Application of hydrolyzed aloe flower protein in skin repair products

Abstract

The present invention discloses the application of hydrolyzed aloe flower protein in skin repair products. The amino acid sequence of the hydrolyzed aloe flower protein is at least one of Leu-Pro-Phe, Leu-Gly, and Ala-Phe. The hydrolyzed aloe flower protein of the present invention improves dermal homeostasis, especially the functional homeostasis of dermal fibroblasts, by enhancing the proliferation and migration abilities of dermal fibroblasts, promoting the synthesis of extracellular matrix components of dermal fibroblasts, and releasing fibroblast growth factors, thereby achieving the purpose of skin repair.

Description

Technical Field

[0001] The present invention relates to the technical field of skin health, and particularly relates to the application of hydrolyzed aloe flower protein in skin repair products. Background Art

[0002] The skin is the largest organ of the human body, mainly composed of three layers: the epidermis, the dermis, and the subcutaneous tissue. The epidermis is the outermost layer, providing a protective barrier. The dermis is located beneath the epidermis and is composed of dense connective tissue, containing various cell types such as fibroblasts, macrophages, mast cells, and vascular endothelial cells. Fibroblasts are the main cell type in the dermis, responsible for synthesizing and secreting collagen, elastin, and other extracellular matrix components, which endow the skin with strength and elasticity. Fibroblasts also play a key role in skin injury repair, promoting wound healing by proliferating, migrating, and synthesizing the extracellular matrix. In addition, various cytokines and growth factors secreted by fibroblasts can participate in regulating immune responses and angiogenesis, which is beneficial to skin repair. It can be seen that the homeostasis of the dermis, especially the functional homeostasis of dermal fibroblasts, is an important link in promoting skin repair.

[0003] When the prior art repairs the skin by improving the dermal environmental homeostasis, it mostly directly supplements extracellular matrix components such as collagen and hyaluronic acid, and the direct supplementation methods of collagen and hyaluronic acid are oral and topical. The oral method decomposes collagen and hyaluronic acid into smaller amino acids and sugar molecules in the digestive system, resulting in low absorption efficiency of collagen and hyaluronic acid, and it is difficult to be directly used for skin repair and regeneration. The topical method is difficult to make large-molecular collagen and hyaluronic acid penetrate through the skin barrier into the dermis layer to play a role in repairing the skin, and it may be limited to moisturizing and temporary improvement of the epidermis layer. It can be seen that there are many limitations and challenges in improving dermal environmental homeostasis and repairing the skin by directly supplementing extracellular matrix components such as collagen and hyaluronic acid.

[0004] Therefore, it is of great significance to develop the application of hydrolyzed aloe flower protein in skin repair products, which can repair the skin by improving dermal homeostasis, especially improving the functional homeostasis of dermal fibroblasts. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the application of hydrolyzed aloe flower protein in skin repair products.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect, the present invention provides an application of hydrolyzed aloe flower protein in skin repair products.

[0008] The way that the hydrolyzed aloe flower protein of the present invention repairs the skin by improving dermal homeostasis, especially improving the functional homeostasis of dermal fibroblasts, is to enhance the proliferation and migration abilities of dermal fibroblasts, promote the synthesis of extracellular matrix components of dermal fibroblasts, and the release of fibroblast growth factors.

[0009] The mechanism of action of the hydrolyzed aloe flower protein of the present invention includes the following aspects:

[0010] (1) Provide absorbable nutrients for dermal fibroblasts, enhance their metabolic activity, and thus enhance their proliferation and migration abilities;

[0011] (2) Activate the signal transduction pathway in dermal fibroblasts, change their metabolic program, which is beneficial to enhancing their proliferation and migration abilities;

[0012] (3) Regulate the gene and protein expression profiles of dermal fibroblasts, promote the activation of their functions, and thus promote the synthesis of extracellular matrix components of dermal fibroblasts;

[0013] (4) Activate molecular receptors on the cell surface or inside the cell, initiate downstream metabolic pathways, and thus promote the release of fibroblast growth factors.

[0014] Preferably, the amino acid sequence of the hydrolyzed aloe flower protein includes at least one of Leu-Pro-Phe (LPF, leucine-proline-phenylalanine), Leu-Gly (LG, leucine-glycine), and Ala-Phe (AF, alanine-phenylalanine).

[0015] Preferably, the product is at least one of daily necessities, drugs, health care products, and biological products.

[0016] In the present invention, the forms or carriers of the daily necessities include but are not limited to toner, moisturizing lotion, facial mask, cream, powder cake, body wash, soap, liquid foundation, cream foundation, essence, hand cream, body lotion, hand sanitizer, shampoo, hair conditioner, facial cleansing paste, facial cleanser, sunscreen, emollient cream, vanishing cream, nutrient solution, perfume, etc.

[0017] Preferably, the dosage form of the product is at least one of liquid dosage form, solid dosage form, semi-solid dosage form, and gas dosage form.

[0018] In the second aspect, the present invention provides an application of a hydrolyzed aloe flower protein in enhancing the proliferation and / or migration of dermal fibroblasts.

[0019] Preferably, the amino acid sequence of the hydrolyzed aloe flower protein comprises at least one of Leu-Pro-Phe (LPF, leucine-proline-phenylalanine), Leu-Gly (LG, leucine-glycine), and Ala-Phe (AF, alanine-phenylalanine).

[0020] In a third aspect, the present invention provides an application of a hydrolyzed aloe flower protein in promoting the synthesis of extracellular matrix components of dermal fibroblasts and / or the release of fibroblast growth factors.

[0021] Preferably, the amino acid sequence of the hydrolyzed aloe flower protein comprises at least one of Leu-Pro-Phe (LPF, leucine-proline-phenylalanine), Leu-Gly (LG, leucine-glycine), and Ala-Phe (AF, alanine-phenylalanine).

[0022] Preferably, the hydrolyzed aloe flower protein contains Leu-Pro-Phe, Leu-Gly, and Ala-Phe in a mass ratio of (93.0-95.3):(1.0-1.7):(3.0-6.0).

[0023] Preferably, the molecular weight of each component in the hydrolyzed aloe flower protein is ≤3 kDa.

[0024] Preferably, the preparation method of the hydrolyzed aloe flower protein comprises the following steps:

[0025] S1. First, extract aloe flowers with an alkali, and then add an acid for precipitation to obtain crude aloe flower protein;

[0026] S2. Enzymatically hydrolyze the crude aloe flower protein with a protease to obtain a protease hydrolysate;

[0027] S3. Ultrafilter the protease hydrolysate, and retain the ultrafiltration fraction with a molecular weight cut-off ≤3 kDa, namely, the hydrolyzed aloe flower protein is obtained.

[0028] In the preparation process of the hydrolyzed aloe flower protein of the present invention, the ultrafiltration fraction with a molecular weight cut-off ≤3 kDa is retained because, compared with the aloe flower hydrolysate in the protease hydrolysate and the components with a molecular weight >3 kDa, the components with a molecular weight ≤3 kDa in the hydrolyzed aloe flower protein are more easily taken up and utilized by fibroblasts and can better improve the functional homeostasis of dermal fibroblasts.

[0029] More preferably, in step S1, the source of the aloe flowers is Aloe vera.

[0030] More preferably, in step S1, the pH of the extraction of aloe flowers with an alkali is 10.0-11.0.

[0031] More preferably, in step S1, the base is at least one of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, and sodium bicarbonate.

[0032] More preferably, in step S1, the pH for adding acid for precipitation is 2.5 - 3.5.

[0033] More preferably, in step S1, the acid is at least one of hydrochloric acid, sulfuric acid, and nitric acid.

[0034] More preferably, in step S2, the protease is at least one of papain, trypsin, flavor protease, and pepsin.

[0035] More preferably, in step S2, the pH for enzymatic hydrolysis is 6.0 - 8.0.

[0036] More preferably, in step S2, the pH regulator for enzymatic hydrolysis is at least one of sodium hydroxide, potassium hydroxide, calcium hydroxide, hydrochloric acid, sulfuric acid, acetic acid, citric acid, and phosphoric acid.

[0037] More preferably, in step S3, the molecular weight of the ultrafiltration component is 0 - 3 kDa.

[0038] More preferably, step S1 is specifically: adding aloe flower and base in water for extraction, taking the supernatant, then adding acid for precipitation, and collecting the precipitate to obtain crude aloe flower protein.

[0039] Even more preferably, the solid - liquid ratio of the aloe flower to water is (0.5 - 1) g:(10 - 20) mL.

[0040] More preferably, step S2 is specifically: adding crude aloe flower protein in water, adjusting the pH, performing enzymatic hydrolysis, then inactivating the enzyme by heating in a water bath, centrifuging, and taking the supernatant to obtain protease - hydrolyzed solution.

[0041] Even more preferably, the solid - liquid ratio of the crude aloe flower protein to water is (1 - 2) g:(3 - 20) mL.

[0042] More preferably, step S3 is specifically: separating the protease - hydrolyzed solution with an ultrafiltration membrane with a molecular weight cut - off ≤ 3 kDa, retaining the ultrafiltration component with a molecular weight ≤ 3 kDa, and drying to obtain hydrolyzed aloe flower protein.

[0043] Compared with the prior art, the beneficial effects of the present invention are:

[0044] The hydrolyzed aloe flower protein of the present invention improves dermal homeostasis, especially the functional homeostasis of dermal fibroblasts, by enhancing the proliferation and migration abilities of dermal fibroblasts, promoting the synthesis of extracellular matrix components of dermal fibroblasts, and releasing fibroblast growth factors, thereby achieving the purpose of skin repair.

[0045] When the hydrolyzed aloe flower protein of the present invention is used for skin repair, since the molecular weights of the components in the hydrolyzed aloe flower protein are all ≤ 3 kDa, the hydrolyzed aloe flower protein is easily absorbed by the skin, has good stability, long-lasting efficacy, and is safe, non-toxic and has no side effects, and can be used for a long time, with wide applicability.

[0046] The preparation process of the hydrolyzed aloe flower protein of the present invention is simple, green and pollution-free, and the raw materials are cheap and easy to obtain, with low production costs. At the same time, the resource utilization rate and economic added value of aloe flowers are significantly improved. Description of the Drawings

[0047] Figure 1 It is the liquid mass BPC diagram of the hydrolyzed aloe flower protein.

[0048] Figure 2 It is the secondary mass spectrum diagram of the amino acid sequence LPF of the hydrolyzed aloe flower protein.

[0049] Figure 3 It is the secondary mass spectrum diagram of the amino acid sequence LG of the hydrolyzed aloe flower protein.

[0050] Figure 4 It is the secondary mass spectrum diagram of the amino acid sequence AF of the hydrolyzed aloe flower protein. Detailed Embodiments

[0051] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0052] Other materials, reagents, etc. used in the embodiments can be obtained from commercial channels without special instructions.

[0053] Human dermal fibroblasts (BJ) were purchased from the Cell Bank of the Chinese Academy of Sciences, and the catalog number is GNHu49;

[0054] The Elisa kit for detecting the content of collagen I was purchased from Shanghai WeiAo Biotechnology Co., Ltd., and the product with the catalog number EH6905M;

[0055] The Elisa kit for detecting the content of collagen III was purchased from Shanghai WeiAo Biotechnology Co., Ltd., and the product with the catalog number EH10891M;

[0056] The Elisa kit for detecting the content of elastin was purchased from Shanghai WeiAo Biotechnology Co., Ltd., and the product with the catalog number EH10759M;

[0057] The ELISA kit for detecting hyaluronic acid content was purchased from Beijing Qisong Biotechnology Co., Ltd., with the product number QS40438-96T;

[0058] The ELISA kit for detecting fibroblast growth factor-1 (FGF-1) level was purchased from Shanghai Hanhong Technology Co., Ltd., with the product number HH-5404H1;

[0059] The ELISA kit for detecting fibroblast growth factor-2 (FGF-2) level was purchased from Shanghai Hanhong Technology Co., Ltd., with the product number HH-05096H1.

[0060] The DMEM medium used in the present invention is DMEM medium containing 10% FBS.

[0061] The technologies not described in detail in the following examples are all common technologies in the art, and reference can be made to "Molecular Biology Experiment Manual" (Ma Wenli, People's Military Medical Press), "Molecular Biology Experiment (Second Edition)" (Zhejiang University Press), "Cell Biology Experiment" (Yang Hongbing, Hou Lixia, Zhang Yuxi, Higher Education Press).

[0062] Example 1

[0063] 1. Preparation of hydrolyzed aloe flower protein

[0064] This example provides a preparation method of hydrolyzed aloe flower protein, which includes the following steps:

[0065] S1. Use a DFY-500 type swing high-speed universal grinder (Zhejiang Wenling Lindaji Machinery Co., Ltd.) to crush dry Aloe vera flowers. Take 100 g and add it to 1000 mL of deionized water. Adjust the pH to 11.0 with 2 mol / L sodium hydroxide solution, extract at 37 °C for 40 min, repeat the extraction 3 times, combine the extracts, centrifuge at 10000 rpm for 10 min (Thermo Sorvall LYNX-4000 high-speed centrifuge), and collect the supernatant; add 2 mol / L HCl solution to the supernatant to adjust the pH to 3.5, precipitate at 4 °C for 24 h, centrifuge at 10000 rpm for 10 min, discard the supernatant, and collect the precipitate to obtain crude aloe flower protein;

[0066] S2. Add 40 g of crude aloe flower protein to 400 mL of deionized water, stir evenly, adjust the pH to 7.0 with 2 mol / L sodium hydroxide solution, place it in a JULABO TW-20 universal water bath, add 6000 U / g of papain (add 6000 U of protease per g of crude aloe flower protein) at 50 °C, and stir with an IKA EUROSTAR-60 stirrer at a speed of 300 r / min for 4 h of enzymatic hydrolysis; after the enzymatic hydrolysis, heat in a water bath at 90 °C for 15 min to inactivate the enzyme, cool to room temperature, centrifuge at 10000 rpm for 10 min, take the supernatant to obtain the protease hydrolysate;

[0067] S3. Separate the protease hydrolysate with a molecular weight cut-off of 3 kDa SUEZ ultrafiltration membrane (material: TFM) through a WTM-1812G-2 laboratory membrane separation device (Hefei Woteng Membrane Separation Equipment Co., Ltd.), retain the ultrafiltration components with a molecular weight cut-off ≤ 3 kDa, and then perform spray drying with an LPG-5 high-speed centrifugal spray dryer at an inlet air temperature of 150 - 180 °C and an outlet air temperature of 80 - 100 °C to obtain hydrolyzed aloe flower protein; the molecular weights of all components in the hydrolyzed aloe flower protein are ≤ 3 kDa.

[0068] 2. Amino acid sequence identification of hydrolyzed aloe flower protein

[0069] Use the HPLC / MS / MS method to identify the amino acid sequence of the hydrolyzed aloe flower protein prepared above. The specific detection method is as follows: Use an X500 LC-ESI-Q-TOF high-resolution liquid chromatography-mass spectrometry instrument (AB SCIEX, USA) to separate and detect the sample; the liquid phase and mass spectrometry control software uses SCIEX OS2.0 integrated version (AB SCIEX, USA); the chromatographic column uses 1×100 mm HSS T3 (1.8 μm, Waters, USA).

[0070] The mobile phase consists of 0.1% (v / v) formic acid aqueous solution (A) and acetonitrile (B). The elution program is as follows: 0 - 4.00 min, 5.0% B; 4.00 - 6.00 min, 5.0 - 10.0% B; 6.00 - 30.00 min, 10.0 - 40.0% B; 30.00 - 34.00 min, 40.0 - 90.0% B; 34.00 - 40.00 min, 90% B; 40.00 - 42.00 min, 90.0 - 5.0% B; 42.00 - 52.00 min, 5.0% B. The flow rate is 0.05 mL / min, the injection volume is 1 μL, and the column temperature is 40 °C. Mass spectrometry detection method: The scan cycle is 0.642 s, the ESI ion source temperature is 500 °C, the positive ion mode is used, the spray voltage is 5500 V, the TOF primary scan range is 100 - 1200 Da, the secondary scan range is 50 - 1200 Da, the working mode is IDA, the maximum number of candidate ions is 4, dynamic exclusion is turned on, and the remaining parameters use the default values of proteomics methods. Note that before using the instrument, its dead volume should be minimized. The experimental results are as Figures 1-4 shown in Table 2:

[0071] Table 1 Identification Results of Amino Acid Sequences of Hydrolyzed Aloe Flower Protein

[0072]

[0073]

[0074] Note: In the above table, "LPF" refers to Leu - Pro - Phe (leucine - proline - phenylalanine), "LG" refers to Leu - Gly (leucine - glycine), "AF" refers to Ala - Phe (alanine - phenylalanine). The relative content of LG = peak area of LG / (peak area of LG + peak area of AF + peak area of LPF) × 100%, the relative content of AF = peak area of AF / (peak area of LG + peak area of AF + peak area of LPF) × 100%, and the relative content of LPF = peak area of LPF / (peak area of LG + peak area of AF + peak area of LPF) × 100%.

[0075] Figure 1 is the LC - MS BPC chromatogram of hydrolyzed aloe flower protein; Figure 2 is the secondary mass spectrometry chromatogram of the amino acid sequence LPF of hydrolyzed aloe flower protein; Figure 3 is the secondary mass spectrometry chromatogram of the amino acid sequence LG of hydrolyzed aloe flower protein; Figure 4 is the secondary mass spectrometry chromatogram of the amino acid sequence AF of hydrolyzed aloe flower protein.

[0076] From Figures 1-4As can be seen from Table 1, the amino acid sequence of the hydrolyzed aloe flower protein of the present invention includes LPF, LG, and AF, and the hydrolyzed aloe flower protein contains Leu-Pro-Phe, Leu-Gly, and Ala-Phe with a mass ratio of 95.3:1.7:3.0.

[0077] Example 2

[0078] Detect the effect of the hydrolyzed aloe flower protein prepared in Example 1 on the proliferation ability of dermal fibroblasts. The specific test method is as follows:

[0079] (1) Seed human dermal fibroblasts in a 96-well plate at a density of 5×10 3 cells / well, and culture them in 100 μL of DMEM medium (Thermo Fisher, C11995500BT) per well for 24 h. Discard the DMEM medium, and then add 100 μL of DMEM medium with hydrolyzed aloe flower protein concentrations of 0 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. Among the added DMEM media, the experiment with a hydrolyzed aloe flower protein concentration of 0 μg / mL serves as the blank control group, the experiment with a hydrolyzed aloe flower protein concentration of 25 μg / mL serves as the low-dose group in the experimental group, the experiment with a hydrolyzed aloe flower protein concentration of 50 μg / mL serves as the medium-dose group in the experimental group, and the experiment with a hydrolyzed aloe flower protein concentration of 100 μg / mL serves as the high-dose group in the experimental group. Each experiment is repeated 6 times. Then, after culturing for 48 h at 37 °C and 5% CO2, add 20 μL of 5 mg / mL MTT solution to each well, continue to culture for 3 h, discard the solution, add 150 μL of DMSO to each well to dissolve the crystals, shake well on a shaker for 5 min, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of each well at 570 nm, and then calculate the cell viability of different experimental groups and the blank control group according to the formula [cell viability of the experimental group (%) = absorbance of the experimental group / average absorbance of the blank control group × 100%]. The larger the cell viability value, the greater the enhancement amplitude of the hydrolyzed aloe flower protein on the proliferation ability of dermal fibroblasts;

[0080] (2) A. Prepare the protease hydrolysate according to steps S1 and S2 in the preparation method of the hydrolyzed aloe flower protein in Example 1, and then dry it under the conditions of an inlet temperature of 150 - 180 °C and an outlet temperature of 80 - 100 °C in an LPG-5 high-speed centrifugal spray dryer to obtain the aloe flower enzymolysis product. Then dissolve it in DMEM medium to prepare DMEM medium containing 100 μg / mL of the aloe flower enzymolysis product;

[0081] B. Prepare the protease hydrolysate according to steps S1 and S2 in the preparation method of hydrolyzed aloe flower protein in Example 1, and then perform ultrafiltration separation on the protease hydrolysate using a SUEZ ultrafiltration membrane with a molecular weight cut-off of 3 kDa. After the components with a molecular weight < 3 kDa pass through, elute the components with a molecular weight > 3 kDa, and dry them under the conditions of an inlet air temperature of 150 - 180 °C and an outlet air temperature of 80 - 100 °C in an LPG-5 high-speed centrifugal spray dryer to obtain hydrolyzed aloe flower protein with a molecular weight > 3 kDa. Then dissolve it in DMEM medium to prepare a DMEM medium containing 100 μg / mL of hydrolyzed aloe flower protein with a molecular weight > 3 kDa;

[0082] C. Using the DMEM medium containing 100 μg / mL of aloe flower hydrolysate and the DMEM medium containing 100 μg / mL of hydrolyzed aloe flower protein with a molecular weight > 3 kDa, test the cell viability of the aloe flower hydrolysate and the hydrolyzed aloe flower protein with a molecular weight > 3 kDa by referring to the method in step (1) of this example. And the experiment using the DMEM medium containing 100 μg / mL of aloe flower hydrolysate is used as the aloe flower hydrolysate group, and the experiment using the DMEM medium containing 100 μg / mL of hydrolyzed aloe flower protein with a molecular weight > 3 kDa is used as the hydrolyzed aloe flower protein group with a molecular weight > 3 kDa;

[0083] The experimental results are shown in the following table:

[0084] Table 2 Results of the proliferation experiment of dermal fibroblasts with hydrolyzed aloe flower protein

[0085]

[0086]

[0087] Note: In the above table, compared with the blank control group, *P < 0.05, ***P < 0.001.

[0088] As can be seen from Table 2, compared with the blank control group, the experimental group containing the hydrolyzed aloe flower protein of the present invention can significantly increase the cell viability of human dermal fibroblasts. And a larger cell viability value indicates a greater enhancement in the proliferation ability of the hydrolyzed aloe flower protein on dermal fibroblasts. Therefore, this shows that the hydrolyzed aloe flower protein of the present invention can provide absorbable nutrients for dermal fibroblasts, enhance their metabolic activity, thereby enhancing their proliferation ability, which is beneficial to improving dermal homeostasis, especially improving the functional homeostasis of dermal fibroblasts, and ultimately achieving the purpose of skin repair; it can also activate the signal transduction pathway in dermal fibroblasts, change their metabolic program, and further enhance their proliferation ability, which is beneficial to improving dermal homeostasis, especially improving the functional homeostasis of dermal fibroblasts, and ultimately achieving the purpose of skin repair.

[0089] In addition, it can be seen that when the concentrations of the test substances are the same, the hydrolyzed aloe flower protein with a molecular weight ≤ 3 kDa has stronger cell viability. This is because compared with aloe flower enzyme hydrolysate and hydrolyzed aloe flower protein with a molecular weight > 3 kDa, the components of the hydrolyzed aloe flower protein with a molecular weight ≤ 3 kDa are more easily taken up and utilized by fibroblasts, can better enhance the proliferation ability of human dermal fibroblasts, and can better improve dermal homeostasis, especially the functional homeostasis of dermal fibroblasts, and are more likely to achieve the purpose of repairing the skin.

[0090] Example 3

[0091] The migration of dermal fibroblasts plays a direct and crucial role in skin physiological and pathological processes. They move to the damaged area, reconstruct the extracellular matrix, and then fill and repair the damaged area to restore the structural integrity and function of the skin. Therefore, the migration ability of dermal fibroblasts directly affects the skin repair speed, wound healing quality, and adaptability to the external environment, and is crucial for maintaining skin health and improving dermal homeostasis, especially the functional homeostasis of dermal fibroblasts.

[0092] The present invention evaluates the effect of the hydrolyzed aloe flower protein prepared in Example 1 on the migration ability of dermal fibroblasts through a cell scratch assay. The specific test method is as follows:

[0093] (1) Human dermal fibroblasts were seeded at 3×10 4The cells were seeded at a density of cells / well in a 6-well plate, and each well was cultured with 2 mL of DMEM medium (Thermo Fisher, C11995500BT). When the cell confluence reached 80%, a 1 mL sterile pipette tip was used to make a vertical scratch. The cells were rinsed twice with 2 mL of PBS (NaCl, 137 mM; KCl, 2.7 mM; Na2HPO4, 10 mM; KH2PO4, 1.8 mM; pH 7.4) to remove the scratched cells. Then, 2 mL of DMEM medium containing hydrolyzed aloe flower protein at concentrations of 0 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL was added to each well. Among the added DMEM media, the experiment with a hydrolyzed aloe flower protein concentration of 0 μg / mL served as the blank control group, the experiment with a hydrolyzed aloe flower protein concentration of 25 μg / mL served as the low-dose group in the experimental group, the experiment with a hydrolyzed aloe flower protein concentration of 50 μg / mL served as the medium-dose group in the experimental group, and the experiment with a hydrolyzed aloe flower protein concentration of 100 μg / mL served as the high-dose group in the experimental group. Subsequently, the cells were continued to be cultured under the conditions of 37 °C and 5% CO2. At 0 h, 12 h, and 24 h of culture, the cell scratch distance in each well was observed and recorded under a microscope. Each experiment was repeated 3 times. The relative scratch distance of the cells was calculated according to the formula [relative scratch distance (%) = scratch distance of the experimental group / scratch distance of the blank control group × 100%]. The smaller the value of the relative scratch distance, the greater the enhancement of the migration ability of dermal fibroblasts by hydrolyzed aloe flower protein.

[0094] (2) A. Prepare the protease hydrolysate according to steps S1 and S2 in the preparation method of hydrolyzed aloe flower protein in Example 1. Then, dry it under the conditions of an inlet air temperature of 150 - 180 °C and an outlet air temperature of 80 - 100 °C in an LPG-5 high-speed centrifugal spray dryer to obtain aloe flower enzyme hydrolysate. Then, dissolve it in DMEM medium to prepare DMEM medium containing 100 μg / mL aloe flower enzyme hydrolysate.

[0095] B. Prepare the protease hydrolysate according to steps S1 and S2 in the preparation method of hydrolyzed aloe flower protein in Example 1. Then, separate the protease hydrolysate with a SUEZ ultrafiltration membrane with a molecular weight cut-off of 3 kDa. After the components with a molecular weight < 3 kDa passed through, the components with a molecular weight > 3 kDa were eluted and dried under the conditions of an inlet air temperature of 150 - 180 °C and an outlet air temperature of 80 - 100 °C in an LPG-5 high-speed centrifugal spray dryer to obtain hydrolyzed aloe flower protein with a molecular weight > 3 kDa. Then, dissolve it in DMEM medium to prepare DMEM medium containing 100 μg / mL hydrolyzed aloe flower protein with a molecular weight > 3 kDa.

[0096] C. Using DMEM medium containing 100 μg / mL aloe flower enzymatic hydrolysate and DMEM medium containing 100 μg / mL hydrolyzed aloe flower protein with a molecular weight > 3 kDa, refer to the method in step (1) of this example to test the relative scratch spacing of aloe flower enzymatic hydrolysate and hydrolyzed aloe flower protein with a molecular weight > 3 kDa. And the experiment using DMEM medium containing 100 μg / mL aloe flower enzymatic hydrolysate was used as the aloe flower enzymatic hydrolysate group, and the experiment using DMEM medium containing 100 μg / mL hydrolyzed aloe flower protein with a molecular weight > 3 kDa was used as the hydrolyzed aloe flower protein group with a molecular weight > 3 kDa;

[0097] The experimental results are shown in the following table:

[0098] Table 3 Results of dermal fibroblast migration experiment of hydrolyzed aloe flower protein

[0099]

[0100]

[0101] Note: In the above table, compared with the blank control group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0102] As can be seen from Table 3, compared with the blank control group, the experimental group containing the hydrolyzed aloe flower protein of the present invention can significantly shorten the relative scratch spacing of cells within the same time, promote the closure of scratches, and ultimately shorten the time for scratch closure; and a smaller value of the relative scratch spacing indicates a greater enhancement in the migration ability of dermal fibroblasts by the hydrolyzed aloe flower protein. Therefore, this shows that the hydrolyzed aloe flower protein of the present invention can provide absorbable nutrients for dermal fibroblasts, enhance their metabolic activity, thereby enhancing their migration ability, which is beneficial to improving dermal homeostasis, especially improving the functional homeostasis of dermal fibroblasts, and ultimately achieving the purpose of repairing the skin; it can also activate the signal transduction pathway in dermal fibroblasts, change their metabolic program, and further enhance their migration ability, which is beneficial to improving dermal homeostasis, especially improving the functional homeostasis of dermal fibroblasts, and ultimately achieving the purpose of repairing the skin.

[0103] In addition, it can be known that when the concentration of the test substance is the same, the hydrolyzed aloe flower protein with a molecular weight ≤ 3 kDa has a smaller relative scratch spacing. This is because compared with aloe flower enzymatic hydrolysate and hydrolyzed aloe flower protein with a molecular weight > 3 kDa, the components of the hydrolyzed aloe flower protein with a molecular weight ≤ 3 kDa are more easily taken up and utilized by fibroblasts, can better enhance the migration ability of human dermal fibroblasts, can better improve dermal homeostasis, especially improve the functional homeostasis of dermal fibroblasts, and are more likely to achieve the purpose of repairing the skin.

[0104] Example 4

[0105] Dermal fibroblasts maintain the structure and function of the skin and improve dermal homeostasis, especially the functional homeostasis of dermal fibroblasts, by synthesizing and regulating extracellular matrix components. These extracellular matrix components include collagen, elastin, and hyaluronic acid, etc. Among them, collagen provides strength and flexibility to the skin; elastin enables the skin to return to its original state and maintain elasticity after stretching; and glycosaminoglycans such as hyaluronic acid have extremely strong water retention capacity, helping the skin to stay moist and elastic.

[0106] This invention detects the effect of the hydrolyzed aloe flower protein prepared in Detection Example 1 of the invention on the synthesis of extracellular matrix components of dermal fibroblasts. The specific test method is as follows:

[0107] Human dermal fibroblasts were inoculated in a 6-well plate at a density of 3×10 4 cells / well, and each well was cultured with 2 mL of DMEM medium (Thermo Fisher, C11995500BT) for 24 h. Then, the DMEM medium was discarded, and 2 mL of DMEM medium with hydrolyzed aloe flower protein concentrations of 0 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL were added respectively. Among the added DMEM media, the experiment with a hydrolyzed aloe flower protein concentration of 0 μg / mL was used as the blank control group, the experiment with a hydrolyzed aloe flower protein concentration of 25 μg / mL was used as the low-dose group in the experimental group, the experiment with a hydrolyzed aloe flower protein concentration of 50 μg / mL was used as the medium-dose group in the experimental group, and the experiment with a hydrolyzed aloe flower protein concentration of 100 μg / mL was used as the high-dose group in the experimental group. Each group of experiments was repeated 3 times. Then, after culturing for 48 h under the conditions of 37 °C and 5% CO2, the medium was discarded, 0.5 mL of trypsin solution with a concentration of 0.25% (w / v) was added to digest the cells for 1 min, the solution was discarded, 2 mL of DMEM medium was added, and the cells were collected by centrifugation at 1000 rpm for 5 min. 0.5 mL of PBS (NaCl, 137 mM; KCl, 2.7 mM; Na2HPO4, 10 mM; KH2PO4, 1.8 mM; pH 7.4) was added for resuspension, and then the cells were lysed using a cell ultrasonic disruptor. The cell lysate was centrifuged at 12000 rpm / min for 10 min, and the supernatant was collected. An Elisa kit was used to detect the contents of collagen I, collagen III, elastin, and hyaluronic acid in the supernatant according to the instructions. The relative contents of collagen I, collagen III, elastin, and hyaluronic acid in different experimental groups and the blank control group were calculated respectively according to the following formula:

[0108] Relative content of collagen I = Content of collagen I in the experimental group / Average content of collagen I in the blank control group;

[0109] Relative content of collagen III = Content of collagen III in the experimental group / Average content of collagen III in the blank control group;

[0110] Relative content of elastin = Content of elastin in the experimental group / Average content of elastin in the blank control group;

[0111] Relative content of hyaluronic acid = Content of hyaluronic acid in the experimental group / Average content of hyaluronic acid in the blank control group;

[0112] The larger the relative content value of each component of the extracellular matrix of dermal fibroblasts, the better the promoting effect of hydrolyzed aloe flower protein on the synthesis of extracellular matrix components of dermal fibroblasts. The experimental results are shown in the following table:

[0113] Table 4 Relative content results of each component of the extracellular matrix of dermal fibroblasts treated with hydrolyzed aloe flower protein

[0114]

[0115] Note: In the above table, compared with the blank control group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0116] As can be seen from Table 4, compared with the blank control group, the experimental group containing the hydrolyzed aloe flower protein of the present invention can significantly increase the relative contents of collagen I, collagen III, elastin and hyaluronic acid, the components of the extracellular matrix of dermal fibroblasts. And the larger the relative content value of each component of the extracellular matrix of dermal fibroblasts indicates the better promoting effect of hydrolyzed aloe flower protein on the synthesis of extracellular matrix components of dermal fibroblasts. Therefore, this shows that the hydrolyzed aloe flower protein of the present invention can regulate the gene and protein expression profiles of dermal fibroblasts, promote the activation of their functions, thereby promoting the synthesis of extracellular matrix components of dermal fibroblasts, which is beneficial to improving dermal homeostasis, especially improving the functional homeostasis of dermal fibroblasts, and finally achieving the purpose of repairing the skin.

[0117] Example 5

[0118] Dermal fibroblasts can also regulate the composition of the extracellular matrix and the functions of skin tissues by releasing fibroblast growth factors.

[0119] The present invention detects the effect of the hydrolyzed aloe flower protein prepared in Example 1 on the release of fibroblast growth factors by dermal fibroblasts. The specific test method is as follows:

[0120] Human dermal fibroblasts were seeded at 3×10 4The cells were inoculated into a 6-well plate at a density of cells / well and cultured in 2 mL of DMEM medium (Thermo Fisher, C11995500BT) per well for 24 h. Then, the DMEM medium was discarded, and 2 mL of DMEM medium containing hydrolyzed aloe flower protein at concentrations of 0 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL was added respectively. Among the added DMEM media, the experiment with a hydrolyzed aloe flower protein concentration of 0 μg / mL served as the blank control group, the experiment with a hydrolyzed aloe flower protein concentration of 25 μg / mL served as the low-dose group in the experimental group, the experiment with a hydrolyzed aloe flower protein concentration of 50 μg / mL served as the medium-dose group in the experimental group, and the experiment with a hydrolyzed aloe flower protein concentration of 100 μg / mL served as the high-dose group in the experimental group. Each experiment was repeated 3 times. Then, after culturing for 48 h under the conditions of 37 °C and 5% CO2, the culture medium was collected, centrifuged at 1000 rpm for 5 min to discard cell debris, and the supernatant was taken. The levels of fibroblast growth factor-1 (FGF-1) and fibroblast growth factor-2 (FGF-2) in the supernatant were detected using an Elisa kit according to the instructions. The higher the level of fibroblast growth factor in the culture medium, the better the promoting effect of hydrolyzed aloe flower protein on the release of fibroblast growth factor by dermal fibroblasts. The experimental results are shown in the following table:

[0121] Table 5 Detection results of fibroblast growth factor release by dermal fibroblasts of hydrolyzed aloe flower protein

[0122]

[0123] Note: In the above table, compared with the blank control group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0124] As can be seen from Table 5, compared with the blank control group, the experimental groups containing the hydrolyzed aloe flower protein of the present invention can significantly increase the levels of FGF-1 and FGF-2 in the supernatant. And a high level of fibroblast growth factor indicates a good promoting effect of hydrolyzed aloe flower protein on the release of fibroblast growth factor by dermal fibroblasts. Therefore, this shows that the hydrolyzed aloe flower protein of the present invention can activate molecular receptors on the cell surface or inside the cell, initiate downstream metabolic pathways, thereby promoting the release of fibroblast growth factors (FGF-1 and FGF-2), which is beneficial to improving dermal homeostasis, especially improving the functional homeostasis of dermal fibroblasts, and ultimately achieving the purpose of repairing the skin.

[0125] In summary, the hydrolyzed aloe flower protein of the present invention improves dermal homeostasis, especially improves the functional homeostasis of dermal fibroblasts, so as to achieve the purpose of repairing the skin, by enhancing the proliferation and migration abilities of dermal fibroblasts, promoting the synthesis of extracellular matrix components of dermal fibroblasts, and the release of fibroblast growth factors and other pathways.

[0126] Example 6

[0127] In order to clarify the skin repair effects of the amino acid sequences Leu-Pro-Phe (LPF), Leu-Gly (LG), and Ala-Phe (AF) in hydrolyzed aloe flower protein, the inventors obtained the corresponding amino acid sequences through chemical synthesis and conducted experimental evaluations. The solid-phase synthesis of the amino acid sequences LPF, LG, and AF was commissioned to a biological company. The effects of the amino acid sequences LPF, LG, and AF on the proliferation and migration of human dermal fibroblasts, as well as the synthesis of extracellular matrix components and the release of fibroblast growth factors, were evaluated according to the experimental procedures described in Examples 2-5. Among them, the dosage of the LPF, LG, and AF amino acid sequences was 5 μg / mL. The experiment using the LPF amino acid sequence was used as the LPF group in the experimental group, the experiment using the LG amino acid sequence was used as the LG group in the experimental group, and the experiment using the AF amino acid sequence was used as the AF group in the experimental group. The experimental results are shown below:

[0128] Table 6 Experimental results of the proliferation of dermal fibroblasts by the amino acid sequences LPF, LG, and AF

[0129]

[0130] Note: In the above table, compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001.

[0131] Table 7 Experimental results of the migration of dermal fibroblasts by the amino acid sequences LPF, LG, and AF

[0132]

[0133] Note: In the above table, compared with the blank control group, **P<0.01, ***P<0.001.

[0134] Table 8 Results of the relative contents of each component of the extracellular matrix of dermal fibroblasts by the amino acid sequences LPF, LG, and AF

[0135]

[0136]

[0137] Note: In the above table, compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001.

[0138] Table 9 Detection results of the release of fibroblast growth factors by dermal fibroblasts by the amino acid sequences LPF, LG, and AF

[0139]

[0140] Note: In the above table, compared with the blank control group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0141] As can be seen from Table 6-9, the amino acid sequences LPF, LG, and AF have a certain promoting function on dermal fibroblasts, can enhance the proliferation and migration ability of dermal fibroblasts, and can also promote the synthesis of extracellular matrix components and the release of fibroblast growth factors in dermal fibroblasts. The above data all suggest that the amino acid sequences LPF, LG, and AF in hydrolyzed aloe flower protein help to improve dermal homeostasis, especially to improve the functional homeostasis of dermal fibroblasts, and ultimately achieve the purpose of repairing the skin.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An application of hydrolyzed aloe flower protein in the preparation of skin repair products, characterized in that: The preparation method of hydrolyzed aloe flower protein comprises the following steps: S1. First, extract the aloe flower with alkali, and then add acid to precipitate to obtain aloe flower crude protein; S2. hydrolyzing the crude protein of aloe flower with protease to obtain a protease hydrolysate; S3. ultrafiltration of the protease hydrolysate, and ultrafiltration fractions with a molecular weight cutoff of ≤3 kDa are obtained to obtain hydrolyzed aloe flower protein; In step S2, the protease is papain.

2. The use according to claim 1, characterized in that: The product is at least one of daily necessities, medicines, health care products, and biological products.

3. Use of hydrolyzed aloe flower protein in the preparation of a product for enhancing the proliferation and / or migration of dermal fibroblasts, characterized in that: The preparation method of hydrolyzed aloe flower protein comprises the following steps: S1. First, extract the aloe flower with alkali, and then add acid to precipitate to obtain aloe flower crude protein; S2. hydrolyzing the crude protein of aloe flower with protease to obtain a protease hydrolysate; S3. ultrafiltration of the protease hydrolysate, and ultrafiltration fractions with a molecular weight cutoff of ≤3 kDa are obtained to obtain hydrolyzed aloe flower protein; In step S2, the protease is papain.

4. Use of hydrolyzed aloe flower protein in the preparation of a product for promoting the synthesis of extracellular matrix components of dermal fibroblasts and / or the release of fibroblast growth factors, characterized in that: The preparation method of hydrolyzed aloe flower protein comprises the following steps: S1. First, extract the aloe flower with alkali, and then add acid to precipitate to obtain aloe flower crude protein; S2. hydrolyzing the crude protein of aloe flower with protease to obtain a protease hydrolysate; S3. ultrafiltration of the protease hydrolysate, and ultrafiltration fractions with a molecular weight cutoff of ≤3 kDa are obtained to obtain hydrolyzed aloe flower protein; In step S2, the protease is papain.

5. The use according to any one of claims 1 to 4, characterized in that: The amino acid sequence of the hydrolyzed aloe flower protein includes at least one of Leu-Pro-Phe, Leu-Gly and Ala-Phe.

6. The use according to any one of claims 1 to 4, characterized in that: The hydrolyzed aloe flower protein contains Leu-Pro-Phe, Leu-Gly and Ala-Phe in a mass ratio of (93.0-95.3):(1.0-1.7):(3.0-6.0).

7. The use according to any one of claims 1 to 4, characterized in that: The molecular weight of each component in the hydrolyzed aloe flower protein is ≤3kDa.

8. The use according to any one of claims 1 to 4, characterized in that: Including at least one of the following (1)-(4): (1) In step S1, the source of the aloe flower is Aloe vera; (2) In step S1, the pH of the aloe flower extracted with alkali is 10.0-11.0; (3) In step S1, the pH of the acid added for precipitation is 2.5-3.5; (4) In step S2, the pH of the enzymatic hydrolysis is 6.0-8.0.

Images