Antibacterial peptide for inhibiting malassezia and preparation method and application thereof
By synthesizing a variety of polypeptides and preparing antimicrobial peptides using solid-phase synthesis, the problems of high cost and low activity of natural antimicrobial peptides have been solved, achieving effective inhibition of Malassezia and therapeutic effects on skin diseases. These peptides are suitable for cosmetics and topical medicines.
Patent Information
- Application Number
- CN202411282295.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-12
- Publication Date
- 2026-02-24
- Estimated Expiration
- 2044-09-12
AI Technical Summary
Existing natural antimicrobial peptides have high production costs, low antimicrobial activity, and poor stability. Furthermore, there is limited research on their use in inhibiting Malassezia, and they suffer from problems such as complex structures and low antimicrobial activity.
We designed and synthesized a variety of peptides, including antimicrobial peptides with substituted, deleted, and modified amino acid sequences, prepared them using solid-phase synthesis, and applied them in cosmetics or topical medicines to treat skin diseases caused by Malassezia fungal infections.
It exhibits significant antibacterial activity against Malassezia, with good safety profile, and is suitable for treating skin diseases such as dandruff, seborrheic dermatitis, tinea versicolor, and Malassezia folliculitis, demonstrating significant application value.
Smart Images
Figure CN118955640B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of antimicrobial peptide technology, specifically relating to an antimicrobial peptide for inhibiting Malassezia, its preparation method, and its application. Background Technology
[0002] Malassezia is an opportunistic pathogen with a lipophilic affinity for lipids. It primarily grows in the stratum corneum of human skin and is associated with sebaceous glands containing triglycerides and free fatty acids on the skin surface. Its density decreases with age due to a reduction in lipid content. It can be detected in human skin and hair samples. Numerous studies have shown it to be an opportunistic pathogen, mostly harmless. However, under certain internal and external conditions, Malassezia can cause tinea versicolor, pityriasis folliculitis, seborrheic dermatitis and dandruff, atopic dermatitis, and psoriasis. With the expansion of research, studies have also confirmed its association with onychomycosis and systemic infections.
[0003] Antimicrobial peptides, as a novel type of antimicrobial agent, are widely available and possess broad-spectrum antimicrobial activity. Compared to traditional antibiotics, most antimicrobial peptides primarily kill bacteria by disrupting their cell membranes, making them less likely to induce antibiotic resistance and thus considered a good alternative. In 1980, Boman et al. successfully isolated and purified cephalosporin from silkworm pupae, marking the first discovery of an antimicrobial peptide. Subsequently, antimicrobial peptides have been discovered in bacteria, fungi, amphibians, higher plants, mammals, and humans.
[0004] Natural antimicrobial peptides suffer from high production costs, low antimicrobial activity, and poor stability, failing to meet practical application needs. With ongoing in-depth research into the properties, structures, and mechanisms of action of existing natural antimicrobial peptides, increasing research focuses on developing synthetically produced antimicrobial peptides. Researchers have successfully utilized various modern biotechnologies to molecularly modify or synthesize novel antimicrobial peptides. Currently, research on antimicrobial peptides for inhibiting Malassezia is limited, and those that exist suffer from complex structures and low antimicrobial activity. Summary of the Invention
[0005] To address the problems in the prior art, the first aspect of the present invention provides an antimicrobial peptide for inhibiting Malassezia, wherein the antimicrobial peptide is any one of the following polypeptides (a)-(d):
[0006] (a) A polypeptide whose amino acid sequence includes the amino acid sequence shown in SEQ ID NO.1, or a polypeptide whose amino acid sequence consists of the amino acid residues shown in SEQ ID NO.1;
[0007] (b) A polypeptide formed by substituting and / or deleting and / or adding one or more amino acid residues into the amino acid sequence defined in (a);
[0008] (c) A polypeptide obtained by modifying the N-terminus and / or C-terminus of the polypeptide defined in (a);
[0009] (d) is a polypeptide that has 99%, 95%, 90%, 85%, or 80% homology with the amino acid sequence defined in (a).
[0010] More preferably, the antimicrobial peptide is P19, and its amino acid sequence is shown in SEQ ID NO.1.
[0011] Preferably, the antimicrobial peptide is P19S, and its amino acid sequence is shown in SEQ ID NO.2.
[0012] Preferably, the antimicrobial peptide is P19K, and its amino acid sequence is shown in SEQ ID NO.3.
[0013] Preferably, the antimicrobial peptide is P19L, and its amino acid sequence is shown in SEQ ID NO.4.
[0014] A second aspect of the present invention provides a method for preparing an antimicrobial peptide for inhibiting Malassezia, wherein the preparation method is a solid-phase synthesis method.
[0015] A third aspect of the present invention provides the application of an antimicrobial peptide for inhibiting Malassezia, for use in the preparation of products for the prevention or treatment of skin diseases caused by Malassezia fungal infections.
[0016] Preferably, the skin diseases include dandruff, seborrheic dermatitis, tinea versicolor, and Malassezia folliculitis caused by Malassezia fungal infection;
[0017] The product in question is a cosmetic or a topical medicine.
[0018] The beneficial effects of this invention are as follows:
[0019] This invention designs and synthesizes an antimicrobial peptide for inhibiting Malassezia, exhibiting good skin safety. It can be used to treat skin diseases caused by Malassezia fungal infections, such as dandruff, seborrheic dermatitis, tinea versicolor, and Malassezia folliculitis. It can be applied in cosmetics or topical medicines for treating dandruff or seborrheic dermatitis, tinea versicolor, and Malassezia folliculitis. The antimicrobial peptide described in this invention has significant inhibitory effects on both Malassezia furfur and Malassezia spp., demonstrating significant application value. Attached Figure Description
[0020] Figure 1A and Figure 1BThe images show the antimicrobial effects of the antimicrobial peptide described in Example 1 after incubation in Malassezia furfur ATCC 44344 at concentrations of 10 μg / mL and 20 μg / mL for 48 h, 72 h, and 96 h, respectively.
[0021] Figure 2A and Figure 2B The figures show the antimicrobial effects of the antimicrobial peptide in Example 1 after incubation in Malassezia restricta ATCC 33081 at concentrations of 10 μg / mL and 20 μg / mL for 48 h, 72 h, and 96 h, respectively. Detailed Implementation
[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0023] The first aspect of the present invention provides an antimicrobial peptide for inhibiting Malassezia, said antimicrobial peptide being any one of the following polypeptides (a)-(d):
[0024] (a) A polypeptide whose amino acid sequence includes the amino acid sequence shown in SEQ ID NO.1, or a polypeptide whose amino acid sequence consists of the amino acid residues shown in SEQ ID NO.1;
[0025] (b) A polypeptide formed by substituting and / or deleting and / or adding one or more amino acid residues into the amino acid sequence defined in (a);
[0026] (c) A polypeptide obtained by modifying the N-terminus and / or C-terminus of the polypeptide defined in (a);
[0027] (d) is a polypeptide that has 99%, 95%, 90%, 85%, or 80% homology with the amino acid sequence defined in (a).
[0028] More preferably, the antimicrobial peptide is P19, and its amino acid sequence is shown in SEQ ID NO.1.
[0029] Preferably, the antimicrobial peptide is P19S, and its amino acid sequence is shown in SEQ ID NO.2.
[0030] Preferably, the antimicrobial peptide is P19K, and its amino acid sequence is shown in SEQ ID NO.3.
[0031] Preferably, the antimicrobial peptide is P19L, and its amino acid sequence is shown in SEQ ID NO.4.
[0032] A second aspect of the present invention provides a method for preparing an antimicrobial peptide for inhibiting Malassezia, wherein the preparation method is a solid-phase synthesis method.
[0033] The third aspect of the present invention provides an application of an antimicrobial peptide for inhibiting Malassezia, which is used in the preparation of products for the prevention or treatment of skin diseases caused by Malassezia fungal infections;
[0034] The product in question is a cosmetic or a topical medicine.
[0035] The above technical solution will be described in detail below with reference to specific embodiments.
[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0037] Example 1
[0038] This embodiment provides an antimicrobial peptide P19 for inhibiting Malassezia, with the amino acid sequence KLKKLIKRILCS, as shown in SEQ ID NO.1.
[0039] This embodiment further provides a method for preparing the antimicrobial peptide, which employs a solid-phase synthesis method, and the specific steps are as follows:
[0040] (1) The peptide synthesis sequence is from C-terminus to N-terminus: 20 g of AM resin was placed in a reaction tube, 15 mL / g of DCM (dichloromethane) was added, and the mixture was shaken for 45 minutes. The DCM solvent was removed by vacuum filtration through a sintered glass core. 3 times the molar excess of Fmoc-Linker-OH (CAS: 145069-56-3) was added, followed by 6.5 times the molar excess of DIEA (N,N-diisopropylethylamine), and the mixture was shaken for 75 minutes. The mixture was blocked with methanol. DMF was removed by rotary evaporation, and 20 mL / g of 20% (V / V) piperidine-DMF solvent was added. After washing for 5 minutes, the solution was removed, and 20 mL / g of 20% (V / V) piperidine-DMF solvent was added again. The mixture was washed for 20 minutes. The resin was rinsed twice with DMF (15 mL / g), DCM (15 mL / g), and DMF (15 mL / g) respectively, and the mixture was shaken for 40 minutes.
[0041] (2) Inoculation of the first amino acid: Remove the solvent DCM by vacuum filtration through a sand core filter, add 3.5 molar excess of Fmoc-Ala-OH, dissolve in DMF (dimethylformamide), then add 6.5 molar excess of DIEA (N,N-diisopropylethylamine), and shake for 70 minutes. Block with methanol.
[0042] (3) Deprotection: Evaporate DMF by rotary evaporation, add 20 mL / g 20% (V / V) piperidine-DMF solvent, wash for 5 minutes, remove the DMF, add 15 mL / g 20% (V / V) piperidine-DMF solvent, and wash for 20 minutes.
[0043] (4) Detection: Take 15 resin grains from the piperidine solution, wash them three times with ethanol, and add the test reagent. Heat at 105-110℃ for 8 minutes. A positive reaction is indicated when the color turns dark blue.
[0044] (5) Rinse the resin: Rinse the resin twice in sequence with DMF (15mL / g), DCM (15mL / g), and DMF (15mL / g).
[0045] (6) Condensation: Add three times the excess of DMF to dissolve the next amino acid derivative (from C-terminus to N-terminus), dissolve 3.5 times the excess of HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate) in DMF in the reaction tube, and then immediately add sixteen times the molar amount of DIEA. React for 40 minutes.
[0046] (7) Detection: Take 15 resin grains, wash them three times with ethanol, add the test reagent, heat at 105℃-110℃ for 5 minutes, and the colorless reaction is a negative reaction.
[0047] (8) Rinse the resin: Rinse the resin twice in sequence with DMF (10mL / g), DCM (10mL / g), and DMF (10mL / g).
[0048] (9) Repeat steps (3) to (8) to connect the amino acids shown in SEQ ID No.1 from right to left.
[0049] (10) Drain and wash the resin as follows: DMF (15 mL / g) twice, methanol (15 mL / g) twice, DMF (15 mL / g) twice, DCM (15 mL / g) twice, and drain for 10 minutes.
[0050] (11) Cutting peptides from resin: The cutting solution used at 25°C consisted of 95% (V / V) TFA (trifluoroacetic acid), 1% (V / V) water, 2% (V / V) EDT (mercaptoethanol), and 2% (V / V) TIS (triisopropylsilane); the cutting time was 120 minutes, and the resulting lysate was obtained.
[0051] (12) Drying and washing: Dry the lysate with nitrogen, wash it six times with ether, and then let it evaporate at room temperature.
[0052] (13) Analysis, purification and freeze drying: The crude peptide was purified by high performance liquid chromatography; the peptide solution was collected and concentrated in a freeze dryer and freeze-dried to produce a white powder, thus obtaining an antimicrobial peptide with the structure KLKKLIKRILCS.
[0053] The detection reagent is ninhydrin.
[0054] Example 2
[0055] This embodiment provides an antimicrobial peptide P19S for inhibiting Malassezia, with the amino acid sequence KLKKLIKRILSS, as shown in SEQ ID NO.2.
[0056] This embodiment further provides a method for preparing the antimicrobial peptide, which employs a solid-phase synthesis method, and the specific steps are as follows:
[0057] (1) The peptide synthesis sequence is from C-terminus to N-terminus: 20 g of AM resin was placed in a reaction tube, 15 mL / g of DCM (dichloromethane) was added, and the mixture was shaken for 45 minutes. The DCM solvent was removed by vacuum filtration through a sintered glass core. 3 times the molar excess of Fmoc-Linker-OH (CAS: 145069-56-3) was added, followed by 6.5 times the molar excess of DIEA (N,N-diisopropylethylamine), and the mixture was shaken for 75 minutes. The mixture was blocked with methanol. DMF was removed by rotary evaporation, and 20 mL / g of 20% (V / V) piperidine-DMF solvent was added. After washing for 5 minutes, the solution was removed, and 20 mL / g of 20% (V / V) piperidine-DMF solvent was added again. The mixture was washed for 20 minutes. The resin was rinsed twice with DMF (15 mL / g), DCM (15 mL / g), and DMF (15 mL / g) respectively, and the mixture was shaken for 40 minutes.
[0058] (2) Inoculation of the first amino acid: Remove the solvent DCM by vacuum filtration through a sand core filter, add 3.5 molar excess of Fmoc-Ala-OH, dissolve in DMF (dimethylformamide), then add 6.5 molar excess of DIEA (N,N-diisopropylethylamine), and shake for 70 minutes. Block with methanol.
[0059] (3) Deprotection: Evaporate DMF by rotary evaporation, add 20 mL / g 20% (V / V) piperidine-DMF solvent, wash for 5 minutes, remove the DMF, add 15 mL / g 20% (V / V) piperidine-DMF solvent, and wash for 20 minutes.
[0060] (4) Detection: Take 15 resin grains from the piperidine solution, wash them three times with ethanol, and add the test reagent. Heat at 105-110℃ for 8 minutes. A positive reaction is indicated when the color turns dark blue.
[0061] (5) Rinse the resin: Rinse the resin twice in sequence with DMF (15mL / g), DCM (15mL / g), and DMF (15mL / g).
[0062] (6) Condensation: Add three times the excess of DMF to dissolve the next amino acid derivative (from C-terminus to N-terminus), dissolve 3.5 times the excess of HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate) in DMF in the reaction tube, and then immediately add sixteen times the molar amount of DIEA. React for 40 minutes.
[0063] (7) Detection: Take 15 resin grains, wash them three times with ethanol, add the test reagent, heat at 105℃-110℃ for 5 minutes, and the colorless reaction is a negative reaction.
[0064] (8) Rinse the resin: Rinse the resin twice in sequence with DMF (10mL / g), DCM (10mL / g), and DMF (10mL / g).
[0065] (9) Repeat steps (3) to (8) to connect the amino acids shown in SEQ ID No.2 from right to left.
[0066] (10) Drain and wash the resin as follows: DMF (15 mL / g) twice, methanol (15 mL / g) twice, DMF (15 mL / g) twice, DCM (15 mL / g) twice, and drain for 10 minutes.
[0067] (11) Cutting peptides from resin: The cutting solution used at 25°C consisted of 95% (V / V) TFA (trifluoroacetic acid), 1% (V / V) water, 2% (V / V) EDT (mercaptoethanol), and 2% (V / V) TIS (triisopropylsilane); the cutting time was 120 minutes, and the resulting lysate was obtained.
[0068] (12) Drying and washing: Dry the lysate with nitrogen, wash it six times with ether, and then let it evaporate at room temperature.
[0069] (13) Analysis, purification and freeze drying: The crude peptide was purified by high performance liquid chromatography; the peptide solution was collected and concentrated in a freeze dryer and freeze-dried to produce a white powder, thus obtaining an antimicrobial peptide with the structure KLKKLIKRILSS.
[0070] The detection reagent is ninhydrin.
[0071] Example 3
[0072] This embodiment provides an antimicrobial peptide P19K for inhibiting Malassezia, with the amino acid sequence KLKKLIKRILKS, as shown in SEQ ID NO.3.
[0073] This embodiment further provides a method for preparing the antimicrobial peptide, which employs a solid-phase synthesis method, and the specific steps are as follows:
[0074] (1) The peptide synthesis sequence is from C-terminus to N-terminus: 20 g of AM resin was placed in a reaction tube, 15 mL / g of DCM (dichloromethane) was added, and the mixture was shaken for 45 minutes. The DCM solvent was removed by vacuum filtration through a sintered glass core. 3 times the molar excess of Fmoc-Linker-OH (CAS: 145069-56-3) was added, followed by 6.5 times the molar excess of DIEA (N,N-diisopropylethylamine), and the mixture was shaken for 75 minutes. The mixture was blocked with methanol. DMF was removed by rotary evaporation, and 20 mL / g of 20% (V / V) piperidine-DMF solvent was added. After washing for 5 minutes, the solution was removed, and 20 mL / g of 20% (V / V) piperidine-DMF solvent was added again. The mixture was washed for 20 minutes. The resin was rinsed twice with DMF (15 mL / g), DCM (15 mL / g), and DMF (15 mL / g) respectively, and the mixture was shaken for 40 minutes.
[0075] (2) Inoculation of the first amino acid: Remove the solvent DCM by vacuum filtration through a sand core filter, add 3.5 molar excess of Fmoc-Ala-OH, dissolve in DMF (dimethylformamide), then add 6.5 molar excess of DIEA (N,N-diisopropylethylamine), and shake for 70 minutes. Block with methanol.
[0076] (3) Deprotection: Evaporate DMF by rotary evaporation, add 20 mL / g 20% (V / V) piperidine-DMF solvent, wash for 5 minutes, remove the DMF, add 15 mL / g 20% (V / V) piperidine-DMF solvent, and wash for 20 minutes.
[0077] (4) Detection: Take 15 resin grains from the piperidine solution, wash them three times with ethanol, and add the test reagent. Heat at 105-110℃ for 8 minutes. A positive reaction is indicated when the color turns dark blue.
[0078] (5) Rinse the resin: Rinse the resin twice in sequence with DMF (15mL / g), DCM (15mL / g), and DMF (15mL / g).
[0079] (6) Condensation: Add three times the excess of DMF to dissolve the next amino acid derivative (from C-terminus to N-terminus), dissolve 3.5 times the excess of HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate) in DMF in the reaction tube, and then immediately add sixteen times the molar amount of DIEA. React for 40 minutes.
[0080] (7) Detection: Take 15 resin grains, wash them three times with ethanol, add the test reagent, heat at 105℃-110℃ for 5 minutes, and the colorless reaction is a negative reaction.
[0081] (8) Rinse the resin: Rinse the resin twice in sequence with DMF (10mL / g), DCM (10mL / g), and DMF (10mL / g).
[0082] (9) Repeat steps (3) to (8) to connect the amino acids shown in SEQ ID No.3 from right to left.
[0083] (10) Drain and wash the resin as follows: DMF (15 mL / g) twice, methanol (15 mL / g) twice, DMF (15 mL / g) twice, DCM (15 mL / g) twice, and drain for 10 minutes.
[0084] (11) Cutting peptides from resin: The cutting solution used at 25°C consisted of 95% (V / V) TFA (trifluoroacetic acid), 1% (V / V) water, 2% (V / V) EDT (mercaptoethanol), and 2% (V / V) TIS (triisopropylsilane); the cutting time was 120 minutes, and the resulting lysate was obtained.
[0085] (12) Drying and washing: Dry the lysate with nitrogen, wash it six times with ether, and then let it evaporate at room temperature.
[0086] (13) Analysis, purification and freeze drying: The crude peptide was purified by high performance liquid chromatography; the peptide solution was collected and concentrated in a freeze dryer and freeze-dried to produce a white powder, thus obtaining an antimicrobial peptide with the structure KLKKLIKRILKS.
[0087] The detection reagent is ninhydrin.
[0088] Example 4
[0089] This embodiment provides an antimicrobial peptide P19L for inhibiting Malassezia, with the amino acid sequence KLKKLIKRILLS, as shown in SEQ ID NO.4.
[0090] This embodiment further provides a method for preparing the antimicrobial peptide, which employs a solid-phase synthesis method, and the specific steps are as follows:
[0091] (1) The peptide synthesis sequence is from C-terminus to N-terminus: 20 g of AM resin was placed in a reaction tube, 15 mL / g of DCM (dichloromethane) was added, and the mixture was shaken for 45 minutes. The DCM solvent was removed by vacuum filtration through a sintered glass core. 3 times the molar excess of Fmoc-Linker-OH (CAS: 145069-56-3) was added, followed by 6.5 times the molar excess of DIEA (N,N-diisopropylethylamine), and the mixture was shaken for 75 minutes. The mixture was blocked with methanol. DMF was removed by rotary evaporation, and 20 mL / g of 20% (V / V) piperidine-DMF solvent was added. After washing for 5 minutes, the solution was removed, and 20 mL / g of 20% (V / V) piperidine-DMF solvent was added again. The mixture was washed for 20 minutes. The resin was rinsed twice with DMF (15 mL / g), DCM (15 mL / g), and DMF (15 mL / g) respectively, and the mixture was shaken for 40 minutes.
[0092] (2) Inoculation of the first amino acid: Remove the solvent DCM by vacuum filtration through a sand core filter, add 3.5 molar excess of Fmoc-Ala-OH, dissolve in DMF (dimethylformamide), then add 6.5 molar excess of DIEA (N,N-diisopropylethylamine), and shake for 70 minutes. Block with methanol.
[0093] (3) Deprotection: Evaporate DMF by rotary evaporation, add 20 mL / g 20% (V / V) piperidine-DMF solvent, wash for 5 minutes, remove the DMF, add 15 mL / g 20% (V / V) piperidine-DMF solvent, and wash for 20 minutes.
[0094] (4) Detection: Take 15 resin grains from the piperidine solution, wash them three times with ethanol, and add the test reagent. Heat at 105-110℃ for 8 minutes. A positive reaction is indicated when the color turns dark blue.
[0095] (5) Rinse the resin: Rinse the resin twice in sequence with DMF (15mL / g), DCM (15mL / g), and DMF (15mL / g).
[0096] (6) Condensation: Add three times the excess of DMF to dissolve the next amino acid derivative (from C-terminus to N-terminus), dissolve 3.5 times the excess of HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate) in DMF in the reaction tube, and then immediately add sixteen times the molar amount of DIEA. React for 40 minutes.
[0097] (7) Detection: Take 15 resin grains, wash them three times with ethanol, add the test reagent, heat at 105℃-110℃ for 5 minutes, and the colorless reaction is a negative reaction.
[0098] (8) Rinse the resin: Rinse the resin twice in sequence with DMF (10mL / g), DCM (10mL / g), and DMF (10mL / g).
[0099] (9) Repeat steps (3) to (8) to connect the amino acids shown in SEQ ID No.4 from right to left.
[0100] (10) Drain and wash the resin as follows: DMF (15 mL / g) twice, methanol (15 mL / g) twice, DMF (15 mL / g) twice, DCM (15 mL / g) twice, and drain for 10 minutes.
[0101] (11) Cutting peptides from resin: The cutting solution used at 25°C consisted of 95% (V / V) TFA (trifluoroacetic acid), 1% (V / V) water, 2% (V / V) EDT (mercaptoethanol), and 2% (V / V) TIS (triisopropylsilane); the cutting time was 120 minutes, and the resulting lysate was obtained.
[0102] (12) Drying and washing: Dry the lysate with nitrogen, wash it six times with ether, and then let it evaporate at room temperature.
[0103] (13) Analysis, purification and freeze drying: The crude peptide was purified by high performance liquid chromatography; the peptide solution was collected and concentrated in a freeze dryer and freeze-dried to produce a white powder, thus obtaining an antimicrobial peptide with the structure KLKKLIKRILLS.
[0104] The detection reagent is ninhydrin.
[0105] Experimental Example
[0106] 1. Antibacterial test:
[0107] The antibacterial properties of the antimicrobial peptide P19 described in Example 1 were tested as follows:
[0108] The lyophilized powder of *Malassezia furfur* / *Malassezia repens* was activated and incubated at 32°C for 72 hours. Several colonies were then picked up with a sterile inoculation loop and added to sterile physiological saline. After several aspirations, the solution was dissolved in sterile culture medium to a specific volume. The turbidity was then measured using a McFarland turbidimeter, and the bacterial suspension was diluted with mDixon medium to a turbidity of 5 × 10⁻⁶. 5CFU / mL bacterial suspension was prepared for later use. Antimicrobial peptide P19 was then diluted with sterile medium to experimental concentrations (10, 20 μg / mL) and set aside. Using a microdilution method, 100 μL of diluted antimicrobial peptide P19 was added to each well of a 96-well plate, followed by 100 μL of bacterial suspension. 100 μL of bacterial suspension and 100 μL of medium were used as growth controls, and 200 μL of medium was used as a blank control. After incubation at 37℃ for 48-72 h, the turbidity of the liquid in the 96-well plate was observed, and the OD600nm value was measured at 48 / 72 h. The inhibition rate of each well was calculated using the formula: Inhibition rate % = [OD growth group - OD sample group - OD blank medium group / OD growth group - OD blank medium group)] × 100%. Finally, after 72 h, 10 μL of CCK-8 was added to each well for incubation for 24 h, and the OD450nm value was measured to calculate bacterial viability.
[0109] The bacteria mentioned were Malassezia furfur ATCC 44344 and Malassezia restricta ATCC 33081, with KCZ being ketoconazole as a control group. All of them were commercially available.
[0110] 2. Experimental Results
[0111] The results of the antibacterial experiment against Malassezia furfur are as follows: Figure 1A and Figure 1B As shown, after incubation with 10 μg / mL of the antimicrobial peptide for 48 hours, the inhibition rate against Malassezia furfur reached 100%; after continuous incubation for 72 hours and 96 hours, the inhibition rate remained unchanged at 100%. Similarly, the addition of 20 μg / mL of the antimicrobial peptide also resulted in a 100% inhibition rate after incubation for 48 hours, 72 hours, and 96 hours, thus demonstrating that the antimicrobial peptide P19 described in this invention has an inhibitory effect on Malassezia furfur.
[0112] The antibacterial results of limiting Malassezia are as follows: Figure 2A and Figure 2B As shown, after incubation with 10 μg / mL of the antimicrobial peptide for 48 hours, the inhibition rate against Malassezia restriction bacteria reached 60.73%; after continuous incubation for 72 and 96 hours, the inhibition rate remained unchanged at 60.73%. Similarly, after incubation with 20 g / mL of the antimicrobial peptide for 48, 72, and 96 hours, an inhibition rate of 80.82% was obtained, indicating that the antimicrobial peptide P19 also has an inhibitory effect on Malassezia restriction bacteria.
[0113] This invention designs and synthesizes an antimicrobial peptide for inhibiting Malassezia, exhibiting good skin safety. It can be used to treat skin diseases caused by Malassezia fungal infections, such as dandruff, seborrheic dermatitis, tinea versicolor, and Malassezia folliculitis. It can be applied in cosmetics or topical medicines for treating dandruff or seborrheic dermatitis, tinea versicolor, and Malassezia folliculitis. The antimicrobial peptide described in this invention has significant inhibitory effects on both Malassezia furfur and Malassezia spp., demonstrating significant application value.
[0114] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. An antimicrobial peptide for inhibiting Malassezia, characterized in that, The antimicrobial peptide is P19, and the amino acid sequence of the antimicrobial peptide P19 is shown in SEQ ID NO.
1.
2. A method for preparing the antimicrobial peptide for inhibiting Malassezia as described in claim 1, characterized in that, The preparation method is a solid-phase synthesis method.
3. The use of the antimicrobial peptide according to claim 1 in the preparation of cosmetics or topical medicines for the prevention or treatment of skin diseases caused by Malassezia fungal infections.
4. The application according to claim 3, characterized in that, The skin conditions mentioned include dandruff, seborrheic dermatitis, tinea versicolor, and Malassezia folliculitis caused by Malassezia fungal infections.
Citation Information
Patent Citations
Antibacterial peptide for maintaining scalp flora health and preparation method and application thereof
CN120058849A