Use of an antibacterial peptide having antifungal activity

Abstract

The application relates to the technical field of antibacterial peptides, in particular to application of an antibacterial peptide with antifungal activity, and discloses antifungal activity, antibiofilm activity and an action mechanism of Jelleine-Ic on Candida albicans. Jelleine-Ic shows very strong antifungal activity on Candida albicans. Jelleine-Ic destroys the integrity of the cell wall of Candida albicans and enhances the permeability of the cell membrane of Candida albicans. In addition, Jelleine-Ic interacts with DNA when entering the fungal cell, influences the expression of genes involved in DNA replication and repair, and induces the generation of active oxygen in the cell, thereby accelerating the death of the fungal cell. According to the above results, Jelleine-Ic can effectively inhibit the growth of Candida albicans and has the potential for treating fungal infections.

Description

Technical Field

[0001] This invention relates to the field of antimicrobial peptide technology, and more specifically to the application of an antimicrobial peptide with antifungal activity. Background Technology

[0002] Candida albicans is one of the most common fungal pathogens in hospital-acquired infections, with a high incidence in humans. Its mortality rate in systemic fungal-associated infections is close to 50%, posing a serious threat to human health. Currently, treatment for candidiasis is limited to a limited number of antifungal compounds, including azoles, echinocandins, polyenes, and flucytosine. Furthermore, drug resistance is a major challenge for effectively treating these infections. Cases of drug resistance in Candida albicans have emerged in hospital settings and are becoming increasingly common. Therefore, the discovery of novel antimicrobial drugs is an urgent need to combat fungal infections. Antimicrobial peptides (AMPs), with their high activity, low resistance rates, and negligible host toxicity, hold promise as potential antifungal agents.

[0003] AMPs, as important innate immune molecules, are widely distributed in nature, including animals, plants, and microorganisms. AMPs range in length from 12 to 50 amino acids, are typically positively charged, and are amphiphilic. AMPs exhibit broad activity against pathogens such as bacteria, fungi, viruses, and parasites. Furthermore, many AMPs show activity against drug-resistant microorganisms. Some AMPs have been investigated as alternative methods for controlling Candida albicans infection. For example, the antimicrobial peptide C16-FengycinA from Bacillus amyloliquefaciens inhibits Candida albicans growth by disrupting its cell wall and accumulating reactive oxygen species (ROS). The antimicrobial peptide AMP-17 from the housefly *Musca domestica* exerts its anti-Candida albicans effect through ROS-mediated apoptosis and necrosis. OctomininII, the antimicrobial peptide octominin derived from the octopus minor, inhibits Candida albicans growth through multiple mechanisms of action.

[0004] The APD database (https: / / aps.unmc.edu / ) currently contains 890 anti-Candida AMPs, most of which have relatively large molecular weights, with an average length of 28.03 amino acid residues. From a research and development perspective, focusing on the development of short peptides is more efficient in terms of both time and cost. Jelleine-I is a short AMP (PFKLSLHL) found in bee royal jelly, consisting of only 8 amino acids. Previous studies have shown that it exerts its anti-Candida activity by targeting cell membranes and intracellular components. Research shows that Jelleine-Ic is a newly designed antimicrobial peptide derived from Jelleine-I, exhibiting stronger antibacterial activity than Jelleine-I. However, the antifungal activity and mechanism of action of Jelleine-Ic against Candida albicans remain unclear.

[0005] In view of the above-mentioned defects, the inventors of this invention have finally obtained this invention after a long period of research and practice. Summary of the Invention

[0006] The purpose of this invention is to solve the problem of how to apply the antimicrobial peptide Jelleine-Ic to anti-Candida albicans activity, and to provide an application of an antimicrobial peptide with antifungal activity.

[0007] To achieve the above objectives, this invention discloses the application of an antimicrobial peptide with antifungal activity in the preparation of antifungal infection drugs, wherein the antimicrobial peptide is Jelleine-Ic, with the amino acid sequence WWKLLRKL-NH2.

[0008] The fungus in question is Candida albicans.

[0009] The minimum inhibitory concentration (MIC) of the antimicrobial peptide against Candida albicans is 12.5 μg / mL.

[0010] This invention also discloses the application of an antimicrobial peptide with antifungal activity in synergistic use with amphotericin B in the preparation of antifungal infection drugs, wherein the antimicrobial peptide is Jelleine-Ic, with the amino acid sequence WWKLLRKL-NH2; and the fungus is Candida albicans.

[0011] The concentration of the antimicrobial peptide Jelleine-Ic is 0.78-3.13 μg / mL, and the concentration of the amphotericin B is 1.25 μg / mL.

[0012] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention discloses the antifungal activity, antibiofilm activity, and mechanism of action of Jelleine-Ic against Candida albicans. Jelleine-Ic exhibits strong antifungal activity against Candida albicans, with a MIC of 12.5 μg / mL. It effectively inhibits the formation of Candida albicans biofilms and disrupts existing biofilms. When used in combination with amphotericin B, it shows a synergistic effect. At concentrations below 125 μg / mL, it has no hemolytic effect on rabbit red blood cells (rRBCs). Jelleine-Ic impairs the integrity of the cell wall, increasing the permeability of the Candida albicans cell membrane. Furthermore, upon entering the cell, Jelleine-Ic interacts with DNA, affecting the expression of genes involved in DNA replication and repair, and inducing the production of intracellular reactive oxygen species, accelerating fungal cell death. In summary, Jelleine-Ic can effectively inhibit the growth of Candida albicans and has the potential to treat fungal infections. Attached Figure Description

[0013] Figure 1 The antifungal activity kinetics and hemolytic activity of Jelleine-Ic were analyzed, wherein (A) was observed in Candida albicans cells (1.0 × 10⁻⁶). 5 (cfu / mL) was incubated with 1 MIC of Jelleine-Ic or amphotericin B at 30°C. After different incubation periods, the OD of each culture was measured. 600 Control: Sterile water; (B) rRBC suspension incubated with different concentrations of Jelleine-I for 1 h, followed by measurement of the OD of the supernatant. 414 Triton X-100 and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. Different lowercase letters indicate significant differences between groups (P<0.05).

[0014] Figure 2 The inhibitory effects of Jelleine-Ic (A), amphotericin B (B), and their combinations (C) on Candida albicans biofilm formation were investigated. Control: Sterile water. Different lowercase letters indicate significant differences between groups (P<0.05).

[0015] Figure 3 The effects of Jelleine-Ic (A), amphotericin B (B), and their combinations (C) on the disruption of Candida albicans biofilm formation were studied. Control: Sterile water. Different lowercase letters indicate significant differences between groups (P<0.05).

[0016] Figure 4 Jelleine-Ic was used to treat the cell wall and cell membrane of *Candida albicans*. (A) Cell wall integrity of *Candida albicans* was observed after incubation with the peptide for 12 h, followed by staining with 5% crystal violet and observation under a microscope. Control: sterile water. Scale bar: 50 μm; (B) Cell membrane permeability of *Candida albicans* was measured after incubation with Jelleine-Ic. Control: PBS; (C) Cell membrane disruption of *Candida albicans* cells was observed after incubation with the peptide for 30 min, followed by fixation with PI (0.1 μg / mL) and observation under a confocal laser scanning microscope (CLSM). Control: sterile water. Scale bar: 10 μm; (D) Expression levels of ergosterol synthesis (ERG1, ERG5, and ERG11), cell wall synthesis (GSL1 and GSL2), and DNA replication and repair-related genes (PSF1, PSF2, and RAD16) in Candida albicans after 12 h of Jelleine-Ic treatment. Control: sterile water. Different lowercase letters indicate significant differences between groups (P<0.05).

[0017] Figure 5 Scanning electron microscopy (AF) and transmission electron microscopy (GL) images of Candida albicans treated with Jelleine-Ic for 12 h, where (A, B, G, and H) are controls (sterile water); (CF and IL) are Jelleine-Ic. All tests were repeated 3 times.

[0018] Figure 6 Jelleine-Ic enters cells, interacts with DNA, and induces intracellular ROS generation. (A) shows the localization of Jelleine-Ic in *Candida albicans* cells. Cells were stained with FITC-Jelleine-Ic and DAPI, and observed under CLSM (scale bar: 10 μm). (B) shows the DNA-binding activity of Jelleine-Ic. Different concentrations of Jelleine-Ic were incubated with *Candida albicans* genomic DNA. The mixture was electrophoresed and observed. (C) shows the effect of Jelleine-Ic on intracellular ROS. *Candida albicans* cells were incubated with Jelleine-Ic for 12 h, and then ROS levels were measured. Control: sterile water. Different lowercase letters indicate significant differences between groups (P < 0.05). Detailed Implementation

[0019] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0020] The experimental data were analyzed using one-way ANOVA with DPS data processing software. The significance of the data was determined using Duncan's new multiple range test, and a p-value < 0.05 was used for statistical analysis. Graphpadprism 5 was used for plotting.

[0021] Example 1: Test Fungi and Their Culture

[0022] Candida albicans was provided by the China Academy of Chinese Medical Sciences (Beijing, China). Candida albicans cells were cultured in Sabouraud glucose broth (1% tryptone, 2% glucose) at 30°C and 160 rpm.

[0023] Example 2: Peptides and Chemicals

[0024] Jelleine-I (PFKLSLHL) and Jelleine-Ic (WWKLLRKL-NH2) were synthesized by Synpeptide (Shanghai, China) using a solid-phase synthesis method. All chemicals were of analytical grade. Amphotericin B was purchased from Solarbio (Beijing, China).

[0025] Example 3: MIC and MFC determination

[0026] C. albicans glycerol bacteria stored at -80℃ were thawed on ice. 100 μL of the glycerol bacteria was transferred to 2 mL of fresh SDB medium and incubated overnight at 30℃ and 160 rpm. The next day, 100 μL of the overnight C. albicans culture was transferred to 2 mL of fresh SDB medium and incubated at 30℃ and 160 rpm until the logarithmic growth phase. A C. albicans bacterial suspension (concentration 1.0 × 10⁻⁶) was prepared using fresh SDB medium. 5 (cfu / mL) Using a two-fold dilution method, 10 μL of antimicrobial peptides diluted with sterile water at different concentrations were added to each well of a 96-well ELISA plate. Then, 90 μL of *C. albicans* bacterial suspension was added to the wells corresponding to the different concentrations of antimicrobial peptides. Bacterial suspension with sterile water served as a positive control, and SDB medium with sterile water served as a negative control. The plates were incubated at 30°C for 24 hours, and the OD values ​​of the 96-well ELISA plates were measured using a microplate reader. 600 value.

[0027] The minimum fungicidal concentration (MFC) was determined using the same method as the MIC assay. After incubating the 96-well microplates at 30°C for 24 hours, 50 μL of bacterial culture (mixed well before use) was transferred from each well to the corresponding SDA medium and spread evenly using a spreader. After incubating at 30°C for 24 hours, the colonies of *C. albicans* on the SDA plates were counted. The lowest concentration at which no colonies grew was taken as the MFC of the antimicrobial peptide against *C. albicans*.

[0028] Jelleine-Ic exhibited strong antifungal activity against Candida albicans, with a MIC of 12.5 μg / mL, superior to the parent peptide Jelleine-I with a MIC of 25 μg / mL. Amphotericin B had a MIC of 5 μg / mL against Candida albicans. The mean fertilization rates (MFCs) of Jelleine-Ic, Jelleine-I, and amphotericin B against Candida albicans were 25 μg / mL, 50 μg / mL, and 10 μg / mL, respectively.

[0029] Example 4: Dynamic antibacterial activity assay and hemolytic activity assay

[0030] Dynamic antibacterial activity assay: The tested *C. albicans* were cultured to the logarithmic growth phase, and a *C. albicans* bacterial suspension (concentration 1.0 × 10⁻⁶) was prepared using fresh SDB medium. 5 The diluted bacterial culture was incubated with Jelleine-Ic or amphotericin B at a final concentration of 1 MIC. Sterile water was used as a negative control. The mixture was incubated at 30°C and 160 rpm for 24 h. The OD of the culture was measured using a microplate reader. 600 value.

[0031] Hemolytic activity assay: Fresh rabbit red blood cells were centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the red blood cells were washed three times with PBS and resuspended. The cells were incubated with shaking at 37℃ for 1 h, centrifuged at 500 × g for 10 min, and 80 μL of the resulting supernatant was measured at 414 nm. Hemolytic activity was calculated.

[0032] Antifungal activity kinetic assays showed that Jelleine-Ic almost completely killed 1 MIC of Candida albicans within 24 hours. Figure 1 A). Amphotericin B showed similar effects. No significant increase in hemolytic activity of Jelleine-Ic was observed at test concentrations below 125 μg / mL compared to the control group (7.63%). Figure 1B). Even at the highest concentration (10 MIC, 125 μg / mL), Jelleine-Ic only exhibited 10.65% hemolytic activity.

[0033] Example 5. Synergy Detection

[0034] The effect of the combined action of Jelleine-Ic and amphotericin B on the growth of C. albicans was detected. The tested C. albicans was cultured to the logarithmic growth phase, and a C. albicans bacterial suspension (concentration of 1.0×10 5 cfu / mL) was prepared with fresh SDB medium. Using the two-fold dilution method, 5 μL of antibacterial peptides at different concentrations (diluted with sterile water) and 5 μL of amphotericin B at different concentrations were added to a 96-well microplate, and then 90 μL of the bacterial suspension was added to the wells corresponding to the different concentration compound mixtures, and it was placed in an incubator at 30 °C for constant temperature culture for 24 h, and the OD 600 value was detected with an enzyme microplate reader. The positive control was the bacterial solution plus sterile water, and the negative control was SDB medium plus sterile water. Synergistic enhancement was evaluated by the fractional inhibitory concentration index (FICI). The combined effect was defined as follows:

[0035] The FICI values obtained from different combinations of antibacterial compounds were interpreted as synergistic effect (FICI ≤ 0.5), additive effect (0.5 < FICI ≤ 1.0), irrelevant effect (1.0 < FICI ≤ 2.0), and antagonistic effect (FICI > 2.0).

[0036] Interactions of synergistic, additive, and irrelevant effects were observed between Jelleine-Ic and amphotericin B, and the FICI values were between 0.31 - 2. As shown in Table 1, Jelleine-Ic at a concentration of 0.78 - 3.13 μg / mL showed a synergistic effect when used in combination with 1.25 μg / mL of amphotericin B.

[0037] Table 1 Combined action of Jelleine-Ic and amphotericin B on Candida albicans

[0038]

[0039] Example 6. Antibiofilm Activity

[0040] The antibiofilm activities of Jelleine-Ic, amphotericin B, and their combinations were determined. To inhibit biofilm formation, 20 μL of continuously diluted antibacterial agents were introduced into a 96-well microplate. Then 180 μL of logarithmic-phase Candida albicans cells (1.0×10 5The solution (cfu / mL) was incubated at 30°C. Sterile water served as a control. After 24 hours of incubation, the inhibitory effect on biofilm formation was assessed by crystal violet staining.

[0041] To disrupt the pre-formed biofilm, 200 μL of Candida albicans cells (1.0 × 10⁻⁶) were used. 4 CFU / mL was added to 96-well microplates and incubated at 30°C for 24 h to form a biofilm. The biofilm was washed with PBS and treated with different concentrations of antibacterial agents. Sterile water was used as a control. The plates were incubated again at 30°C for 24 h. The pre-formed biofilm disruption effect was quantified by crystal violet staining.

[0042] Jelleine-Ic and amphotericin B were found to possess excellent biofilm inhibitory activity. At 1 MIC, Jelleine-Ic and amphotericin B almost completely inhibited biofilm formation. Figure 2 A and 2B). When used in combination with 0.25 MIC of amphotericin B, all tested concentrations of Jelleine-Ic inhibited >90% of biofilm formation. Figure 2 C).

[0043] Jelleine-Ic can also disrupt existing biofilms. After treatment with 1 MIC of Jelleine-Ic and amphotericin B for 24 hours, the biofilm percentage decreased from 100% to 41.90% and 26.93%, respectively. Figure 3 A and 3B). When bound to amphotericin B at 0.25 MIC, Jelleine-Ic reduced biofilm to 24.71% at 0.5 MIC. Figure 3 C).

[0044] Example 7: Cell Wall Integrity

[0045] Take C. albicans in the logarithmic growth phase and prepare a C. albicans bacterial suspension (concentration 2.0 × 10⁻⁶) using fresh SDB medium. 7 C. albicans bacterial suspensions were treated with Jelleine-Ic and amphotericin B at a final concentration of 2 MIC (cfu / mL), with sterile water treatment serving as a control. Each group was incubated at 30℃ for 10 h, then centrifuged at 3000 rpm for 10 min to collect C. albicans cells, and washed three times with PBS. 5% crystal violet was added for staining for 10 min, followed by PBS resuscitation and centrifugation until colorless. 5 μL of the bacterial suspension was precipitated onto a glass slide, fixed with 10% tannic acid, covered with a coverslip, and observed under an inverted microscope. One hundred cells were randomly selected for observation, and the cell wall integrity rate of C. albicans was calculated. Each experiment was repeated three times.

[0046] like Figure 4 As shown in Figure A, the control group (Control) Candida albicans cells were round or oval, with their intact cell walls stained light purple or remaining colorless, and the cell wall integrity rate was 92.83%. In contrast, the cell walls and cytoplasm of the Jelleine-Ic-treated Candida albicans cells were both dark purple, and the cell integrity rate was significantly reduced to 57.19%, which was significantly lower than that of the control group.

[0047] Example 8: Cell Membrane Permeability

[0048] C. albicans was removed from a -80°C cryogenic freezer and added to fresh SDB medium. It was then incubated overnight on a shaker at 30°C and 160 rpm. After centrifugation at 3000 rpm for 10 min, the precipitate was collected and resuspended in PBS to a final concentration of 2.0 × 10⁷ CFU / mL. The C. albicans suspension was treated with Jelleine-Ic and amphotericin B at a final concentration of 1 MIC, respectively. Treatment of the C. albicans suspension with 10% Triton X-100 served as a positive control, and PBS served as a negative control. The OD of the filtrate was measured using a UV spectrophotometer. 260 The value was calculated, and the cell membrane permeability was determined.

[0049] Treatment with Jelleine-Ic rapidly enhanced the membrane permeability of Candida albicans cells. Figure 4 B). Following Jelleine-Ic treatment, cell membrane permeability increased from 5.45% to 15.11% at 0.5 h and further to 27.37% at 12 h. This increase was significantly greater than that observed in the control group. These findings suggest that Jelleine-Ic specifically targets and permeates fungal cell membranes.

[0050] Example 9: Propidine Iodide Uptake Assay

[0051] The extent of cell membrane disruption in *Candida albicans* was assessed by measuring propidium iodide (PI) uptake. *Candida albicans* (2.0 × 10⁻⁶) 7 Cells were cultured at 30°C with different concentrations of Jelleine-Ic (cfu / mL), with sterile water as a control. After incubation for 30 min, the cells were stored in a light-protected environment with PI (0.1 μg / mL). Cells were then observed using a Carl Zeiss LSM800 confocal laser scanning microscope (CLSM).

[0052] Further assessment of Candida albicans cell membrane disruption was conducted via PI uptake. Figure 4C). Control Candida albicans cells did not show red fluorescence. In contrast, fungal cells treated with Jelleine-Ic exhibited red fluorescence of PI. Furthermore, the number of red fluorescent cells increased with increasing peptide concentration, indicating that Jelleine-Ic enhanced cell membrane disruption in Candida albicans.

[0053] Jelleine-Ic treatment affected the expression levels of genes associated with ergosterol (ERG1, ERG5, and ERG11) and cell wall (GSL1 and GSL2) synthesis. Figure 4 D). In Candida albicans treated with Jelleine-Ic, the expression levels of ERG1, ERG5, and ERG11 were significantly increased by 6.89-fold, 5.91-fold, and 11.22-fold, respectively, compared with the control. Furthermore, Jelleine-Ic treatment led to a significant upregulation of GSL1 and GSL2 expression by 8.27-fold and 23-fold, respectively, compared with the control group (52-fold, respectively).

[0054] Example 10: Scanning Electron Microscopy and Transmission Electron Microscopy

[0055] Fungal cells (2.0 × 10⁻⁶) 7 Cells were cultured at 30°C with cfu / mL and Jelleine-Ic (1 MIC) as a control, with sterile water as a control. After 12 h of incubation, cell samples for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were prepared according to a previously established method (K. Ding, P. Shen, Z. Xie, L. Wang, X. Dang, In vitro and in vivo antifungal activity of two peptides with the same composition and different distribution, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 252(2022)109243). The samples were examined under a Hitachi SU8100 scanning electron microscope and a Hitachi HT-7700 transmission electron microscope, respectively.

[0056] The surface morphological changes of *C. albicans* after 10 h of Jelleine-Ic treatment were analyzed using scanning electron microscopy (SEM). The images show that the control group *C. albicans* cells were round or oval, plump, and had smooth cell surfaces. Figure 5(A and 5B). In contrast, treatment with Jelleine-Ic for 12 hours resulted in significant damage and morphological changes (A and 5B). Figure 5 C), for example, cell surface depressions ( Figure 5 D) Perforation ( Figure 5 E) and deformation ( Figure 5 F).

[0057] The changes in the internal morphology and structure of *C. albicans* cells after treatment with Jelleine-Ic were analyzed using transmission electron microscopy (TEM). In the control group, *C. albicans* cells were round or oval in shape and had intact cell structure. Figure 5 G and 5H). On the other hand, Jelleine-Ic-treated cells showed many ultrastructural changes (G and 5H). Figure 5 I), including deformation of extracellular structures ( Figure 5 The red arrow in J), loosely arranged cell walls ( Figure 5 (blue arrows in K and 5L), electron density inhomogeneity, and intracellular vacuolar formation ( Figure 5 (Red arrows in K and 5L).

[0058] Example 11: Fluorescence Microscopy

[0059] To assess the localization of peptides in Candida albicans cells. Fungal cells (2.0 × 10⁻⁶) 7 Cells were cultured with fluorescein isothiocyanate (FITC)-Jelleine-Ic (1 MIC) at 30 °C for 1 h, with sterile water as a control. Cells were then harvested and washed with PBS. Subsequently, they were treated with 1 μg / mL of 4',6-diamidinyl-2-phenylindole (DAPI) and examined under a Carl Zeiss LSM800 CLSM.

[0060] Green fluorescence was observed in Candida albicans cells exposed to FITC-Jelleine-Ic compared to control cells. Figure 6 A) indicates that Jelleine-Ic successfully enters the cell by penetrating the cell wall and cell membrane. When the cells were stained with both dyes simultaneously, co-localization of FITC-Jelleine-Ic and DAPI was observed, indicating that the peptide interacts with fungal DNA.

[0061] Example 12: Measurement of Electrophoretic Mobility Changes

[0062] Genomic DNA was isolated from Candida albicans cells. 40 ng of genomic DNA was incubated with different concentrations of Jelleine-Ic (15.63–1000 μg / mL) at 25 °C. Sterile water was used as a control. After 1.5 hours of incubation, the DNA-binding activity of Jelleine-Ic was assessed by evaluating DNA migration on a 0.8% agarose gel.

[0063] The results showed that 125 μg / mL of Jelleine-Ic almost completely inhibited the migration of Candida albicans genomic DNA. Figure 6 B). This finding indicates that Jelleine-Ic interacts with the genomic DNA of Candida albicans. Furthermore, compared to the control, the expression levels of the DNA replication and repair genes PSF1, PSF2, and RAD16 in Candida albicans treated with Jelleine-Ic were 2.13-fold, 2.82-fold, and 1.56-fold higher, respectively. Figure 4 D), which indicates that Jelleine-Ic disrupts DNA structure and affects its function.

[0064] Example 13: RT-qPCR

[0065] Candida albicans cells (2.0 × 10⁻⁶) 7 Cells were cultured with CFU / mL and Jelleine-Ic (1 MIC) at 30°C. After 12 h of incubation, cells were harvested and total RNA was extracted. Sterile water served as a control. The expression levels of Candida albicans cell wall synthesis genes (GSL1 and GSL2), ergosterol synthesis genes (ERG1, ERG5, and ERG11), and genes involved in DNA replication and repair (PSF1, PSF2, and RAD16) were analyzed. The 18S gene was used as an internal reference gene. Primer sequences are listed in Table 2.

[0066] Table 2 Primer sequences

[0067]

[0068] Example 14: Detection of Intracellular ROS in Candida albicans

[0069] Logarithmic growth phase *C. albicans* were collected, and a *C. albicans* bacterial suspension with a concentration of 2.0 × 10⁷ cfu / mL was prepared using fresh SDB medium. The suspension was then treated with Jelleine-Ic and amphotericin B at final concentrations of 1 / 2 MIC, 1 MIC, and 2 MIC, respectively. Sterile water served as a control. After incubation at 30℃ for 12 h, the cells were collected by centrifugation at 3500 rpm for 10 min, washed twice with extracellular fluid, and resuspended in PBS to a bacterial concentration of 2.0 × 10⁷ cfu / mL. 7Add 100 μL of 2',7'-dichlorofluorescein diacetate (DCFH-DA) solution to a final concentration of 10 μM, cfu / mL, and incubate at 30°C in the dark for 30 min. Take 200 μL of bacterial culture from each group and place it in a black plate of a 96-well plate. Detect the fluorescence intensity of 2',7'-dichlorofluorescein (DCF) (excitation / emission wavelength: 488 / 525 nm) using a multi-functional microplate reader.

[0070] Detection revealed that Jelleine-Ic enhanced the fluorescence intensity of DCF. Figure 6 C). Compared with control cells, cells exposed to 2MIC, 1MIC, and 0.5MIC peptides showed fluorescence intensities of 5.30, 3.43, and 1.71 times, respectively, indicating that Jelleine-Ic promotes intracellular ROS production.

[0071] Example 15: Effect of ROS scavengers on peptide activity

[0072] A solution of 10 mM glutathione (GSH), N-acetylcysteine ​​(NAC), and vitamin C (VC) was pre-packed into 96-well microplates, followed by the addition of fungal cells at a concentration of 1.0 × 10⁵ CFU / mL, and incubated at 30 °C for 30 min. Then, peptide dilution buffer (6.25 to 400 μg / mL) was added, and the plates were incubated at 30 °C for another 24 h. After incubation, the MIC values ​​for each group were assessed. Sterile water served as a control.

[0073] The results showed that the presence of ROS scavengers significantly reduced peptide activity. The MIC value increased from 12.5 μg / mL to 50 μg / mL after the addition of 10 mMVC, GSH, or NAC, suggesting that the antifungal activity of Jelleine-Ic may be mediated by inducing ROS accumulation.

[0074] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. The application of Jelleine-Ic in the preparation of antifungal infection drugs, characterized in that, The amino acid sequence of Jelleine-Ic is WWKLLRKL-NH2, and the fungus is Candida albicans.

2. The application of Jelleine-Ic combined with amphotericin B in the preparation of antifungal drugs, characterized in that, The amino acid sequence of Jelleine-Ic is WWKLLRKL-NH2, and the fungus is Candida albicans.

3. The application of Jelleine-Ic combined with amphotericin B as described in claim 2 in the preparation of antifungal drugs, characterized in that, The concentration of Jelleine-Ic is 0.78-3.13 μg / mL, and the concentration of amphotericin B is 1.25 μg / mL.

Images