Application of combination of tannic acid and triazole medicines in preparation of medicines for treating diseases caused by fungi

Abstract

The invention discloses application of tannic acid combined triazole medicines in preparation of medicines for treating diseases caused by fungi, and belongs to the technical field of antifungal. A series of drug sensitive tests show that tannic acid lower than 3 [mu] g / ml has no influence on the activity of human vaginal epithelial cells, and after the tannic acid is combined with fluconazole, the tannic acid can significantly inhibit the growth activity of drug-resistant candida (including drug-resistant candida albicans and drug-resistant candida glabrata). In view of the natural property, wide source and low toxicity of tannic acid, the invention provides a new antifungal treatment strategy, and the tannic acid has important clinical application value for treating infection (such as vaginal candidiasis) caused by drug-resistant fungi, especially under the condition that the existing antifungal drugs are poor in effect. Therefore, the tannic acid combined triazole medicine can be used for preparing the medicine for treating diseases caused by fungi.

Description

Technical Field

[0001] The invention belongs to the technical field of antifungal drugs, and particularly relates to an application of tannic acid combined with triazole drugs in preparing drugs for treating diseases caused by fungi. Background Art

[0002] In recent years, the incidence of invasive fungal infections has been increasing year by year, becoming one of the leading causes of mortality in high-risk patients. Common pathogenic fungi include Candida, Aspergillus, and Cryptococcus neoformans. Of all invasive infections, 90% are caused by opportunistic Candida species, including Candida albicans, Candida glabrata, Candida tropicalis, Candida, and Candida krusei.

[0003] Traditional antifungal drugs mainly include polyenes, azoles, echinocandins, and the pyrimidine analog 5-fluorocytosine. Due to the increasing resistance of fungi to these drugs, treatment failure has become common. Azoles are currently the first-line drugs for the treatment of various deep and superficial fungal diseases. Among them, fluconazole is a new triazole broad-spectrum antifungal drug that can inhibit the biosynthesis of ergosterol in the fungal cell membrane, thereby destroying the integrity of the fungal cell membrane and leading to fungal cell death. However, with the long-term and widespread use of azoles, drug-resistant strains have gradually appeared in clinical practice, and there is a clear trend of increase.

[0004] Tannic acid, also known as tannic acid, is a natural polyphenolic compound found in a variety of plants. It exhibits a wide range of biological activities, including antioxidant, anti-inflammatory, and antibacterial properties. Tannic acid also exhibits potential antifungal activity, with strong inhibitory effects against Escherichia coli, Staphylococcus aureus, Salmonella, and Helicobacter pylori.

[0005] Currently, there are no reports on the antifungal effects of tannic acid combined with triazole drugs. Summary of the Invention

[0006] Purpose of the Invention: This invention aims to address the problem of Candida resistance by providing a method for the use of tannic acid in combination with a triazole drug in the preparation of a drug for treating fungal diseases. Using the triazole drug fluconazole as an example, this invention evaluated the in vitro antifungal activity of tannic acid combined with fluconazole against drug-resistant Candida albicans and Candida glabrata, providing insights into addressing the clinical challenge of fungal resistance.

[0007] The present invention provides a treatment plan that enhances antifungal effects and reduces the development of drug resistance by combining the natural polyphenol compound tannic acid with fluconazole.

[0008] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0009] The present invention provides an application of tannic acid combined with triazole drugs in the preparation of medicines for treating diseases caused by fungi.

[0010] The present invention also provides an application of tannic acid combined with triazole drugs in the preparation of antifungal products.

[0011] Tannic acid is a natural compound widely found in plants. It is widely available, easily accessible, has low toxicity, and exhibits strong antimicrobial activity. Tannic acid's natural origin and low toxicity make it an attractive candidate for combination therapy. The present invention employs a strategy of combining tannic acid with a triazole drug. Compared to monotherapy, the combined use of two synergistic antifungal drugs offers significant advantages in reducing drug resistance and helps prevent the development of multidrug-resistant bacteria.

[0012] Tannic acid may improve the therapeutic effect by enhancing the penetration of triazole drugs or interfering with the fungal resistance mechanism.

[0013] Preferably, the triazole drug is fluconazole, voriconazole, itraconazole and posaconazole.

[0014] More preferably, the triazole drug is fluconazole.

[0015] Preferably, the fungus is Candida. Polymorphic opportunistic Candida can cause fungal infections including vaginal candidiasis.

[0016] Further preferably, the Candida albicans is drug-resistant Candida albicans or drug-resistant Candida glabrata.

[0017] The present invention has experimentally found that the combination of tannic acid and fluconazole has a synergistic effect on drug-resistant Candida albicans and drug-resistant Candida glabrata, which may indicate that the commonality of triazole drugs in structure or mechanism of action leads to similar synergistic effects.

[0018] In the present invention, the clinical drug-resistant Candida albicans (06-37) strain has a preservation number of 06-37; the drug-resistant Candida albicans (09-07) strain has a preservation number of 09-07; the drug-resistant Candida albicans (C1-17) strain has a preservation number of C1-17; the drug-resistant Candida albicans (C1-19) strain has a preservation number of C1-19; and the clinical drug-resistant Candida glabrata (Y10-5) strain has a preservation number of Y10-5. The above-mentioned Candida albicans is preserved in the Fungal Center of the Pathogenic Microorganisms (Viruses) Collection Management Center of the Chinese Academy of Medical Sciences. The clinical resistant Candida albicans (06-37) strain and the resistant Candida albicans (09-07) strain are preserved in May 2021; the resistant Candida albicans (C1-17) strain and the resistant Candida albicans (C1-19) strain are preserved in March 2009; the clinical resistant Candida glabrata (Y10-5) strain is preserved in June 2011.

[0019] The medicine comprises tannic acid, triazole drugs and pharmaceutically acceptable carriers or excipients.

[0020] The excipients include one or more of antioxidants, lubricants, emulsifiers, diluents, preservatives, wetting agents, binders or disintegrants.

[0021] The dosage form of the drug is tablets, granules, capsules, pills, oral liquids, injections, infusions, gels, ointments, patches or creams.

[0022] The medicament of the present invention can be administered in various known ways, such as orally, by injection, or by transdermal administration. Oral compositions can be in any orally acceptable dosage form, including but not limited to tablets, granules, capsules, pills, and oral liquids. Dosage forms for transdermal administration include but are not limited to gels, ointments, patches, or creams.

[0023] Sterile injectable compositions can be formulated using suitable dispersing agents or wetting agents and suspending agents according to techniques known in the art. Pharmaceutically acceptable carriers and solvents that can be used include water, sodium chloride solution, and the like.

[0024] The antifungal products include antifungal drugs and antifungal inhibition model tools. These antifungal inhibition model drugs mainly include azoles, polyenes, and echinocandins. By targeting cell membranes, cell walls, or nucleic acid metabolic pathways, they play a key role in drug resistance mechanism research, combination drug evaluation, and new drug screening.

[0025] The maximum concentration of tannic acid that does not damage human vaginal epithelial cells is 3 μg / ml. Tannic acid is used at low concentrations and has no toxic effects on human vaginal epithelial cells. The concentration of tannic acid used has no toxic effects on mammalian cells in vitro.

[0026] The present invention also provides a drug combination of tannic acid and fluconazole, wherein the effective concentration ratio of tannic acid to fluconazole is 3 μg / ml: 0.25-0.5 μg / ml.

[0027] The present invention also provides use of the above-mentioned drug combination of tannic acid and fluconazole in reducing fungal drug resistance.

[0028] The effective concentration ratio of the tannic acid and fluconazole for significantly inhibiting the activity of drug-resistant Candida albicans or drug-resistant Candida glabrata in vitro is 3 μg / ml: 0.25-0.5 μg / ml.

[0029] The tannic acid significantly improves the sensitivity of drug-resistant Candida albicans to fluconazole in vitro.

[0030] The tannic acid significantly improves the sensitivity of drug-resistant Candida glabrata to fluconazole in vitro.

[0031] The disease is a skin candidiasis, a mucosal candidiasis, a visceral candidiasis or a central nervous system candidiasis.

[0032] The skin candidiasis includes candidal infections of the fossa, groin, under the breast, anus, nail groove, and between the fingers.

[0033] Beneficial effects:

[0034] The present invention provides a new approach for preparing drugs for treating drug-resistant Candida albicans and drug-resistant Candida glabrata infections by using tannic acid in combination with fluconazole. The present invention shows through a series of drug sensitivity tests that tannic acid below 3 μg / ml has no effect on the activity of human vaginal epithelial cells, while in combination with fluconazole it can significantly inhibit the growth activity of drug-resistant Candida (including drug-resistant Candida albicans and drug-resistant Candida glabrata), thereby increasing the sensitivity of drug-resistant Candida to triazole drugs. In addition, tannic acid is used at a low concentration and has no toxic effect on human vaginal epithelial cells. Therefore, in view of the natural properties, wide sources and low toxicity of tannic acid, the present invention provides a new antifungal treatment strategy, which has important clinical application value for treating infections caused by drug-resistant fungi (such as vaginal candidiasis), especially when existing antifungal drugs are ineffective. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The effect of different concentrations of tannic acid on the activity of human vaginal epithelial cells.

[0036] Figure 2 This is a growth dynamic diagram of drug-resistant Candida albicans 06-37 under the combined action of tannic acid and fluconazole.

[0037] Figure 3 This is a growth dynamic diagram of drug-resistant Candida albicans 09-07 under the combined action of tannic acid and fluconazole.

[0038] Figure 4 This is a growth dynamic diagram of drug-resistant Candida albicans C1-17 under the combined action of tannic acid and fluconazole.

[0039] Figure 5 This is a growth dynamic diagram of drug-resistant Candida albicans C1-19 under the combined action of tannic acid and fluconazole.

[0040] Figure 6 This is a growth dynamic diagram of drug-resistant Candida glabrata Y10-5 under the combined action of tannic acid and fluconazole. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0042] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0043] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0044] The bacterial culture medium used in the following examples is as follows:

[0045] Solid yeast extract peptone dextrose medium (YPD): peptone 10 g / L, glucose 20 g / L, yeast extract 5 g / L, agar powder 15 g / L, autoclave at 121°C for 20 min and set aside.

[0046] The inoculation method of the test bacteria in the following examples is as follows:

[0047] Before the experiment, irradiate the clean bench with UV light for 30 minutes. Turn on the alcohol lamp to ensure a sterile environment within the working range. Remove the Candida albicans for experimental use from the -80°C freezer and thaw at room temperature. In the clean bench, use a disposable sterile inoculating loop to gently dip a small amount of bacterial solution. Using the four-zone method, apply the inoculating loop containing the bacterial solution to YPD agar medium. Place the medium in a 35°C incubator for 48 hours.

[0048] In the following examples, the tannic acid solution is as follows:

[0049] Precision weigh tannic acid powder (purity >99%) (manufacturer: Wenzhou Liren Technology) using an analytical balance. RPMI1640 medium (Wuhan Punosai) was used as the solvent for the tannic acid. Filter-sterilize the prepared tannic acid solution using a 0.22 μm filter. Store the tannic acid solution at -20°C in the dark.

[0050] Example 1 Effect of Tannic Acid on the Activity of Human Vaginal Epithelial Cells

[0051] Human vaginal epithelial cells VK2 / E6E7 were derived from normal female vaginal mucosa and immortalized with HPV16E6E7. The ATCC number of this cell line is CRL-2616. Cells were cultured in DefinedK-SFM (Gibco) medium at a concentration of 1×10 cells per well. 5The cells were inoculated into 96-well plates, and tannic acid was added at final concentrations of 0, 0.125 μg / ml, 0.25 μg / ml, 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 2.5 μg / ml and 3 μg / ml, respectively, and placed in a 37°C incubator for 24 h, 48 h and 72 h. Subsequently, 20 μl of CCK8 solution reagent (Biyuntian) was added to each well, mixed well and placed in an incubator for 4 h. Finally, the absorbance of each well was measured at a wavelength of 490 nm using a microplate reader. The experimental results are shown in Figure 1 .

[0052] like Figure 1 As shown in the results, after 24h, 48h, and 72h of treatment with different concentrations of tannic acid, at low concentrations, the activity of human vaginal epithelial cells was significantly promoted compared to the control group without tannic acid. Moreover, at a concentration of 3μg / ml, tannic acid had no effect on cell activity. This indicates that tannic acid does not inhibit the activity of human vaginal epithelial cells at concentrations below 3μg / ml and has no toxic effect on the cells.

[0053] Example 2 Antifungal Susceptibility Test

[0054] Antifungal susceptibility testing was performed using the broth microdilution method for the test drugs in accordance with CLSI (Clinical and Laboratory Standards Institute) document M27-A3. The MIC was visually defined as the lowest drug concentration that caused a significant decrease compared to the growth control. All experiments were repeated three times with three replicates each time.

[0055] This experiment selected Candida isolated from the blood of clinical patients: clinical fluconazole-resistant Candida albicans strains (06-37, 09-07, C1-17 and C1-19), clinical fluconazole-resistant Candida glabrata (Y10-5). The Candida mentioned above are preserved at the Fungal Center of the Pathogenic Microbial Collection Management Center of the Chinese Academy of Medical Sciences. The clinical drug-resistant Candida albicans (06-37) strain and the drug-resistant Candida albicans (09-07) strain were preserved in May 2021; the drug-resistant Candida albicans (C1-17) strain and the drug-resistant Candida albicans (C1-19) strain were preserved in March 2009; the clinical drug-resistant Candida glabrata (Y10-5) strain was preserved in June 2011. The sample was inoculated onto YPD solid culture medium, cultured at 35°C for 48 hours, and then the colonies were picked. Prepare a concentration of 3×10 3 CFU / ml of bacterial suspension.

[0056] The concentrations of fluconazole (FCZ) (RPMI 1640 medium as solvent) were 0.25 μg / ml, 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml, 8 μg / ml, 16 μg / ml, 32 μg / ml, 64 μg / ml and 128 μg / ml, which were added to columns 2 to 11 of a 96-well plate.

[0057] The concentrations of tannic acid and fluconazole combined were as follows: 3 μg / ml tannic acid (TA) + fluconazole, in descending order of concentration, 0.25 μg / ml, 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml, 8 μg / ml, 16 μg / ml, 32 μg / ml, 64 μg / ml, and 128 μg / ml, which were added sequentially to columns 2 through 11 of a 96-well plate. 200 μl of RPMI-1640 was added to columns 1 and 12 of the 96-well plate to prevent the wells from drying out.

[0058] The growth control group contained only the test bacteria without any drug, and the blank control group contained only RPMI-1640 liquid. The treated 96-well plates were placed in a 35°C incubator for 24 hours. The minimum inhibitory concentration (MIC) was observed visually and recorded. The experimental results are shown in Table 1:

[0059] Table 1 Inhibitory effect of tannic acid combined with fluconazole on clinical drug-resistant Candida

[0060]

[0061] As can be seen from Table 1, the significant inhibitory concentration of fluconazole against clinically isolated Candida albicans and Candida glabrata needs to reach 128 μg / ml. When combined with 3 μg / ml of tannic acid, only 0.25 μg / ml or 0.5 μg / ml of fluconazole is required to achieve the effect of significantly inhibiting the growth of Candida.

[0062] Example 3 Effect of Tannic Acid Combined with Fluconazole on the Proliferation of Drug-Resistant Candida

[0063] The drug-resistant Candida albicans 06-37, 09-07, C1-17, C1-19 and the drug-resistant Candida glabrata Y10-5 were cultured in RPMI 1640 medium at a concentration of 3 × 10 3 The cells were cultured at a density of CFU / ml in a 30°C incubator for 48 h.

[0064] After the test bacteria were grown on YPD solid medium for 48 h, 3-5 colonies were picked and prepared with RPMI 1640 medium at a concentration of 3×10 3CFU / ml bacterial suspension. 100 μl of the test bacterial suspension was inoculated into each well of a 96-well cell culture plate. 100 μl of 128 μg / mL fluconazole alone, 100 μl of 3 μg / mL tannic acid solution, or 100 μl (0.5 μg / mL to 0.25 μg / mL fluconazole + 3 μg / mL tannic acid solution) FCZ+TA was added to the wells containing the test bacterial suspension. The final volume in each well was 200 μL. The 96-well cell culture plate was then placed in a real-time label-free cell dynamics analyzer (Nanoanalytics). Data was captured every 6 hours, and the growth dynamics of the test bacteria were monitored for a total of 48 hours.

[0065] The experimental results are shown in Figure 2-Figure 6 In the figure, FCZ is fluconazole; FCZ+TA means the combined use of fluconazole and tannic acid, TA is tannic acid, and the control group only has the test bacteria without any drug treatment.

[0066] like Figure 2-6 As shown, the hyphal morphology of the 128 μg / ml FCZ alone group slightly changed, with hyphae growing in clusters and becoming long and dense. The 3 μg / ml TA alone group had a larger hyphal coverage area. Although not as dense as the control group, there were still many closely clustered hyphae. In the FCZ + TA combination group, only 0.25 μg / ml or 0.5 μg / ml FCZ was needed to significantly inhibit hyphal growth, with distinct yeast-like cells visible within the field of view. A small number of hyphae were also significantly shortened, with loose hyphal growth and few clusters. This phenomenon indicates that the combination of FCZ and TA can significantly inhibit the transition of Candida albicans and Candida glabrata from the yeast phase to the hyphal phase, thereby reducing the growth and proliferation of the test bacteria.

[0067] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. Application of tannic acid combined with triazole drugs in the preparation of drugs for treating diseases caused by fungi.

2. Application of tannic acid combined with triazole drugs in the preparation of antifungal products.

3. The use according to claim 1 or 2, characterized in that The triazole drugs are fluconazole, voriconazole, itraconazole and posaconazole.

4. The use according to claim 1 or 2, characterized in that The fungus is Candida.

5. The use according to claim 4, characterized in that The Candida albicans is drug-resistant Candida albicans or drug-resistant Candida glabrata.

6. The use according to claim 1, characterized in that The medicine comprises tannic acid, triazole drugs and pharmaceutically acceptable carriers or excipients.

7. The use according to claim 2, characterized in that The antifungal products include antifungal drugs and antifungal inhibition model tool drugs.

8. The use according to claim 3, characterized in that The effective concentration ratio of the tannic acid and fluconazole for significantly inhibiting the activity of drug-resistant Candida albicans or drug-resistant Candida glabrata in vitro is 3 μg / ml: 0.25-0.5 μg / ml.

9. The use according to claim 1, characterized in that The tannic acid significantly increases the sensitivity of drug-resistant Candida albicans to fluconazole in vitro; the tannic acid significantly increases the sensitivity of drug-resistant Candida glabrata to fluconazole in vitro.

10. The use according to claim 1, characterized in that The disease is a skin candidiasis, a mucosal candidiasis, or a visceral or central nervous system candidiasis; the skin candidiasis includes candidiasis infections of the fossa, groin, under the breast, anus, nail groove, and between the fingers.

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