Use of larch mushroom acid in the preparation of antifungal drugs

Abstract

This invention discloses the application of larvicin or its pharmaceutical salt in the preparation of antifungal drugs. The structure of larvicin is shown below. This invention discovers an effective antifungal compound with pharmaceutical potential: larvicin. The compound larvicin of this invention exhibits clear antifungal activity against 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). 90 The range is 2–8 μg / mL.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to application of larch mushroom acid in preparation of antifungal drugs. BACKGROUND

[0002] Pathogenic fungi can cause serious harm to the human body, and the infection ranges from common, mild superficial infection to life-threatening invasive infection (IFIs). Most of the pathogenic fungi in IFIs are opportunistic, and usually occur in immunocompromised populations, such as HIV patients, cancer patients, radiotherapy and chemotherapy, organ transplant and immunosuppressive drug users. IFIs are a difficult problem worldwide, and the overall incidence and mortality continue to rise, with the number of people dying from fungal diseases worldwide rising to 3.75 million per year, equivalent to 7 people dying from IFIs every minute. Candida species is one of the main pathogenic bacteria of IFIs, and about 740,000 cases of invasive candidiasis (IC) are diagnosed worldwide each year, with a mortality rate of 35%-90%. Among them, Candida albicans is the most common pathogenic bacterium of IC, causing more than 40% of infections. In addition to being able to cause superficial and deep fungal infections in humans in yeast and hyphal states, Candida albicans can also attach to the surface of internal organs or biological materials by forming a structurally dense biofilm, and the biofilm is not only a virulence factor of Candida albicans but also an important mechanism for drug resistance and repeated infection of Candida albicans. There are limited drugs for the treatment of fungal infections in the clinic, mainly including azoles, echinocandins, polyenes and nucleosides, etc. With the increase of the use of drugs and the unreasonable use of antifungal drugs, the phenomenon of fungal drug resistance gradually increases in the clinic, which has become one of the main reasons for the failure of fungal infection treatment. In addition, some traditional antifungal drugs also have problems of high toxicity and high price, and the existing antifungal drugs are difficult to meet the clinical needs, and it is of great significance to develop new antifungal drugs with high efficiency and low toxicity.

[0003] Larch mushroom acid (2-hydroxy-1,2,3-nonadecanetricarboxylic acid, hereinafter referred to as AGA) is a main component in traditional Chinese medicine Sanghuang, and its structural formula is shown as (I), the molecular formula is C 22 H 40 O7, and the molecular weight is 416.5.

[0004]

[0005] Previous studies have shown that AGA has a significant antibiofilm effect on bacterial pathogens. AGA can down-regulate the transcription of genes responsible for flagellar movement, inhibit the formation of Salmonella biofilm by interfering with the motile phenotype, and also reduce the formation of Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli biofilms. Fungi and bacteria are different biological kingdoms, and there are huge differences in structure and physiological functions. Previous studies have mostly been reported for bacteria, and there is currently no relevant report on the research of anti-pathogenic fungi at home and abroad. SUMMARY

[0006] The purpose of the present application is to provide a use of larch mushroom acid in the preparation of an antifungal drug.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] In a first aspect of the present application, a use of larch mushroom acid or a pharmaceutically acceptable salt thereof in the preparation of an antifungal drug is provided.

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

[0010]

[0011] The pharmaceutically acceptable salt refers to the following salts formed by larch mushroom acid and the corresponding base: sodium salt, calcium salt, potassium salt, zinc salt and meglumine salt, etc.

[0012] The pharmaceutically acceptable salt refers to the salt formed after larch mushroom acid reacts 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; the organic acid is at least one of formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalene disulfonic acid, suberoylanilide hydroxamic acid, glycyrrhizic acid, glycyrrhetinic acid, oleanolic acid, maslinic acid, ursolic acid, colosseum acid, white birch acid, olibanic 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, sulfamic 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 fungi are selected from the group consisting of Candida albicans, Candida auris, Candida tropicalis, Candida parapsilosis, Candida krusei, Cryptococcus neoformans, Trichophyton mentagrophytes, Trichophyton rubrum, etc., and specifically are: 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 T5a; Trichophyton rubrum CMCC FTLA.

[0014] The effective concentration of the larch mushroom acid or the pharmaceutically acceptable salt thereof in the anti-Candida albicans drug is ≥4 μg / mL; the effective concentration of the larch mushroom acid or the pharmaceutically acceptable salt thereof in the anti-Candida auris drug is ≥4 μg / mL; the effective concentration of the larch mushroom acid or the pharmaceutically acceptable salt thereof in the anti-Candida tropicalis drug is ≥2 μg / mL; the effective concentration of the larch mushroom acid or the pharmaceutically acceptable salt thereof in the anti-Candida parapsilosis drug is ≥2 μg / mL; the effective concentration of the larch mushroom acid or the pharmaceutically acceptable salt thereof in the anti-Candida krusei drug is ≥2 μg / mL; the effective concentration of the larch mushroom acid or the pharmaceutically acceptable salt thereof in the anti-Cryptococcus neoformans drug is ≥4 μg / mL; the effective concentration of the larch mushroom acid or the pharmaceutically acceptable salt thereof in the anti-Trichophyton mentagrophytes drug is ≥4 μg / mL; and the effective concentration of the larch mushroom acid or the pharmaceutically acceptable salt thereof in the anti-Trichophyton rubrum drug is ≥4 μg / mL.

[0015] The application provides the use of the larch mushroom acid or the pharmaceutically acceptable salt thereof in the preparation of an anti-fungal drug, wherein the larch mushroom acid serves as the only active ingredient.

[0016] The application further provides a pharmaceutical preparation made of the larch mushroom acid or the pharmaceutically acceptable salt thereof and a medically acceptable auxiliary.

[0017] The dosage form of the pharmaceutical preparation is injection, capsule, tablet, granule, pill, micro-capsule preparation, micro-sphere preparation, nano-preparation, etc.

[0018] In the second aspect of the application, the use of the larch mushroom acid or the pharmaceutically acceptable salt thereof in the preparation of an anti-fungal drug in cooperation with a clinically commonly used anti-fungal drug is provided.

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

[0020] The pharmaceutical salt refers to the following salts of larch mushroom acid and corresponding bases: sodium salt, calcium salt, potassium salt, zinc salt and meglumine salt and the like.

[0021] The pharmaceutical salt refers to the salt formed after larch mushroom acid reacts 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; the organic acid is at least one of formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalene disulfonic acid, aspartic acid, carbenicillin, glycyrrhizic acid, oleanolic acid, maslinic acid, ursolic acid, corosolic acid, betulinic acid, latic 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, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, stearic acid, nicotinic acid, isonicotinic acid, benzene sulfonic acid, methyl sulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid or amino acid.

[0022] The fungi are selected from Candida albicans, Candida auris, Candida tropicalis, Candida parapsilosis, Candida krusei, Cryptococcus neoformans, Trichophyton mentagrophytes and Trichophyton rubrum; 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 T5a; Trichophyton rubrum CMCC FTLA.

[0023] The application also provides a pharmaceutical preparation made of larch mushroom acid or a pharmaceutical salt thereof in combination with a clinical commonly used antifungal drug and a medically acceptable excipient.

[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 application finds that, for fluconazole-resistant Candida albicans, AGA combined with 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 combination regimen is AGA concentration 0.5 μg / mL + fluconazole concentration 0.5 μg / mL or AGA concentration 1 μg / mL + fluconazole concentration 0.5 μg / mL.

[0026] The present application finds that larch mushroom acid has very low toxicity at an effective antifungal concentration, and is toxic to mammalian cells when the concentration is greater than or equal to 64 μg / mL.

[0027] In the present application, when using wax moth larvae (Example 8) as an in vivo experimental model of fungal infection, larch mushroom acid at a concentration of 0.25 mg / kg can significantly improve the survival rate of fungal-infected wax moth larvae.

[0028] Due to the use of the above technical solutions, the present application has the following advantages and beneficial effects:

[0029] The present application uses micro-liquid-based dilution method to widely screen natural compound libraries and finds that larch mushroom acid has good antifungal activity, and has good antifungal effect on pathogenic fungi such as Candida albicans, Candida auris, Candida krusei, Candida tropicalis, Candida parapsilosis, Cryptococcus neoformans, Trichophyton mentagrophytes, and Trichophyton rubrum. Larch mushroom acid has obvious synergistic effect on azole drugs against drug-resistant fungi. Larch mushroom acid can inhibit the formation process of fungal biofilm and destroy mature biofilm. Larch mushroom acid has in vivo antifungal activity and low toxicity. Larch mushroom acid is expected to be developed as a new antifungal drug, and the present application provides a new strategy for antifungal.

[0030] The present application finds an effective and drug-potential antifungal compound: larch mushroom acid (AGA). The compound larch mushroom acid in the present application has clear antifungal effect on pathogenic fungi such as Candida albicans, Candida auris, Candida krusei, Candida tropicalis, Candida parapsilosis, Cryptococcus neoformans, Trichophyton mentagrophytes, and Trichophyton rubrum, and the minimum inhibitory concentration MIC 90The range of AGA was 2-8 μg / mL. Larch mushroom acid can inhibit the growth of Candida albicans, inhibit the adhesion (0.5 μg / mL) and hyphal formation (0.25 μg / mL) process of Candida albicans, inhibit the formation process of Candida albicans biofilm (4 μg / mL) and destroy the mature biofilm (16 μg / mL). Larch mushroom acid also has good antifungal activity on fluconazole-resistant strains of Candida albicans, other naturally resistant strains of fluconazole (such as Candida auris, Candida krusei, Trichophyton mentagrophytes), and the combination of larch mushroom acid and fluconazole shows synergistic effect on resistant fungi. Larch mushroom acid can also effectively improve the survival rate of wax moth larvae infected with fungi in vivo, and has low toxicity to mammalian cells. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0033] Figure 3 is the inhibitory effect of AGA on the hyphal formation ability of Candida albicans SC5314.

[0034] Figure 4 is the expression change of adhesion and hyphal formation related genes in Candida albicans SC5314 after AGA treatment compared with the blank control group.

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

[0036] Figure 6 is the result of the inhibitory effect of AGA combined with fluconazole on drug-resistant strain 904.

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

[0038] Figure 8 is the result of the toxic effect of AGA on mammalian cells (mouse embryonic fibroblast NIH / 3T3). DETAILED DESCRIPTION

[0039] In order to make the present application clearer, the present application will be further described in conjunction with preferred embodiments. Those skilled in the art should understand that the specific description below is illustrative rather than limiting and should not limit the scope of the present application.

[0040] In the present application, it is found that the compound larch mushroom acid has good antifungal activity and synergistic antifungal activity with fluconazole against drug-resistant fungi, and the larch mushroom acid has low toxicity to mammalian cells.

[0041] In the present application, the fungi used can be obtained or cultured by conventional methods, and can also be obtained by commercial channels, such as isolation from somatic cells or tissues of patients with corresponding diseases, or purchased from any commercial standard strain agency or company, such as ATCC. It should be understood that the strain samples used in the embodiments of the present application are only exemplary examples, and any fungi that can form mycelium or biofilm or are resistant to fluconazole can be used to test the antifungal activity of the compounds of the present application, without limiting the scope of the embodiments. It should be understood that in the embodiments of the present application, only the minimum concentration of the active ingredient when effectively inhibiting the formation of fungal mycelium, biofilm, and destroying mature biofilm is written. It can be known by combining the common knowledge in the art that when the concentration of the antifungal active ingredient increases, the antifungal effect will be improved to a certain extent. Therefore, any person skilled in the art can use a dose higher than the minimum concentration to inhibit the growth of fungal mycelium or biofilm or destroy the mature biofilm after reading the present specification by combining the common knowledge in the art, and such changes also fall within the scope of the present application.

[0042] Example 1

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

[0044] Materials and methods

[0045] 1. Experimental strain

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

[0047] 2. Medium and drug preparation

[0048] YPD medium: 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of glucose were precisely weighed into a 1 L measuring cylinder, 200 mL of ultrapure water was added, a glass rod was used to stir the mixture until the powders were dissolved, and then ultrapure water was added to make up to 1 L. The mixture was sterilized by high-temperature high-pressure steam at 121 °C for 15 min, and then naturally cooled to room temperature. The solution was stored at 4 °C for later use.

[0049] PBS buffer solution: 8.0 g of sodium chloride, 3.57 g of disodium hydrogen phosphate, 0.2 g of potassium chloride, and 0.24 g of potassium dihydrogen phosphate were dissolved in 1 L of ultrapure water, and then sterilized by high-temperature high-pressure steam at 121 °C for 15 min. The solution was stored at room temperature for later use.

[0050] RPMI 1640 medium: 10.0 g of RPMI 1640, 2.0 g of sodium bicarbonate, and 34.5 g of 3-morpholino propanesulfonic acid, and 2.7 g of sodium hydroxide were precisely weighed into a measuring cylinder, and then 200 mL of ultrapure water was added. A glass rod was used to stir the mixture until the powders were dissolved, and then ultrapure water was added to make up to 1 L. The mixture was sterilized by filtration through a 0.22 μM microporous filter, and then stored at 4 °C for later use.

[0051] AGA and fluconazole stock solution preparation: DMSO was used as the solvent to prepare a 20 mg / mL stock solution of the drug for later use.

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

[0053] The frozen sample of the desired strain was quickly taken out from the ultra-low temperature freezer at -80°C, 10 μL of the bacterial solution was taken and added to a 15 mL capacity test tube containing 1 mL of YPD culture solution, and placed in a 30°C air bath shaking incubator, using a shaking speed of 200 rpm for shaking culture. After 24 h, 10 μL of the bacterial solution was taken from the YPD culture solution and added to a new 1 mL YPD culture solution, and again placed in a 30°C air bath shaking incubator, using a shaking speed of 200 rpm for continued shaking culture overnight, so that the concentration of the bacterial solution reached 1-2 x 10 8 cells / mL. Then the bacterial solution was diluted to 5 x 10 5 -2 x 10 6 cells / mL (OD 600 about 0.2) with YPD culture solution, and placed in a 30°C shaking incubator, using a shaking speed of 200 rpm for culture, after about 4-6 h, the bacterial solution would reach 2 x 10 7 cells / mL, after 3-4 divisions, the fungus was in the late logarithmic (exponential) growth phase.

[0054] The cultured bacterial solution was transferred to a 1.5 mL centrifuge tube, centrifuged at 5000 g for 1 min, and the supernatant was discarded, the bacterial cells were washed with PBS buffer solution for 2-3 times to remove residual culture medium. The bacterial solution was resuspended, 10 μL of the bacterial solution was taken and mixed with 1 mL of PBS buffer solution, and 10 μL was taken and counted under a biological microscope using a blood cell counting plate for subsequent dilution to different concentrations of bacterial solution.

[0055] 4. Micro-liquid based dilution method for determining minimum inhibitory concentration (MIC)

[0056] In this experiment, the micro-liquid based dilution method was used to determine the minimum inhibitory concentration according to the standard experimental method of CLSI micro-liquid based dilution method experimental manual M27-A3. In the experiment, the test strain was cultured to the end of the exponential growth phase, and the strain was diluted to 1 x 10 3 CFU / mL using RPMI 1640 culture medium, and then transferred to a 96-well cell culture plate, 200 μL of the bacterial solution was added to column 1, 100 μL of the bacterial solution was added to columns 3-12, and 100 μL of RPMI 1640 culture medium was added to the last column as a blank control. Then 6.4 μL of drug solution was added to each well of column 1 and mixed by blowing, and then diluted by a factor of 2 to column 10, and column 11 was not added with drug as a growth control. Then the 96-well plate was placed in a 30°C constant temperature incubator for 24 h or 48 h, and the absorbance value at a wavelength of 630 nm was measured using a microplate reader. The minimum drug concentration at which more than 90% of the fungus was inhibited was the MIC90 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 concentration of AGA and fluconazole against 21 strains of fungi

[0058]

[0059]

[0060] Conclusion: AGA showed good antifungal effect on 21 strains of test fungi. Stable inhibitory effect was observed on C. albicans SC5314 and 6 strains of fluconazole-sensitive clinical strains, with MIC 90 range of 4-8 μg / mL. At the same time, AGA also showed antifungal effect on three strains of fluconazole-resistant C. albicans (901, 904, 632), with MIC 90 similar to that of sensitive strains, i.e. 4 μg / mL; AGA also showed good antifungal activity against C. auris and C. krusei, which are naturally resistant to fluconazole, with MIC 90 of 4 μg / mL and 2 μg / mL, respectively. In addition, the MIC 90 of AGA against two strains of C. tropicalis and two strains of C. parapsilosis was 2 μg / mL. AGA also showed broad and stable antibacterial effect against non-Candida fungi such as C. neoformans, T. rubrum, and T. mentagrophytes, with MIC 90 of 4 μg / mL against T. mentagrophytes, while the control drug fluconazole still had no inhibitory effect at a concentration of 64 μg / mL. In summary, AGA showed good and stable antifungal activity against common pathogenic fungi.

[0061] Example 2

[0062] Effect of Larch Fungic Acid (AGA) on Inhibition of Growth of C. albicans

[0063] Materials and Methods

[0064] 1. Experimental Strains

[0065] C. albicans experimental strain SC5314.

[0066] 2. Culture Medium and Drug Preparation

[0067] YPD culture solution: 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of glucose were precisely weighed into a 1 L measuring cylinder, 200 mL of ultrapure water was added, a glass rod was used to stir thoroughly to dissolve the powder, and ultrapure water was added to make up to 1 L. The solution was sterilized by high-temperature high-pressure steam at 121°C for 15 min, naturally cooled to room temperature, and stored at 4°C for standby use.

[0068] PBS buffer solution: sodium chloride 8.0 g, disodium hydrogen phosphate 3.57 g, potassium chloride 0.2 g, potassium dihydrogen phosphate 0.24 g, dissolved with ultrapure water and constant volume to 1 L, sterilized at 121 ℃ high temperature and high pressure for 15 min, and stored at room temperature.

[0069] RPMI 1640 medium: precisely weigh RPMI 1640 10.0 g, sodium bicarbonate 2.0 g, 3-morpholinopropanesulfonic acid 34.5 g, sodium hydroxide 2.7 g, transfer to a graduated cylinder, add ultrapure water 200 mL, use a glass rod to stir thoroughly to dissolve the powder, continue to add ultrapure water to constant volume to 1 L, filter sterilization through 0.22 μM microporous filter, then store at 4 ℃ for standby use.

[0070] AGA, fluconazole stock solution: using DMSO as solvent, the drug is configured into a 20 mg / mL stock solution for subsequent experimental use.

[0071] 3. Activation of the strain and preparation of the bacterial suspension

[0072] The frozen sample of the required strain was quickly taken out from the -80 ℃ ultra-low temperature refrigerator, 10 μL of the bacterial solution was taken from it and added to a 15 mL capacity test tube containing 1 mL of YPD culture solution, which was placed in a 30 ℃ air bath shaking incubator, and the shaking culture was carried out using a shaking speed of 200 rpm. After 24 h, 10 μL of the bacterial solution was taken from the YPD culture solution and added to a new 1 mL of YPD culture solution, and was again placed in a 30 ℃ air bath shaking incubator, and the shaking culture was continued using a shaking speed of 200 rpm overnight, so that the concentration of the bacterial solution reached 1-2 × 10 8 cells / mL. Then the bacterial solution was diluted to 5 × 10 5 -2 × 10 6 cells / mL (OD 600 about 0.2) with YPD culture solution, and was placed in a 30 ℃ shaking incubator using a shaking speed of 200 rpm, and after about 4-6 h, the bacterial solution reached 2 × 10 7 cells / mL. After 3-4 divisions, the fungus was in the late logarithmic (exponential) growth phase.

[0073] The cultured bacterial solution was transferred to a 1.5 mL centrifuge tube, centrifuged at 5000 g for 1 min, and the supernatant was discarded, and the bacterial cells were washed with PBS buffer solution 2-3 times to remove residual culture medium. The bacterial solution was resuspended, 10 μL of the bacterial solution was taken and mixed with 1 mL of PBS buffer solution, and 10 μL was taken and counted under a biological microscope using a blood cell counting plate, for subsequent dilution to different concentrations of bacterial solution.

[0074] 4. Determination of growth curve

[0075] The test strain in the late exponential growth phase was diluted with RPMI 1640 medium to 2 x 10 5 CFU / mL of bacterial suspension. Take 7 small conical flasks, each add 10 mL of bacterial suspension, then add AGA mother liquor to 3 of them, respectively, to prepare a bacterial suspension containing 2, 4, 8 μg / mL of AGA, and add fluconazole with a final concentration of 2, 4, 8 μg / mL to the other 3 as a positive control group, and add DMSO with the same volume as the 8 μg / mL AGA group to the 7th conical flask as a blank control group. Place the conical flasks in a 30°C water bath shaker incubator at 200 rpm. At 2h, 4h, 6h, 8h, 12h, 24h, take 100 μL of bacterial solution from each conical flask into a 96-well plate (three wells per group), and use a microplate reader to measure the absorbance value of each well at a wavelength of 630 nm. The results are shown in Figure 1 Figure 1 The results of the inhibition of the growth of C. albicans SC5314 by AGA at different concentrations (2, 4, 8 μg / mL) and fluconazole are shown in the figure.

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

[0077] Example 3

[0078] Laricin (AGA) inhibits the adhesion of C. albicans

[0079] Materials and methods

[0080] 1. Test strain

[0081] C. albicans test 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 into a 1 L graduated cylinder, add 200 mL of ultrapure water, stir thoroughly with a glass rod to dissolve the powder, continue to add ultrapure water to 1 L, sterilize with 121°C high-temperature high-pressure steam for 15 min, naturally cool to room temperature, and store at 4°C for standby use.

[0084] ​PBS buffer solution: sodium chloride 8.0 g, sodium phosphate dibasic 3.57 g, potassium chloride 0.2 g, potassium phosphate dibasic 0.24 g, dissolved with ultrapure water and constant volume to 1 L, sterilized at 121 ℃ high temperature and high pressure for 15 min, and stored at room temperature.

[0085] RPMI 1640 medium: precisely weigh RPMI 1640 10.0 g, sodium bicarbonate 2.0 g, 3-morpholinopropanesulfonic acid 34.5 g, sodium hydroxide 2.7 g, transfer to a graduated cylinder, add ultrapure water 200 mL, use a glass rod to stir thoroughly to dissolve the powder, continue to add ultrapure water to constant volume to 1 L, filter sterilization through 0.22 μM microporous filter, then store at 4 ℃ for standby use.

[0086] AGA, fluconazole stock solution: using DMSO as solvent, the drug is configured into a 20 mg / mL stock solution for subsequent experimental use.

[0087] 3. Activation of the strain and preparation of the bacterial suspension

[0088] The frozen sample of the required strain was quickly taken out from the -80 ℃ ultra-low temperature refrigerator, 10 μL of the bacterial solution was taken from it and added to a 15 mL capacity test tube containing 1 mL of YPD culture solution, which was placed in a 30 ℃ air bath shaking incubator, and the shaking culture was carried out using a shaking speed of 200 rpm. After 24 h, 10 μL of the bacterial solution was taken from the YPD culture solution and added to a new 1 mL of YPD culture solution, and was again placed in a 30 ℃ air bath shaking incubator, and the shaking culture was continued overnight using a shaking speed of 200 rpm, so that the concentration of the bacterial solution reached 1-2 × 10 8 cells / mL. Then the bacterial solution was diluted to 5 × 10 5 -2 × 10 6 cells / mL (OD 600 about 0.2) with YPD culture solution, and was placed in a 30 ℃ shaking incubator using a shaking speed of 200 rpm, and after about 4-6 h, the bacterial solution reached 2 × 10 7 cells / mL. After 3-4 divisions, the fungus was in the late logarithmic (exponential) growth phase.

[0089] The cultured bacterial solution was transferred to a 1.5 mL centrifuge tube, centrifuged at 5000 g for 1 min, and the supernatant was discarded, and the bacterial cells were washed with PBS buffer solution 2-3 times to remove residual culture medium. The bacterial solution was resuspended, 10 μL of the bacterial solution was taken and mixed with 1 mL of PBS buffer solution, and 10 μL was taken and counted under a biological microscope using a blood cell counting plate, for subsequent dilution to different concentrations of bacterial solution.

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

[0091] The fungi mainly adhere to the surface of organs, macromolecular biological materials and the like in the host body. The stronger the hydrophobicity of the surface of the fungal cells, i.e. the stronger the lipophilic ability, the stronger the adhesion ability in the host body. Therefore, the adhesion ability of the fungal cells can be reflected by measuring the hydrophobicity of the surface of the fungal cells.

[0092] The fungal strain to be tested at the end of the exponential growth phase was resuspended in fresh RPMI 1640 medium to a concentration of 1 x 10 6 CFU / mL. Three 6-well cell culture plates treated with tissue culture (TC) were taken, 2 mL of the diluted fungal solution was added to each well, and incubation was performed at 37°C in an incubator for 90 min to allow the fungi to adhere to the bottom of the cell culture plate. The 6-well plate was taken out, the supernatant was slowly aspirated, and PBS buffer was slowly washed to wash away the residual medium and the unadhered suspended fungal cells. Then, 4 mL of RPMI 1640 medium containing drugs was added to each well, and the drug concentrations were 4, 2, 1, 0.5, 0.25 μg / mL, respectively. The same volume of DMSO was added to the blank control group. Each different drug concentration group was repeated three times, and the 6-well plate was placed in a 37°C incubator for 24 h of drug action culture.

[0093] Hydrophobicity determination: the 6-well plate was taken out, the supernatant was discarded, and the biofilm was completely washed down with PBS buffer solution as much as possible. The biofilm was washed multiple times to obtain as much biofilm as possible. Finally, the obtained biofilm suspension was centrifuged and washed twice, the biofilm was resuspended with PBS buffer solution, and placed in a 5 mL centrifuge tube for mixing. After mixing, 2 mL of the bacterial solution was transferred to a new 5 mL centrifuge tube and 0.5 mL of n-octane was added. After the water and hydrocarbon two phases were mixed thoroughly for several minutes, they were allowed to separate completely for 15 min. The upper n-octane was aspirated, and the lower water phase was taken to determine the absorbance at 630 nm. Each group had at least three repeated wells. The absorbance value of the group without drugs was taken as the negative control. The relative cell surface hydrophobicity calculation formula was (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 ANOVA (ANOVA), n = 3. The experimental results are shown in Figure 2 , Figure 2 is a schematic diagram of the inhibitory effect of AGA on the cell surface hydrophobicity of Candida albicans SC5314 from 0.25 μg / mL. The horizontal coordinate represents the AGA concentration, and the vertical coordinate indicates the size 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 C. albicans. 0.5 μg / mL of AGA can significantly reduce the relative cell surface hydrophobicity of C. albicans from 0.8 to 0.64 (P < 0.01, see Figure 2 ). With the increasing concentration of AGA, the decreasing trend of cell surface hydrophobicity gradually increases. Under the action of 4 μg / mL of AGA, the relative value of cell surface hydrophobicity of C. albicans decreases to 0.02 (P < 0.001, see Figure 2 ).

[0095] Example 4

[0096] Inhibitory effect of agaric acid (AGA) on hyphal formation of Candida albicans

[0097] Materials and methods

[0098] 1. Experimental strain

[0099] C. albicans experimental strain SC5314.

[0100] 2. Culture medium and drug preparation

[0101] YPD culture solution: 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of glucose were precisely weighed into a 1 L measuring cylinder, 200 mL of ultrapure water was added, a glass rod was used to fully stir to dissolve the powder, and then ultrapure water was added to make up to 1 L. After sterilization by high-temperature high-pressure steam at 121 °C for 15 min, it was naturally cooled to room temperature and stored at 4 °C for standby use.

[0102] PBS buffer solution: 8.0 g of sodium chloride, 3.57 g of disodium hydrogen phosphate, 0.2 g of potassium chloride, and 0.24 g of potassium dihydrogen phosphate were dissolved and made up to 1 L with ultrapure water, and then sterilized by high-temperature high-pressure steam at 121 °C for 15 min and stored at room temperature.

[0103] RPMI 1640 medium: 10.0 g of RPMI 1640, 2.0 g of sodium bicarbonate, 34.5 g of 3-morpholinopropanesulfonic acid, and 2.7 g of sodium hydroxide were precisely weighed and transferred to a measuring cylinder, 200 mL of ultrapure water was added, a glass rod was used to fully stir to dissolve the powder, and then ultrapure water was added to make up to 1 L. After sterilization by filtration with a 0.22 μM microporous filter, it was stored at 4 °C for standby use.

[0104] Spider liquid medium: 10 g of nutrient broth powder, 2.0 g of potassium phosphate dibasic, and 10 g of mannitol were precisely weighed into a measuring cylinder, 200 mL of ultrapure water was added, a glass rod was used to fully stir to dissolve the powder, and then made up to 1 L. After sterilization by high-temperature high-pressure steam at 121 °C for 15 min, it was stored in a 4 °C refrigerator under seal.

[0105] Lee's liquid medium: 0.0714 g of ornithine, 0.5 g of alanine, 0.5 g of threonine, 1.3 g of leucine, 0.5 g of proline, 1.0 g of lysine, 0.5 g of phenylalanine, 0.1 g of methionine, 0.5 g of ammonium sulfate, 0.357 g of magnesium sulfate heptahydrate, 0.25 g of potassium phosphate dibasic, 5.0 g of sodium chloride, 0.001 g of biotin, and 12.5 g of glucose were precisely weighed, dissolved in ultrapure water by ultrasonic dissolution, and then added with 0.5 g of ammonium sulfate. The mixture was stirred to completely dissolve. Then, water was added to make up to 1 L, and the pH value was adjusted to about 6.8. Finally, the mixture was sterilized by high-pressure sterilization, and then stored at room temperature after being sealed.

[0106] AGA, fluconazole mother liquor: DMSO was used as a solvent to prepare a 20 mg / mL mother liquor of the drug for subsequent experiments.

[0107] 3. Activation of the strain and preparation of the bacterial suspension

[0108] The frozen sample of the desired strain was quickly taken out from the -80°C ultra-low temperature freezer, and 10 μL of the bacterial solution was taken and added to a 15 mL capacity test tube containing 1 mL of YPD culture solution. The test tube was placed in a 30°C air bath shaking incubator, and the shaking speed was set to 200 rpm. After 24 h, 10 μL of the bacterial solution was taken from the YPD culture solution and added to a new 1 mL YPD culture solution, which was again placed in a 30°C air bath shaking incubator, and the shaking speed was set to 200 rpm. The bacterial solution was shaken overnight to make the concentration of the bacterial solution reach 1-2 x 10 8 cells / mL. Then, the bacterial solution was diluted to 5 x 10 5 -2 x 10 6 cells / mL (OD 600 about 0.2) with YPD culture solution, and was placed in a 30°C shaking incubator with a shaking speed of 200 rpm. After about 4-6 h, the bacterial solution reached 2 x 10 7 cells / mL. After 3-4 divisions, the fungus was in the late logarithmic (exponential) growth phase.

[0109] The cultured bacterial solution was transferred to a 1.5 mL centrifuge tube, and after centrifugation at 5000 g for 1 min, the upper culture medium was discarded. The bacterial cells were washed with PBS buffer solution for 2-3 times to remove the residual culture medium. The bacterial solution was resuspended, and 10 μL of the bacterial solution was taken and mixed with 1 mL of PBS buffer solution. Then, 10 μL of the mixture was taken and counted under a biological microscope using a blood cell counting plate for subsequent dilution to different concentrations of the bacterial solution.

[0110] 4. Mycelium culture

[0111] The strains to be tested at the end of the exponential growth phase were diluted with RPMI 1640 medium, Spider liquid medium and Lee's liquid medium to prepare a bacterial suspension of 2 x 10 5 CFU / mL. The diluted bacterial solution was added to a 24-well plate, 2 mL per well. AGA stock solution was added to each well to a final concentration of 1, 2, 4, 8, and 16 μg / mL, respectively, and the drug was mixed well with the bacterial solution. Each group had three replicates. In the blank control group, the same volume of DMSO as the drug solution was added. The plate was incubated in a 37°C incubator for 3 h. Then, photographs were taken under an inverted microscope to observe the growth of mycelium at different drug concentrations. ImageJ was used to measure the mycelium length. Five fields of view were selected for each group, and 20 mycelia were randomly measured in each field of view, for a total of n = 100. The measured lengths were statistically analyzed, and the experimental results were subjected to one-way ANOVA. The results are shown in Figure 3 , Figure 3 Figure 1 is a schematic diagram of the inhibitory effect of AGA on the mycelial formation of C. albicans SC5314. The photographs show the mycelial formation observed under an inverted microscope (the scale bar is 100 μm). The columnar statistical chart shows the average length of the mycelium in each group. * represents P < 0.05, and **** represents P < 0.0001.

[0112] Conclusion: AGA has a clear inhibitory effect on liquid mycelial formation. Without drug action, C. albicans SC5314 can form long, nodular-free mycelia at 37°C in RPMI 1640, Lee's, Spider, and other mycelial induction media. Figure 3 In RPMI 1640 medium, 0.25 μg / mL of AGA can significantly inhibit the formation of C. albicans mycelium, and 4 μg / mL of AGA can completely inhibit the formation of C. albicans mycelium, making it completely in the yeast state. This inhibitory effect on the formation of C. albicans mycelium is positively correlated with the concentration of AGA (P < 0.0001). In Lee's liquid medium and Spider liquid medium, C. albicans mycelium also begins to shorten under the action of 0.25 μg / mL of AGA, and the mycelium is significantly inhibited as the concentration of AGA increases. At a concentration of 4 μg / mL of AGA, most of the mycelium is inhibited, and the cells are in the yeast state (P < 0.0001). In summary, AGA can inhibit the formation of mycelium in three different mycelial induction media, and the effective concentration is lower than the minimum effective concentration required to inhibit biofilm formation.

[0113] Example 5

[0114] Effect of agaric acid (AGA) on the expression of adhesion and mycelium-related genes in C. albicans

[0115] Materials and methods

[0116] 1. Experimental strains

[0117] C. albicans experimental strain SC5314.

[0118] 2. Medium and drug preparation

[0119] YPD medium: precisely weigh 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of glucose into a 1 L measuring cylinder, add 200 mL of ultrapure water, and use a glass rod to stir thoroughly to dissolve the powder. Continue to add ultrapure water to make up to 1 L, and sterilize with 121 °C high-temperature high-pressure steam for 15 min. After natural cooling to room temperature, store at 4 °C for standby use.

[0120] PBS buffer solution: sodium chloride 8.0 g, disodium hydrogen phosphate 3.57 g, potassium chloride 0.2 g, potassium dihydrogen phosphate 0.24 g, dissolve with ultrapure water and make up to 1 L, sterilize at 121 °C high-temperature high-pressure for 15 min, and store at room temperature.

[0121] RPMI 1640 medium: precisely weigh 10.0 g of RPMI 1640, 2.0 g of sodium bicarbonate, 34.5 g of 3-morpholinopropanesulfonic acid, and 2.7 g of sodium hydroxide into a measuring cylinder, add 200 mL of ultrapure water, and use a glass rod to stir thoroughly to dissolve the powder. Continue to add ultrapure water to make up to 1 L, and sterilize by suction filtration with a 0.22 μM microporous filter. Then store at 4 °C for standby use.

[0122] AGA, fluconazole stock solution preparation: use DMSO as solvent to prepare a 20 mg / mL stock solution for subsequent experiments.

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

[0124] Rapidly remove the frozen sample of the desired strain from the -80 °C ultra-low temperature freezer, and take 10 μL of the bacterial solution and add it to a 15 mL capacity test tube containing 1 mL of YPD medium. Place it in a 30 °C air bath shaking incubator, and use a shaking speed of 200 rpm for shaking culture. After 24 h, take 10 μL of the bacterial solution from the YPD medium and add it to a new 1 mL of YPD medium, and again place it in a 30 °C air bath shaking incubator, and continue to shake culture overnight at a shaking speed of 200 rpm, so that the concentration of the bacterial solution reaches 1-2 × 10 8 cells / mL. Then dilute the bacterial solution to 5 × 10 5 -2 × 10 6 cells / mL (OD 600about 0.2), and put it back into the 30 °C shaking incubator, using a shaking speed of 200 rpm, after about 4-6 h, the bacterial solution will reach 2 x 10 7 cells / mL, after 3-4 divisions, the fungus is in the late logarithmic (exponential) growth phase.

[0125] The cultured bacterial solution is transferred to a 1.5 mL centrifuge tube, and after 5000g centrifugation for 1 min, the supernatant is discarded, and the bacterial cells are washed with PBS buffer solution for 2-3 times to remove residual culture medium. Resuspend the bacterial solution, take 10 μL of the bacterial solution into 1 mL of PBS buffer solution, mix well, and then take 10 μL under a biological microscope using a blood cell counting plate for counting, for subsequent dilution into different concentrations of bacterial solution.

[0126] 4. Design and synthesis of primers

[0127] First, search for the relevant gene sequence in the CGD database (http: / / www.candidagenome.org / ), and according to the obtained gene sequence, use Primer 3Input online design corresponding primers. The synthesized primers are purified by HAP and stored at -20 °C.

[0128] Table 2 Primer sequence

[0129]

[0130] 5. Extraction of total RNA of fungus

[0131] Take the late exponential growth phase of Candida albicans, wash it with PBS buffer solution for 3 times, and dilute the bacterial solution to 1 x 10 6 CFU / mL with fresh RPMI 1640 medium. Add 80 mL of diluted bacterial solution to a cell culture dish with a diameter of 150 mm, and incubate it in a 37 °C incubator for 90 min to form a biofilm. Then, slowly add RPMI 1640 medium containing 4 μg / mL AGA, and add an equal volume of DMSO to the blank control group, then continue to incubate the culture dish in a 37 °C incubator for 1 h. Then, carefully discard the supernatant, and use a cell scraper to scrape off the biofilm and collect it in a 50 mL centrifuge tube, and use PBS buffer solution to rinse the residual biofilm in the culture dish. Finally, centrifuge the biofilm at 5000g for 10 min, resuspend the collected biofilm with PBS buffer solution, and transfer it to an RNase-free 1.5 mL centrifuge tube for RNA extraction.

[0132] According to the steps of the fungal RNA extraction kit, the extracted RNA was detected for concentration. When the A260 / A280 value was between 1.8 and 2.1, it indicated that the RNA was of good purity, and the concentration value of the RNA should be above 20 ng / μL for subsequent experiments. The concentration value of each group of RNA was recorded for subsequent reverse transcription for RNA quantification.

[0133] 6. Reverse transcription to cDNA

[0134] The entire experimental process was operated on ice. Eight multi-tubes were taken, 8 μL of 5x PrimeScript RT Master Mix was added to each tube, and then the volume of RNA sample needed to be added was calculated according to the concentration of each tube of RNA sample determined previously. 500 ng of RNA was added to each tube, and then the total system was supplemented with DEPC water to 20 μL. After centrifugation of each multi-tube for 30 s to mix the liquid on the tube wall completely in the tube bottom, a PCR instrument was used for reverse transcription. The reverse transcription program was set as follows: 37℃ for 15 min, 85℃ for 5 s, and 4℃ for 5 min. The cDNA after completion of reverse transcription could be directly used for subsequent experiments or stored at -20℃.

[0135] 7. Real-time fluorescent quantitative PCR

[0136] β-actin was used as an internal reference gene, and the relative expression amount of the gene was calculated by ΔΔC T t method. First, the common reaction liquid for each well was configured. 10 μL of SYBR Green and 7.2 μL of deionized water should be added to each well. The two were mixed uniformly before being added to the 96-well PCR plate. Next, 0.8 μL of each pair of primer working solution was added, and 2.0 μL of diluted cDNA sample was added to each well. The total reaction liquid was 20 μL. After the addition was completed, the 96-well PCR plate was sealed and centrifuged for 30 s to centrifuge the liquid on the tube wall to the tube bottom and mix thoroughly. It was placed in a real-time fluorescent quantitative PCR instrument, and the PCR amplification program was set as follows: pre-denaturation temperature 95℃ for 30 s; followed by 40 cycles, the process being: 95℃ for 5 s; 60℃ for 34 s; and finally melting, the temperature range being 60-95℃.

[0137] The success of amplification was identified by the continuity and completeness of the amplification curve and the reaching degree of the amplification amount. The curve shape of the melting curve could reflect the purity of the PCR product. If the melting curve showed a single peak, it indicated that the amplification product had good specificity, and if there were miscellaneous peaks, the specificity was poor, and there was non-specific amplification, such as primer dimer. Under the condition that both parameters were investigated and met the requirements, the C TThe differences in values ​​were analyzed and compared using the relative expression levels of each gene in the AGA treatment group (with the expression levels of all corresponding genes in the blank control group set to 1). Each experiment was repeated three times.

[0138] The calculation formula is:

[0139] ΔC T =C T Test gene-C T Internal reference gene

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

[0141] fold change (mRNA fold change) = 2 -ΔΔCT

[0142] Experimental results are as follows Figure 4 As shown, Figure 4 The diagram shows the changes in the expression of adhesion and hyphal formation-related genes in Candida albicans SC5314 after AGA treatment compared to the blank control group. The horizontal axis represents the related genes, and the vertical axis represents the fold change in gene expression (in logarithmic form).

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

[0144] Example 6

[0145] Larvic acid (AGA) has an inhibitory effect on the biofilm of Candida albicans.

[0146] Materials and methods

[0147] 1. Experimental strains

[0148] C. albicans experimental strain SC5314.

[0149] 2. Medium and drug preparation

[0150] YPD medium: 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of glucose were precisely weighed into a 1 L measuring cylinder, 200 mL of ultrapure water was added, a glass rod was used to fully stir the powder to dissolve it, and then ultrapure water was added to make up to 1 L. The solution was sterilized by high-temperature high-pressure steam at 121 °C for 15 min, and then naturally cooled to room temperature before being stored at 4 °C for later use.

[0151] PBS buffer solution: 8.0 g of sodium chloride, 3.57 g of disodium hydrogen phosphate dodecahydrate, 0.2 g of potassium chloride, and 0.24 g of potassium dihydrogen phosphate were dissolved in 1 L of ultrapure water, and then sterilized by high-temperature high-pressure steam at 121 °C for 15 min before being stored at room temperature.

[0152] RPMI 1640 medium: 10.0 g of RPMI 1640, 2.0 g of sodium bicarbonate, and 34.5 g of 3-morpholinopropanesulfonic acid, and 2.7 g of sodium hydroxide were precisely weighed into a measuring cylinder, 200 mL of ultrapure water was added, a glass rod was used to fully stir the powder to dissolve it, and then ultrapure water was added to make up to 1 L. The solution was sterilized by filtration through a 0.22 μM microporous filter, and then stored at 4 °C for later use.

[0153] AGA, fluconazole stock solution preparation: DMSO was used as the solvent to prepare a 20 mg / mL stock solution of the drug for later use in experiments.

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

[0155] The frozen sample of the desired strain was quickly removed from the -80 °C ultra-low temperature freezer, and 10 μL of the bacterial solution was taken and added to a 15 mL capacity test tube containing 1 mL of YPD medium. The test tube was placed in a 30 °C air bath shaking incubator, and the shaking speed was set to 200 rpm. After 24 h, 10 μL of the bacterial solution was taken from the YPD medium and added to a new 1 mL of YPD medium, and then placed back in the 30 °C air bath shaking incubator, and the shaking speed was set to 200 rpm. The bacterial solution was continuously shaken overnight 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 600about 0.2), and put it back into the 30 °C shaking incubator, using a shaking speed of 200 rpm, after about 4-6 h, the bacterial solution will reach 2 x 10 7 After 3-4 divisions, the fungi are in the late logarithmic (exponential) growth phase.

[0156] The cultured bacterial solution is transferred to a 1.5 mL centrifuge tube, and after centrifugation at 5000 g for 1 min, the supernatant is discarded, and the bacterial cells are washed with PBS buffer solution 2-3 times to remove residual culture medium. Resuspend the bacterial solution, and after mixing 10 μL of the bacterial solution in 1 mL of PBS buffer solution, take 10 μL for counting under a biological microscope using a blood cell counting plate, for subsequent dilution to different concentrations of bacterial solution.

[0157] 4. XTT method for determining AGA antibiofilm activity

[0158] 4.1 Anti-biofilm formation experiment

[0159] Biofilm adhesion: Take the test strain in the late exponential growth phase and dilute it with RPMI 1640 medium to prepare a bacterial suspension of 2 x 10 5 CFU / mL. Transfer it to the ABC three rows of wells in the 96-well cell culture plate treated with TC, 100 μL of bacterial solution per well, excluding the last column, and place the 96-well plate in a 37 °C incubator for 1.5 h of incubation.

[0160] Drug preparation during biofilm incubation: Take a new 96-well cell culture plate, first add 300 μL of RPMI 1640 medium to the first column, and 150 μL to the remaining columns. Add drugs to each well in the first column to make the drug concentration in each well 64 μg / mL, and then dilute by half 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 gently wash the biofilm with PBS buffer solution 2-3 times to thoroughly wash away the residual culture medium and suspended non-adherent cells. Take the previously prepared drug solution, and aspirate 100 μL into each well of the 96-well plate containing the cultured biofilm. Add 100 μL of fresh RPMI 1640 medium to each well in the last column without biofilm as a blank control, and add 100 μL of fresh RPMI 1640 medium to each well in the second-to-last column with biofilm as a negative control. Continue to incubate the 96-well plate at 37 °C for 24 h.

[0162] The XTT method was used to determine the biofilm formation ability. The XTT reagent was prepared into a solution with a concentration of 0.5 mg / mL using PBS buffer, 10 mL of the XTT solution was added to 1 μL of the previously prepared 10 mM menadione acetone solution. Subsequently, the bacteria were filtered using a 0.22 μM microporous filter to remove bacteria, and the XTT reaction solution was prepared immediately before use.

[0163] The 96-well cell culture plate after 24 h of drug action was taken out, the upper culture medium was slowly sucked and discarded, and the PBS buffer solution was washed 2-3 times to wash the drug solution and suspended cells in the hole. The prepared XTT solution was slowly added to each well of the 96-well plate at 120 μL, the 96-well plate was wrapped with tin foil to avoid light, and the reaction was carried out in a 37°C constant temperature incubator for 3 h. After the reaction was completed, the 96-well plate was taken out, a new 96-well plate was taken, and 70 μL of the supernatant of the reaction solution was transferred to the new 96-well blank plate (note that the biofilm at the bottom of the hole should not be sucked). The multifunctional enzyme label instrument was used to determine the absorbance of each well at 492 nm wavelength.

[0164] The biofilm formation rate was analyzed by the obtained absorbance value, and the calculation method was: (experimental group OD 492 - background control OD 492 ) / (growth control OD 492 - background control OD 492 ) x 100%. The experiment was carried out at least three biological repeats, and the obtained data was analyzed by one-way ANOVA (ANOVA), n = 3. The results are shown in Figure 5 A of Figure 5 Figure 6 shows the inhibitory effect of different concentrations (2, 4, 8, 16, 32, 64 μg / mL) of AGA on the biofilm of Candida albicans SC5314, wherein A is the inhibitory effect of AGA on the formation process of the biofilm of Candida albicans SC5314. The horizontal coordinate is the AGA concentration (μg / mL), and the vertical coordinate 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 biofilm culture time was different from the above biofilm formation, the rest of the experimental operation was basically the same. Mature biofilm culture: after 1.5 h of adhesion of Candida albicans, the 96-well plate was taken out, the upper culture solution was discarded, and the PBS buffer solution was slowly washed to wash away the upper fungal cells that were not adhered, then 150 μL of fresh RPMI 1640 medium was added to each well, and the incubation was continued in a 37°C incubator for 24 h to form mature biofilm. The subsequent experimental operation was referred to the anti-biofilm formation experiment. The results are shown in Figure 5Figure 2A shows the inhibitory effect of AGA on the formation of mature biofilm. Figure 2B shows the inhibitory effect of AGA on the destruction of mature biofilm. The horizontal axis represents the concentration of AGA, and the vertical axis represents 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, which is very strong in drug resistance, and common antifungal drugs are difficult to remove in clinical application. This experiment investigated the inhibitory effect of AGA on biofilm at two different growth stages: one is the initial stage of biofilm formation, when Candida albicans adheres for 90 min, the drug is added to evaluate the inhibitory effect of AGA on the formation of biofilm; the other is after the biofilm is fully matured, that is, after the biofilm is preliminarily formed, the drug is added after 24 h of continuous culture to evaluate the destructive effect of AGA on the mature biofilm. The experimental results show that AGA can not only inhibit the formation of Candida albicans biofilm, but also destroy the mature biofilm in the above two cases. The experimental results are shown in Figure 5 As shown in Figure 2A, AGA can significantly inhibit the formation of Candida albicans biofilm from 4 μg / mL, with an inhibition rate of about 40% (P<0.05, Figure 2A), and the inhibition rate of AGA on biofilm formation is greater than 90% (P<0.0001, Figure 2A) from 8 μg / mL, and it is in a concentration-dependent manner. The results of the destruction of mature biofilm show that the destructive effect of AGA on mature biofilm also shows obvious concentration dependence. More specifically, when the concentration reaches 16 μg / mL, the inhibition rate of AGA on mature biofilm is 52%, with a significant level of P<0.01 (Figure 2B); and the inhibition rate of the 64 μg / mL AGA group is about 70%, with a significant level of P<0.0001 (Figure 2B). In general, AGA has excellent anti-biofilm effect. Figure 5 Figure 5 Example 7 Figure 5 Figure 5 Synergistic effect of AGA and fluconazole on drug-resistant Candida albicans

[0168] Materials and methods

[0169] Example 7

[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 solution: precisely weigh 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of glucose into a 1 L measuring cylinder, add 200 mL of ultrapure water, use a glass rod to stir thoroughly to dissolve the powder, continue to add ultrapure water to make up to 1 L, sterilize at 121°C high temperature and high pressure steam for 15 min, naturally cool to room temperature, and store at 4°C for standby use.

[0175] PBS buffer solution: sodium chloride 8.0 g, disodium hydrogen phosphate 3.57 g, potassium chloride 0.2 g, potassium dihydrogen phosphate 0.24 g, dissolve with ultrapure water and make up to 1 L, sterilize at 121°C high temperature and high pressure for 15 min, and store at room temperature.

[0176] RPMI 1640 medium: precisely weigh 10.0 g of RPMI 1640, 2.0 g of sodium bicarbonate, 34.5 g of 3-morpholinopropanesulfonic acid, and 2.7 g of sodium hydroxide into a measuring cylinder, add 200 mL of ultrapure water, use a glass rod to stir thoroughly to dissolve the powder, continue to add ultrapure water to make up to 1 L, sterile filter through a 0.22 μM microporous filter, and store at 4°C for standby use.

[0177] Sandcastle glucose agar medium (SDA): 10 g of peptone, 40 g of glucose, and 18 g of agar are added to 900 mL of triple distilled water, 50 mL of 2 mg / mL chloramphenicol aqueous solution is added, the pH is adjusted to 7.0, and the volume is made up to 1000 mL, and it is stored at 4°C after high pressure sterilization.

[0178] Drug-containing SDA plate: precisely weigh 18 g of agar, 10 g of peptone, and 40 g of glucose into a measuring cylinder, add 200 mL of ultrapure water, use a glass rod to stir thoroughly to dissolve the powder, continue to add ultrapure water to make up to 1 L, adjust the pH to 7.0, and sterilize at 121°C high pressure steam for 15 min. When cooled to 50-55°C, add AGA drug solution to the liquid SDA culture solution to make the final concentration of the drug 8 μg / mL, mix well, and quickly pour into a culture dish, and store in a 4°C refrigerator after cooling and solidification.

[0179] AGA, fluconazole stock solution: use DMSO as solvent to prepare a 20 mg / mL stock solution for subsequent experiments.

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

[0181] The frozen sample of the desired strain was quickly taken out from the ultra-low temperature freezer at -80°C, 10 μL of the bacterial solution was taken and added to a 15 mL capacity test tube containing 1 mL of YPD culture solution, and it was placed in a 30°C air-bath shaking incubator, using a shaking speed of 200 rpm for shaking culture. After 24 h, 10 μL of the bacterial solution was taken from the YPD culture solution and added to a new 1 mL YPD culture solution, and it was again placed in a 30°C air-bath shaking incubator, using a shaking speed of 200 rpm for continued shaking culture overnight, so that the concentration of the bacterial solution reached 1-2 x 10 8 cells / mL. Then the bacterial solution was diluted to 5 x 10 5 -2 x 10 6 cells / mL (OD 600 about 0.2) with YPD culture solution, and it was placed in a 30°C shaking incubator, using a shaking speed of 200 rpm for culture, after about 4-6 h, the bacterial solution would reach 2 x 10 7 cells / mL, after 3-4 divisions, the fungus was in the late logarithmic (exponential) growth phase.

[0182] The cultured bacterial solution was transferred to a 1.5 mL centrifuge tube, after centrifugation at 5000 g for 1 min, the supernatant was discarded, and the bacterial cells were washed with PBS buffer solution for 2-3 times to remove the residual culture medium. The bacterial solution was resuspended, 10 μL of the bacterial solution was taken and mixed with 1 mL of PBS buffer solution, and 10 μL of the mixture was taken and counted under a biological microscope using a blood cell counting plate, for subsequent dilution of the bacterial solution to different concentrations.

[0183] 4. Checkerboard microdilution method to investigate the synergistic antifungal effect of AGA and fluconazole

[0184] The strain to be tested in the late exponential growth phase was diluted to 1 x 10 3CFU / mL. Take 6 5 mL centrifuge tubes to transfer the bacterial suspension into the centrifuge tubes, 1 of which is added with 2.6 mL of the bacterial solution, and the other 5 tubes are added with 1.3 mL of the bacterial solution. Add the AGA mother liquor into the first tube to make the drug concentration 64 μg / mL, and then carry out the dilution by the ratio of 2 after mixing evenly, so that the drug concentrations in the other 5 tubes are 32, 16, 8, 4, and 2 μg / mL respectively. Add the bacterial suspensions with different concentrations of drugs into the A-F rows of the 96-well cell culture plate respectively, and the drug concentration is reduced by the ratio of 2, 200 μL of the bacterial solution with drugs is added into the first column of the 96-well plate, and 100 μL is added into the second to tenth columns. Add the fluconazole mother liquor into the A-G holes of the first column to make the drug concentration in each hole 64 μg / mL. Carry out the dilution by the ratio of 2 from the first column to the ninth column, so that the fluconazole concentrations are 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL respectively. The blank holes should be added with the blank bacterial solution and the blank RPMI 1640 culture medium, 100 μL of each is added into each hole as the negative control and the blank control. After the 96-well cell culture plate is placed in a 30°C incubator for incubation for 48 h, the absorbance value at 630 nm is measured by using an enzyme marker. The fractional inhibitory concentration index (FICI) is calculated by using the formula.

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

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

[0187] 5. Paper disc diffusion experiment for investigating the synergistic antifungal effect of AGA and fluconazole

[0188] Take the washed fungi to be tested, and dilute the bacterial solution to 1 x 10 6 CFU / mL with PBS buffer solution. Take 100 μL of the bacterial solution and evenly spread it on a drug-containing SDA plate and a drug-free SDA plate respectively. Place the sterilized paper discs equidistantly in the culture medium, and prepare different concentrations of fluconazole solution and add 5 μL of the solution to each of the paper discs, so that the drug content in the paper discs is 3.125, 6.25, 12.5, 25, and 50 μg respectively. Place the drug-containing SDA plate in a 30°C incubator for culture for 48 h, observe the growth of the colonies, and take photos for record. The experimental results are shown in Figure 6 Figure 6 The figure shows the results of the antibacterial effect of AGA combined with fluconazole on the drug-resistant bacteria 904, in which fluconazole is represented by FLC. ​

[0189] Conclusion: FICI is the main parameter to evaluate the interaction mode of two drugs. When FICI is less than 0.5, it represents that the two drugs have synergistic effect, and the smaller the value, the stronger the synergistic effect. When FICI is between 0.5 and 1, it represents that the two drugs have additive effect. When FICI is greater than 1 and less than or equal to 4, it represents that the two drugs have no effect. When FICI is greater than 4, it represents that the two drugs have antagonistic effect. The experiment selected three strains of fluconazole-resistant C. albicans 901, 904 and 632, and the results showed that the FICI of AGA combined with fluconazole was less than 0.5, and the two drugs had synergistic effect (Table 3).

[0190] Paper disc diffusion experiment can more directly verify the synergistic antifungal effect of AGA and fluconazole. One of the resistant strains 904 was selected for paper disc diffusion experiment on AGA-containing agar plates to investigate the synergistic antifungal effect of AGA and fluconazole against resistant fungi. The sparse area of colonies around the filter paper is the inhibition zone of the drug. Figure 6 The paper disc diffusion experiment results showed that in the plates without AGA, there were inhibition zones around the paper discs of different concentrations of fluconazole. Figure 6 In the agar plates containing 8 μg / mL AGA, the inhibition zones produced by the same concentration of fluconazole were further enlarged, and the inhibition effect was more obvious. Figure 6 The above experimental results showed that AGA combined with fluconazole had synergistic effect against resistant fungi.

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

[0192]

[0193] Example 8

[0194] In vivo verification of antifungal effect of agaric acid (AGA)

[0195] Materials and methods

[0196] 1. Experimental strains

[0197] C. albicans experimental strain SC5314.

[0198] 2. Culture medium and drug preparation

[0199] YPD culture solution: accurately weigh 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of glucose into a 1 L graduated cylinder, add 200 mL of ultrapure water, stir thoroughly with a glass rod until the powder is dissolved, continue to add ultrapure water to 1 L, sterilize by 121 ℃ high-temperature high-pressure steam for 15 min, naturally cool to room temperature, and store at 4 ℃ for standby.

[0200] PBS buffer solution: sodium chloride 8.0 g, sodium phosphate dibasic 3.57 g, potassium chloride 0.2 g, potassium phosphate dibasic 0.24 g, dissolved with ultrapure water and constant volume to 1 L, high temperature and high pressure sterilization at 121 ℃ for 15 min, and room temperature preservation.

[0201] RPMI 1640 medium: precisely weigh RPMI 1640 10.0 g, sodium bicarbonate 2.0 g, 3-morpholinopropanesulfonic acid 34.5 g, sodium hydroxide 2.7 g, transfer to a graduated cylinder, add ultrapure water 200 mL, use a glass rod to stir thoroughly to dissolve the powder, continue to add ultrapure water to constant volume to 1 L, filter sterilization through a 0.22 μM microporous filter, and then store at 4 ℃ for standby use.

[0202] Drug injection solution preparation method: dissolve 0.9 g of NaCl in 100 mL of distilled water to prepare a clear 0.9% saline solution. Weigh 2 g of dry SBE-β-CD and dissolve it in 0.9% saline to prepare a 10 mL SBE-β-CD physiological saline solution with a concentration of 20%. Take 100 μL of 20 mg / mL clear DMSO drug stock solution and add it to 900 μL of 20% SBE-β-CD physiological saline solution, mix well, and obtain a uniform suspension with a maximum concentration of 2 mg / mL.

[0203] 3. Activation of the strain and preparation of the bacterial suspension

[0204] The frozen sample of the desired strain was quickly taken out from the -80 ℃ ultra-low temperature freezer, 10 μL of the bacterial solution was taken from it and added to a 15 mL capacity test tube containing 1 mL of YPD culture solution, which was placed in a 30 ℃ air bath shaking incubator, and the shaking speed was 200 rpm. After 24 h, 10 μL of the bacterial solution was taken from the YPD culture solution and added to a new 1 mL of YPD culture solution, and was again placed in a 30 ℃ air bath shaking incubator, and the shaking speed was 200 rpm. The bacterial solution was continuously shaken overnight 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 about 0.2) with YPD culture solution, and was placed in a 30 ℃ shaking incubator with a shaking speed of 200 rpm. After about 4-6 h, the bacterial solution reached 2 × 10 7 cells / mL. After 3-4 divisions, the fungus was in the late logarithmic (exponential) growth phase.

[0205] The cultured bacteria solution was transferred to a 1.5 mL centrifuge tube, centrifuged at 5000g for 1 min, and the supernatant was discarded. The bacterial cells were washed with PBS buffer solution for 2-3 times to remove the residual culture medium. The bacterial solution was resuspended, 10 μL of the bacterial solution was taken and mixed with 1 mL of PBS buffer solution, and then 10 μL of the mixture was taken and counted under a biological microscope using a blood cell counting plate for subsequent dilution of the bacterial solution to different concentrations.

[0206] 4. Modeling and drug administration observation of galleria mellonella fungal infection model

[0207] The galleria mellonella was first acclimated at 37°C for 2 days, then 32 healthy galleria mellonella larvae with similar size were selected and placed in the same dish, and the average weight was calculated as a group. According to this operation, a total of 5 groups of galleria mellonella larvae were prepared (blank control group, fluconazole 0.5 mg / kg group, AGA 0.25 mg / kg group, AGA 0.5 mg / kg group, sham group, and fluconazole group with 16 galleria mellonella larvae). The Candida albicans at the end of the exponential growth phase was washed with PBS buffer solution for 2-3 times, and the bacterial solution was resuspended to a concentration of 1×10 8 CFU / mL. Then, 5 μL of the bacterial solution was injected into the left hind leg of each group of galleria mellonella larvae (sham group injected with equal volume of PBS), and 5 μL of the corresponding concentration of drug injection solution diluent was injected into the right hind leg 30 min after the injection of the bacterial solution (the drug injection solution should be diluted to the required concentration according to the average body weight of each group of galleria mellonella) (sham group injected with equal volume of PBS). The syringe was washed with ethanol and water before and after use. After injection, each dish was placed in a 37°C incubator, and the death of galleria mellonella larvae was observed every day, and the survival curve was drawn and analyzed by Log-rank statistical analysis. The experimental results are shown in Figure 7 Figure 7 The results show the effect of AGA on the survival rate of galleria mellonella infected with Candida albicans. The horizontal axis represents the number of days, and the vertical axis represents the survival rate. *** represents P<0.001, and **** represents P<0.0001.

[0208] ​Conclusion: The concentration of AGA at 0.25, 0.5 mg / kg can significantly improve the survival time of larvae (P < 0.001, P < 0.0001). The mortality rate of the control group of C. bombix infected with C. albicans was as high as 31% within 1 day, while no larvae died in the drug administration group. The mortality rate of the control group within 2 days was as high as 63%, while the mortality rate of the drug administration group was not higher than 20%. On the 8th day after modeling, all larvae in the control group died, while the survival rate of the group with AGA concentration of 0.5 mg / kg was still more than 16%. By the end of the observation period, there were still larvae surviving in the AGA administration group. The above experiments show that AGA can protect C. bombix infected with C. albicans, showing in vivo antifungal activity, and there is a certain dose-dependent relationship.

[0209] Example 9

[0210] Investigation of the cytotoxicity of AGA

[0211] Materials and methods

[0212] 1. Experimental cell line

[0213] Mouse embryonic fibroblasts (NIH / 3T3) were purchased from the China Academy of Sciences Typical Culture Preservation Committee Cell Library.

[0214] 2. Culture medium and drug preparation

[0215] DMEM complete medium: 5 mL of calf serum was added to 45 mL of DMEM high-sugar medium and mixed thoroughly, then stored at 4°C.

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

[0217] 3. Investigation of the toxicity of the drug to mammalian cells by CCK-8 method

[0218] Mouse embryonic fibroblasts (NIH / 3T3) were trypsinized, centrifuged to collect the cells, and resuspended with DMEM complete medium. After counting with a hemocytometer, the cell density was adjusted to 5 x 10 4 cells / mL. A 96-well plate was divided into experimental, control, and cell-free zones. 100 μL of cell solution was added to each well in the experimental and control zones, and 100 μL of DMEM complete medium was added to the cell-free zone. Care was taken to ensure uniform cell distribution in the wells. The cells were incubated overnight at 37°C in an incubator.

[0219] The next day, sterile EP tubes were used to serially dilute the test drug with DMEM complete medium. The 96-well plate was then removed, and the medium was discarded. 100 μL of DMEM complete medium containing different concentrations of the drug (AGA 8, 16, 32, 64 μg / mL) was added to each well, and the plate was incubated at 37°C for 24 h. The 96-well plate was then removed, the medium was discarded, and 110 μL of DMEM complete medium containing CCK-8 (CCK-8:DMEM complete medium = 1:10) was added to each well, and the plate was incubated at 37°C for 2 h. The OD values ​​of each well were measured. 450 When processing the results, use the OD values ​​of the experimental and control areas. 450 Value minus OD of cell-free region 450 The cell viability of each group was calculated using a value of 1.0, with the control group's cell viability set at 1.0. The experiment was repeated three times, and the results were plotted and statistically analyzed. The experimental results are shown below. Figure 8 As shown, Figure 8 This diagram illustrates the toxic effects of AGA on mammalian cells (mouse embryonic fibroblasts NIH / 3T3). **** represents P < 0.0001.

[0220] Conclusion: The results showed that AGA exhibited some toxicity at a concentration of 64 μg / mL, while AGA did not show significant cytotoxic effects below 64 μg / mL. Figure 8 This indicates that AGA has relatively low toxicity to mammalian cells, represented by NIH / 3T3, and is highly safe with specific antifungal activity.

[0221] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. Use of larch mushroom acid or a pharmaceutically acceptable salt thereof in combination with fluconazole for the preparation of a drug for resisting fluconazole-resistant Candida albicans.

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