain a reaction solution;

[0074] (5) Add 4 times the volume of anhydrous ethanol to the reaction solution obtained in step (4), let it stand for half an hour until precipitation occurs, centrifuge at 8000 r / min for 15 min, collect the precipitate, and freeze-dry (the freeze-drying temperature is -56°C; the freeze-drying vacuum is 20 Pa) to obtain a yak skin peptide-anthocyanin complex.

[0075] Example 5A method for preparing a yak hide peptide-anthocyanin complex for anti-skin aging comprises the following steps:

[0076] (1) dissolving blueberry anthocyanins in distilled water to prepare a 5 mg / ml anthocyanin solution;

[0077] (2) passing the anthocyanin solution obtained in step (1) through a 220 μm aqueous microporous membrane to remove insoluble impurities, thereby obtaining an anthocyanin stock solution for standby use;

[0078] (3) adding yak skin peptide (the molecular weight of yak skin collagen oligopeptide is <2 kDa) to the anthocyanin stock solution obtained in step (2) at a mass ratio of 5:1, and maintaining the mass concentration of yak skin peptide at 1% to obtain a mixed solution;

[0079] (4) adjusting the pH value of the mixed solution obtained in step (3) to 5 using 1 mol / L HCl, reacting for 2 h to obtain a reaction solution;

[0080] (5) Add 4 times the volume of anhydrous ethanol to the reaction solution obtained in step (4), let it stand for half an hour until precipitation occurs, centrifuge at 8000 r / min for 15 min, collect the precipitate in step (5), and freeze-dry (the freeze-drying temperature is -56°C; the freeze-drying vacuum is 20 Pa) to obtain a yak skin peptide-anthocyanin complex.

[0081] Comparative Example 1 A method for preparing a yak skin peptide-anthocyanin complex for resisting skin aging

[0082] Same as Example 1, except that:

[0083] Step (3) adding yak skin peptide to anthocyanin stock solution at a mass ratio of 4:1, and maintaining the concentration of yak skin peptide at 1.5%.

[0084] Comparative Example 2 A method for preparing a yak skin peptide-anthocyanin complex for resisting skin aging

[0085] Same as Example 1, except that:

[0086] Step (3) adding yak skin peptide to anthocyanin stock solution at a mass ratio of 7:1, and maintaining the concentration of yak skin peptide at 1.5%.

[0087] Comparative Example 3 A method for preparing a yak skin peptide-anthocyanin complex for resisting skin aging

[0088] Same as Example 1, except that:

[0089] Step (3) adding yak skin peptide to anthocyanin stock solution at a mass ratio of 5:1, and maintaining the concentration of yak skin peptide at 0.5%.

[0090] Comparative Example 4 A method for preparing a yak skin peptide-anthocyanin complex for resisting skin aging

[0091] Same as Example 1, except that:

[0092] Step (3) adding yak skin peptide to anthocyanin stock solution at a mass ratio of 5:1, and maintaining the concentration of yak skin peptide at 3%.

[0093] Comparative Example 5 A method for preparing a yak skin peptide-anthocyanin complex for resisting skin aging

[0094] Same as Example 1, except that:

[0095] Step (4) Use 1 mol / L HCl to adjust the pH value of the solution in step (3) to 3 and react for 2 hours.

[0096] Comparative Example 6 A method for preparing a yak skin peptide-anthocyanin complex for resisting skin aging

[0097] Same as Example 1, except that:

[0098] Step (4) The pH value of the solution in step (3) was adjusted to 8 using 1 mol / L NaOH and the reaction was continued for 2 h.

[0099] Comparative Example 7 A method for preparing a yak skin peptide-anthocyanin complex for resisting skin aging

[0100] Same as Example 1, except that:

[0101] Step (4) The pH value of the solution in step (3) was adjusted to 5 using 1 mol / L HCl and the reaction was continued for 0.5 h.

[0102] Comparative Example 8 A method for preparing a yak skin peptide-anthocyanin complex for resisting skin aging

[0103] Same as Example 1, except that:

[0104] Step (4) The pH value of the solution in step (3) was adjusted to 5 using 1 mol / L HCl and the reaction was continued for 5 h.

[0105] Comparative Example 9

[0106] Dissolve blueberry anthocyanin in distilled water to prepare a 5 mg / ml anthocyanin solution for later use.

[0107] Comparative Example 10

[0108] The yak skin peptide was dissolved in distilled water to prepare a yak skin peptide solution with a mass concentration of 1.5%.

[0109] Comparative Example 11 A method for preparing a yak skin peptide-anthocyanin complex for resisting skin aging

[0110] Same as Example 1, except that:

[0111] In step (3), the molecular weight of the yak skin peptide is >3 kDa.

[0112] Test Example 1 Yak skin peptide-anthocyanin complex binding ability test

[0113] After adding four times the volume of anhydrous ethanol, centrifuge at 8000r / min for 15min, take the supernatant, and determine the anthocyanin content in the supernatant by pH differential method, according to the following formula:

[0114]

[0115] The combined rate of the two was calculated and the results are shown in Table 1.

[0116] Table 1 Comparison of the binding ability between yak skin peptide and blueberry anthocyanin

[0117]

[0118] It can be seen from Table 1 that the binding rate of the yak skin peptide-anthocyanin complex prepared in the example of the present invention is significantly higher than the binding rate of the yak skin peptide-anthocyanin complex prepared in the comparative example.

[0119] By comparing Example 1, Example 2, Comparative Example 1, and Comparative Example 2, it can be seen that when the mass ratio of yak skin peptide to anthocyanin is too high or too low, the binding rate of the two is significantly reduced to 13.36% and 19.37%, respectively, which indicates that the appropriate mass ratio is crucial to its binding ability.

[0120] By comparing Example 1, Example 5, Example 4, Comparative Example 3 and Comparative Example 4, it can be seen that appropriate peptide concentration is conducive to the binding of anthocyanins thereto, too low a concentration may result in insufficient binding of the peptide and anthocyanins, while too high a concentration may be detrimental to the dispersion of the solute, resulting in a decrease in the binding rate.

[0121] By comparing Example 1, Example 3, Comparative Example 5 and Comparative Example 6, it can be seen that when the reaction pH of yak skin peptide and anthocyanin is changed, the binding ability of yak skin peptide and anthocyanin is significantly reduced. Under acidic conditions, because the hydrogen ions on –COOH are more easily ionized when the pH increases, there are more opportunities to bind to anthocyanin. When the pH is greater than 7, anthocyanin is easily degraded in the environment, so the binding rate is low.

[0122] By comparing Example 1, Example 4, Comparative Example 7 and Comparative Example 8, it can be seen that when the reaction time is changed, too short a reaction time may lead to insufficient reaction between the two, while too long a reaction time may destroy the interaction between the complexes due to too long stirring, resulting in a decrease in the binding rate.

[0123] By comparing Example 1 and Comparative Example 11, it can be seen that the yak skin peptide with a large molecular weight has a weaker binding ability with anthocyanins. This may be because the yak skin peptide with a large molecular weight has a more complex structure, resulting in fewer binding sites with anthocyanins, so its binding rate is not as good as the low molecular weight yak skin peptide in Example 1.

[0124] Therefore, the binding ability of the two can be effectively changed by adjusting the mass ratio of yak skin peptide to anthocyanin, reaction pH, reaction time and yak skin peptide concentration.

[0125] Test Example 2 Structural characterization of yak skin peptide-anthocyanin complex

[0126] The yak skin peptide-anthocyanin complex obtained in Example 1 was characterized by UV-visible spectroscopy and infrared spectroscopy to prove the formation of the yak skin peptide-anthocyanin complex and the interaction between yak skin peptide and anthocyanin.

[0127] 1. UV-visible spectroscopy analysis

[0128] The composite (CPA) obtained in Example 1 and the yak skin peptide (CP) obtained in Comparative Example 10 were analyzed by ultraviolet spectroscopy, wherein the abscissa represents the wavelength and the ordinate represents the absorbance. Figure 1 The anthocyanins in the medium caused the ultraviolet absorption of yak skin peptide to blue shift, and its absorption peak shifted from 214 nm to 209 nm. This indicates that the binding of anthocyanins to yak skin peptides causes changes in its skeleton or conformation. Most protein molecules have an absorption peak near the wavelength of 270 nm, which is the characteristic peak of aromatic amino acids such as tryptophan (Trp) and tyrosine. Changes in the microenvironment of amino acid residues in protein molecules can cause a red shift or blue shift in the maximum absorption wavelength of the protein, leading to changes in the protein conformation. The peak intensity of yak skin peptides at 270nm-280nm increased and slightly red shifted, indicating that the peptide structure changed after the interaction and the hydrophobicity of the microenvironment around the aromatic amino acid residues increased.

[0129] 2. Fourier infrared spectroscopy analysis

[0130] Infrared spectroscopy was performed on the composite (CPA) in Example 1, the yak skin peptide (CP) obtained in Comparative Example 10, and the anthocyanin (ANC) obtained in Comparative Example 9. Figure 2 The infrared spectrum shows that the FT-IR spectrum of anthocyanin shows 1071 cm -1 The peaks at 1637 and 1446 cm-1 correspond to the ring CH deformation. -1 The bands at 1235 cm-1 correspond to the vibration of the benzopyran aromatic ring (CO) and the stretching vibration of the aromatic ring (CC). -1 The band at 1334 cm-1 corresponds to the stretching of the pyran ring, which is a typical feature of flavonoids. -1 The band at 1647 cm -1 and 1554 cm -1 The characteristic peaks at 3371.02 cm-1 are attributed to the amide I band (CO stretching vibration of peptide bond) and the amide II band (CN stretching vibration and NH bending vibration of amino group). -1 The -OH peak shifted to 3374.74 cm -1 This indicates that anthocyanins and yak skin peptides form hydrogen bonds. At the same time, yak skin peptides at 1647.16 cm -1 The characteristic peak of 1084 cm-1 shifts to a higher wave number after the complex is formed, indicating that a hydrogen bond is formed between the two substances. -1 The characteristic peaks shift to lower wavenumbers, indicating the existence of CN stretching and NH bending in the system.

[0131] Test Example 3 Anthocyanin stability

[0132] 1. Thermal stability

[0133] The composite in Example 1 and the anthocyanin in Comparative Example 9 were prepared into 5 mg / ml solutions, respectively, and heated at 80°C for 5 hours. The effect of the composite on the thermal stability of anthocyanin was determined by the retention rate of anthocyanin. Figure 3 As shown in a, anthocyanin aqueous solution degrades rapidly at high temperature, and the retention rate of anthocyanin is only 24.69% after heating at 80°C for 5 h. However, the retention rate of anthocyanin in the yak skin peptide-anthocyanin complex reaches 40.54% after heating at 80°C for 5 h. The experimental results of thermal stability show that the thermal stability of anthocyanin is significantly improved after the yak skin peptide is combined with anthocyanin.

[0134] 2. pH stability

[0135] Environmental pH also has an important influence on the stability of anthocyanins. Anthocyanins are easily degraded under neutral to alkaline conditions. The changes in the pH stability of anthocyanins in the complex were determined by preparing 5 mg / ml solutions of the complex in Example 1 and the anthocyanins in Comparative Example 9 and keeping them at different pH values ​​for 2 hours. Figure 3 b It can be seen that under acidic conditions, the retention rate of free anthocyanins and anthocyanins in the complex does not change much, but when the pH is closer to neutral, the retention rate of anthocyanins in the complex is significantly higher than that of free anthocyanins. Therefore, the pH stability of anthocyanins in the complex is significantly better than that of free anthocyanins.

[0136] 3. In vitro digestion simulation

[0137] (1) Simulate oral digestion

[0138] Simulated saliva: Dissolve 32.5 mg of α-amylase in 25 mL of 1 mmol / L CaCl2 solution at pH 7.

[0139] Oral digestion process: Take the complex in Example 1 and 0.1 g of the raw material anthocyanin in a 100 mL conical flask, add 20 mL of Na Cl solution (9 g / L) and 1 mL of simulated saliva, shake in a water bath shaker at 37°C and 120 rpm / min for 10 min, and then take out. Calculate the anthocyanin retention rate in the digestion solution. The result is Figure 3 The value corresponding to M in c.

[0140] (2) Simulated gastric digestion

[0141] Simulated gastric fluid: Dissolve 1.2 g pepsin in 30 mL 0.1 mol / L HCl solution.

[0142] After simulating oral digestion, the pH value of the sample solution was adjusted to 3.0 with HCl solution (6 mol / L), and then 2.5 mL of simulated gastric fluid was added. The samples were shaken and digested at 37°C and 120 rpm / min for 1 h and 2 h, and the anthocyanin concentration was measured and the anthocyanin retention rate in the digestive fluid was calculated. The results were Figure 3 The values ​​corresponding to G1h and G2h in c.

[0143] (3) Simulated intestinal digestion

[0144] Simulated intestinal fluid: Dissolve 0.8 g pancreatic enzyme and 4.8 g bile salt in 40 mL 0.1 mol / L NaHCO3 solution.

[0145] After simulated gastric digestion, the pH value of the sample solution was adjusted to 7.5 with NaHCO3 solution (0.9 mol / L), and 5 mL of intestinal fluid was added. The samples were taken out after 1 h and 2 h of shaking digestion at 37°C and 120 rpm / min, and the concentration of anthocyanins was measured and the retention rate of anthocyanins in the digestive fluid was calculated. The results were Figure 3 The corresponding values ​​of I1h, I2h, I3h, and I4h in c.

[0146] In vitro gastrointestinal simulation showed that ( Figure 3 c) During the oral and gastric digestion stages, there was no significant difference in the retention rate of anthocyanins between free anthocyanins and yak skin peptide-anthocyanin complexes. Since the polyhydroxy structure of ANC has poor stability and is easily degraded in an alkaline environment, after 1 hour of intestinal digestion, the retention rate of anthocyanins in the complex was significantly higher than that of free anthocyanins after 1 hour of simulated intestinal fluid, indicating that anthocyanins compounded with yak skin peptides are more stable than free anthocyanins in the gastrointestinal environment. The complex significantly improves the stability of anthocyanins in the intestinal digestion stage and has a significant protective effect on anthocyanins.

[0147] Test Example 4 Test on the anti-skin aging performance of yak skin peptide-anthocyanin complex

[0148] Fifty male KM mice (6 weeks old) were housed in plastic cages covered with sawdust bedding. All mice were placed in an air-conditioned room (25°C) with normal light for 12 h and relative humidity of 40%-60% for 1 week of adaptive feeding.

[0149] (1) Grouping and drug administration

[0150] The mice were randomly divided into 5 groups: blank control (NC) group, model group, yak skin peptide (CP) group obtained in comparative example 10, anthocyanin (ANC) group obtained in comparative example 9, and complex (CPA) group obtained in Example 1, and complex (LCP) group obtained in comparative example 6. The experimental period was 6 weeks, with 10 mice in each group. Except for the blank group, the remaining groups were subcutaneously injected with D-galactose at the back of the neck at 1000 mg / kg / d daily. The body weight was measured weekly to adjust the injection and gavage doses. The dosage of the CP group, LCP group and CPA group was 200 mg / kg / d, and the dosage of the ANC group was 46 mg / kg / d.

[0151] (2) Macroscopic appearance of the skin

[0152] from Figure 4It can be seen that the model group has the deepest and largest back wrinkles, indicating that the skin aging model was successfully established. The yak skin peptide group and anthocyanin group had less back wrinkles than the model group, and the complex group had the best effect on skin aging mice, which was basically close to the skin appearance of the normal group. It can be seen that the synergistic effect of the two substances in the complex makes it more effective for skin aging. In addition, the anti-aging effect of the LCP group is not as good as that of the CPA group, so the combination rate of the two has a certain influence on their synergistic effect.

[0153] (3) Skin relaxation test

[0154] Use your left hand to gently lift the back skin about 1 cm away from the mouse's coccyx until the mouse's hind limbs slightly leave the table. Hold for 1 second and then release your hand to see how long it takes for the mouse's skin to return to its initial state. The shorter the time, the better the elasticity of the mouse's skin. Figure 5 It can be seen that the recovery time of skin lifting in the model group mice was significantly different from that in the other groups, indicating that each treatment group had a significant effect on skin elasticity. Among them, the complex group had the best therapeutic effect on mice with aging skin, indicating that the yak skin peptide-anthocyanin complex has a synergistic effect on preventing skin aging. The recovery time of the LCP group was higher than that of the CPA group, indicating that the binding rate of yak skin peptides and anthocyanins affects their synergistic anti-skin aging effect.

[0155] (4) Determination of skin moisture content

[0156] The skin moisture content was determined by drying the back skin samples in an oven at 105°C for 4 hours until they reached a constant weight. Figure 6 The skin moisture of the model group was significantly reduced compared with the normal group and the CPA group, while the use of a single substance had no significant effect on the skin surface moisture of the skin aging mice, which further illustrates the synergistic effect of the complex on skin aging.

[0157] (5) Skin histological analysis

[0158] After killing the mice, appropriate amounts of dorsal skin tissue were obtained, fixed in 4% paraformaldehyde for 24 h, then embedded in paraffin, made into 4 μm thick sections, and stained. Hematoxylin-eosin (H&E) and Masson trichrome staining were used for staining. Structural features and images were observed and obtained using an optical microscope. Figure 7 The epidermal thickness of the model group was significantly increased compared with the blank control group, while the epidermal thickness was significantly reduced after treatment with yak skin peptide, anthocyanin and compound, indicating that each group has a therapeutic effect on aging skin, among which the compound has the best anti-skin aging effect. Masson staining can clearly show that the collagen fibers in the model group are loosely arranged and not as tightly arranged as the collagen fibers in the other groups. The state of the collagen fibers in the compound group is basically consistent with that in the blank control group, while the state of the collagen fibers in the single substance treatment group is improved compared with the model group.

[0159] (6) Determination of skin hydroxyproline, hyaluronic acid and antioxidant indexes

[0160] Hydroxyproline (HYP) is a major amino acid in collagen. The reduction of HYP content can affect collagen synthesis and reflect the degree of skin aging. Hyaluronic acid (HA) is a moisturizer present in connective tissue that can lock in moisture and delay aging. Mouse skin tissue was made into 10% tissue homogenate, centrifuged at 4000r / min for 10 minutes, and the supernatant was taken. The ELISA kit was used to measure the HYP and HA content in mouse skin tissue. The results are shown in Figure 8 .Depend on Figure 8 It was found that after oral administration, the skin HYP and HA contents in each group increased, but the HYP content of yak skin peptide increased to 174.64 μg / mgprot compared with the model group, which was better than anthocyanins, and anthocyanins had a better effect on HA content.

[0161] Oxidative stress is an important feature of skin aging. The activity of antioxidant enzymes (SOD, GSH-Px) and the content of oxidative product MDA in mouse skin tissue were measured. Mouse skin tissue was made into 10% tissue homogenate, centrifuged at 4000r / min for 10 minutes, and the supernatant was taken. The SOD, MDA and GSH-Px in mouse skin tissue were measured using a kit. The results are shown in Fig. 9 .Depend on Fig. 9 It can be seen that among all the drug-administered groups, the complex group had the greatest effect on the antioxidant indicators of mouse skin, basically restoring them to the level of the normal group, while the yak skin peptide group had a better effect than the anthocyanin group on the MDA level, and the anthocyanin group had a better effect than the yak skin peptide on the SOD and GSH-Px levels.

[0162] In summary, yak skin peptides and anthocyanins in the complex group may achieve complementarity by promoting or inhibiting different endogenous components and antioxidant indicators of the skin to exert their synergistic effects and obtain the best anti-skin aging effect.

[0163] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a yak skin peptide-anthocyanin complex for anti-skin aging, characterized in that: The steps include: (1) dissolving blueberry anthocyanins in water to obtain an anthocyanin solution, and filtering the solution through an aqueous microporous membrane to obtain an anthocyanin stock solution; (2) After adding yak skin peptide to the above-mentioned anthocyanin stock solution, the pH value is adjusted to 4-6, and the reaction is carried out for 1-2 hours to obtain a reaction solution; wherein the mass ratio of yak skin peptide to anthocyanin is 5-6:1, and the mass concentration of yak skin peptide is 1%-2%; the yak skin peptide is selected from yak skin collagen oligopeptide; the molecular weight of the yak skin collagen oligopeptide is less than 2kDa; (3) adding anhydrous ethanol to the above reaction solution to precipitate the yak skin peptide-anthocyanin complex; collecting the precipitate after centrifugation and freeze-drying to obtain the yak skin peptide-anthocyanin complex.

2. The preparation method according to claim 1, characterized in that: In step (1), the concentration of the anthocyanin solution is 1-10 mg / ml.

3. The preparation method according to claim 1, characterized in that: In step (1), the pore size of the aqueous microporous membrane is 220 μm.

4. The preparation method according to claim 1, characterized in that: In step (3), the centrifugal speed is 6000-10000 r / min, and the centrifugal time is 10-20 min.

5. The preparation method according to claim 1, characterized in that: In step (3), the freeze-drying temperature is -40 to -60°C; the freeze-drying vacuum degree is 10 to 30 Pa.

6. The anti-skin aging yak skin peptide-anthocyanin complex prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the yak skin peptide-anthocyanin complex according to claim 6 in the preparation of medicines for improving skin aging.

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