Application of hydrolyzed aloe flower protein in skin repair product

Abstract

The invention discloses an application of a hydrolyzed aloe flower protein in a skin repair product. 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 improves the dermal homeostasis, especially improves the functional homeostasis of dermal fibroblasts by enhancing the proliferation and migration capabilities of the dermal fibroblasts, promoting the synthesis of extracellular matrix components of the dermal fibroblasts and the release of fibroblast growth factors and the like; therefore, the purpose of repairing the skin is achieved.

Description

Technical Field

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

[0002] The skin is the largest organ in the human body and is mainly composed of three layers: the epidermis, dermis, and subcutaneous tissue. The epidermis is the outermost layer and provides a protective barrier. The dermis is located below the epidermis and is composed of dense connective tissue, containing a variety of cell types, such as fibroblasts, macrophages, mast cells, and vascular endothelial cells. Fibroblasts are the main cell type in the dermis and are responsible for the synthesis and secretion of collagen, elastin, and other extracellular matrix components, which give the skin strength and elasticity. Fibroblasts also play a key role in the repair of skin injuries, promoting wound healing by proliferation, migration, and synthesis of extracellular matrix. In addition, a variety of cytokines and growth factors secreted by fibroblasts can participate in the regulation of 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 existing technology repairs the skin by improving the homeostasis of the dermis environment, most of them are through direct supplementation of 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 causes collagen and hyaluronic acid to be broken down into smaller amino acids and sugar molecules in the digestive system, resulting in low absorption efficiency of collagen and hyaluronic acid, which is difficult to be directly used for skin repair and regeneration. It is difficult for the topical method to make large molecules of collagen and hyaluronic acid penetrate 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. It can be seen from this that there are many limitations and challenges in improving the homeostasis of the dermis environment 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 a method for repairing the skin by improving dermal homeostasis, especially improving the functional homeostasis of dermal fibroblasts, and applying the hydrolyzed aloe flower protein in skin repair products. Summary of the invention

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

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

[0007] In a first aspect, the present invention provides a use of hydrolyzed aloe flower protein in a skin repair product.

[0008] The hydrolyzed aloe flower protein of the present invention improves dermal homeostasis, especially improves dermal fibroblast functional homeostasis to repair skin by enhancing the proliferation and migration ability of dermal fibroblasts, promoting the synthesis of extracellular matrix components of dermal fibroblasts and the release of fibroblast growth factor.

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

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

[0011] (2) Activate signal transduction pathways in dermal fibroblasts, change their metabolic programs, and enhance 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 factor.

[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, medicines, 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, facial cream, pressed powder, shower gel, soap, liquid foundation, foundation cream, essence, hand cream, body lotion, hand soap, shampoo, conditioner, facial cleanser, facial cleanser, sunscreen, moisturizing cream, vanishing cream, nutrient solution, perfume, etc.

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

[0018] In a second aspect, the present invention provides a use of 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 includes 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 a use of hydrolyzed aloe flower protein in promoting the synthesis of extracellular matrix components of dermal fibroblasts and / or the release of fibroblast growth factor.

[0021] 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).

[0022] Preferably, the hydrolyzed aloe flower protein contains Leu-Pro-Phe, Leu-Gly, and Leu-Gly 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 hydrolyzed aloe flower protein comprises the following steps:

[0025] S1. First, extract the aloe flower with alkali, and then add acid to precipitate to obtain aloe flower crude protein;

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

[0027] S3. Ultrafiltration is performed on the protein hydrolysate to retain the ultrafiltration component with a molecular weight of ≤3 kDa to obtain hydrolyzed aloe flower protein.

[0028] In the preparation process of hydrolyzed aloe flower protein of the present invention, the reason why the ultrafiltration component with a molecular weight of ≤3kDa is retained is that, compared with the aloe flower hydrolysate in the protein hydrolyzate and the component with a molecular weight >3kDa, the component with a molecular weight of ≤3kDa in the hydrolyzed aloe flower protein is 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 aloe flower extracted with alkali is 10.0-11.0.

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

[0032] More preferably, in step S1, the pH of the acid added 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 of the enzymatic hydrolysis is 6.0-8.0.

[0036] More preferably, in step S2, the pH adjuster 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, the step S1 specifically comprises: adding aloe flowers and alkali to water for extraction, taking the supernatant, adding acid for precipitation, collecting the precipitate, and obtaining aloe flower crude protein.

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

[0040] More preferably, the step S2 specifically comprises: adding aloe flower crude protein to water, adjusting the pH, performing enzymatic hydrolysis, then inactivating the enzyme by heating the water bath, centrifuging, taking the supernatant, and obtaining an enzymatic hydrolyzate.

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

[0042] More preferably, the step S3 specifically comprises: separating the protein hydrolysate with an ultrafiltration membrane with a molecular weight of ≤3 kDa, retaining the ultrafiltration component with a molecular weight of ≤3 kDa, and drying to obtain hydrolyzed aloe flower protein.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

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

[0045] When the hydrolyzed aloe flower protein of the present invention is used for skin repair, since the molecular weight of each component in the hydrolyzed aloe flower protein is ≤3kDa, the hydrolyzed aloe flower protein is easily absorbed by the skin, has good stability, and has a lasting therapeutic effect. It is also safe, has no toxic side effects, can be used for a long time, and has wide applicability.

[0046] The preparation process of the hydrolyzed aloe flower protein of the invention is simple, green and pollution-free, and the raw materials are cheap and easily available, the production cost is low, and at the same time, the resource utilization rate and economic added value of the aloe flower are significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0050] Figure 4 This is the secondary mass spectrum of the amino acid sequence AF of hydrolyzed aloe flower protein. DETAILED DESCRIPTION

[0051] In order 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] Unless otherwise specified, other materials, reagents, etc. used in the examples can be obtained from commercial sources.

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

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

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

[0056] The Elisa kit for detecting elastin content was purchased from Shanghai Weiao Biotechnology Co., Ltd. with the product 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) levels 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) levels was purchased from Shanghai Hanhong Technology Co., Ltd. with product number HH-05096H1.

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

[0061] The techniques not described in detail in the following examples are all commonly used techniques in the art. Please refer to "Molecular Biology Experiment Manual" (Ma Wenli, People's Military Medical Publishing House), "Molecular Biology Experiment (Second Edition)" (Zhejiang University Press), and "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 embodiment provides a method for preparing hydrolyzed aloe flower protein, comprising the following steps:

[0065] S1. Use DFY-500 swing high-speed universal grinder (Zhejiang Wenling Linda Machinery Co., Ltd.) to grind dried 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 SorvallLYNX-4000 high-speed centrifuge), 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, collect the precipitate, and obtain the crude protein of Aloe vera flowers;

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

[0067] S3. The protein hydrolysate is separated by a SUEZ ultrafiltration membrane (material: TFM) with a molecular weight of 3 kDa using a WTM-1812G-2 laboratory membrane separation equipment (Hefei Woteng Membrane Separation Equipment Co., Ltd.), and the ultrafiltration component with a molecular weight of ≤3 kDa is retained. The product is then spray-dried using 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 weight of each component in the hydrolyzed aloe flower protein is ≤3 kDa.

[0068] 2. Identification of amino acid sequence of hydrolyzed aloe flower protein

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

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

[0071] Table 1 Amino acid sequence identification results 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), and "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%, 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 This is the liquid BPC diagram of hydrolyzed aloe flower protein; Figure 2 It is the secondary mass spectrum of the amino acid sequence LPF of hydrolyzed aloe flower protein; Figure 3 The second mass spectrum of the amino acid sequence LG of hydrolyzed aloe flower protein; Figure 4 This is the secondary mass spectrum of the amino acid sequence AF of hydrolyzed aloe flower protein.

[0076] from Figure 1-4As shown in 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 Leu-Gly in a mass ratio of 95.3:1.7:3.0.

[0077] Example 2

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

[0079] (1) Human dermal fibroblasts were cultured at 5×10 3 The cells were inoculated in a 96-well plate at a density of cells / well, and each well was cultured with 100 μL of DMEM culture medium (Thermo Fisher, C11995500BT) for 24 h, the DMEM culture medium was discarded, and then 100 μL of DMEM culture medium with hydrolyzed aloe flower protein concentrations of 0 μg / mL, 25 μg / mL, 50 μg / mL and 100 μg / mL were added respectively. In the added DMEM culture medium, the experiment with hydrolyzed aloe flower protein concentration of 0 μg / mL was used as a blank control group, the experiment with hydrolyzed aloe flower protein concentration of 25 μg / mL was used as a low-dose group in the experimental group, the experiment with hydrolyzed aloe flower protein concentration of 50 μg / mL was used as a medium-dose group in the experimental group, and the experiment with hydrolyzed aloe flower protein concentration of 100 μg / mL was used as a high-dose group in the experimental group. Each experiment was repeated 6 times; then, after culturing for 48 h at 37°C and 5% CO2, 20 μL of 5 mg / mL MTT solution, continue to culture for 3 hours, discard the solution, add 150 μL DMSO to each well to dissolve the crystals, place on an oscillator and shake thoroughly for 5 minutes, use a microplate 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 of the proliferation ability of dermal fibroblasts by hydrolyzed aloe flower protein;

[0080] (2) A. Prepare a protease hydrolysate according to steps S1 and S2 of the method for preparing aloe flower protein hydrolyzate in Example 1, and then dry it in an LPG-5 high-speed centrifugal spray dryer under conditions of an inlet air temperature of 150-180° C. and an outlet air temperature of 80-100° C. to obtain an aloe flower hydrolysate, which is then dissolved in a DMEM culture medium to prepare a DMEM culture medium containing 100 μg / mL of the aloe flower hydrolysate;

[0081] B. Prepare a proteolytic solution according to steps S1 and S2 of the method for preparing hydrolyzed aloe flower protein in Example 1, and then separate the proteolytic solution using a SUEZ ultrafiltration membrane with a molecular weight of 3 kDa. After the components with a molecular weight of less than 3 kDa pass through, the components with a molecular weight of more than 3 kDa are eluted, and dried in an LPG-5 high-speed centrifugal spray dryer under conditions of an inlet air temperature of 150-180° C. and an outlet air temperature of 80-100° C. to obtain a hydrolyzed aloe flower protein with a molecular weight of more than 3 kDa, and then dissolve the hydrolyzed aloe flower protein in a DMEM culture medium to prepare a DMEM culture medium containing 100 μg / mL of the hydrolyzed aloe flower protein with a molecular weight of more than 3 kDa;

[0082] C. Using DMEM medium containing 100 μg / mL aloe flower hydrolysate and DMEM medium containing 100 μg / mL hydrolyzed aloe flower protein with a molecular weight greater than 3 kDa, the cell viability of the aloe flower hydrolysate and the hydrolyzed aloe flower protein with a molecular weight greater than 3 kDa was tested with reference to the method in step (1) of this embodiment, and the experiment using the DMEM medium containing 100 μg / mL aloe flower hydrolysate was used as the aloe flower hydrolysate group, and the experiment using the DMEM medium containing 100 μg / mL hydrolyzed aloe flower protein with a molecular weight greater than 3 kDa was used as the hydrolyzed aloe flower protein group with a molecular weight greater than 3 kDa;

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

[0084] Table 2 Results of the dermal fibroblast proliferation experiment of hydrolyzed aloe vera 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 large cell viability value indicates that the hydrolyzed aloe flower protein has a large enhancement on the proliferation ability of 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 repairing the skin; it can also activate the signal transduction pathway in dermal fibroblasts, change their metabolic program, and then 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 repairing the skin.

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

[0090] Example 3

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

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

[0093] (1) Human dermal fibroblasts were cultured at 3×10 4The cells were inoculated at a density of 1.5 cells / well in a 6-well plate, and each well was cultured with 2 mL of DMEM medium (Thermo Fisher Scientific, C11995500BT). When the cell confluence grew to 80%, a 1 mL sterile pipette tip was used to vertically draw a line, and 2 mL of PBS (NaCl, 137 mM; KCl, 2.7 mM; Na2HPO4, 10 mM; KH2PO4, 1.8 mM; pH 7.4) Wash the cells twice, remove the scratched cells, and add 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 to each well, and in the added DMEM medium, the experiment with hydrolyzed aloe flower protein concentration of 0 μg / mL was used as the blank control group, the experiment with hydrolyzed aloe flower protein concentration of 25 μg / mL was used as the low-dose group in the experimental group, the experiment with hydrolyzed aloe flower protein concentration of 50 μg / mL was used as the medium-dose group in the experimental group, and the experiment with hydrolyzed aloe flower protein concentration of 100 μg / mL was used as the medium-dose group in the experimental group. The experiment with aloe vera protein concentration of 100 μg / mL was used as the high-dose group in the experimental group; then the culture was continued at 37°C and 5% CO2. The cell scratch spacing of each well was observed and recorded under a microscope at 0h, 12h and 24h of culture. Each group of experiments was repeated 3 times. The relative scratch spacing of the cells was calculated according to the formula [relative scratch spacing (%) = scratch spacing of the experimental group / scratch spacing of the blank control group × 100%]. The smaller the value of the relative scratch spacing, the greater the enhancement of the migration ability of dermal fibroblasts by hydrolyzed aloe vera flower protein.

[0094] (2) A. Prepare a protease hydrolysate according to steps S1 and S2 of the method for preparing aloe flower protein hydrolyzate in Example 1, and then dry it in an LPG-5 high-speed centrifugal spray dryer under conditions of an inlet air temperature of 150-180° C. and an outlet air temperature of 80-100° C. to obtain an aloe flower hydrolysate, which is then dissolved in a DMEM culture medium to prepare a DMEM culture medium containing 100 μg / mL of the aloe flower hydrolysate;

[0095] B. Prepare a proteolytic solution according to steps S1 and S2 of the method for preparing hydrolyzed aloe flower protein in Example 1, and then separate the proteolytic solution using a SUEZ ultrafiltration membrane with a molecular weight of 3 kDa. After the components with a molecular weight of less than 3 kDa pass through, the components with a molecular weight of more than 3 kDa are eluted, and dried in an LPG-5 high-speed centrifugal spray dryer under conditions of an inlet air temperature of 150-180° C. and an outlet air temperature of 80-100° C. to obtain a hydrolyzed aloe flower protein with a molecular weight of more than 3 kDa, and then dissolve the hydrolyzed aloe flower protein in a DMEM culture medium to prepare a DMEM culture medium containing 100 μg / mL of the hydrolyzed aloe flower protein with a molecular weight of more than 3 kDa;

[0096] C. Using DMEM medium containing 100 μg / mL aloe flower hydrolysate and DMEM medium containing 100 μg / mL hydrolyzed aloe flower protein with a molecular weight greater than 3 kDa, the relative scratch spacing between the aloe flower hydrolysate and the hydrolyzed aloe flower protein with a molecular weight greater than 3 kDa was tested with reference to the method in step (1) of this embodiment, and the experiment using the DMEM medium containing 100 μg / mL aloe flower hydrolysate was used as the aloe flower hydrolysate group, and the experiment using the DMEM medium containing 100 μg / mL hydrolyzed aloe flower protein with a molecular weight greater than 3 kDa was used as the hydrolyzed aloe flower protein group with a molecular weight greater than 3 kDa;

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

[0098] Table 3 Results of dermal fibroblast migration experiment of hydrolyzed aloe vera 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 in the same time, promote the closure of scratches, and ultimately shorten the time of scratch closure; and the small value of the relative scratch spacing indicates that the hydrolyzed aloe flower protein has a large enhancement on the migration ability of 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 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 then 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 seen that when the concentration of the test substances is the same, the hydrolyzed aloe flower protein with a molecular weight ≤3kDa has a smaller relative scratch spacing. This is because compared with aloe flower enzymatic hydrolysate and hydrolyzed aloe flower protein with a molecular weight >3kDa, the components of hydrolyzed aloe flower protein with a molecular weight ≤3kDa are more easily taken up and utilized by fibroblasts, which can better enhance the migration ability of human dermal fibroblasts, better improve dermal homeostasis, especially improve the functional homeostasis of dermal fibroblasts, and more easily 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 by synthesizing and regulating extracellular matrix components, especially improving the functional homeostasis of dermal fibroblasts, thereby achieving the purpose of repairing the skin. The extracellular matrix components include collagen, elastin and hyaluronic acid. Among them, collagen provides strength and flexibility for the skin; elastin enables the skin to return to its original shape 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] The present invention tests the effect of the hydrolyzed aloe flower protein prepared in Example 1 on the synthesis of extracellular matrix components of dermal fibroblasts. The specific test method is as follows:

[0107] Human dermal fibroblasts were cultured at 3×10 4 The cells / well were inoculated in a 6-well plate, and each well was inoculated with 2 mL DMEM medium (Thermo Fisher, C11995500BT) was used for culturing for 24 h, the DMEM medium was discarded, and then 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, and in the added DMEM medium, the experiment with hydrolyzed aloe flower protein concentration of 0 μg / mL was used as a blank control group, the experiment with hydrolyzed aloe flower protein concentration of 25 μg / mL was used as a low-dose group in the experimental group, the experiment with hydrolyzed aloe flower protein concentration of 50 μg / mL was used as a medium-dose group in the experimental group, and the experiment with hydrolyzed aloe flower protein concentration of 100 μg / mL was used as a high-dose group in the experimental group, and each experiment was repeated 3 times; then, after culturing for 48 h at 37° C. and 5% CO2, the medium was discarded, 0.5 mL of 0.25% (w / v) trypsin solution was added to digest the cells for 1 min, the solution was discarded, and 2 mL DMEM culture medium, centrifuge at 1000rpm for 5min to collect cells, add 0.5mL PBS (NaCl, 137mM; KCl, 2.7mM; Na2HPO4, 10mM; KH2PO4, 1.8mM; pH 7.4) for resuspending, and then lyse the cells using a cell ultrasonic disruptor. Centrifuge the cell lysate at 12000rpm / min for 10min, collect the supernatant, and use Elisa kit according to the instructions to detect the content of collagen I, collagen III, elastin and hyaluronic acid in the supernatant. Calculate the relative content of collagen I, collagen III, elastin and hyaluronic acid in different experimental groups and the blank control group 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 greater the relative content of each component of the extracellular matrix of dermal fibroblasts, the better the 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 of each component of the extracellular matrix of dermal fibroblasts hydrolyzed with aloe vera 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 extracellular matrix components of dermal fibroblasts, and the large relative content values ​​of the extracellular matrix components of dermal fibroblasts indicate that the hydrolyzed aloe flower protein has a good promoting effect 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 profile 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 the homeostasis of the dermis, especially improving the functional homeostasis of dermal fibroblasts, and ultimately achieving the purpose of repairing the skin.

[0117] Example 5

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

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

[0120] Human dermal fibroblasts were cultured at 3×10 4The cells were inoculated 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) for 24 h, the DMEM medium was discarded, and then 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, and in the added DMEM medium, the experiment with hydrolyzed aloe flower protein concentration of 0 μg / mL was used as the blank control group, the experiment with hydrolyzed aloe flower protein concentration of 25 μg / mL was used as the low-dose group in the experimental group, the experiment with hydrolyzed aloe flower protein concentration of 50 μg / mL was used as the medium-dose group in the experimental group, and the experiment with hydrolyzed aloe flower protein concentration of 100 μg / mL was used as the medium-dose group. The experiment with 100 μg / mL was used as the high-dose group in the experimental group, and each experiment was repeated 3 times; then, after culturing for 48 hours at 37°C and 5% CO2, the culture medium was collected, centrifuged at 1000 rpm for 5 minutes to discard the 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 effect of hydrolyzed aloe flower protein on promoting the release of fibroblast growth factor in dermal fibroblasts. The experimental results are shown in the following table:

[0121] Table 5 Fibroblast growth factor release test results of dermal fibroblasts from 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 group 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 the high level of fibroblast growth factor indicates that the hydrolyzed aloe flower protein has a good promoting effect on the release of fibroblast growth factor of dermal fibroblasts. Therefore, this shows that the hydrolyzed aloe flower protein of the present invention can activate molecular receptors on the cell surface or in the cell, start the downstream metabolic pathway, thereby promoting the release of fibroblast growth factor (FGF-1 and FGF-2), which is beneficial to improve the homeostasis of the dermis, especially to improve the functional homeostasis of dermal fibroblasts, and finally achieve the purpose of repairing the skin.

[0125] In summary, the hydrolyzed aloe flower protein of the present invention improves dermal homeostasis, especially improves functional homeostasis of dermal fibroblasts, by enhancing the proliferation and migration ability of dermal fibroblasts, promoting the synthesis of extracellular matrix components of dermal fibroblasts and the release of fibroblast growth factor, thereby achieving the purpose of repairing the skin.

[0126] Example 6

[0127] In order to clarify the skin repair effect of the amino acid sequences Leu-Pro-Phe (LPF), Leu-Gly (LG), and Ala-Phe (AF) in the hydrolyzed aloe flower protein, the inventor obtained the corresponding amino acid sequences by chemical synthesis and conducted experimental evaluation. 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, the synthesis of extracellular matrix components, and the release of fibroblast growth factor were evaluated according to the experimental procedures described in Examples 2-5, respectively. The dosage of the LPF, LG, and AF amino acid sequences was 5 μg / mL, and 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 as follows:

[0128] Table 6 Results of dermal fibroblast proliferation experiments of 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 Results of dermal fibroblast migration experiments of 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 Relative content of each component of the extracellular matrix of dermal fibroblasts with 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 Fibroblast growth factor release test results of dermal fibroblasts of amino acid sequences LPF, LG, 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 shown in Tables 6-9, the amino acid sequences LPF, LG, and AF have certain promoting functions on dermal fibroblasts, can enhance the proliferation and migration ability of dermal fibroblasts, and can also promote the synthesis of extracellular matrix components of dermal fibroblasts and the release of fibroblast factors. The above data all indicate that the amino acid sequences LPF, LG, and AF in the hydrolyzed aloe flower protein are helpful to improve the homeostasis of the dermis, 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 solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Application of hydrolyzed aloe flower protein in skin repair products.

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. Application of hydrolyzed aloe flower protein in enhancing the proliferation and / or migration of dermal fibroblasts.

4. Use of hydrolyzed aloe flower protein in promoting the synthesis of extracellular matrix components and / or the release of fibroblast growth factor in dermal fibroblasts.

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 Leu-Gly 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: 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 is performed on the protein hydrolysate to retain the ultrafiltration component with a molecular weight of ≤3 kDa to obtain hydrolyzed aloe flower protein.

9. The use according to claim 8, 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.

10. The use according to claim 8, characterized in that In step S2, the protease is at least one of papain, trypsin, flavor protease and pepsin.

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