Royal jelly polypeptide CMKI-21 and application thereof

Abstract

The invention provides royal jelly polypeptide CMKI-21 capable of promoting wound healing and resisting skin photoaging and application of the royal jelly polypeptide CMKI-21, and belongs to the technical field of biological medicine. The amino acid sequence of the royal jelly polypeptide is KKELVREVGFEICFKDHHEKI, and the royal jelly polypeptide is obtained by a solid-phase chemical synthesis method. The royal jelly polypeptide CMKI-21 disclosed by the invention is small in molecular weight, simple and feasible in preparation method, high in purity and good in biocompatibility, and can remarkably promote proliferation and migration of HaCaT cells, accelerate the healing rate of full-thickness skin defect wounds of mice, relieve light damage of the HaCaT cells and relieve skin light aging and sunburn caused by ultraviolet (UVB). The royal jelly polypeptide has the potential to be applied to various clinical skin wounds as a wound healing agent, and can also be applied to medicines and cosmetics for resisting skin photoaging and repairing sunburn.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a royal jelly polypeptide CMKI-21 and an application thereof. Background Art

[0002] The skin is exposed to some harmful physical, chemical and biological environments every day. When skin cells cannot defend against these external stimuli, they can no longer maintain their integrity and homeostasis. Skin damage and premature aging are the result of continuous exposure to harmful environments. In order to enable skin cells to respond to these external stimuli more actively, we need to provide the skin with some additional nutrients to improve cell activity. At present, people have discovered some substances that can treat skin wounds and prevent aging. For example, polyphenols can not only reduce inflammation and promote wound healing, but also reduce the photoaging of the skin caused by ultraviolet radiation. Lipids and fatty acids can prevent water loss in the skin and are essential for maintaining the integrity of the epidermal barrier. Peptides have multiple pharmacological activities such as antibacterial, antioxidant and immunomodulatory, and have good specificity and stability, which has aroused people's interest in the research of peptides in the treatment of skin injuries.

[0003] As the main cell type constituting the epidermis, keratinocytes not only serve as structural cells, but also play an important role in wound healing and anti-aging of the skin. The proliferation and migration of keratinocytes can accelerate the wound healing rate and restore the barrier function of the epidermis. UVB is the main cause of skin sunburn, and it mainly acts on epidermal keratinocytes. Long-term ultraviolet radiation not only accelerates the aging and death of keratinocytes, but the cytokines secreted and released by aging keratinocytes also indirectly promote the expression of extracellular matrix metalloproteinases (MMP-1) in fibroblasts in a paracrine manner, ultimately making the skin rough and loose.

[0004] Royal jelly is a milky white or light yellow pulpy substance secreted by the subpharyngeal gland (also known as royal jelly gland) and maxillary gland of 5-15 day old worker bees. It is rich in proteins, peptides and free amino acids, and has multiple biological activities such as antibacterial, anti-inflammatory, wound healing and antioxidant. It is a natural health food. Researchers have found that royal jelly peptides have more ideal pharmacological functions than royal jelly proteins and are easier to store. At present, there are few studies on royal jelly peptides to promote wound healing and resist skin photoaging. Therefore, it is of great clinical significance to explore royal jelly peptides that can promote wound healing and prevent and repair skin ultraviolet damage. Summary of the invention

[0005] The purpose of the present invention is to provide an application of royal jelly polypeptide as a functional component in promoting wound healing and resisting skin photoaging.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: The present invention provides a royal jelly polypeptide CMKI-21, wherein the amino acid sequence of the royal jelly polypeptide CMKI-21 is Lys-Lys-Glu-Leu-Val-Arg-Glu-Val-Gly-Phe-Glu-Ile-Cys-Phe-Lys-Asp-His-His-Glu-Lys-Ile (KKELVREVGFEICFKDHHEKI), the molecular weight is 2584.8 Da, and the structure thereof is as follows:

[0007] The present invention also provides the use of the royal jelly polypeptide CMKI-21 in promoting the proliferation and migration of skin cells.

[0008] Furthermore, the skin cells are human immortalized keratinocytes, and the concentration of royal jelly polypeptide CMKI-21 is 10 -11 ~10 -6 M.

[0009] The present invention also provides the use of the royal jelly polypeptide CMKI-21 in the preparation of a preparation for promoting skin wound healing.

[0010] Furthermore, the skin wounds include wounds caused by physical and chemical damage.

[0011] Furthermore, the preparation for promoting skin wound healing also contains pharmaceutically acceptable excipients.

[0012] The present invention also provides the use of the royal jelly polypeptide CMKI-21 in the preparation of cosmetics for resisting skin photoaging or sunburn repair.

[0013] Furthermore, the cosmetics include sprays, gels, emulsions, and water solutions, and the concentration of royal jelly polypeptide CMKI-21 in the cosmetics is 10 -9 ~10 -5 M.

[0014] Beneficial effects of the present invention: The polypeptide of the present invention is derived from royal jelly protein, can promote the proliferation and migration of human immortalized keratinocytes (HaCaT), and has no cytotoxicity. Experiments have shown that the polypeptide of the present invention can significantly inhibit the apoptosis of keratinocytes damaged by ultraviolet light, promote wound repair of full-thickness skin damage in mice, and reduce photoaging of mouse skin caused by ultraviolet light irradiation. Therefore, the royal jelly polypeptide CMKI-21 has good application prospects in the fields of medicine and cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1This is the high performance liquid chromatogram of royal jelly polypeptide CMKI-21.

[0016] Figure 2 This is the mass spectrum of royal jelly polypeptide CMKI-21.

[0017] Figure 3 The results of the effect of royal jelly polypeptide CMKI-21 on HaCaT cell activity are shown in Figure 2; Control represents the control well, 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 , 10 -7 , 10 -6 , 10 -5 M represents the wells with intervention of royal jelly peptides at different concentrations; (A) is the cell activity graph of HaCaT cells treated with royal jelly peptide CMKI-21 at different concentrations for 48 h; (B) is the cell activity graph of HaCaT cells treated with royal jelly peptide CMKI-21 at different concentrations for 72 h; * p <0.05,** p <0.01.

[0018] Figure 4 The results of the effect of royal jelly polypeptide CMKI-21 on the scratch healing ability of HaCaT cells; Control represents the control well, 10 -11 , 10 -10 , 10 -9 , 10 -8 M represents the wells intervened by royal jelly peptides at different concentrations; (A) is a micrograph of cell scratch healing at different time points after adding royal jelly peptides at different concentrations to the HaCaT cell scratch model; (B) is an analysis of the scratch healing rate of HaCaT cells after being treated with CMKI-21 at different concentrations for a certain period of time; * p <0.05,*** p <0.001.

[0019] Figure 5 The results of the effect of royal jelly polypeptide CMKI-21 on the migration ability of HaCaT cells are shown in Figure 2; Control represents the control well, 10 -11 , 10 -10 , 10 -9 , 10 -8 M indicates wells with intervention of royal jelly peptides at different concentrations; (A) is a micrograph of HaCaT cell migration 24 hours after adding royal jelly peptides at different concentrations to the lower chamber of Transwell; (B) is a graph of cell migration analysis after HaCaT cells were treated with CMKI-21 for 24 hours; * p <0.05,** p<0.01.

[0020] Figure 6 The effect of royal jelly polypeptide CMKI-21 on UVB irradiation (30 mj / cm 2 ×3) HaCaT cell activity; Control represents the control group, 10 -11 , 10 -10 , 10 -9 , 10 -8 M represents the intervention wells of royal jelly polypeptide at different concentrations; Model represents the model group; * p <0.05, **** p <0.0001.

[0021] Figure 7 The results of the royal jelly peptide CMKI-21 on the full-thickness skin defect of mice; Control means the control group, EGF (10 -7 M) represents the positive control group, 10 -10 , 10 -9 , 10 -8 M represents the intervention groups of royal jelly peptides at different concentrations; (A) is the macroscopic evaluation diagram of mouse skin wounds after treatment with royal jelly peptide CMKI-21; (B) is the statistical diagram of the healing rate of full-thickness skin defects in mice after treatment with royal jelly peptide CMKI-21; * p <0.05,** p <0.01.

[0022] Figure 8 is the repair effect of royal jelly peptide CMKI-21 on mouse skin photoaging; Control represents the control group, Model represents the model group, 10 -8 , 10 -7 , 10 -6 , 10 -5 M represents the royal jelly peptide intervention groups at various concentrations. DETAILED DESCRIPTION

[0023] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific embodiments. The embodiments are intended to illustrate the present invention by way of example, rather than to limit the present invention in any form.

[0024] Example 1 Synthesis of royal jelly polypeptide CMKI-21 by solid phase chemical synthesis The amino acid sequence of royal jelly polypeptide CMKI-21 is as follows: KKELVREVGFEICFKDHHEKI (SEQ ID NO.1), with a molecular weight of 2584.8 Da. Royal jelly polypeptide CMKI-21 was synthesized by Jiangsu GenScript Biotechnology Co., Ltd.

[0025] The HPLC separation results are as follows Figure 1 As shown in the HPLC analysis, a single peak appeared in the royal jelly protein sample at 7.725 min, with an area accounting for more than 98%, indicating that the purity of the synthetic polypeptide met the requirements. Therefore, the component corresponding to this peak was selected for mass spectrometry analysis, and its molecular weight was verified by mass spectrometry analysis.

[0026] The results of mass spectrometry analysis are as follows Figure 2 As shown in Figure 2, mass spectrometry analysis showed that in the positive ion mode, the primary mass spectrum of CMKI-21 showed different valence ion peaks, m / z = 517.9 ([M+5H] 5+ )、m / z=627.2([M+4H] 4+ ) etc. According to the molecular weight derivation formula: molecular weight = (m / s value × charge number - charge number × proton mass), the calculated molecular weight is 2584.8 Da, which is consistent with the theoretical value, proving the accuracy of the molecular weight of CMKI-21.

[0027] Example 2 Effect of royal jelly polypeptide CMKI-21 on HaCaT cell activity Human immortalized keratinocytes (HaCaT) were cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at a concentration of 3.5 × 10 4 Cell suspensions of 10 cells / mL were evenly inoculated in 96-well plates, with 100 μL per well. After culturing for 24 h, the cells were starved with serum-free medium for 12 h, and royal jelly polypeptide CMKI-21 was dissolved in serum-free medium. 100 μL of royal jelly polypeptide CMKI-21 (0, 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 , 10 -7 , 10 -6 , 10 -5 M), and cultured in a CO2 incubator. After 48 h and 72 h of treatment with royal jelly peptide CMKI-21, 10 μL of MTT was added to each well, incubated in a CO2 incubator in the dark for 4 h, the supernatant was discarded, 100 μL of dimethyl sulfoxide (DMSO) was added to each well, shaken at room temperature for 15 min, and the absorbance was detected at 490 nm using a microplate reader.

[0028] According to cell viability = OD 试验组 / OD 对照组 ×100% to calculate cell viability.

[0029] The results are as follows Figure 3 As shown, compared with the Control group, 10-9 , 10 -8 and 10 -6 M royal jelly peptide CMKI-21 can significantly promote the proliferation of HaCaT cells after 48 hours of action. -11 , 10 -10 , 10 -9 , 10 -8 and 10 -7 M's royal jelly peptide CMKI-21 can significantly promote the proliferation of HaCaT cells after 72 h of action.

[0030] Example 3 Healing activity of royal jelly polypeptide CMKI-21 on HaCaT cell scratch model The plug (ibidi) was placed in a 24-well cell culture plate, and 70 μL of HaCaT cell suspension (3.5×10 4 cells / chamber), the insert was removed after culturing for 12 h, a regular scratch area was formed, and then 1 mL of royal jelly peptide (0, 10 -11 , 10 -10 , 10 -9 , 10 -8 M), The scratch area was recorded under a microscope at 0, 12 and 24 h after treatment with royal jelly peptide CMKI-21.

[0031] The results are as follows Figure 4 As shown, 10 -11 , 10 -10 and 10 -9 M's royal jelly peptide CMKI-21 can significantly reduce the cell scratch area after 24 hours of action.

[0032] Example 4 Effect of royal jelly polypeptide CMKI-21 on HaCaT cell migration Each Transwell chamber was inoculated with 1 × 10 5 HaCaT cells were added to the lower chamber at the same time, 600 μL of royal jelly peptide CMKI-21 (0, 10 -11 , 10 -10 , 10 -9 , 10 -8 M), and placed in a CO2 incubator for 24 h. Fix the cells with paraformaldehyde for 20 min, then stain with 0.1% crystal violet solution for 20 min. Wash twice with PBS (pH=7.4), gently wipe the unmigrated cells in the upper chamber with a cotton swab, observe the bottom of the chamber under a microscope, and take pictures.

[0033] The results are as follows Figure 5 As shown, 10 -11 and 10 -10M's royal jelly peptide CMKI-21 can significantly promote the migration of HaCaT cells.

[0034] Example 5 Effect of royal jelly polypeptide CMKI-21 on the activity of UVB-irradiated HaCaT cells HaCaT cells were prepared with DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) to a volume of 4 × 10 3 Cell suspensions of 10 cells / mL were evenly inoculated into 96-well plates, 100 μL per well, and cultured in a CO2 incubator for 24 h. -11 , 10 -10 , 10 -9 , 10 -8 ) with royal jelly polypeptide CMKI-21 (10 -11 , 10 -10 , 10 -9 , 10 -8 M) serum-free medium for 12 h, and the model group (Model) and the control group (Control) were cultured with serum-free medium without royal jelly peptides. After 12 h, the medium was removed and washed twice with PBS (pH=7.4) buffer, leaving 35 μL PBS (pH=7.4) buffer in each well. The 96-well plate was placed under UVB (313 nm) light, the wells of the control group were covered with tin foil, and the model group and the drug-added group were given 30 mj / cm 2 Ultraviolet irradiation. Irradiation was performed twice a day with an interval of 12 h for 3 consecutive times. After each irradiation, the PBS (pH = 7.4) buffer in each well was discarded, and 100 μL of royal jelly peptide CMKI-21 (10 -11 , 10 -10 , 10 -9 , 10 -8 The model group and the control group were recovered with serum-free medium without royal jelly peptides, and the cell activity was detected with CCK8 kit 24 h after the third irradiation.

[0035] The results are as follows Figure 6 As shown in the figure, compared with the control group, the activity of HaCaT cells in the model group was significantly reduced, indicating that UVB irradiation caused serious damage to the cells. -10 and 10 -9 M's royal jelly peptide CMKI-21 can significantly increase the activity of HaCaT cells.

[0036] Example 6 Effect of royal jelly polypeptide CMKI-21 on full-thickness skin defects in mice After 1 week of adaptive feeding, 35 5-6-week-old female Kunming mice were shaved with an electric shaver. Then, depilatory cream was applied to the exposed skin and wiped with gauze 10 minutes later to completely remove the back hair. The next day, the mice were anesthetized with 5% (w / v) sutane solution (50 mg / kg) under a sterile environment. Two circular wounds with a diameter of about 6 mm were made on the back of the mice with a hole punch. After the wounds dried, they were photographed and the area of ​​the mouse skin wound was calculated using Image J software, which was the initial wound area.

[0037] After the mouse full-thickness skin defect model was successfully established, the mice were randomly divided into 5 groups, with 7 mice in each group. -7 M, positive control group) and royal jelly polypeptide CMKI-21 group (10 -10 , 10 -9 , 10 -8 M). The control group was smeared with 20 μL PBS (pH=7.4) buffer, and the positive control group was smeared with 20 μL epidermal growth factor (EGF, 10 -7 M), royal jelly peptide group smear 20 μL containing CMKI-21 (10 -10 , 10 -9 and 10 -8 M) PBS buffer.

[0038] Determination of wound healing rate: Wound changes were observed every day, and the wounds of mice were photographed on the 1st, 3rd, 5th and 7th days. The wound area was calculated using Image J software, which is the unhealed wound area. The calculation formula is as follows:

[0039] The results are as follows Figure 7 As shown, after treatment with royal jelly polypeptide CMKI-21, the royal jelly polypeptide group (10 -9 , 10 -8 The wound healing rate of mice was significantly improved.

[0040] Example 7 Repair effect of royal jelly polypeptide CMKI-21 on photoaging of mouse skin Thirty female Kunming mice aged 5-6 weeks were randomly divided into 6 groups after adaptive feeding for 1 week, namely, control group, model group and royal jelly peptide CMKI-21 group (10 -8 , 10 -7 , 10 -6 , 10 -5M). The back hair was shaved with an electric shaver before UV irradiation, and then the depilatory cream was applied to the exposed skin and wiped with gauze after 10 minutes to completely remove the back hair. The control group was not given UV irradiation, while the model group and the royal jelly peptide CMKI-21 group were irradiated with UVB (313 nm) every other day, 3 times a week, and the single irradiation dose in the first week was 60 mj / cm 2 , and then the weekly single irradiation dose increases by 30 mj / cm 2 After UV irradiation, 200 μL PBS (pH=7.4) buffer was evenly applied on the back of the mice in the Model group, and 200 μL royal jelly peptide CMKI-21 (concentration 10 -8 , 10 -7 , 10 -6 , 10 -5 M) PBS buffer once a day. The skin changes of mice were observed daily, and the newly grown mouse hair was removed. The skin damage of mice in each group was evaluated 24 hours after the 28th application of active peptides.

[0041] The results are as follows Figure 8 As shown in the figure, the skin of mice in the Control group was firm, smooth, wrinkle-free, and healthy. The skin of mice in the Model group was reddish, dry, flaky, with obvious sun spots, wrinkles, and hyperkeratosis. -7 , 10 -6 , 10 -5 M) The skin color of mice was slightly reddish, and the characteristics of light damage such as sun spots, dryness and desquamation were significantly improved compared with the Model group.

[0042] In summary, the present invention provides a royal jelly polypeptide CMKI-21 having wound healing and anti-skin photoaging activities. The polypeptide is easy to synthesize and has good biocompatibility, and has good application prospects in skin wound treatment and sunburn repair.

[0043] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A royal jelly polypeptide CMKI-21, characterized in that: The amino acid sequence of the royal jelly polypeptide CMKI-21 is KKELVREVGFEICFKDHHEKI, and the molecular weight is 2584.8 Da.

2. Use of the royal jelly polypeptide CMKI-21 as claimed in claim 1 in promoting skin cell proliferation and migration.

3. Use of the royal jelly polypeptide CMKI-21 as claimed in claim 1 in the preparation of a preparation for promoting skin wound healing.

4. Use of the royal jelly polypeptide CMKI-21 as claimed in claim 1 in the preparation of cosmetics for resisting skin photoaging or sunburn repair.

5. The use according to claim 2, characterized in that: The skin cells are human immortalized keratinocytes, and the concentration of royal jelly polypeptide CMKI-21 is 10 -11 ~10 -6 M.

6. The use according to claim 3, characterized in that: The skin wounds include wounds caused by physical and chemical damage.

7. The use according to claim 3, characterized in that: The skin wound healing treatment preparation further comprises a pharmaceutically acceptable auxiliary material.

8. The use according to claim 4, characterized in that: The cosmetics include sprays, gels, emulsions, and water solutions. The concentration of royal jelly polypeptide CMKI-21 in the cosmetics is 10 -9 ~10 -5 M.

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