Blood protein peptide for promoting hair follicle reconstruction and application thereof

Abstract

The invention relates to a blood protein peptide for promoting hair follicle reconstruction and application thereof, and belongs to the technical field of bioactive peptides. The blood protein peptide disclosed by the invention is selected from one or more of an MLL peptide fragment and a WDL peptide fragment; the amino acid sequence of the MLL peptide fragment is as follows: methionine-leucine-leucine; the amino acid sequence of the WDL peptide fragment is as follows: tryptophan-aspartic acid-leucine. The invention also provides a preparation method of the blood protein peptide and application of the blood protein peptide in hair follicle reconstruction. The blood protein peptide prepared by the technical scheme of the invention can obviously improve local blood supply around hair follicles, and has a wide application prospect in the field of treatment of alopecia.

Description

Technical Field

[0001] The present application relates to the field of bioactive peptides, and in particular to a blood protein peptide for promoting hair follicle reconstruction and its application. Background Art

[0002] my country has abundant blood resources, but they have not been truly utilized, resulting in a large waste of resources. Blood is rich in protein, and improving the utilization rate of protein is the key to the current research and utilization of blood. Blood protein peptides contain biologically active peptides, which have unique biological activities beyond basic nutritional needs. They also have small molecular weight, easy absorption and simple preparation, so they have received more and more attention, but there is no research on promoting hair follicle reconstruction.

[0003] Hair follicles are skin organs that grow hair. Under normal circumstances, there are 100,000 to 150,000 hair follicles on the human head. However, if hair loss reaches the late stage, the blood supply to the hair follicles is reduced due to various reasons, and the hair follicles are malnourished, causing the hair follicles in the hair loss area to basically necrotize.

[0004] Therefore, in order to improve the local blood supply around the hair follicles and / or promote hair growth, it is urgent to develop a blood protein peptide that promotes hair follicle reconstruction. Summary of the invention

[0005] The purpose of the present application is to improve the local blood supply around the hair follicles and / or promote hair growth, and to propose a blood protein peptide that promotes hair follicle reconstruction and its application.

[0006] In the first aspect, the present application provides a blood protein peptide, using the following technical solution: A blood protein peptide, wherein the blood protein peptide is selected from one or more of an MLL peptide segment and a WDL peptide segment; the amino acid sequence of the MLL peptide segment is: methionine-leucine-leucine; the amino acid sequence of the WDL peptide segment is: tryptophan-aspartic acid-leucine.

[0007] Optionally, the hemoprotein peptide has one or more of the following characteristics: (a) activates inflammatory cells; (b) promotes differentiation and / or proliferation of vascular endothelial cells; and (c) promotes hair growth.

[0008] Optionally, the inflammatory cells are mast cells.

[0009] By adopting the above technical scheme, the blood protein MLL peptide segment and / or WDL peptide segment activates inflammatory cells such as mast cells, promotes the expression of angiogenic factors, causes the migration, differentiation and / or proliferation of vascular endothelial cells, improves the local blood supply around the hair follicles, and thus promotes hair growth.

[0010] In a second aspect, the present application provides a method for preparing the above-mentioned hemoprotein peptide, wherein the preparation method is a solid phase synthesis method.

[0011] By adopting the above technical solution, the purity of blood protein MLL peptide segment and / or WDL peptide segment is higher than 95%, with high biological activity and less impurities.

[0012] In a third aspect, the present application provides a use of the above-mentioned hemoprotein peptide in the preparation of a product that promotes hair growth and / or hair follicle reconstruction.

[0013] Optionally, the product is a medicine, food, health product or cosmetic.

[0014] Optionally, the product comprises the above-mentioned hemoprotein peptide and an acceptable carrier.

[0015] By adopting the above technical solution, the blood protein MLL peptide segment and / or WDL peptide segment can be prepared into medicine, food, health care product or cosmetics for promoting hair follicle reconstruction and treating hair loss.

[0016] In summary, the present application includes at least one of the following beneficial technical effects: 1. The blood protein MLL peptide segment and / or WDL peptide segment has the characteristics of small molecular weight, easy absorption and simple preparation, and therefore is suitable for preparation into various products for the treatment of hair loss; 2. The biological activity of blood protein MLL peptide and / or WDL peptide is high. It activates inflammatory cells such as mast cells, promotes the expression of angiogenic factors, causes the migration, differentiation and / or proliferation of vascular endothelial cells, improves the local blood supply around the hair follicles, and thus promotes hair growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It shows the release rate of tryptase after the blood protein peptide of the present application acts on mast cells; Figure 2 It shows the release rate of histamine after the hemoprotein peptide of the present application acts on mast cells; Figure 3 It shows the effect of the hemoprotein peptide of the present application on the mRNA level of VEGF expression in HUVECs; Figure 4 The results show that the blood protein peptide of the present invention is effective in treating hair loss mice. DETAILED DESCRIPTION Example 1 Screening of blood protein peptides

[0018] Fresh bovine blood was added with an anticoagulant (0.2% sodium citrate), and sterile water was added at a mass ratio of 1:1 (bovine blood: water). Ultrasonic wave assisted hemolysis was performed, and after the cells were broken, the cells were centrifuged for 15 minutes to remove the cell walls and obtain bovine blood protein solution.

[0019] Take the bovine blood protein solution and add distilled water to dilute it to 5.57% of the bovine blood protein content, sterilize it in a 90℃ water bath for 20 minutes, adjust the pH value to 8.0, add alkaline protease according to the amount of bovine blood protein, the addition amount is 5870U / g, and enzymolysis is carried out at 50℃ for 3.9h; boil to inactivate the enzyme, adjust the pH value to 6.5, add papain according to the amount of bovine blood protein, the addition amount is 2130U / g, and enzymolysis is carried out at 55℃ for 1.2h. Boil to inactivate the enzyme, adjust the pH value to 7.0, and the bovine blood protein hydrolyzate is obtained. The supernatant was obtained by centrifugation at 6000r / min for 20min, and the supernatant was first filtered through a 0.45μm filter membrane to collect the filtrate; the filtrate was separated by 10kDa, 5kDa, and 3kDa ultrafiltration membranes to collect peptides with different molecular weights (>10kDa, 5-10kDa, 3-5kDa, and <3kDa); the peptide components with molecular weight <3kDa were subjected to LC-MS / MS liquid mass analysis, LC-MS / MS analysis, and database search and comparison, and finally two WDL (TrpAspLeu) peptides and MLL (MetLeuLeu) peptides that did not match the known bioactive peptide sequences in the BIOPEP-UWM database were screened out and artificially synthesized. Example 2 Preparation of blood protein peptides

[0020] WDL peptide and MLL peptide (synthesized by Nanjing Yuanpeptide Biotechnology Co., Ltd.) were synthesized by solid phase synthesis using an automatic peptide synthesizer, purified by high performance liquid chromatography (HPLC), and identified by mass spectrometry (MS) and amino acid sequence analysis to confirm their sequence and purity, ultimately obtaining peptide powder with a purity greater than 95%. Example 3 In vitro functional testing of hemoprotein peptides

[0021] 3.1 Effects of hemoprotein peptides on mast cell activation The cell suspension density of HMC-1 cells (human mast cell line) was adjusted to 5×10 5 cells / mL, added to a new 12-well cell culture plate at 180 μL per well, and pre-incubated at 37°C for 5 minutes. Then add 20 μL of 5 nM hemoprotein peptides (WDL peptides, MLL peptides, 1:1 mixture of WDL peptides and MLL peptides prepared in Example 2) and incubate for 15 minutes. The reaction was terminated by adding 4°C culture medium (1.8 mL), and then the cells and supernatant were separated by centrifugation (400 g, 5 minutes). The concentrations of tryptase and histamine were detected by ELISA, and their release rates were calculated. Release rate (%) = (supernatant content - blank content) / (cell content - blank content), and the cell content was the cell lysate content plus the supernatant content.

[0022] The results are as follows Figure 1 and Figure 2 As shown, the hemoprotein peptide WDL peptide, MLL peptide and their combination have a certain activation effect on mast cells, and the effect of the combination is more significant than that of the single WDL peptide and MLL peptide.

[0023] 3.2 HUVECs migration assay Human umbilical vein endothelial cell line (HUVECs) (purchased from Beina Biotechnology) was revived, and cells that had been passaged 2-4 times and were in good growth condition were used in the experiment. The cells were resuspended after trypsin digestion, centrifuged at room temperature and 300 g to remove the supernatant, and then the cells were resuspended in EBM-2 medium containing 0.5% FBS. After counting, the cell density was adjusted to 1×10 6 cells / mL, and 100 μL per well was added to the upper chamber of the Transwell chamber. 600 μL of the supernatant of Example 3.1 and PBS were added to the lower chamber, and the cells were cultured in a 37°C cell culture incubator overnight. The liquid in the chamber was removed, and the non-migrated cells on the membrane were carefully wiped clean with a cotton swab, and stained with 0.1% crystal violet for 10 minutes. Six fields of view were randomly selected, and the migrated cells were counted under a microscope.

[0024] The results showed that the number of cells migrating in the supernatant of the hemoprotein peptide of Example 3.1 was significantly increased compared with the blank control group, p<0.05.

[0025] 3.3 Detection of cytokine VEGF secretion Human umbilical vein endothelial cell line (HUVEC) (purchased from Beina Biotechnology) was revived, and cells that had been passaged 2-4 times and were in good growth condition were used in the experiment. The cells were resuspended after trypsin digestion, centrifuged at room temperature and 300 g to remove the supernatant, and then the cells were resuspended in EBM-2 medium containing 0.5% FBS. After counting, the cell density was adjusted to 1×10 6 cells / mL, 200 μL per well was added to a new 24-well cell culture plate, and cultured at 37°C, 5% CO2 for 24 h. The supernatant was discarded and 200 μL of the supernatant of Example 3.1 was added, wherein PBS was used as the blank group (abbreviated as NC in the figure), and each group had 4 replicate wells. After 48 h of culture, total RNA was extracted using the Total RNA Kit II extraction kit (OMEGA), and the results were determined using Real-time RT-PCR technology. Human GAPDH was used as the internal reference gene to perform relative quantitative data analysis on the mRNA expression of the target gene VEGF.

[0026] The results are as follows Figure 3 As shown, the angiogenic factors produced in the supernatant after the hemoprotein peptides WDL peptide and MLL peptide activated mast cells promoted the expression of VEGF in endothelial cells, indicating that the hemoprotein peptides had a differentiation and / or proliferation effect on endothelial cells HUVEC. Example 4 In vivo functional testing of hemoprotein peptides

[0027] The hair loss model was established in 7-week-old male C57BL / 6 mice using the steroid corticosteroid dexamethasone (modeling period 23 days).

[0028] The hair in the resting phase on the back was removed and depilatory cream was used. The mice in the control group (NC in the figure) were topically applied with dexamethasone solution (100nM, 0.1mL, dissolved in propylene glycol) to the depilatory area once a day for 40 consecutive days. The mice in the treatment group were gavaged with blood protein peptides, including WDL peptide, MLL peptide and WDL+MLL peptide, at a concentration of 0.5mg / kg / day while applying the drug every day.

[0029] The experimental results are shown in Figure 4 The results were analyzed by skin color score, the higher the score, the darker the color, as follows: Dark Grey 6 points; Grey 5 points; Greyish White 4 points; White 3 points; Pinky White 2 points; Pink 1 point. By comparing the results, it can be seen that the blood protein peptide of the present application can alleviate the hair loss phenomenon of mice induced by local skin steroid corticosteroid dexamethasone.

[0030] Those skilled in the art will further recognize that the present invention may be implemented in other specific forms without departing from its spirit or central features. Since the foregoing description of the present disclosure discloses only exemplary embodiments thereof, it should be understood that other variations are considered to be within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. On the contrary, the scope and content of the present invention should be indicated with reference to the appended claims.

Claims

1. A blood protein peptide, characterized in that The blood protein peptide is selected from one or more of MLL peptide segments and WDL peptide segments; the amino acid sequence of the MLL peptide segment is: methionine-leucine-leucine; the amino acid sequence of the WDL peptide segment is: tryptophan-aspartic acid-leucine.

2. A hemoprotein peptide according to claim 1, characterized in that: The hemoprotein peptide has one or more of the following characteristics: (a) activates inflammatory cells; (b) promotes the migration, differentiation and / or proliferation of vascular endothelial cells; and (c) promotes hair growth.

3. A hemoprotein peptide according to claim 2, characterized in that: The inflammatory cells are mast cells.

4. A method for preparing the hemoprotein peptide according to claim 1, characterized in that: The preparation method is a solid phase synthesis method.

5. Use of the hemoprotein peptide according to claim 1 in the preparation of a product for promoting hair growth and / or hair follicle reconstruction.

6. The use according to claim 5, characterized in that: The product is a medicine, food, health product or cosmetic.

7. The use according to claim 5, characterized in that: The product comprises the hemoprotein peptide according to claim 1 and an acceptable carrier.

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