Application of laricilic acid in preparation of antifungal drugs

Abstract

The invention discloses application of laricilic acid or pharmaceutical salt thereof in preparation of antifungal drugs. The structure of the laricilic acid is shown as follows: # imgabs0. The invention finds an effective antifungal compound with patent medicine potential, namely the laricilic acid. The compound laricilic acid has a definite antifungal effect on pathogenic fungi such as candida albicans, candida auricula, candida tropicalis, candida parapsilosis, candida krusei, cryptococcus neoformans, trichophyton mentagrophytes and trichophyton rubrum, and the range of the minimum inhibitory concentration MIC90 is 2-8 micrograms per milliliter.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to an application of agaricic acid in the preparation of antifungal drugs. Background Art

[0002] Pathogenic fungi can cause serious harm to the human body, ranging from common, mild superficial infections to life-threatening invasive fungal infections (IFIs). Most pathogenic fungi in IFIs are opportunistic and usually occur in people with compromised immune function, such as HIV patients, cancer patients, radiotherapy and chemotherapy, organ transplantation, and immunosuppressive drug users. IFIs are a difficult problem to solve worldwide, with overall morbidity and mortality continuing to rise. The number of people who die from fungal diseases worldwide has risen to 3.75 million per year, equivalent to an average of 7 people dying from IFIs every minute. Candida species are one of the main pathogens of IFIs, with approximately 740,000 cases of invasive candidiasis (IC) diagnosed worldwide each year, with a mortality rate of 35%-90%. Among them, Candida albicans is the most important pathogen of IC, causing more than 40% of infections. In addition to causing superficial and deep fungal infections in the human body in yeast and hyphae states, Candida albicans can also attach to the surface of internal organs or the surface of biological materials by forming a dense biofilm. The biofilm is not only a virulence factor of Candida albicans, but also an important mechanism for Candida albicans to develop drug resistance and repeated infections. At present, there are limited drugs used in the clinic to treat fungal infections, mainly azoles, echinocandins, polyenes and nucleosides. With the increase in the years of drug use and the irrational use of antifungal drugs, the phenomenon of fungal resistance has gradually increased in the clinic, becoming one of the main reasons for the failure of fungal infection treatment. In addition, some traditional antifungal drugs still have problems such as high toxicity and high prices. Existing antifungal drugs are difficult to meet clinical needs. It is of great significance to develop new antifungal drugs with high efficiency and low toxicity.

[0003] Agaricin (2-hydroxy-1,2,3-nonadecanetricarboxylic acid, hereinafter referred to as AGA) is a main component of the traditional Chinese medicine Phellinus igniarius. Its structural formula is shown in (I). 22 H 40 O7, molecular weight 416.5.

[0004]

[0005] Existing studies have shown that AGA has a significant anti-biofilm effect on bacterial pathogens. AGA can downregulate the transcription of genes responsible for flagellar motility, thereby inhibiting the formation of Salmonella biofilm by interfering with the motility phenotype. It can also reduce the formation of biofilms of Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli. Fungi and bacteria belong to different biological kingdoms and have great differences in structure and physiological functions. Existing studies are mostly reports on bacteria, while there are no relevant reports on research against pathogenic fungi at home and abroad. Summary of the invention

[0006] The purpose of the present invention is to provide an application of agaricic acid in the preparation of antifungal drugs.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a use of agaricic acid or a pharmaceutically acceptable salt thereof in the preparation of an antifungal drug.

[0009] The structure of the agaric acid (2-hydroxy-1,2,3-nonadecanetricarboxylic acid, hereinafter referred to as AGA, CAS No.: 666-99-9) is shown below:

[0010]

[0011] The pharmaceutically acceptable salts refer to the following salts formed by agaric acid and corresponding bases: sodium salt, calcium salt, potassium salt, zinc salt and meglumine salt.

[0012] The pharmaceutically acceptable salt refers to a salt formed by the reaction of agaric acid with a pharmaceutically acceptable inorganic acid or organic acid; wherein the inorganic acid is at least one of hydrochloric acid, hydrobromic acid, phosphoric acid, carbonic acid, nitric acid or sulfuric acid; and the organic acid is at least one of formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalenedisulfonic acid, asiatic acid, carbenoxolone acid, glycyrrhetinic acid, oleanolic acid, hawthorn acid, ursolic acid, corosolic acid, betulinic acid, boswellic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, stearic acid, nicotinic acid, isonicotinic acid, benzenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid or amino acid.

[0013] The fungus is selected from Candida albicans, Candida auris, Candida tropicalis, Candida parapsilosis, Candida krusei, Cryptococcus neoformans, Trichophyton mentagrophytes, Trichophyton rubrum, etc.; specifically: Candida albicans SC5314, 9173, 7781, 9871, 9893, 7654, 9161, 901, 904, 632; Candida auris 0029; Candida tropicalis 8915, 409; Candida parapsilosis 22019, 90018; Candida krusei 4996, 62588, 10153; Cryptococcus neoformans H99; Trichophyton mentagrophytes mentagrophytes)T5a; Trichophyton rubrum (Trichophyton rubrum)CMCCFTLA.

[0014] The effective concentration of the agaric acid or its pharmaceutical salt in the anti-Candida albicans drug is ≥4 μg / mL; the effective concentration of the agaric acid or its pharmaceutical salt in the anti-Candida auris drug is ≥4 μg / mL; the effective concentration of the agaric acid or its pharmaceutical salt in the anti-Candida tropicalis drug is ≥2 μg / mL; the effective concentration of the agaric acid or its pharmaceutical salt in the anti-Candida parapsilosis drug is ≥2 μg / mL; the effective concentration of the agaric acid or its pharmaceutical salt in the anti-Candida krusei drug is ≥2 μg / mL; the effective concentration of the agaric acid or its pharmaceutical salt in the anti-Cryptococcus neoformans drug is ≥4 μg / mL; the effective concentration of the agaric acid or its pharmaceutical salt in the anti-Trichophyton mentagrophytes drug is ≥4 μg / mL; the effective concentration of the agaric acid or its pharmaceutical salt in the anti-Trichophyton rubrum drug is ≥4 μg / mL.

[0015] The invention provides an application of agaricic acid or a pharmaceutically acceptable salt thereof in the preparation of an antifungal drug, wherein the agaricic acid is used as the sole active ingredient.

[0016] The present invention also provides a pharmaceutical preparation, which is prepared from agaricic acid or its pharmaceutically acceptable salt and medically acceptable auxiliary materials.

[0017] The dosage form of the pharmaceutical preparation is injection, capsule, tablet, granule, pill, microcapsule preparation, microsphere preparation, nano preparation and the like.

[0018] The second aspect of the present invention provides a use of agaricic acid or a pharmaceutically acceptable salt thereof in conjunction with commonly used clinical antifungal drugs in the preparation of antifungal drugs.

[0019] The commonly used clinical antifungal drugs are selected from fluconazole, ketoconazole, itraconazole, voriconazole, amphotericin B, caspofungin, terbinafine and other commonly used clinical antifungal drugs.

[0020] The pharmaceutically acceptable salts refer to the following salts formed by agaric acid and corresponding bases: sodium salt, calcium salt, potassium salt, zinc salt and meglumine salt.

[0021] The pharmaceutically acceptable salt refers to a salt formed by the reaction of agaric acid with a pharmaceutically acceptable inorganic acid or organic acid; wherein the inorganic acid is at least one of hydrochloric acid, hydrobromic acid, phosphoric acid, carbonic acid, nitric acid or sulfuric acid; and the organic acid is at least one of formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalenedisulfonic acid, asiatic acid, carbenoxolone acid, glycyrrhetinic acid, oleanolic acid, hawthorn acid, ursolic acid, corosolic acid, betulinic acid, boswellic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, stearic acid, nicotinic acid, isonicotinic acid, benzenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid or amino acid.

[0022] The fungus is selected from Candida albicans, Candida auris, Candida tropicalis, Candida parapsilosis, Candida krusei, Cryptococcus neoformans, Trichophyton mentagrophytes, Trichophyton rubrum, etc.; specifically: Candida albicans SC5314, 9173, 7781, 9871, 9893, 7654, 9161, 901, 904, 632; Candida auris 0029; Candida tropicalis 8915, 409; Candida parapsilosis 22019, 90018; Candida krusei 4996, 62588, 10153; Cryptococcus neoformans H99; Trichophyton mentagrophytes mentagrophytes)T5a; Trichophyton rubrum (Trichophyton rubrum)CMCCFTLA.

[0023] The present invention also provides a pharmaceutical preparation, which is prepared from agaricic acid or its pharmaceutically acceptable salt in combination with clinically commonly used antifungal drugs and medically acceptable excipients.

[0024] The dosage form of the pharmaceutical preparation is injection, capsule, tablet, granule, pill, microcapsule preparation, microsphere preparation, nano preparation and the like.

[0025] The present invention finds that for fluconazole-resistant Candida albicans, the combined use of AGA and fluconazole can reduce the effective concentration of the drug, reducing the effective concentration of fluconazole from 64 μg / mL to 0.5 μg / mL. The preferred combined use scheme is AGA concentration of 0.5 μg / mL + fluconazole concentration of 0.5 μg / mL or AGA concentration of 1 μg / mL + fluconazole concentration of 0.5 μg / mL.

[0026] The present invention finds that agaricic acid has very low toxicity at an effective antifungal concentration, and is toxic to mammalian cells when the concentration is ≥64 μg / mL.

[0027] In the present invention, when wax moth larvae (Example 8) are used as an in vivo experimental model of fungal infection, when the concentration of agaricic acid reaches 0.25 mg / kg, the survival rate of wax moth larvae infected with fungi can be significantly improved.

[0028] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0029] The present invention uses a micro-liquid dilution method to extensively screen a natural compound library and finds that agaric acid has good antifungal activity, and has good antifungal effects on pathogenic fungi such as Candida albicans, Candida auris, Candida krusei, Candida tropicalis, Candida parapsilosis, Cryptococcus neoformans, Trichophyton mentagrophytes, and Trichophyton rubrum. Agaric acid and azole drugs have obvious synergistic anti-resistant fungi effects. Agaric acid can inhibit the formation process of fungal biofilms and destroy mature biofilms. Agaric acid has in vivo antifungal activity and low toxicity. Agaric acid is expected to be developed as a new antifungal drug, and the present invention provides a new strategy for antifungal.

[0030] The present invention has discovered an effective antifungal compound with drug potential: agaric acid (AGA). The compound agaric acid in the present invention has a clear antifungal effect on pathogenic fungi such as Candida albicans, Candida auris, Candida tropicalis, Candida parapsilosis, Candida krusei, Cryptococcus neoformans, Trichophyton mentagrophytes, and Trichophyton rubrum, with a minimum inhibitory concentration (MIC) of 1.17. 90The range is 2-8μg / mL. Agaricin can inhibit the growth of Candida albicans, inhibit the adhesion (0.5μg / mL) and hyphae formation (0.25μg / mL) of Candida albicans, inhibit the formation of Candida albicans biofilm (4μg / mL) and destroy the mature biofilm (16μg / mL). Agaricin also has good antifungal activity against fluconazole-resistant strains of Candida albicans and other strains that are naturally resistant to fluconazole (such as Candida auris, Candida krusei, and Trichophyton mentagrophytes), and the combination of agaric acid and fluconazole shows a synergistic anti-resistant fungal effect. Agaricin can also effectively increase the survival rate of fungal-infected wax moth larvae in in vivo experiments, and has low toxicity to mammalian cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the inhibitory effects of different concentrations (2, 4, 8 μg / mL) of AGA and fluconazole on the growth of Candida albicans SC5314.

[0032] Figure 2 Schematic diagram of the inhibitory effect of AGA on the cell surface hydrophobicity of Candida albicans SC5314 starting from 0.25 μg / mL.

[0033] Figure 3 This is a schematic diagram of the inhibitory effect of AGA on the hyphae formation ability of Candida albicans SC5314.

[0034] Figure 4 Shown is a schematic diagram of the expression changes of adhesion and hyphae formation related genes in Candida albicans SC5314 after AGA treatment compared with the blank control group.

[0035] Figure 5 This is a schematic diagram of the inhibitory effect of different concentrations (2, 4, 8, 16, 32, 64 μg / mL) of AGA on the biofilm of Candida albicans SC5314 in Example 6.

[0036] Figure 6 Shown is a schematic diagram of the antibacterial effect of AGA combined with fluconazole on drug-resistant bacteria 904.

[0037] Figure 7 This is a schematic diagram showing the effect of AGA in Example 8 on the survival rate of wax moth larvae infected with Candida albicans.

[0038] Figure 8 Shown is a schematic diagram of the toxic effects of AGA on mammalian cells (mouse embryonic fibroblasts NIH / 3T3). DETAILED DESCRIPTION

[0039] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0040] The present invention finds that the compound agaric acid has good antifungal activity and synergistic anti-drug-resistant fungal activity with fluconazole, and agaric acid has low toxicity to mammalian cells.

[0041] In the present invention, the fungi used can be obtained or cultured by conventional methods, or can be obtained through commercial channels, such as separation from somatic cells or tissues of patients with corresponding diseases, or purchased from any institution or company that sells standard strains, such as ATCC. It should be understood that the strain samples used in the embodiments of the present invention are only exemplary examples, and any fungi that can form hyphae or biofilm or are resistant to fluconazole can be used to test the antifungal activity of the compounds of the present invention, without being limited by the scope of the embodiments. It should be understood that in the embodiments of the present invention, only the minimum concentration of the active ingredient is stated to effectively inhibit the formation of fungal hyphae, biofilm formation, and destroy mature biofilm. In combination with the common knowledge in the art, it can be known that when the concentration of the antifungal active molecular component increases, the antifungal effect will be improved to a certain extent. Therefore, after reading this specification, any technician in the field can use a dosage higher than the minimum concentration to inhibit the formation of fungal hyphae or the growth of biofilm or destroy mature biofilm by combining the common knowledge in the art, and this change also falls within the scope of the disclosure of the present invention.

[0042] Example 1

[0043] Determination of the antifungal spectrum of agaric acid (AGA):

[0044] Materials and methods

[0045] 1. Experimental strains

[0046] Candida albicans experimental strain SC5314, Candida albicans clinical strains 9173, 7781, 9871, 9893, 7654, 9161, 901, 904, 632, Candida auris 0029, Candida krusei 4996, 62588, 10153, Candida tropicalis 409, 8915, Cryptococcus neoformans H99, Candida parapsilosis 22019, 90018, Trichophyton mentagrophytes T5a and Trichophyton rubrum CMC CFTLA were purchased from Shanghai Changzheng Hospital.

[0047] 2. Culture medium and drug preparation

[0048] YPD culture medium: Accurately weigh 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of glucose and add them into a 1-liter measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make up to 1 L. Stir with 121°C high-temperature and high-pressure steam for 15 min. Cool naturally to room temperature and store at 4°C for later use.

[0049] PBS buffer solution: 8.0 g sodium chloride, 3.57 g disodium hydrogen phosphate dodecahydrate, 0.2 g potassium chloride, 0.24 g dipotassium hydrogen phosphate, dissolved in ultrapure water and fixed to 1 L, sterilized at 121°C and high temperature and high pressure for 15 min, and then stored at room temperature.

[0050] RPMI 1640 medium: Accurately weigh 10.0 g of RPMI 1640, 2.0 g of sodium bicarbonate, 34.5 g of 3-morpholinepropanesulfonic acid, and 2.7 g of sodium hydroxide and transfer them to a measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make the volume up to 1 L. Filter and sterilize through a 0.22 μM microporous filter membrane and store at 4°C for later use.

[0051] Preparation of AGA and fluconazole stock solutions: Use DMSO as solvent to prepare the drug into 20 mg / mL stock solution for subsequent experiments.

[0052] 3. Activation of strains and preparation of bacterial suspension

[0053] Quickly take out the frozen sample of the desired strain from the -80℃ ultra-low temperature freezer, take 10μL of the bacterial solution and add it to a 15mL test tube containing 1mL YPD culture medium, place it in a 30℃ air bath shaking incubator, and shake it at 200rpm. After 24h, take out 10μL of the bacterial solution from the YPD culture medium and add it to a new 1mL YPD culture medium, and put it back into the 30℃ air bath shaking incubator again, and continue shaking and culturing overnight at 200rpm to make the concentration of the bacterial solution reach 1-2×10 8 cells / mL. Then, the bacterial solution was diluted to 5×10 5 -2×10 6 cells / mL(OD 600 The culture medium is about 0.2), and then placed back in a shaking incubator at 30°C and cultured at a shaking speed of 200 rpm. After about 4-6 hours, the bacterial solution will reach 2×10 7 cells / mL, after 3-4 divisions, the fungus is at the end of the logarithmic (exponential) growth phase.

[0054] Transfer the cultured bacterial solution to a 1.5mL centrifuge tube, centrifuge at 5000g for 1min, discard the upper culture medium, wash the bacteria 2-3 times with PBS buffer solution to remove the residual culture medium. Resuspend the bacterial solution, pipette 10μL of the bacterial solution into 1mL PBS buffer solution and mix well, take 10μL and count it under a biological microscope using a hemocytometer to prepare for subsequent dilution into bacterial solutions of different concentrations.

[0055] 4. Determination of minimum inhibitory concentration (MIC) by microdilution method

[0056] This experiment uses the micro-liquid dilution method, and the minimum inhibitory concentration can be determined according to the standard experimental method of the CLSI micro-liquid dilution method manual M27-A3. In the experiment, the strain to be tested was cultured to the end of the exponential growth phase, and the strain was diluted to 1×10 using RPMI 1640 medium. 3 CFU / mL, and then transfer it to a 96-well cell culture plate, add 200μL of bacterial solution to the first column, 100μL of bacterial solution to columns 3-12, and 100μL of RPMI 1640 medium to the last column as a blank control. Then add the drug, add 6.4μL of drug solution to each well in the first column and mix by pipetting, then dilute in multiples to the tenth column, and the 11th column is the bacterial solution without drug as a growth control. Then place the 96-well plate in a 30℃ constant temperature incubator and culture for 24h or 48h, and use an enzyme reader to measure the absorbance value at a wavelength of 630nm. The lowest drug concentration at which more than 90% of fungal growth is inhibited is the MIC.90 To improve the accuracy and stability of the results, the experiment was repeated at least three times. The experimental results are shown in Table 1:

[0057] Table 1 Minimum inhibitory concentrations of AGA and fluconazole against 21 fungi

[0058]

[0059]

[0060] Conclusion: AGA showed good antifungal effect against 21 tested fungi. It had stable inhibitory effect against Candida albicans SC5314 and 6 fluconazole-sensitive strains from clinical sources. 90 The range was 4-8 μg / mL. At the same time, AGA also had antifungal effects on three fluconazole-resistant Candida albicans (901, 904, and 632), and the MIC 90 Similar to the sensitive strains, it was 4 μg / mL; AGA also showed good antifungal activity against Candida auris and Candida krusei, which are naturally resistant to fluconazole. 90 The MICs of AGA against two strains of Candida tropicalis and two strains of Candida parapsilosis were 4 μg / mL and 2 μg / mL, respectively. 90 AGA also has a wide range of stable antibacterial effects on non-Candida fungi such as Cryptococcus neoformans, Trichophyton rubrum, and Trichophyton mentagrophytes. The MIC of AGA against Trichophyton mentagrophytes is 90 At 4 μg / mL, the control drug fluconazole still had no inhibitory effect at a concentration of 64 μg / mL. In summary, AGA showed good antifungal activity against common pathogenic fungi and its effect was stable.

[0061] Example 2

[0062] Inhibitory effect of agaric acid (AGA) on the growth of Candida albicans

[0063] Materials and methods

[0064] 1. Experimental strains

[0065] Candida albicans experimental strain SC5314.

[0066] 2. Culture medium and drug preparation

[0067] YPD culture medium: Accurately weigh 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of glucose and add them into a 1-liter measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make up to 1 L. Stir with 121°C high-temperature and high-pressure steam for 15 min. Cool naturally to room temperature and store at 4°C for later use.

[0068] PBS buffer solution: 8.0 g sodium chloride, 3.57 g disodium hydrogen phosphate dodecahydrate, 0.2 g potassium chloride, 0.24 g dipotassium hydrogen phosphate, dissolved in ultrapure water and fixed to 1 L, sterilized at 121°C and high temperature and high pressure for 15 min, and then stored at room temperature.

[0069] RPMI 1640 medium: Accurately weigh 10.0 g of RPMI 1640, 2.0 g of sodium bicarbonate, 34.5 g of 3-morpholinepropanesulfonic acid, and 2.7 g of sodium hydroxide and transfer them to a measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make the volume up to 1 L. Filter and sterilize through a 0.22 μM microporous filter membrane and store at 4°C for later use.

[0070] Preparation of AGA and fluconazole stock solutions: Use DMSO as solvent to prepare the drug into 20 mg / mL stock solution for subsequent experiments.

[0071] 3. Activation of strains and preparation of bacterial suspension

[0072] Quickly take out the frozen sample of the desired strain from the -80℃ ultra-low temperature freezer, take 10μL of the bacterial solution and add it to a 15mL test tube containing 1mL YPD culture medium, place it in a 30℃ air bath shaking incubator, and shake it at 200rpm. After 24h, take out 10μL of the bacterial solution from the YPD culture medium and add it to a new 1mL YPD culture medium, and put it back into the 30℃ air bath shaking incubator again, and continue shaking and culturing overnight at 200rpm to make the concentration of the bacterial solution reach 1-2×10 8 cells / mL. Then, the bacterial solution was diluted to 5×10 5 -2×10 6 cells / mL(OD 600 The culture medium is about 0.2), and then placed back in a shaking incubator at 30°C and cultured at a shaking speed of 200 rpm. After about 4-6 hours, the bacterial solution will reach 2×10 7 cells / mL, after 3-4 divisions, the fungus is at the end of the logarithmic (exponential) growth phase.

[0073] Transfer the cultured bacterial solution to a 1.5mL centrifuge tube, centrifuge at 5000g for 1min, discard the upper culture medium, wash the bacteria 2-3 times with PBS buffer solution to remove the residual culture medium. Resuspend the bacterial solution, pipette 10μL of the bacterial solution into 1mL PBS buffer solution and mix well, take 10μL and count it under a biological microscope using a hemocytometer to prepare for subsequent dilution into bacterial solutions of different concentrations.

[0074] 4. Determination of growth curve

[0075] Take the strain to be tested at the end of exponential growth and dilute it into 2×10 5 CFU / mL of bacterial suspension. Take 7 small conical flasks, add 10mL of bacterial suspension to each, and then add AGA mother solution to 3 of the conical flasks respectively, and finally prepare bacterial suspensions containing 2, 4, and 8μg / mL AGA. Take another 3 and add fluconazole with a final concentration of 2, 4, and 8μg / mL as the positive control group. Add DMSO with the same volume as the 8μg / mL AGA group to the 7th conical flask as the blank control group. Place the small conical flask stably in a 30°C air bath shaking incubator with a speed of 200rpm. And at 2h, 4h, 6h, 8h, 12h, and 24h, respectively, draw 100μL of bacterial solution from each conical flask into a 96-well culture plate (three sets of wells in each group), and use an enzyme reader to measure the absorbance value of each well at a wavelength of 630nm. See the results. Figure 1 As shown, Figure 1 This is a schematic diagram of the inhibitory effects of different concentrations (2, 4, 8 μg / mL) of AGA and fluconazole on the growth of Candida albicans SC5314.

[0076] Conclusion: The experimental results show that within 24 hours, compared with the blank control group, the low concentration of AGA at 2μg / mL can significantly reduce the growth rate of SC5314. When the AGA concentration is 8μg / mL, the growth of SC5314 is completely inhibited, indicating that AGA has a good antibacterial effect at high concentrations and shows a dose-dependent relationship. In the comparison between AGA and the positive control fluconazole at the same concentration, both showed a better inhibitory effect on growth.

[0077] Example 3

[0078] Agaricin (AGA) has an inhibitory effect on the adhesion ability of Candida albicans

[0079] Materials and methods

[0080] 1. Experimental strains

[0081] Candida albicans experimental strain SC5314.

[0082] 2. Culture medium and drug preparation

[0083] YPD culture medium: Accurately weigh 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of glucose and add them into a 1-liter measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make up to 1 L. Stir with 121°C high-temperature and high-pressure steam for 15 min. Cool naturally to room temperature and store at 4°C for later use.

[0084] PBS buffer solution: 8.0 g sodium chloride, 3.57 g disodium hydrogen phosphate dodecahydrate, 0.2 g potassium chloride, 0.24 g dipotassium hydrogen phosphate, dissolved in ultrapure water and fixed to 1 L, sterilized at 121°C and high temperature and high pressure for 15 min, and then stored at room temperature.

[0085] RPMI 1640 medium: Accurately weigh 10.0 g of RPMI 1640, 2.0 g of sodium bicarbonate, 34.5 g of 3-morpholinepropanesulfonic acid, and 2.7 g of sodium hydroxide and transfer them to a measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make the volume up to 1 L. Filter and sterilize through a 0.22 μM microporous filter membrane and store at 4°C for later use.

[0086] Preparation of AGA and fluconazole stock solutions: Use DMSO as solvent to prepare the drug into 20 mg / mL stock solution for subsequent experiments.

[0087] 3. Activation of strains and preparation of bacterial suspension

[0088] Quickly take out the frozen sample of the desired strain from the -80℃ ultra-low temperature freezer, take 10μL of the bacterial solution and add it to a 15mL test tube containing 1mL YPD culture medium, place it in a 30℃ air bath shaking incubator, and shake it at 200rpm. After 24h, take out 10μL of the bacterial solution from the YPD culture medium and add it to a new 1mL YPD culture medium, and put it back into the 30℃ air bath shaking incubator again, and continue shaking and culturing overnight at 200rpm to make the concentration of the bacterial solution reach 1-2×10 8 cells / mL. Then, the bacterial solution was diluted to 5×10 5 -2×10 6 cells / mL(OD 600 The culture medium is about 0.2), and then placed back in a shaking incubator at 30°C and cultured at a shaking speed of 200 rpm. After about 4-6 hours, the bacterial solution will reach 2×10 7 cells / mL, after 3-4 divisions, the fungus is at the end of the logarithmic (exponential) growth phase.

[0089] Transfer the cultured bacterial solution to a 1.5mL centrifuge tube, centrifuge at 5000g for 1min, discard the upper culture medium, wash the bacteria 2-3 times with PBS buffer solution to remove the residual culture medium. Resuspend the bacterial solution, pipette 10μL of the bacterial solution into 1mL PBS buffer solution and mix well, take 10μL and count it under a biological microscope using a hemocytometer to prepare for subsequent dilution into bacterial solutions of different concentrations.

[0090] 4. Determination of the inhibitory effect of AGA on the adhesion ability of Candida albicans

[0091] Fungi mainly adhere to the surface of organs, macromolecular biomaterials, etc. in the host body. The stronger the hydrophobicity of the fungal cell surface, that is, the stronger the lipophilicity, the stronger the adhesion ability in the host body. Therefore, the adhesion ability of fungal cells can be reflected by measuring the hydrophobicity of the fungal cell surface.

[0092] Take the strain to be tested at the end of exponential growth and resuspend the bacterial solution to 1×10 6 CFU / mL. Take 3 6-well cell culture plates treated with TC (Tissue-culture treated), add 2mL of diluted bacterial solution to each well, and incubate in a 37°C constant temperature incubator for 90 minutes to make it adhere to the bottom of the cell culture plate. Take out the 6-well plate and slowly aspirate the upper culture solution, and slowly rinse with PBS buffer to wash away the residual culture medium and non-adherent suspended fungal cells. Then, add 4mL of drug-containing RPMI 1640 culture medium to each well, and the drug concentrations are 4, 2, 1, 0.5, and 0.25μg / mL, respectively. The blank control group is added with the same volume of DMSO. Make three replicate wells for each different drug concentration group, and place the 6-well plate in a 37°C incubator for 24h of drug action culture.

[0093] Hydrophobicity determination: Take out the 6-well plate, discard the supernatant and rinse the biofilm as completely as possible with PBS buffer solution, and rinse several times to obtain as many membranes as possible. Finally, centrifuge the obtained membrane suspension and wash twice, resuspend the membrane with PBS buffer solution, and place it in a 5mL centrifuge tube for mixing. After mixing, transfer 2mL of bacterial solution from each tube to a new 5mL centrifuge tube and add 0.5mL of n-octane. After the water and hydrocarbon phases are fully mixed for a few minutes, let it stand for 15 minutes to completely separate the two phases, discard the upper n-octane, take the lower aqueous phase and measure the absorbance at a wavelength of 630nm, and perform at least three replicates for each group. The absorbance value of the group without drug addition was used as the negative control. The formula for calculating relative cell surface hydrophobicity is (OD 630阴性对照 -OD 630实验组 ) / OD 630阴性对照 The experimental results were analyzed by one-way analysis of variance (ANOVA), n = 3. Figure 2 As shown, Figure 2 This is a schematic diagram of the inhibitory effect of AGA on the cell surface hydrophobicity of Candida albicans SC5314 starting from 0.25 μg / mL. The horizontal axis represents the AGA concentration, the vertical axis indicates the magnitude of the cell surface hydrophobicity, ** represents P < 0.01, and *** represents P < 0.001.

[0094] Conclusion: AGA can significantly reduce the cell surface hydrophobicity of Candida albicans. 0.5 μg / mL AGA can significantly reduce the relative cell surface hydrophobicity of Candida albicans from 0.8 to 0.64 (P < 0.01, see Figure 2 ). With the continuous increase of AGA concentration, the decreasing trend of cell surface hydrophobicity gradually increased. Under the action of 4 μg / mL AGA, the relative value of cell surface hydrophobicity of Candida albicans decreased to 0.02 (P < 0.001, see Figure 2 ).

[0095] Example 4

[0096] Agaricin (AGA) inhibits hyphae formation of Candida albicans

[0097] Materials and methods

[0098] 1. Experimental strains

[0099] Candida albicans experimental strain SC5314.

[0100] 2. Culture medium and drug preparation

[0101] YPD culture medium: Accurately weigh 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of glucose and add them into a 1-liter measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make up to 1 L. Stir with 121°C high-temperature and high-pressure steam for 15 min. Cool naturally to room temperature and store at 4°C for later use.

[0102] PBS buffer solution: 8.0 g sodium chloride, 3.57 g disodium hydrogen phosphate dodecahydrate, 0.2 g potassium chloride, 0.24 g dipotassium hydrogen phosphate, dissolved in ultrapure water and fixed to 1 L, sterilized at 121°C and high temperature and high pressure for 15 min, and then stored at room temperature.

[0103] RPMI 1640 medium: Accurately weigh 10.0 g of RPMI 1640, 2.0 g of sodium bicarbonate, 34.5 g of 3-morpholinepropanesulfonic acid, and 2.7 g of sodium hydroxide and transfer them to a measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make the volume up to 1 L. Filter and sterilize through a 0.22 μM microporous filter membrane and store at 4°C for later use.

[0104] Spider liquid culture medium: Accurately weigh 10 g of nutrient broth powder, 2.0 g of potassium dihydrogen phosphate, and 10 g of mannitol into a measuring cylinder, add 200 mL of ultrapure water, stir thoroughly with a glass rod to dissolve the powder, then make up to 1 L, sterilize at 121°C and autoclave for 15 min, then seal and store in a refrigerator at 4°C.

[0105] Lee's liquid medium: accurately weigh 0.0714g of ornithine, 0.5g of alanine, 0.5g of threonine, 1.3g of leucine, 0.5g of proline, 1.0g of lysine, 0.5g of phenylalanine, and 0.1g of methionine. All the above amino acids should be dissolved by ultrasonic in ultrapure water, and then 0.5g of ammonium sulfate, 0.357g of magnesium sulfate heptahydrate, 0.25g of dipotassium hydrogen phosphate, 5.0g of sodium chloride, 0.001g of biotin, and 12.5g of glucose should be added respectively, and stirred until completely dissolved. Then, add water to make the volume 1L and adjust the pH value to about 6.8, and finally sterilize it by high pressure, and store it in a sealed container at room temperature.

[0106] Preparation of AGA and fluconazole stock solutions: Use DMSO as solvent to prepare the drug into 20 mg / mL stock solution for subsequent experiments.

[0107] 3. Activation of strains and preparation of bacterial suspension

[0108] Quickly take out the frozen sample of the desired strain from the -80℃ ultra-low temperature freezer, take 10μL of the bacterial solution and add it to a 15mL test tube containing 1mL YPD culture medium, place it in a 30℃ air bath shaking incubator, and shake it at 200rpm. After 24h, take out 10μL of the bacterial solution from the YPD culture medium and add it to a new 1mL YPD culture medium, and put it back into the 30℃ air bath shaking incubator again, and continue shaking and culturing overnight at 200rpm to make the concentration of the bacterial solution reach 1-2×10 8 cells / mL. Then, the bacterial solution was diluted to 5×10 5 -2×10 6 cells / mL(OD 600 The culture medium is about 0.2), and then placed back in a shaking incubator at 30°C and cultured at a shaking speed of 200 rpm. After about 4-6 hours, the bacterial solution will reach 2×10 7 cells / mL, after 3-4 divisions, the fungus is at the end of the logarithmic (exponential) growth phase.

[0109] Transfer the cultured bacterial solution to a 1.5mL centrifuge tube, centrifuge at 5000g for 1min, discard the upper culture medium, wash the bacteria 2-3 times with PBS buffer solution to remove the residual culture medium. Resuspend the bacterial solution, pipette 10μL of the bacterial solution into 1mL PBS buffer solution and mix well, take 10μL and count it under a biological microscope using a hemocytometer to prepare for subsequent dilution into bacterial solutions of different concentrations.

[0110] 4. Mycelium culture

[0111] Take the strain to be tested at the end of exponential growth and dilute it with RPMI 1640 medium, Spider liquid medium and Lee's liquid medium to make 2×10 5 CFU / mL bacterial suspension. Add the diluted bacterial solution to a 24-well plate, 2mL per well. Add AGA mother solution to each well to make the final concentrations of 1, 2, 4, 8, and 16μg / mL, respectively, and ensure that the drug and the bacterial solution are fully mixed. Each group has three replicate wells. Add DMSO of the same volume as the drug solution to the blank control group, and place the well plate in a constant temperature incubator at 37°C for incubation for 3h. Subsequently, take pictures with an inverted microscope to observe the growth of hyphae under different drug concentrations, and use ImageJ to measure the length of hyphae. Five fields of view were selected for each group, and 20 hyphae were randomly measured in each field of view, with a total of n=100. The measured lengths were statistically analyzed, and the experimental results were subjected to one-way analysis of variance (ANOVA). The results are shown in Figure 3 , Figure 3 It is a schematic diagram of the inhibitory effect of AGA on the hyphae formation ability of Candida albicans SC5314. The photo shows the hyphae formation observed under an inverted microscope (the scale length in the figure is 100 μm). The bar graph shows the average hyphae length of each group. * represents P < 0.05, and **** represents P < 0.0001.

[0112] Conclusion: AGA has a clear inhibitory effect on the formation of liquid hyphae. In the absence of drug effects, Candida albicans SC5314 can form slender, non-noded hyphae in RPMI 1640, Lee's, Spider and other hyphae induction media at 37°C ( Figure 3 ). In RPMI 1640 medium, 0.25μg / mL of AGA can significantly inhibit the formation of Candida albicans hyphae, and 4μg / mL of AGA can completely inhibit the formation of Candida albicans hyphae, making it completely in the yeast state. This inhibitory effect on the formation of Candida albicans hyphae is positively correlated with the concentration of AGA (P<0.0001). In Lee's liquid medium and Spider liquid medium, Candida albicans hyphae also began to shorten under the action of 0.25μg / mL of AGA, and the hyphae were significantly inhibited with the increase of AGA concentration. When the AGA concentration was 4μg / mL, most of the hyphae were inhibited and the cells were in the yeast state (P<0.0001). In summary, AGA can inhibit the formation of hyphae in three different hyphae induction media, and the effective concentration is lower than the minimum concentration required to inhibit biofilm formation.

[0113] Example 5

[0114] Agaricin (AGA) affects the expression of adhesion- and hyphae-related genes in Candida albicans

[0115] Materials and methods

[0116] 1. Experimental strains

[0117] Candida albicans experimental strain SC5314.

[0118] 2. Culture medium and drug preparation

[0119] YPD culture medium: Accurately weigh 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of glucose and add them into a 1-liter measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make up to 1 L. Stir with 121°C high-temperature and high-pressure steam for 15 min. Cool naturally to room temperature and store at 4°C for later use.

[0120] PBS buffer solution: 8.0 g sodium chloride, 3.57 g disodium hydrogen phosphate dodecahydrate, 0.2 g potassium chloride, 0.24 g dipotassium hydrogen phosphate, dissolved in ultrapure water and fixed to 1 L, sterilized at 121°C and high temperature and high pressure for 15 min, and then stored at room temperature.

[0121] RPMI 1640 medium: Accurately weigh 10.0 g of RPMI 1640, 2.0 g of sodium bicarbonate, 34.5 g of 3-morpholinepropanesulfonic acid, and 2.7 g of sodium hydroxide and transfer them to a measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make the volume up to 1 L. Filter and sterilize through a 0.22 μM microporous filter membrane and store at 4°C for later use.

[0122] Preparation of AGA and fluconazole stock solutions: Use DMSO as solvent to prepare the drug into 20 mg / mL stock solution for subsequent experiments.

[0123] 3. Activation of strains and preparation of bacterial suspension

[0124] Quickly take out the frozen sample of the desired strain from the -80℃ ultra-low temperature freezer, take 10μL of the bacterial solution and add it to a 15mL test tube containing 1mL YPD culture medium, place it in a 30℃ air bath shaking incubator, and shake it at 200rpm. After 24h, take out 10μL of the bacterial solution from the YPD culture medium and add it to a new 1mL YPD culture medium, and put it back into the 30℃ air bath shaking incubator again, and continue shaking and culturing overnight at 200rpm to make the concentration of the bacterial solution reach 1-2×10 8 cells / mL. Then, the bacterial solution was diluted to 5×10 5 -2×10 6 cells / mL(OD 600The culture medium is about 0.2), and then placed back in a shaking incubator at 30°C and cultured at a shaking speed of 200 rpm. After about 4-6 hours, the bacterial solution will reach 2×10 7 cells / mL, after 3-4 divisions, the fungus is at the end of the logarithmic (exponential) growth phase.

[0125] Transfer the cultured bacterial solution to a 1.5mL centrifuge tube, centrifuge at 5000g for 1min, discard the upper culture medium, wash the bacteria 2-3 times with PBS buffer solution to remove the residual culture medium. Resuspend the bacterial solution, pipette 10μL of the bacterial solution into 1mL PBS buffer solution and mix well, take 10μL and count it under a biological microscope using a hemocytometer to prepare for subsequent dilution into bacterial solutions of different concentrations.

[0126] 4. Primer design and synthesis

[0127] First, the relevant gene sequences were searched in the CGD database (http: / / www.candidagenome.org / ), and the corresponding primers were designed online using Primer 3Input based on the obtained gene sequences. The synthesized primers were purified by HAP and stored at -20°C.

[0128] Table 2 Primer sequences

[0129]

[0130] 5. Extraction of fungal total RNA

[0131] Take the Candida albicans at the end of exponential growth, wash it three times with PBS buffer solution, and dilute the bacterial solution to 1×10 6 CFU / mL. 80mL of the diluted bacterial solution was added to a cell culture dish with a diameter of 150mm and incubated in a 37℃ incubator for 90min to form a biofilm. Then, RPMI 1640 medium containing 4μg / mL AGA was slowly added, and the blank control group was added with a culture solution containing an equal volume of DMSO as the drug, and then the culture dish was continued to stand in a 37℃ incubator for 1h. Subsequently, the upper culture solution was carefully discarded, and the biofilm was scraped off and collected in a 50mL centrifuge tube using a cell scraper, and the residual biofilm in the culture dish was rinsed with PBS buffer solution. Finally, the membrane was centrifuged at 5000g for 10min, and the collected membrane was resuspended with PBS buffer solution and transferred to a 1.5mL centrifuge tube without RNase for RNA extraction.

[0132] Follow the instructions of the fungal RNA extraction kit and test the concentration of the extracted RNA. When the A260 / A280 value is between 1.8 and 2.1, it indicates that the RNA purity is good. The RNA concentration must reach above 20 ng / μL before subsequent experiments can be carried out. Record the RNA concentration value of each group for subsequent reverse transcription and RNA quantification.

[0133] 6. Reverse transcription to cDNA

[0134] The entire experiment needs to be performed on ice. Take eight tubes and add 8μL 5× PrimeScript RTMaster Mix to each tube. Then calculate the volume of RNA sample to be added based on the concentration of each tube of RNA sample previously measured. Add 500ng RNA to each tube, and then fill the total system to 20μL with DEPC water. Centrifuge each eight-tube for 30s to mix all the liquid on the tube wall at the bottom of the tube, and use a PCR instrument for reverse transcription. The reverse transcription program is set as: 37℃15min, 85℃5sec, 4℃5min. After reverse transcription, the cDNA can be used directly in subsequent experiments or stored at -20℃.

[0135] 7. Real-time fluorescence quantitative PCR

[0136] β-actin was used as the internal reference gene and ΔΔC T The relative expression of genes was calculated by the method. First, the common reaction solution for each well was prepared. 10 μL SYBR Green and 7.2 μL deionized water should be added to each well. The two should be mixed evenly before adding to the 96-well PCR plate. Next, 0.8 μL of each primer working solution was added, and 2.0 μL of the diluted cDNA sample was added to each well, with a total reaction solution of 20 μL. After the addition of the sample, the 96-well PCR plate was sealed and centrifuged for 30 seconds, so that all the liquid on the tube wall was centrifuged to the bottom of the tube and fully mixed, and placed in a real-time fluorescence quantitative PCR instrument. The PCR amplification program was set as follows: pre-denaturation temperature 95°C, duration 30 seconds; followed by 40 cycles, the process of which was: 95°C, duration 5 seconds; 60°C, duration 34 seconds; finally, melting, the temperature range was 60-95°C.

[0137] Real-time fluorescence quantitative PCR technology is used to identify the success of amplification by the degree of continuity and integrity of the amplification curve and the degree of amplification. The shape of the melting curve can reflect the purity of the PCR product. If the melting curve presents a single peak, it means that the amplification product has good specificity. If there is a mixed peak, the specificity is poor and there is non-specific amplification, such as primer dimers. When both parameters meet the requirements, according to the measured C TThe relative expression levels of each gene in the AGA-treated group (the corresponding gene expression levels in the blank control group were all set as 1) were used for analysis and comparison, and each group of experiments was repeated three times.

[0138] The calculation formula is:

[0139] ΔC T =C T Gene to be tested-C T Internal reference gene

[0140] ΔΔC T =ΔC T Treatment group-ΔC T Control group

[0141] mRNA fold change = 2 -ΔΔCT

[0142] The experimental results are as follows Figure 4 As shown, Figure 4 Shown is a schematic diagram of the expression changes of adhesion and hyphae formation related genes in Candida albicans SC5314 after AGA treatment compared with the blank control group. The horizontal axis is the related gene, and the vertical axis represents the multiple of gene expression difference (expressed in logarithmic form).

[0143] Conclusion: AGA at a concentration of 4 μg / mL can reduce the expression of genes related to adhesion and hyphae formation in Candida albicans SC5314, such as SAP5, ECE1, ALS3, HWP1, HGC1, CYR1, EFG1, RAS1, IFF4, ALS1 and YWP1( Figure 4 ). The average expression of RAS1, a common upstream gene of Cek-MAPK and cAMP-PKA pathways, decreased by 1.3 times, the average expression of CYR1 and EFG1, genes related to the cAMP-PKA pathway, decreased by 1.4 times and 2.2 times, respectively, the average expression of ALS3, ALS1 and IFF4, genes specific to adhesion, decreased by 6.2 times, 2.3 times and 1.8 times, respectively, and the average expression of hyphae-specific genes such as HGC1, SAP5, ECE1 and HWP1 decreased by 2.1 times, 1.3 times, 3.4 times and 2.5 times, respectively, compared with the untreated group. The results showed that AGA could inhibit the expression of genes related to Cek-MAPK, cAMP-PKA pathways, hyphae-specific genes and adhesion-specific genes in Candida albicans, further verifying that AGA could inhibit the transformation of Candida albicans to hyphae and its adhesion ability.

[0144] Example 6

[0145] Agaricin (AGA) has an inhibitory effect on Candida albicans biofilm

[0146] Materials and methods

[0147] 1. Experimental strains

[0148] Candida albicans experimental strain SC5314.

[0149] 2. Culture medium and drug preparation

[0150] YPD culture medium: Accurately weigh 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of glucose and add them into a 1-liter measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make up to 1 L. Stir with 121°C high-temperature and high-pressure steam for 15 min. Cool naturally to room temperature and store at 4°C for later use.

[0151] PBS buffer solution: 8.0 g sodium chloride, 3.57 g disodium hydrogen phosphate dodecahydrate, 0.2 g potassium chloride, 0.24 g dipotassium hydrogen phosphate, dissolved in ultrapure water and fixed to 1 L, sterilized at 121°C and high temperature and high pressure for 15 min, and then stored at room temperature.

[0152] RPMI 1640 medium: Accurately weigh 10.0 g of RPMI 1640, 2.0 g of sodium bicarbonate, 34.5 g of 3-morpholinepropanesulfonic acid, and 2.7 g of sodium hydroxide and transfer them to a measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make the volume up to 1 L. Filter and sterilize through a 0.22 μM microporous filter membrane and store at 4°C for later use.

[0153] Preparation of AGA and fluconazole stock solutions: Use DMSO as solvent to prepare the drug into 20 mg / mL stock solution for subsequent experiments.

[0154] 3. Activation of strains and preparation of bacterial suspension

[0155] Quickly take out the frozen sample of the desired strain from the -80℃ ultra-low temperature freezer, take 10μL of the bacterial solution and add it to a 15mL test tube containing 1mL YPD culture medium, place it in a 30℃ air bath shaking incubator, and shake it at 200rpm. After 24h, take out 10μL of the bacterial solution from the YPD culture medium and add it to a new 1mL YPD culture medium, and put it back into the 30℃ air bath shaking incubator again, and continue shaking and culturing overnight at 200rpm to make the concentration of the bacterial solution reach 1-2×10 8 cells / mL. Then, the bacterial solution was diluted to 5×10 5 -2×10 6 cells / mL(OD 600The culture medium is about 0.2), and then placed back in a shaking incubator at 30°C and cultured at a shaking speed of 200 rpm. After about 4-6 hours, the bacterial solution will reach 2×10 7 cells / mL, after 3-4 divisions, the fungus is at the end of the logarithmic (exponential) growth phase.

[0156] Transfer the cultured bacterial solution to a 1.5mL centrifuge tube, centrifuge at 5000g for 1min, discard the upper culture medium, wash the bacteria 2-3 times with PBS buffer solution to remove the residual culture medium. Resuspend the bacterial solution, pipette 10μL of the bacterial solution into 1mL PBS buffer solution and mix well, take 10μL and count it under a biological microscope using a hemocytometer to prepare for subsequent dilution into bacterial solutions of different concentrations.

[0157] 4. XTT method to determine the anti-biofilm activity of AGA

[0158] 4.1 Anti-biofilm formation test

[0159] Adhesion of biofilm: The strain to be tested was taken at the end of exponential growth and diluted to 2×10 5 CFU / mL of bacterial suspension was transferred to the wells of rows ABC in a 96-well cell culture plate treated with TC, with 100 μL of bacterial suspension in each well, excluding the last column, and the 96-well plate was placed in a 37°C incubator for 1.5 h.

[0160] During the incubation of the biofilm, the drug was prepared: Take a new 96-well cell culture plate, first add 300 μL of RPMI 1640 medium to the first column, and then add 150 μL to each of the remaining columns. Add the drug to each well in the first column so that the drug concentration in each well is 64 μg / mL, mix well, and then dilute in series from the first column to the tenth column.

[0161] Take out the incubated biofilm culture plate, slowly aspirate the RPMI 1640 medium in each well, and slowly wash the biofilm 2-3 times with PBS buffer solution to fully wash away the residual medium and suspended non-attached cells. Take the previously prepared drug solution, and aspirate 100 μL per well to each well of the 96-well plate where the biofilm is cultured. Add 100 μL of fresh RPMI 1640 medium to each well in the last column without biofilm as a blank control, and also add 100 μL of fresh RPMI 1640 medium to each well in the penultimate column with biofilm as a negative control. Continue to place the 96-well plate at 37°C for 24 hours.

[0162] The XTT method was used to determine the biofilm formation ability. First, the XTT reagent was prepared into a 0.5 mg / mL solution with PBS buffer, and 1 μL of the 10 mM menadione acetone solution prepared in advance was added to 10 mL of the XTT solution. Subsequently, the solution was sterilized by filtering with a 0.22 μM microporous filter membrane before use. Note that the XTT reaction solution needs to be prepared before use.

[0163] After 24 hours of drug action, take out the 96-well cell culture plate, slowly remove the upper culture medium, and wash 2-3 times with PBS buffer solution to clean the drug solution and suspended cells in the wells. Slowly add 120 μL of the prepared XTT solution to each well of the 96-well plate, wrap the 96-well plate with tin foil and place it in a 37°C constant temperature incubator to react for 3 hours. After the reaction is completed, take out the 96-well plate, take a new 96-well plate, absorb 70 μL of the supernatant of the reaction solution and transfer it to a new 96-well blank plate (be careful not to absorb the biofilm at the bottom of the well), and use a multifunctional microplate reader to measure the absorbance of each well at a wavelength of 492 nm.

[0164] The biofilm formation rate was analyzed by the absorbance value obtained, and the calculation method was: (OD 492 - Background control OD 492 ) / (Growth control OD 492 - Background control OD 492 )×100%. The experiment was repeated at least three times, and the data were analyzed by one-way analysis of variance (ANOVA), n=3. Figure 5 As shown in A, Figure 5 Schematic diagram of the inhibitory effect of AGA at different concentrations (2, 4, 8, 16, 32, 64 μg / mL) on the biofilm of Candida albicans SC5314 in Example 6, wherein A is a schematic diagram of the inhibitory effect of AGA on the formation process of the biofilm of Candida albicans SC5314. The horizontal axis is the AGA concentration (μg / mL), and the vertical axis is the biofilm formation rate (%). * represents P < 0.05, and **** represents P < 0.0001.

[0165] 4.2 Destruction of mature biofilm

[0166] Except that the time of biofilm culture is different from the above-mentioned biofilm formation, the rest of the experimental operations are basically the same. Cultivation of mature biofilm: After allowing Candida albicans to adhere for 1.5 hours, remove the 96-well plate, discard the upper culture solution, and slowly rinse with PBS buffer solution to wash away the non-adherent upper fungal cells, then add 150 μL of fresh RPMI 1640 culture medium to each well, and continue to incubate in a 37°C incubator for 24 hours to generate a mature biofilm. Subsequent experimental operations refer to the anti-biofilm formation experiment. Results are shown in Figure 5As shown in B, B is a schematic diagram of the destructive effect of AGA on the mature biofilm of Candida albicans SC5314. The horizontal axis is the AGA concentration, and the vertical axis is the biofilm formation rate. ** represents P < 0.01, and **** represents P < 0.0001.

[0167] Conclusion: Biofilm is considered to be the main growth state of many microorganisms. It has very strong drug resistance, and commonly used antifungal drugs are difficult to eliminate it in clinical applications. This experiment investigated the inhibitory effect of AGA on biofilms at two different growth and development stages: one is the initial stage of biofilm formation, when the drug was added after Candida albicans adhered for 90 minutes to evaluate the inhibitory effect of AGA on the biofilm formation process; the other is after the biofilm is fully mature, that is, after the initial formation of the film, the culture is continued for 24 hours and then the drug is added to evaluate the destructive effect of AGA on the mature biofilm that has been formed. The experimental results show that in the above two cases, AGA can not only inhibit the formation of Candida albicans biofilm, but also destroy the mature biofilm. The experimental results are as follows Figure 5 As shown in the results, compared with the group without drug addition, AGA at 4 μg / mL could significantly inhibit the formation of Candida albicans biofilm, with an inhibition rate of about 40% (P<0.05, Figure 5 The inhibition rate of AGA on biofilm formation was greater than 90% (P<0.0001, Figure 5 The results of the mature biofilm destruction experiment showed that the destructive effect of AGA on mature biofilm also showed obvious concentration dependence. More specifically, when the concentration reached 16μg / mL, the inhibition rate of AGA on mature biofilm was 52%, and the significance level was P<0.01 ( Figure 5 The inhibition rate of the 64μg / mLAGA group reached about 70%, with a significance level of P<0.0001 ( Figure 5 In general, AGA has an excellent anti-biofilm effect.

[0168] Example 7

[0169] Synergistic effect of agaric acid (AGA) and fluconazole against drug-resistant Candida albicans

[0170] Materials and methods

[0171] 1. Experimental strains

[0172] Candida albicans (C.albicans) clinical strains 901, 904, and 632 were purchased from Shanghai Changzheng Hospital.

[0173] 2. Culture medium and drug preparation

[0174] YPD culture medium: Accurately weigh 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of glucose and add them into a 1-liter measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make up to 1 L. Stir with 121°C high-temperature and high-pressure steam for 15 min. Cool naturally to room temperature and store at 4°C for later use.

[0175] PBS buffer solution: 8.0 g sodium chloride, 3.57 g disodium hydrogen phosphate dodecahydrate, 0.2 g potassium chloride, 0.24 g dipotassium hydrogen phosphate, dissolved in ultrapure water and fixed to 1 L, sterilized at 121°C and high temperature and high pressure for 15 min, and then stored at room temperature.

[0176] RPMI 1640 medium: Accurately weigh 10.0 g of RPMI 1640, 2.0 g of sodium bicarbonate, 34.5 g of 3-morpholinepropanesulfonic acid, and 2.7 g of sodium hydroxide and transfer them to a measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make the volume up to 1 L. Filter and sterilize through a 0.22 μM microporous filter membrane and store at 4°C for later use.

[0177] Sandburg Dextrose Agar (SDA): 10 g of peptone, 40 g of glucose, and 18 g of agar were dissolved in 900 mL of triple-distilled water, and 50 mL of 2 mg / mL chloramphenicol aqueous solution was added to adjust the pH to 7.0, and the volume was made up to 1000 mL. After autoclaving, the culture was stored at 4 °C.

[0178] Drug-containing SDA plate: accurately weigh 18g agar, 10g peptone, and 40g glucose into a measuring cylinder, add 200mL ultrapure water, stir thoroughly with a glass rod to dissolve the powder, continue to add ultrapure water to 1L, adjust the pH to 7.0, and sterilize at 121℃ with high pressure steam for 15min. After cooling to 50℃-55℃, add the AGA drug solution to the liquid SDA culture medium to make the final drug concentration 8μg / mL, mix well, and quickly pour into the culture dish. After cooling and solidifying, invert it in a 4℃ refrigerator for storage.

[0179] Preparation of AGA and fluconazole stock solutions: Use DMSO as solvent to prepare the drug into 20 mg / mL stock solution for subsequent experiments.

[0180] 3. Activation of strains and preparation of bacterial suspension

[0181] Quickly take out the frozen sample of the desired strain from the -80℃ ultra-low temperature freezer, take 10μL of the bacterial solution and add it to a 15mL test tube containing 1mL YPD culture medium, place it in a 30℃ air bath shaking incubator, and shake it at 200rpm. After 24h, take out 10μL of the bacterial solution from the YPD culture medium and add it to a new 1mL YPD culture medium, and put it back into the 30℃ air bath shaking incubator again, and continue shaking and culturing overnight at 200rpm to make the concentration of the bacterial solution reach 1-2×10 8 cells / mL. Then, the bacterial solution was diluted to 5×10 5 -2×10 6 cells / mL(OD 600 The culture medium is about 0.2), and then placed back in a shaking incubator at 30°C and cultured at a shaking speed of 200 rpm. After about 4-6 hours, the bacterial solution will reach 2×10 7 cells / mL, after 3-4 divisions, the fungus is at the end of the logarithmic (exponential) growth phase.

[0182] Transfer the cultured bacterial solution to a 1.5mL centrifuge tube, centrifuge at 5000g for 1min, discard the upper culture medium, wash the bacteria 2-3 times with PBS buffer solution to remove the residual culture medium. Resuspend the bacterial solution, pipette 10μL of the bacterial solution into 1mL PBS buffer solution and mix well, take 10μL and count it under a biological microscope using a hemocytometer to prepare for subsequent dilution into bacterial solutions of different concentrations.

[0183] 4. Investigating the synergistic antifungal effect of AGA and fluconazole by chessboard micro-liquid dilution method

[0184] Take the strain to be tested at the end of exponential growth and dilute it to 1×10 3CFU / mL bacterial suspension. Take 6 5mL centrifuge tubes and transfer the bacterial suspension to the centrifuge tubes, add 2.6mL bacterial solution to one of them, and add 1.3mL bacterial solution to the other 5 tubes. Add AGA mother solution to the first tube to make the drug concentration 64μg / mL, mix well and dilute in turn, so that the drug concentrations in the other 5 tubes are 32, 16, 8, 4, and 2μg / mL respectively. Add 6 tubes of bacterial suspension containing different concentrations of drugs to the AF rows of the 96-well cell culture plate, and the drug concentration is reduced exponentially. Add 200μL of drug-containing bacterial solution to the first column of the 96 wells, and 100μL to each of the 2nd to 10th columns. Add fluconazole mother solution to the AG wells in the first column, so that the drug concentration in each well is 64μg / mL. Dilute in turn from the first column to the 9th column, so that the concentrations of fluconazole are 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25μg / mL respectively. Blank bacterial solution and blank RPMI 1640 medium should be added to the blank wells, and 100 μL should be added to each well as negative control and blank control. After the 96-well cell culture plate is placed in a 30°C incubator for 48 hours, the absorbance value at a wavelength of 630 nm is measured using an ELISA reader. The synergy index (fractional inhibitory concentration index, FICI) is calculated using the formula.

[0185] The calculation formula of FICI is: FICI = MIC 90 AGA (combined) / MIC 90 AGA(single use)+MIC 90 FLC(combined) / MIC 90 FLC(single use)

[0186] The experimental results are shown in Table 3.

[0187] 5. Disc diffusion experiment to investigate the synergistic antifungal effect of AGA and fluconazole

[0188] Take the washed fungi to be tested and dilute the bacterial solution to a concentration of 1×10 6 CFU / mL, 100μL of bacterial solution was evenly applied to a drug-containing SDA plate and a drug-free SDA plate. Sterilized paper discs were placed equidistantly in the culture medium, and different concentrations of fluconazole solution were prepared and 5μL of drug solution was added to each paper disc, so that the drug content in the paper disc was 3.125, 6.25, 12.5, 25, and 50μg, respectively. The drug-containing SDA plate was placed in a 30℃ incubator for 48h, and the growth of the colony was observed and photographed for record. The experimental results are as follows Figure 6 As shown, Figure 6 Shown is a schematic diagram of the antibacterial effect of AGA combined with fluconazole on drug-resistant bacteria 904, in which fluconazole is represented by FLC.

[0189] Conclusion: FICI is the main parameter for evaluating the interaction between two drugs. When FICI is less than 0.5, it means that the two drugs have a synergistic effect, and the smaller the value, the stronger the synergistic effect; FICI values ​​between 0.5-1 indicate that the two drugs have an additive effect; when the FICI value is greater than 1 and less than or equal to 4, it means that the two drugs have an unrelated effect; when the FICI value is greater than 4, it means that the two drugs have an antagonistic effect. The experiment selected three strains of Candida albicans resistant to fluconazole, 901, 904 and 632. The results showed that the FICI of the combination of AGA and fluconazole was less than 0.5, and the combination of the two drugs had a synergistic effect (Table 3).

[0190] The disc diffusion experiment can more intuitively verify the synergistic antifungal effect of AGA and fluconazole. In the experiment, one of the resistant bacteria 904 was selected for a disc diffusion experiment on an agar plate containing AGA to examine the synergistic antifungal effect of AGA and fluconazole. The sparse colony area around the filter paper is the drug inhibition zone ( Figure 6 The results of the paper diffusion experiment showed that inhibition zones appeared around the paper discs with different concentrations of fluconazole in the plate without AGA ( Figure 6 ), the size of the inhibition zone showed a significant concentration-dependent relationship with the content of fluconazole. In the agar plate containing 8 μg / mL AGA, the inhibition zone produced by the same concentration of fluconazole was further expanded, and the antibacterial effect was more obvious ( Figure 6 The above experimental results show that the combined use of AGA and fluconazole has a synergistic effect against drug-resistant bacteria.

[0191] Table 3. Synergistic effect of AGA and fluconazole against fluconazole-resistant Candida albicans

[0192]

[0193] Example 8

[0194] Verification of the antifungal effect of agaric acid (AGA) in vivo

[0195] Materials and methods

[0196] 1. Experimental strains

[0197] Candida albicans experimental strain SC5314.

[0198] 2. Culture medium and drug preparation

[0199] YPD culture medium: Accurately weigh 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of glucose and add them into a 1-liter measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make up to 1 L. Stir with 121°C high-temperature and high-pressure steam for 15 min. Cool naturally to room temperature and store at 4°C for later use.

[0200] PBS buffer solution: 8.0 g sodium chloride, 3.57 g disodium hydrogen phosphate dodecahydrate, 0.2 g potassium chloride, 0.24 g dipotassium hydrogen phosphate, dissolved in ultrapure water and fixed to 1 L, sterilized at 121°C and high temperature and high pressure for 15 min, and then stored at room temperature.

[0201] RPMI 1640 medium: Accurately weigh 10.0 g of RPMI 1640, 2.0 g of sodium bicarbonate, 34.5 g of 3-morpholinepropanesulfonic acid, and 2.7 g of sodium hydroxide and transfer them to a measuring cylinder. Add 200 mL of ultrapure water and stir thoroughly with a glass rod to dissolve the powder. Continue to add ultrapure water to make the volume up to 1 L. Filter and sterilize through a 0.22 μM microporous filter membrane and store at 4°C for later use.

[0202] Preparation method of drug injection: Dissolve 0.9g NaCl in 100mL distilled water to make a clear 0.9% saline solution. Weigh 2g dry SBE-β-CD and dissolve it in 0.9% saline to make 10mL of 20% SBE-β-CD saline solution. Taking 1mL working solution as an example, take 100μL 20mg / mL clear DMSO drug stock solution and add it to 900μL 20% SBE-β-CD saline solution, mix well, and a uniform suspension with a maximum concentration of 2mg / mL can be obtained.

[0203] 3. Activation of strains and preparation of bacterial suspension

[0204] Quickly take out the frozen sample of the desired strain from the -80℃ ultra-low temperature freezer, take 10μL of the bacterial solution and add it to a 15mL test tube containing 1mL YPD culture medium, place it in a 30℃ air bath shaking incubator, and shake it at 200rpm. After 24h, take out 10μL of the bacterial solution from the YPD culture medium and add it to a new 1mL YPD culture medium, and put it back into the 30℃ air bath shaking incubator again, and continue shaking and culturing overnight at 200rpm to make the concentration of the bacterial solution reach 1-2×10 8 cells / mL. Then, the bacterial solution was diluted to 5×10 5 -2×10 6 cells / mL(OD 600 The culture medium is about 0.2), and then placed back in a shaking incubator at 30°C and cultured at a shaking speed of 200 rpm. After about 4-6 hours, the bacterial solution will reach 2×10 7 cells / mL, after 3-4 divisions, the fungus is at the end of the logarithmic (exponential) growth phase.

[0205] Transfer the cultured bacterial solution to a 1.5mL centrifuge tube, centrifuge at 5000g for 1min, discard the upper culture medium, wash the bacteria 2-3 times with PBS buffer solution to remove the residual culture medium. Resuspend the bacterial solution, pipette 10μL of the bacterial solution into 1mL PBS buffer solution and mix well, take 10μL and count it under a biological microscope using a hemocytometer to prepare for subsequent dilution into bacterial solutions of different concentrations.

[0206] 4. Modeling and drug administration of the wax moth fungus infection model

[0207] The wax moth should be first adapted to 37°C for 2 days, then 32 healthy wax moth larvae of similar size were selected and placed in the same plate, and their average weight was calculated as one group. According to this operation, a total of 5 groups of wax moth larvae were prepared (divided into blank control group, fluconazole 0.5 mg / kg group, AGA 0.25 mg / kg group, AGA 0.5 mg / kg group, and sham group, of which the fluconazole group had 16 wax moth larvae). Take the Candida albicans at the end of the exponential growth stage, wash it 2-3 times with PBS buffer solution, and resuspend the bacterial solution to a concentration of 1×10 8 CFU / mL. Then, 5 μL of bacterial solution was injected into the last leg on the left side of each group of wax moth larvae (the sham group was injected with an equal volume of PBS), and 30 minutes after the bacterial solution injection, 5 μL of drug injection solution diluent of corresponding concentration was injected into the last leg on the right side (the dosage of the drug solution here should be calculated based on the average weight of each group of wax moths, and diluted to the required concentration with drug injection solution) (the sham group was injected with an equal volume of PBS). Be sure to clean the syringe in ethanol and water before and after use. After the injection, place each dish in an incubator at 37°C, observe the death of wax moth larvae every day, and observe for 10 consecutive days. Record and draw the survival curve in detail, and perform Log-rank statistical analysis. The experimental results are as follows Figure 7 As shown, Figure 7 The figure shows the effect of AGA on the survival rate of G. mellonella larvae infected with Candida albicans. The horizontal axis represents the experimental days and the vertical axis represents the survival rate. *** represents P < 0.001, and **** represents P < 0.0001.

[0208] Conclusion: AGA concentrations of 0.25 and 0.5 mg / kg can significantly increase the survival time of larvae (P<0.001, P<0.0001). The mortality rate of wax moth larvae infected with Candida albicans in the control group was as high as 31% within 1 day, while no larvae died in the drug-treated group. The mortality rate of the control group was as high as 63% on the second day, and the mortality rate of each drug-treated group was no higher than 20%. On the 8th day after modeling, all the larvae in the control group died, while the survival rate was still greater than 16% when the concentration of AGA was 0.5 mg / kg. By the end of the observation period, there were still larvae surviving in the AGA-treated group. The above experiments show that AGA can protect wax moth larvae infected with Candida albicans, showing antifungal activity in vivo, and has a certain dose-dependent relationship.

[0209] Example 9

[0210] Study on the Cytotoxicity of Agaricus Acid (AGA)

[0211] Materials and methods

[0212] 1. Experimental cell lines

[0213] Mouse embryonic fibroblasts (NIH / 3T3) were purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences.

[0214] 2. Culture medium and drug preparation

[0215] DMEM complete medium: Add 5 mL of calf serum to 45 mL of DMEM high glucose medium, mix thoroughly, and store at 4°C.

[0216] Preparation of AGA stock solution: Use DMSO as solvent to prepare the drug into a 20 mg / mL stock solution for subsequent experiments.

[0217] 3. CCK-8 method to investigate drug toxicity to mammalian cells

[0218] Mouse embryonic fibroblasts (NIH / 3T3) were obtained, digested with trypsin, collected by centrifugation and resuspended in DMEM complete medium. The cell density was adjusted to 5×10 4 cells / mL. Take a 96-well plate and divide it into experimental area, control area, and cell-free area. Add 100μL of cell solution to each well of the experimental area and control area, and add 100μL of DMEM complete medium to the cell-free area. Pay attention to make the cells in the wells as evenly distributed as possible. The cells adhere to the wall in a 37℃ incubator overnight.

[0219] On the second day, take a sterile EP tube and dilute the drug to be tested with DMEM complete medium. Then take out the 96-well plate and discard the medium. Add 100 μL of DMEM complete medium containing different concentrations of drugs (AGA 8, 16, 32, 64 μg / mL) to each well and continue incubation at 37°C for 24 hours. Take out the 96-well plate, discard the medium, add 110 μL of DMEM complete medium containing CCK-8 to each well (CCK-8: DMEM complete medium = 1:10), and incubate at 37°C for 2 hours. Measure the OD of each well. 450 . When processing the results, use the experimental area and control area OD 450 The OD of the cell-free area was subtracted from the value 450 The survival of cells in each group was calculated by taking the survival of cells in the control area as 1.0. The experiment was repeated 3 times, and the graphs were drawn and statistically analyzed. Figure 8 As shown, Figure 8 The figure shows the toxic effect of AGA on mammalian cells (mouse embryonic fibroblasts NIH / 3T3). **** represents P < 0.0001.

[0220] Conclusion: The results showed that AGA showed certain toxicity when the concentration was 64 μg / mL, but AGA did not show significant cytotoxicity when the concentration was lower than 64 μg / mL ( Figure 8 ), which indicates that AGA has relatively low toxicity to mammalian cells represented by NIH / 3T3, high safety and specific antifungal effect.

[0221] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. Use of agaric acid or a pharmaceutically acceptable salt thereof in the preparation of an antifungal drug.

2. The use according to claim 1, characterized in that: The pharmaceutically acceptable salts refer to the following salts formed by agaricic acid and corresponding bases: sodium salt, calcium salt, potassium salt, zinc salt, and meglumine salt.

3. The use according to claim 1, characterized in that: The pharmaceutically acceptable salt refers to a salt formed by the reaction of agaricic acid with a pharmaceutically acceptable inorganic acid or organic acid.

4. The use according to claim 1, characterized in that: The fungus is selected from Candida albicans, Candida auris, Candida tropicalis, Candida parapsilosis, Candida krusei, Cryptococcus neoformans, Trichophyton mentagrophytes, and Trichophyton rubrum.

5. The use according to claim 1, characterized in that: In the application, agaricic acid is used as the only active ingredient.

6. A pharmaceutical preparation, characterized in that The invention is prepared from agaric acid or its pharmaceutical salt and medically acceptable auxiliary materials.

7. Use of agaric acid or a pharmaceutically acceptable salt thereof in combination with commonly used clinical antifungal drugs in the preparation of antifungal drugs.

8. The use according to claim 7, characterized in that: The commonly used clinical antifungal drugs are selected from fluconazole, ketoconazole, itraconazole, voriconazole, amphotericin B, caspofungin and terbinafine.

9. The use according to claim 7, characterized in that: The fungus is selected from Candida albicans, Candida auris, Candida tropicalis, Candida parapsilosis, Candida krusei, Cryptococcus neoformans, Trichophyton mentagrophytes, and Trichophyton rubrum.

10. A pharmaceutical preparation, characterized in that The invention is prepared from agaric acid or its pharmaceutical salt in combination with clinically commonly used antifungal drugs and medically acceptable auxiliary materials.

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