A royal jelly polypeptide CMKI-21 and application thereof

Abstract

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

Description

Technical Field

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

[0002] The skin is exposed to a host of physical, chemical, and biological hazards daily. When skin cells are unable to defend themselves against these external stimuli, they can no longer maintain their integrity and homeostasis. Skin damage and premature aging are the result of continued exposure to these harmful environments. To enable skin cells to more actively respond to these external stimuli, we need to provide the skin with additional nutrients to boost cell activity. Currently, several substances have been discovered that can treat skin wounds and prevent aging. For example, polyphenols not only reduce inflammation and promote wound healing, but also mitigate photoaging caused by ultraviolet radiation. Lipids and fatty acids prevent water loss in the skin and are crucial for maintaining the integrity of the epidermal barrier. Peptides possess a variety of pharmacological activities, including antimicrobial, antioxidant, and immunomodulatory activities, along with good specificity and stability. Consequently, they have sparked research interest in the treatment of skin injuries.

[0003] As the primary cell type constituting the epidermis, keratinocytes not only serve as structural cells but also play a vital role in wound healing and anti-aging. The proliferation and migration of keratinocytes can accelerate wound healing and restore the barrier function of the epidermis. UVB is the primary cause of sunburn, primarily acting on epidermal keratinocytes. Long-term UV radiation not only accelerates keratinocyte aging and death, but the cytokines released by aging keratinocytes also indirectly promote the expression of extracellular matrix metalloproteinases (MMP-1) in fibroblasts in a paracrine manner, ultimately causing the skin to become rough and sagging.

[0004] Royal jelly is a milky white or pale yellow paste secreted by the subpharyngeal and maxillary glands of 5-15 day-old worker bees. Rich in proteins, peptides, and free amino acids, it possesses numerous biological activities, including antibacterial, anti-inflammatory, wound healing, and antioxidant properties, making it a natural health food. Researchers have found that royal jelly peptides possess more desirable pharmacological properties than royal jelly proteins and are easier to store. Currently, research on the role of royal jelly peptides in promoting wound healing and combating photoaging is limited. Therefore, exploring royal jelly peptides that can promote wound healing and prevent and repair UV-induced skin damage is of great clinical significance. 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 solutions:

[0007] The present invention provides a royal jelly polypeptide CMKI-21. 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 shown below:

[0008]

[0009] The present invention also provides the use of the royal jelly polypeptide CMKI-21 in promoting the proliferation and migration of skin cells.

[0010] Furthermore, the skin cells are human immortalized keratinocytes, and the concentration of royal jelly polypeptide CMKI-21 is 10 -11 ~10 -6 M.

[0011] The present invention also provides the use of the royal jelly polypeptide CMKI-21 in preparing a preparation for promoting skin wound healing.

[0012] Furthermore, the skin wounds include wounds caused by physical and chemical damage.

[0013] Furthermore, the skin wound healing promoting preparation further comprises pharmaceutically acceptable excipients.

[0014] 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.

[0015] Furthermore, the cosmetics include sprays, gels, emulsions and lotions, and the concentration of royal jelly polypeptide CMKI-21 in the cosmetics is 10 -9 ~10 -5 M.

[0016] Beneficial effects of the present invention:

[0017] The polypeptide of this invention, derived from royal jelly protein, can promote the proliferation and migration of human immortalized keratinocytes (HaCaT) without cytotoxicity. Experimental studies have shown that the polypeptide can significantly inhibit apoptosis of UV-induced keratinocytes, promote wound repair in full-thickness skin damage in mice, and mitigate photoaging of mouse skin induced by UV radiation. Therefore, this royal jelly polypeptide, CMKI-21, has promising application prospects in the pharmaceutical and cosmetic fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the high performance liquid chromatogram of royal jelly polypeptide CMKI-21.

[0019] Figure 2 This is the mass spectrum of royal jelly polypeptide CMKI-21.

[0020] Figure 3 The results of the effect of royal jelly polypeptide CMKI-21 on HaCaT cell activity are shown in the figure; 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 treated with royal jelly peptides at different concentrations; (A) is the cell viability graph of HaCaT cells treated with royal jelly peptides CMKI-21 at different concentrations for 48 h; (B) is the cell viability graph of HaCaT cells treated with royal jelly peptides CMKI-21 at different concentrations for 72 h; * p <0.05,** p <0.01.

[0021] Figure 4 The results of the effect of royal jelly polypeptide CMKI-21 on the scratch healing ability of HaCaT cells are shown in the figure; Control represents the control well, 10 -11 , 10 -10 , 10 -9 , 10 -8 M represents the intervention wells of 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.

[0022] Figure 5 The results of the effect of royal jelly polypeptide CMKI-21 on the migration ability of HaCaT cells are shown in the figure; Control represents the control well, 10 -11 , 10 -10 , 10 -9 , 10 -8 M represents the wells treated with 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 the Transwell; (B) is a graph showing the cell migration rate analysis of HaCaT cells after treatment with CMKI-21 for 24 hours; *p <0.05,** p <0.01.

[0023] 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.

[0024] Figure 7 The results of the effect of royal jelly peptide CMKI-21 on full-thickness skin defects in mice are shown in the figure; Control represents 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 a macroscopic evaluation diagram of mouse skin wounds after treatment with royal jelly peptide CMKI-21; (B) is a statistical diagram of the healing rate of full-thickness skin wounds in mice after treatment with royal jelly peptide CMKI-21; * p <0.05,** p <0.01.

[0025] Figure 8 The repair effect of royal jelly polypeptide CMKI-21 on photoaging of mouse skin; 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

[0026] 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 and are not intended to limit the present invention in any form.

[0027] Example 1 Synthesis of royal jelly polypeptide CMKI-21 by solid phase chemical synthesis

[0028] The amino acid sequence of the royal jelly peptide CMKI-21 is as follows: KKELVREVGFEICFKDHHEKI (SEQ ID NO. 1), with a molecular weight of 2584.8 Da. The royal jelly peptide CMKI-21 was synthesized by Jiangsu GenScript Biotechnology Co., Ltd.

[0029] The HPLC separation results are as follows Figure 1 As shown in the HPLC analysis, a single peak appeared at 7.725 min for the royal jelly protein sample, accounting for over 98% of the total area, indicating that the synthetic peptide met the purity requirements. Therefore, the component corresponding to this peak was selected for mass spectrometry analysis, and its molecular weight was verified by mass spectrometry.

[0030] Mass spectrometry analysis results Figure 2 As shown, mass spectrometry analysis showed that in 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.

[0031] Example 2 Effect of Royal Jelly Peptide CMKI-21 on HaCaT Cell Activity

[0032] Human immortalized keratinocytes (HaCaT) were cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) to a volume of 3.5 × 10 4 Cell suspensions of 100 cells / mL were evenly inoculated into 96-well plates, with 100 μL per well. After culturing for 24 h, the cells were starved with serum-free medium for 12 h. Royal jelly polypeptide CMKI-21 was dissolved in serum-free medium, and 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 treatment with royal jelly peptide CMKI-21 for 48 and 72 hours, 10 μL of MTT was added to each well and incubated in a CO2 incubator in the dark for 4 hours. The supernatant was discarded, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. The cells were shaken at room temperature for 15 minutes, and the absorbance was measured at 490 nm using a microplate reader.

[0033] According to cell activity = OD 试验组 / OD 对照组 ×100% to calculate cell viability.

[0034] The results are as follows Figure 3 As shown, compared with the Control group, 10 -9 , 10 -8 and 10 -6 M royal jelly polypeptide CMKI-21 can significantly promote the proliferation of HaCaT cells after 48 hours of treatment. -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 hours of treatment.

[0035] Example 3 Healing activity of royal jelly polypeptide CMKI-21 on HaCaT cell scratch model

[0036] 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), and the plug was removed after culturing for 12 h to form a regular scratch area. 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.

[0037] 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.

[0038] Example 4 Effect of Royal Jelly Peptide CMKI-21 on HaCaT Cell Migration

[0039] Each Transwell chamber was inoculated with 1 × 10 5 At the same time, 600 μL of royal jelly peptide CMKI-21 (0, 10 -11 , 10 -10 , 10 -9 , 10 -8M) and incubated in a CO2 incubator for 24 hours. Fix the cells with paraformaldehyde for 20 minutes and then stain with 0.1% crystal violet solution for 20 minutes. Wash twice with PBS (pH 7.4). Unmigrated cells in the upper chamber were gently removed with a cotton swab. The bottom of the chamber was observed under a microscope and photographed.

[0040] The results are as follows Figure 5 As shown, 10 -11 and 10 -10 M's royal jelly peptide CMKI-21 can significantly promote the migration of HaCaT cells.

[0041] Example 5 Effect of Royal Jelly Peptide CMKI-21 on the Activity of UVB-Irradiated HaCaT Cells

[0042] HaCaT cells were diluted with DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) to a volume of 4 × 10 3 Cell suspensions of 100 μL / mL were evenly inoculated into 96-well plates, with 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 The model and control groups were pretreated with serum-free medium containing royal jelly peptides for 12 hours. The model and control groups were cultured in serum-free medium without royal jelly peptides. After 12 hours, the medium was removed and the cells were washed twice with PBS (pH 7.4), leaving 35 μL of PBS (pH 7.4) 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. The model and drug-treated groups were exposed to 30 mJ / cm 2 UV 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 M) serum-free medium, the model group and the control group were recovered with serum-free medium without royal jelly polypeptide, and the cell activity was detected using CCK8 kit 24 h after the third irradiation.

[0043] The results are as follows Figure 6As 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.

[0044] Example 6 Effect of Royal Jelly Peptide CMKI-21 on Full-Thickness Skin Injuries in Mice

[0045] After one week of acclimatization, 35 5- to 6-week-old female Kunming mice were shaved with an electric shaver. Depilatory cream was then applied to the exposed skin and wiped off with gauze 10 minutes later to completely remove the hair. The next day, the mice were anesthetized with 5% (w / v) Zotazone solution (50 mg / kg) under sterile conditions. Two circular wounds approximately 6 mm in diameter were created on the backs of the mice using a hole punch. After the wounds dried, they were photographed and the area of ​​the skin wounds was calculated using Image J software, which was used to calculate the initial wound area.

[0046] After the full-thickness skin defect model was successfully established in mice, 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 smeared 20 μL containing CMKI-21 (10 -10 , 10 -9 and 10 -8 M) PBS buffer.

[0047] Determination of wound healing rate: Wound changes were observed daily, and the wounds of mice were photographed on days 1, 3, 5, and 7. The wound area was calculated using Image J software, which is the area of ​​unhealed wounds. The calculation formula is as follows:

[0048]

[0049] 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.

[0050] Example 7 Repair effect of royal jelly polypeptide CMKI-21 on photoaging of mouse skin

[0051] Thirty female Kunming mice aged 5-6 weeks were randomly divided into 6 groups after adaptive feeding for 1 week, 5 mice in each group, namely Control group, Model group and Royal Jelly Peptide CMKI-21 group (10 -8 , 10 -7 , 10 -6 , 10 -5 M). Before UV irradiation, the back hair was shaved with an electric shaver. Then, depilatory cream was applied to the exposed skin and wiped off with gauze 10 minutes later to completely remove the back hair. The control group was not given UV irradiation. The model group and the royal jelly peptide CMKI-21 group were irradiated with UVB (313 nm) every other day, three times a week. 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 solution 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 daily. The mice's skin was observed daily for changes, and newly grown hair was removed. Skin damage in each group was evaluated 24 hours after the 28th application of the active peptide.

[0052] 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 presented a healthy complexion. The skin of mice in the model group was reddish, dry, scaly, 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 photodamage such as sun spots, dryness and desquamation were significantly improved compared with the Model group.

[0053] In summary, the present invention provides a royal jelly polypeptide, CMKI-21, with wound healing and anti-photoaging activities. This polypeptide is easy to synthesize and has good biocompatibility, showing promising application prospects in skin wound treatment and sunburn repair.

[0054] 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 by: 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 preparing a preparation for promoting skin wound healing.

3. Use of the royal jelly polypeptide CMKI-21 according to claim 1 in the preparation of cosmetics for resisting skin photoaging or sunburn repair.

4. The use according to claim 2, characterized in that: The skin wounds include wounds caused by physical and chemical damage.

5. The use according to claim 2, characterized in that: The skin wound healing promoting preparation further comprises a pharmaceutically acceptable excipient.

6. The use according to claim 3, characterized in that: The cosmetics include sprays, gels, emulsions and aqueous solutions. The concentration of royal jelly polypeptide CMKI-21 in the cosmetics is 10 -9 ~10 -5 M.

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