Application of dehydrogenated epicatechin gallate combined with fluconazole in preparation of antifungal drugs

Abstract

This invention relates to the field of pharmaceutical technology, specifically to the application of dehydronuciolin and fluconazole in the preparation of antifungal drugs. The in vitro synergistic effect of the combination of dehydronuciolin and fluconazole was determined using a checkerboard assay, showing a fractional inhibitory concentration (FIC) ≤0.5, confirming a strong synergistic effect. The in vivo efficacy of this drug combination was verified using a large wax moth infection model. Phenotypic experiments explored the synergistic mechanism, showing that the combination of dehydronuciolin and fluconazole significantly inhibited the hyphal growth, biofilm formation, and adhesion to host cells of drug-resistant Candida albicans. Transcriptome sequencing analysis confirmed these findings, showing that treatment with the combination of dehydronuciolin and fluconazole downregulated hyphae-specific genes, GPI-anchored cell wall proteins, and the ergosterol biosynthesis pathway, collectively weakening the virulence, surface integrity, and host recognition ability of Candida albicans, thereby reversing its drug resistance.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of dehydronusine combined with fluconazole in the preparation of antifungal drugs. Background Technology

[0002] Clinically, the majority of fungal strains isolated from blood, sputum, or urine samples from patients with fungal infections are Candida, with Candida albicans accounting for over 40%. Therefore, the prevention and treatment of Candida albicans infections, especially invasive Candida albicans infections, is crucial in clinical practice. Currently, the number of effective antifungal drugs available clinically is very limited compared to other antibacterial or antiviral drugs, and long-term use of antifungal drugs (such as azoles) can easily lead to fungal resistance. As eukaryotes, fungi share many similarities with mammalian cells, making antifungal drugs prone to toxic side effects in humans. This has slowed the progress of antifungal drug research, highlighting the urgent clinical need to develop new and effective drugs against drug-resistant fungi. Summary of the Invention

[0003] To overcome the above problems, the present invention provides the application of dehydronusine combined with fluconazole in the preparation of antifungal drugs.

[0004] To achieve the above technical objectives, the present invention adopts the following technical solution: A first aspect of the invention provides the use of dehydronusine in combination with fluconazole in the preparation of an antifungal drug; wherein the fungus is a fluconazole-resistant Candida albicans.

[0005] In one or more embodiments, the fungus includes drug-resistant Candida albicans CA10, drug-resistant Candida albicans CA16, drug-resistant Candida albicans CA103, drug-resistant Candida albicans CA137, drug-resistant Candida albicans CA632 and drug-resistant Candida albicans CA20003.

[0006] In one or more embodiments, the mass ratio of dehydronusine to fluconazole is (0.25~2):(0.25~8).

[0007] A second aspect of the invention provides the use of dehydronusine in the preparation of a medicament for reducing fluconazole resistance.

[0008] In one or more embodiments, the mass ratio of dehydronusine to fluconazole is (0.25~2):(0.25~8).

[0009] A third aspect of the invention provides the use of dehydronusine in the preparation of a medicament that enhances the antifungal effect of fluconazole, wherein the fungus is a fluconazole-resistant Candida albicans.

[0010] In one or more embodiments, the fungus includes drug-resistant Candida albicans CA10, drug-resistant Candida albicans CA16, drug-resistant Candida albicans CA103, drug-resistant Candida albicans CA137, drug-resistant Candida albicans CA632 and drug-resistant Candida albicans CA20003.

[0011] In one or more embodiments, the mass ratio of dehydronusine to fluconazole is (0.25~2):(0.25~8).

[0012] In a fourth aspect, the present invention provides an antifungal drug comprising dehydronusine and fluconazole; wherein the fungus is a fluconazole-resistant Candida albicans.

[0013] In one or more embodiments, the fungus includes drug-resistant Candida albicans CA10, drug-resistant Candida albicans CA16, drug-resistant Candida albicans CA103, drug-resistant Candida albicans CA137, drug-resistant Candida albicans CA632 and drug-resistant Candida albicans CA20003.

[0014] In one or more embodiments, the mass ratio of dehydronusine to fluconazole is (0.25~2):(0.25~8).

[0015] In one or more embodiments, the drug further includes pharmaceutically acceptable carriers, excipients, and diluents.

[0016] The non-pharmaceutical active ingredients that may be included, such as carriers, excipients, and diluents, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0017] Preferably, the carrier, excipients, and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0018] Preferably, the dosage form of the drug is a suspension, emulsion, granules, spray, injection, transdermal absorbent, a dosage form suitable for transfection, tablet, powder, granules or capsule.

[0019] A fifth aspect of the present invention provides a pharmaceutical composition comprising the antibacterial agent described in the fourth aspect.

[0020] The beneficial effects of this invention are as follows: In this invention, the in vitro synergistic effect of dehydronuciferine and fluconazole was determined using a checkerboard assay. The results showed that the fractional inhibitory concentration index (FICI) was ≤0.5, confirming the synergistic effect of the two drugs. Subsequently, the in vivo efficacy of this drug combination was verified using a large wax moth infection model. Furthermore, phenotypic experiments explored the synergistic mechanism, showing that the combination of dehydronuciferine and fluconazole significantly inhibited the hyphal growth, biofilm formation, and adhesion to host cells of drug-resistant Candida albicans. Transcriptome sequencing (RNA-seq) analysis further confirmed the above findings, showing that treatment with the combination of dehydronuciferine and fluconazole downregulated hyphae-specific genes, GPI-anchored cell wall proteins, and the ergosterol biosynthesis pathway, collectively weakening the virulence, surface integrity, and host recognition ability of Candida albicans, thereby reversing its drug resistance. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 Figure 1 shows the results of an agar disc diffusion experiment for the combined use of dehydronusin and fluconazole against drug-resistant Candida albicans CA10. Figure 2 Time-survival curves for the combined treatment of drug-resistant Candida albicans CA10 infection with giant wax moth using dehydronusin and fluconazole; Figure 3 Periodic acid-Schiff (PAS) staining image of dehydronusin and fluconazole in combination for the treatment of drug-resistant Candida albicans CA10 infection of the large wax moth; Figure 4 The image shows the results of using dehydronusin and fluconazole in combination to inhibit the growth of drug-resistant Candida albicans CA10 hyphae. Figure 5 Figure showing the results of the combined use of dehydronusin and fluconazole to inhibit biofilm formation; Figure 6 The combined use of dehydronusine and fluconazole inhibited the adhesion of drug-resistant Candida albicans CA10 to bronchial epithelial cells; where a is a fluorescence microscopy image, b is the average fluorescence intensity, and c is the fungal adhesion area ratio; in b and c, * indicates p<0.05, and *** indicates p<0.001. Figure 7 Venn diagram of gene number; Figure 8 Heatmap of the main upregulated and downregulated genes in the dehydronuciferine and fluconazole combination group; Figure 9 A schematic diagram illustrating the mechanism of action of dehydronusine and fluconazole in synergistic inhibition of drug-resistant Candida albicans. Detailed Implementation

[0023] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0025] Currently, the number of effective antifungal drugs available clinically is very limited compared to other antibacterial or antiviral drugs. Fluconazole is widely used clinically due to its effectiveness against Candida albicans, its relatively low price, and its low toxicity. However, with the increased clinical use of fluconazole, Candida albicans resistance to it is constantly increasing. Strategies for addressing fungal resistance can be summarized into two types: one is to avoid the development of fungal resistance by rationally using existing antifungal drugs, and the other is to strengthen the research and development of new antifungal agents. However, developing a new drug with a completely novel chemical structure and antifungal activity is extremely time-consuming and costly, while the rate of increase in fungal resistance is accelerating. The speed at which new drugs are launched cannot keep up with the rate of increase in fungal resistance. Therefore, combination therapy targeting fluconazole resistance pathways has become a current hot topic in antifungal resistance research.

[0026] In this invention, the in vitro synergistic effect of dehydronuciferine and fluconazole was determined using a checkerboard assay. The results showed that the fractional inhibitory concentration index (FICI) was ≤0.5, confirming the synergistic effect of the two drugs. Subsequently, the in vivo efficacy of this drug combination was verified using a large wax moth infection model. Furthermore, phenotypic experiments explored the synergistic mechanism, showing that the combination of dehydronuciferine and fluconazole significantly inhibited the hyphal growth, biofilm formation, and adhesion to host cells of drug-resistant Candida albicans. Transcriptome sequencing (RNA-seq) analysis further confirmed the above findings, showing that treatment with the combination of dehydronuciferine and fluconazole downregulated hyphae-specific genes, GPI-anchored cell wall proteins, and the ergosterol biosynthesis pathway, collectively weakening the virulence, surface integrity, and host recognition ability of Candida albicans, thereby reversing its drug resistance.

[0027] Terminology Explanation: YPD: Yeast extract-peptone-glucose.

[0028] The structural formula of the dehydrolotus leaf alkaloid described in this invention is shown below: ; The structural formula of fluconazole is shown below: .

[0029] The drug-resistant Candida albicans CA10, CA16, CA103, CA137, CA632, and CA20003 strains used in this invention were all derived from the First Affiliated Hospital of Shandong First Medical University (Jinan, Shandong Province). All strains were cultured on YPD agar. Before each experiment, fungal cells were cultured overnight at 35°C with shaking (200 rpm) in YPD liquid medium. RPMI 1640 liquid medium was used in subsequent experiments.

[0030] Dehydronusine was prepared into a stock solution with a concentration of 5120 μg / mL using sterile distilled water; fluconazole was prepared into a stock solution with a concentration of 2560 μg / mL using sterile distilled water; both were stored at -20℃.

[0031] The weight of the wax moth larvae used in the experiment was 0.25±0.03 g.

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0033] Example 1 Referring to the Clinical and Laboratory Standards Institute (CLSI) standards for broth dilution antifungal susceptibility testing of yeast (CLSI M27-Ed4), the checkerboard method was used to evaluate the antifungal activity of dehydronuciferine in combination with fluconazole in suspension.

[0034] The drug solutions were diluted with RPMI 1640 liquid medium to create a serial dilution gradient, with the final concentrations of dehydronuciferine ranging from 32 to 0.5 μg / mL and fluconazole from 128 to 0.25 μg / mL. 50 μL of fluconazole solution was pipetted into columns 2-11 of a 96-well plate in ascending order of concentration, and 50 μL of dehydronuciferine solution was pipetted into rows G-A of the same plate in ascending order of concentration. Except for column 12, 100 μL of fungal cell culture (concentration 2 × 10⁻⁶) was added to each well. 3 (cells / mL), and the remaining wells with less than 200 μL were supplemented with RPMI 1640 liquid medium. H1 is the growth control, containing only bacterial culture and no drug, and column 12 is the blank control, containing only RPMI 1640 liquid medium.

[0035] After static culture in an incubator at 35 °C for 24 h, the growth of fungal cells was then observed. The minimal inhibitory concentration (MIC) was defined as the drug concentration that could inhibit more than 90% of the bacteria.

[0036] Evaluation of synergistic effect: The results of the combined drug sensitivity test were used to judge the interaction after the combination of two drugs by the fractional inhibitory concentration index (FICI). The calculation formula of FICI is as follows: FICI = FIC A + FIC B = C A / MIC A + C B / MIC B ; Among them, FICI is the fractional inhibitory concentration index, FIC A and FIC B respectively represent the MIC value of the drug when drugs A and B are combined divided by the MIC value of the drug when drugs A and B are used alone. MIC A and MIC B are the minimum inhibitory concentrations of drugs A and B when used alone respectively. C A and C B are the respective concentrations of the two drugs when the same drug efficacy is achieved when the two drugs are combined. FICI ≤ 0.5 indicates that the combination of the two drugs has a synergistic effect; 0.5 < FICI ≤ 1 indicates that the combination of the two drugs has an additive effect; 1 < FICI ≤ 2 indicates that the combination of the two drugs has no interaction; FICI > 2 indicates that the combination of the two drugs has an antagonistic effect.

[0037] The results of the in vitro static antifungal activity of the combination of dehydro-nuciferine and fluconazole are shown in Table 1.

[0038] Table 1 Antifungal effect of the combination of dehydro-nuciferine and fluconazole (in vitro static)

[0039] In Table 1, drug A is fluconazole and drug B is dehydro-nuciferine. MIC A is the minimum inhibitory concentration of fluconazole when used alone, MIC B is the minimum inhibitory concentration of dehydro-nuciferine when used alone, C A is the minimum inhibitory concentration of fluconazole when fluconazole and dehydro-nuciferine are combined, C BFICI represents the minimum inhibitory concentration of dehydronucidium when fluconazole and dehydronucidium are used in combination.

[0040] As can be seen from Table 1, dehydronuciferine has limited antifungal activity when used alone, but it exhibits a synergistic effect when used in combination with fluconazole (dehydronuciferine 0.25~2 μg / mL, fluconazole 0.25~8 μg / mL), with a fractionated inhibitory concentration index (FICI) ranging from 0.063 to 0.265.

[0041] Example 2 Freshly cultured, overnight-old, drug-resistant Candida albicans CA10 was collected by centrifugation and resuspended in phosphate-buffered saline (PBS) solution to a final volume of 1.0 × 10⁻⁶. 6 Cells / mL, 200 μL of bacterial suspension was spread onto YPD agar plates. To assess antifungal activity, cellulose paper discs impregnated with 0.5 μg / mL fluconazole, 4 μg / mL dehydronucidin, and simultaneously impregnated with 0.5 μg / mL fluconazole and 4 μg / mL dehydronucidin were placed on YPD agar plates. After incubation at 35°C for 48 h, the plates were photographed.

[0042] The results are as follows Figure 1 As shown, the results indicate that dehydronuciferine or fluconazole alone produced only weak inhibition zones, while the combination of dehydronuciferine and fluconazole produced significantly larger inhibition zones against drug-resistant Candida albicans CA10.

[0043] The in vitro experiments in Examples 1 and 2 confirmed that the combination of dehydronusine and fluconazole has a clear synergistic antibacterial effect against drug-resistant Candida albicans, laying the foundation for subsequent in vivo and mechanism studies.

[0044] Example 3 The in vivo efficacy of dehydronusine combined with fluconazole was evaluated using a large wax moth infection model. Before the experiment, larvae weighing 0.25 ± 0.03 g were stored in sawdust at 25℃, protected from light. After culturing the drug-resistant Candida albicans CA10 on YPD agar medium for 24 h, they were then injected with 5 × 10⁻⁶... 6Larvae were inoculated with a dose of 1 cell / larva. 2.5 h after infection, four groups were established: fluconazole monotherapy group, dehydronucidium monotherapy group, dehydronucidium and fluconazole combination group, and a control group. Fifteen larvae were randomly selected from each group. The fluconazole monotherapy group received fluconazole (final in vivo concentration: 0.5 μg / larva), the dehydronucidium monotherapy group received dehydronucidium (final in vivo concentration: 4 μg / larva), and the dehydronucidium and fluconazole combination group received both fluconazole (final in vivo concentration: 0.5 μg / larva) and dehydronucidium (final in vivo concentration: 4 μg / larva). The control group received the same volume of PBS solution. Larvae were placed in culture dishes and incubated at 35°C. Survival was monitored daily. Larvae were considered dead if they did not react to touch with metal tweezers. All experiments were repeated three times.

[0045] Two days after infection and treatment of the large wax moth, one larva was randomly selected from each group, and 8 μm frozen tissue sections were prepared using a cryostat. The sections were then stained with PAS, and the histopathological differences of the large wax moth sections in each group were observed under a microscope.

[0046] Figure 2 The time-survival curves for the combined treatment of dehydronusine and fluconazole for drug-resistant Candida albicans CA10 infection of the large wax moth were obtained from... Figure 2 As can be seen, compared with the control group, neither fluconazole nor dehydronucidium alone significantly improved the survival rate, while the combination of dehydronucidium and fluconazole showed a significantly higher survival rate, especially on the 4th and 5th days after treatment.

[0047] Figure 3 PAS staining images of dehydronusine and fluconazole used in combination to treat drug-resistant Candida albicans CA10 infection of the large wax moth. Figure 3 It can be seen that the fungal load in the group treated with dehydronusine and fluconazole was significantly lower than that in other groups.

[0048] In vivo experiments further verified the synergistic efficacy of dehydronucidin and fluconazole, which not only significantly improved the survival rate of Candida albicans CA10 infected with giant wax moth, but also effectively reduced the fungal load in vivo, indicating that the combination of dehydronucidin and fluconazole also has anti-infective effects in vivo.

[0049] Example 4 The synergistic mechanism of action of dehydronusine and fluconazole in combination: 4.1 Mycelial Growth Experiment Overnight cultured drug-resistant Candida albicans CA10 cells were diluted to 2 × 10⁻⁶ using RPMI 1640 liquid medium. 5Cells / mL: 200 μL of drug-resistant Candida albicans CA10 cell suspension was added to 96-well microplates. Four treatment groups were established: fluconazole monotherapy group, dehydronuciferine monotherapy group, dehydronuciferine and fluconazole combination group, and a control group. Fluconazole monotherapy group received fluconazole (0.5 μg / mL), dehydronuciferine monotherapy group received dehydronuciferine (4 μg / mL), and dehydronuciferine and fluconazole combination group received both fluconazole (0.5 μg / mL) and dehydronuciferine (4 μg / mL). The control group received no drugs. The microplates were incubated at 37°C. Morphological changes of the drug-resistant Candida albicans CA10 cells were observed and recorded under a microscope at preset time points (0, 3, 6, 9, and 24 h).

[0050] The results are as follows Figure 4 As shown, from Figure 4 The study showed that the control group developed dense, long, and interwoven hyphae, while the group treated with the combination of dehydronucidin and fluconazole showed a significantly reduced number and shorter hyphae compared to either single-drug group, indicating a stronger inhibitory effect. These results demonstrate that the combination of dehydronucidin and fluconazole synergistically inhibits hyphal formation in drug-resistant Candida albicans CA10, a crucial ability for host invasion and infection establishment. This finding reveals the potential mechanism by which the combination of dehydronucidin and fluconazole weakens pathogen virulence.

[0051] 4.2 Biofilm formation experiment Biofilm inhibition is a key virulence factor and resistance mechanism that confers high levels of antifungal drug resistance, and it was assessed by crystal violet staining.

[0052] Overnight cultured drug-resistant Candida albicans CA10 cells were diluted to 1×10⁻⁶ using RPMI 1640 liquid medium. 6Cells / mL were collected, and 100 μL of cell suspension was added to each well of a 96-well plate. The plates were incubated at 37°C for 24 h to allow for the formation of a stable biofilm. The culture medium was discarded. Then, a two-fold serial dilution of the drug (dehydronuciferine concentration 0.25–4 μg / mL; fluconazole concentration 0.125–1 μg / mL) was prepared in RPMI 1640 and added to each well containing the biofilm, 50 μL per well. Wells with less than 100 μL were supplemented with RPMI 1640 liquid medium. The 96-well plates were incubated again at 37°C for 24 h. Biofilms without the drug served as a control group. After incubation, the supernatant was carefully removed, and the adherent cells were washed three times with PBS. For crystal violet staining, cells were first fixed in 100 μL of methanol for 15 min in each well, then the methanol was discarded. The plates were air-dried at room temperature for 30 min. Next, 50 μL of 1% crystal violet solution was added to each well, and staining was performed at room temperature for 5 min. After removing the staining solution, wash thoroughly with PBS to remove unbound dye. Invert the plate onto absorbent paper to remove residual liquid and dry in a 37°C oven for 2–3 h or at room temperature overnight. Once completely dry, add 100 μL of 30% glacial acetic acid to each well to dissolve the bound dye. Cover the plate and incubate at 37°C for 30 min to ensure complete dissolution. Finally, measure the optical density (OD) of each well at 570 nm using a microplate reader.

[0053] The results are as follows Figure 5 As shown, the results indicate that the combination of dehydronucidin and fluconazole reduces optical density in a dose-dependent manner and significantly inhibits biofilm formation compared to either drug alone. The combination of dehydronucidin and fluconazole synergistically inhibits the formation of drug-resistant Candida albicans biofilms, which are a major cause of persistent infections and drug resistance that are difficult to eradicate clinically. This result suggests that the combination of dehydronucidin and fluconazole holds promise for overcoming biofilm-related refractory infections.

[0054] 4.3 Bronchial Epithelial Cell Adhesion Assay Adhesion to host epithelial cells is a crucial initial step in colonization and infection, and was assessed using human bronchial epithelial cells (16HBE). A standard in vitro adhesion assay was performed using human bronchial epithelial cells (16HBE cells). After culturing human bronchial epithelial cells to a confluent monolayer in 24-well plates, the cells were infected with drug-resistant Candida albicans CA10 in RPMI 1640 liquid medium at a multiplicity of infection (MOI) ratio of 1:10. Infected cells were randomly assigned to four groups: fluconazole monotherapy, dehydronuciferine monotherapy, dehydronuciferine and fluconazole combination therapy, and a control group. The fluconazole monotherapy group received fluconazole (0.5 μg / mL), the dehydronuciferine monotherapy group received dehydronuciferine (4 μg / mL), the dehydronuciferine and fluconazole combination therapy group received both fluconazole (0.5 μg / mL) and dehydronuciferine (4 μg / mL), and the control group received no drugs. After incubation at 37°C for 2 h, unadhered CA10 cells were washed with PBS to remove them. Adhered fungal cells were fixed with 4% paraformaldehyde and stained with Calcofluor White (CFW, 10 μg / mL) to visualize the fungal cell walls. Fluorescence images were acquired using a fluorescence microscope (Nikon Eclipse Ti2) under a 40x objective lens. Adhesion was quantitatively analyzed by measuring the mean fluorescence intensity (MFI) of three replicates in each group and the ratio of fungal adhesion area (CFW-positive area of ​​fungi in the experimental group / CFW-positive area of ​​fungi in the control group).

[0055] The results are as follows Figure 6 As shown, the results indicated that the control group exhibited dense and extensive fungal adhesion, while fluconazole and dehydronuciferine monotherapy groups produced only slight inhibitory effects. However, the combination of dehydronuciferine and fluconazole significantly and markedly inhibited adhesion. Quantitative analysis confirmed ( Figure 6 In groups b and c), the mean fluorescence intensity and fungal adhesion area ratio of the dehydronuciferine and fluconazole combination group were significantly lower than those of the control group, the fluconazole monotherapy group, and the dehydronuciferine monotherapy group, showing a strong synergistic effect in blocking adhesion.

[0056] The combined use of dehydronucidin and fluconazole can synergistically inhibit the adhesion ability of drug-resistant Candida albicans to host epithelial cells. Adhesion is the initiation step of infection. This finding indicates that the combined use of dehydronucidin and fluconazole can effectively block the interaction between pathogens and hosts in the early stage of infection, thereby preventing subsequent colonization and invasion.

[0057] 4.4 RNA-seq analysis To elucidate the molecular mechanism by which dehydronuciferine and fluconazole synergistically inhibit drug-resistant Candida albicans, transcriptome analysis was performed using RNA-Seq. Overnight cultured Candida albicans CA10 cells were diluted to 1×10⁻⁶ using RPMI 1640 liquid medium. 6Cells were incubated at 35°C for 6 h under four different conditions: fluconazole monotherapy group, dehydronuciferine monotherapy group, dehydronuciferine and fluconazole combination group, and control group. The fluconazole monotherapy group received fluconazole (0.5 μg / mL), the dehydronuciferine monotherapy group received dehydronuciferine (4 μg / mL), the dehydronuciferine and fluconazole combination group received both fluconazole (0.5 μg / mL) and dehydronuciferine (4 μg / mL), and the control group received no drugs. After incubation, cells were collected by centrifugation, washed twice with PBS, and total RNA was extracted using a yeast RNA kit (Omega Bio-Tek, USA). RNA quality was verified by yield and purity measurements (NanoDrop ND-1000), and only samples with an RNA integrity index exceeding 7.0 (Agilent 2100 Bioanalyzer) were included in subsequent analyses.

[0058] A unique differential gene expression profile emerged after the combined use of dehydronuciferine and fluconazole. Figure 7 Significantly altered genes were primarily enriched in three key functional categories: hyphal morphogenesis (e.g., ALS family genes, ECE1, UME6), GPI-anchored cell wall protein biosynthesis (e.g., PGA family genes), and ergosterol biosynthesis (e.g., ERG family genes). Figure 8 Transcriptome results of the synergistic effect of dehydronuciferine and fluconazole further validated the phenotypic results of this drug combination at the gene level. Downregulation of hyphae-specific genes (such as ALS3, HWP1, and ECE1) was directly associated with in vitro hyphae defects and reduced biofilm biomass. Alterations in the expression of GPI-anchoring protein genes, crucial for adhesion, corresponded to decreased adhesion to epithelial cells, potentially weakening the immune evasion ability of Candida albicans. Perturbation of ergosterol biosynthesis not only enhanced the activity of azole drugs but also disrupted the membrane integrity and stress homeostasis of drug-resistant Candida albicans, making them more sensitive to environmental stress.

[0059] RNA-seq analysis revealed at the gene level the synergistic mechanism of the combined use of dehydronucidin and fluconazole: by simultaneously inhibiting hyphal development, adhesion-related factors, and ergosterol synthesis pathways, it synergistically weakened the virulence, structural integrity, and drug resistance of drug-resistant Candida albicans at multiple levels. Figure 9 This provides a systematic explanation for reversing drug resistance.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of dehydronusine in combination with fluconazole in the preparation of antifungal drugs; wherein the fungus is a fluconazole-resistant Candida albicans.

2. The application as described in claim 1, characterized in that, The fungi include drug-resistant Candida albicans CA10, drug-resistant Candida albicans CA16, drug-resistant Candida albicans CA103, drug-resistant Candida albicans CA137, drug-resistant Candida albicans CA632 and drug-resistant Candida albicans CA20003.

3. The application as described in claim 1, characterized in that, The mass ratio of dehydronusine to fluconazole is (0.25~2):(0.25~8).

4. Application of dehydronusine in the preparation of drugs that reduce fluconazole resistance.

5. The application as described in claim 4, characterized in that, The mass ratio of dehydronusine to fluconazole is (0.25~2):(0.25~8).

6. The application of dehydronusine in the preparation of drugs that enhance the antifungal effect of fluconazole, wherein the fungus is a fluconazole-resistant Candida albicans.

7. The application as described in claim 6, characterized in that, The fungi include drug-resistant Candida albicans CA10, drug-resistant Candida albicans CA16, drug-resistant Candida albicans CA103, drug-resistant Candida albicans CA137, drug-resistant Candida albicans CA632 and drug-resistant Candida albicans CA20003; The mass ratio of dehydronusine to fluconazole is (0.25~2):(0.25~8).

8. An antifungal drug, characterized in that, It includes dehydronusine and fluconazole; the fungus is Candida albicans resistant to fluconazole.

9. The antifungal drug as described in claim 8, characterized in that, The fungi include drug-resistant Candida albicans CA10, drug-resistant Candida albicans CA16, drug-resistant Candida albicans CA103, drug-resistant Candida albicans CA137, drug-resistant Candida albicans CA632 and drug-resistant Candida albicans CA20003; The mass ratio of dehydronusine to fluconazole is (0.25~2):(0.25~8).

10. A pharmaceutical composition, characterized in that, Including the antibacterial drug as described in claim 8 or 9.

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