An antifungal composition and its use in the treatment of fungal infections in diabetics

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an antifungal composition and application thereof in antifungal and treatment of diabetes combined with fungal infection. The betaine and 4-acetylamino benzoic acid in the application have a broad-spectrum synergistic bacteriostatic effect on Candida albicans, Cryptococcus neoformans, Aspergillus flavus and Aspergillus fumigatus, the FIC index is 0.292, and the concentration is reduced by 7 times compared with single drug; in a diabetes DB (db / db) mouse infection model, the survival period is significantly prolonged, which is better than voriconazole, and the application has the function of regulating sugar metabolism. The antibacterial composition has high safety, raw materials are cheap and easy to obtain, and can be prepared into oral or external preparations, and is used for preparing a medicine for treating diabetes combined with fungal infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antifungal composition and its application in antifungal treatment and the treatment of fungal infections in diabetic patients. Background Technology

[0002] Invasive fungal infections are common and critical illnesses in clinical practice, frequently affecting individuals with weakened immune function or metabolic abnormalities, such as those with immunodeficiency, long-term use of hormones, diabetes, or tumors. Systemic candidiasis and invasive aspergillosis, in particular, have mortality rates as high as 30%–80%, seriously threatening patients' lives and health. Among numerous high-risk factors, diabetic patients are considered the highest-risk group for fungal infections. The pathological mechanism lies in the fact that prolonged hyperglycemia significantly inhibits the bactericidal ability of phagocytes, while the high-glucose microenvironment provides favorable conditions for fungal proliferation, resulting in a 3–4 times higher incidence of fungal infections in diabetic patients compared to healthy individuals. Furthermore, diabetic patients with fungal infections often experience multiple organ damage, further increasing the difficulty of clinical treatment.

[0003] Currently, the mainstream drugs used clinically to treat invasive fungal infections are mainly divided into three categories: first, azole drugs (such as fluconazole and voriconazole), but the fungal resistance rate continues to rise after long-term use; second, polyene drugs (such as amphotericin B), which have strong hepatotoxicity and nephrotoxicity, and their use in diabetic patients carries a significantly increased risk; and third, echinocandins, which, although relatively less toxic, are expensive and have a narrow antibacterial spectrum. Regarding combination therapy, existing regimens involve combining the aforementioned marketed antifungal drugs. While this can enhance efficacy to some extent, the risk of cross-resistance remains, failing to fundamentally solve the resistance problem.

[0004] In summary, existing antifungal treatment systems face several pressing technical challenges: First, single antifungal drugs have limited inhibitory capabilities, and long-term use easily induces multidrug resistance in fungi; second, traditional antifungal drugs exhibit significant hepatotoxicity and nephrotoxicity, while diabetic patients often have microvascular complications and liver and kidney damage, making medication safety difficult to guarantee; third, existing combination regimens are all combinations of clinical antifungal drugs, resulting in a high risk of cross-resistance, and there is a lack of novel, low-toxicity small molecule combination systems; fourth, there is currently no specific treatment regimen for fungal infections in diabetic patients, and existing drugs can only inhibit fungal growth, failing to simultaneously improve underlying diabetic conditions such as hyperglycemia and organ damage, leading to a persistently high recurrence rate of infection. It is noteworthy that existing research on betaine focuses primarily on liver protection, metabolic regulation, and improvement of insulin resistance, with no reports of its combination with antifungal drugs; and 4-acetaminobenzoic acid is currently only used as a chemical synthesis intermediate, with no research involving antifungal or diabetic infection treatment. Therefore, developing a novel combination therapy strategy that can synergistically combat fungi, reduce drug toxicity, and improve underlying diabetic complications has significant clinical implications and promising application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide an antifungal composition and its application in antifungal treatment and treatment of fungal infections in diabetic patients. The antifungal composition has excellent antifungal effects and good therapeutic effects on fungal infections in diabetic patients.

[0006] The present invention provides an antifungal composition comprising betaine and 4-acetaminobenzoic acid.

[0007] Preferably, the molar ratio of betaine to 4-acetaminobenzoic acid is (1~10):(1~10).

[0008] The present invention also provides the application of the antifungal composition described above in the preparation of antibacterial agents.

[0009] Preferably, the antibacterial properties include antifungal properties.

[0010] Preferably, the fungus includes yeast and / or filamentous fungi.

[0011] Preferably, the yeast includes Candida albicans and / or Cryptococcus neoformans; the filamentous fungus includes Aspergillus flavus and / or Aspergillus fumigatus.

[0012] The present invention also provides the use of the antifungal composition described above in the preparation of a medicament for treating fungal infections complicated with diabetes.

[0013] Preferably, the diabetes includes type 2 diabetes.

[0014] Preferably, the diabetes mellitus complicated with fungal infection includes type 2 diabetes mellitus complicated with Candida albicans infection.

[0015] The present invention also provides a medicament, which is an antibacterial drug or a drug for treating diabetic comorbidities with fungal infection, wherein the active ingredient of the medicament comprises the antifungal composition described in the above-described technical solution. As one embodiment, the medicament of the present invention can be an oral formulation and / or a topical formulation.

[0016] Beneficial effects: This invention is the first to discover that the combined use of betaine and 4-acetaminobenzoic acid exhibits broad-spectrum synergistic in vitro antibacterial activity, significantly superior to that of a single component. It demonstrates significant synergistic inhibitory effects against four clinically prevalent pathogenic fungi: Candida albicans, Cryptococcus neoformans, Aspergillus flavus, and Aspergillus fumigatus. Furthermore, the two compounds have complementary targets, simultaneously interfering with fungal cell wall synthesis and effectively compensating for the insufficient antibacterial strength of single compounds. This provides a novel, low-toxicity combination for clinical antifungal treatment, avoiding the risk of cross-resistance with existing drugs. Simultaneously, the antifungal composition of this invention has excellent therapeutic effects against fungal infections in diabetic patients, assisting in the regulation of glucose metabolism and lowering blood glucose levels. It achieves a dual effect of antifungal activity and improvement of basal metabolic damage in diabetes, solving the problems of traditional antifungal drugs that only inhibit fungi, cannot intervene in primary diabetic damage, and have a high recurrence rate. Furthermore, the antifungal composition of this invention boasts high safety and readily available, inexpensive raw materials, making it suitable for long-term use by diabetic patients. Betaine is a naturally occurring endogenous alkaloid with good physiological compatibility; 4-acetaminobenzoic acid is low in toxicity and has minimal irritation. The combined use of these two ingredients can reduce the dosage of single-drug administration and minimize adverse liver and kidney reactions, making it suitable for long-term administration by diabetic patients. Both chemical raw materials are mature for industrial mass production and have low procurement costs, demonstrating promising prospects for the industrialization of topical and oral formulations. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The effects of betaine, 4-acetaminobenzoic acid, and their combination on the colony diameter of four fungi in Example 2; Figure 2 This is a statistical chart showing the antibacterial effects of betaine, 4-acetaminobenzoic acid, and their combination on four fungi in Example 2. Figure 3 This is the result of the verification of the effect of 4-acetaminobenzoic acid combined with betaine on the ultrastructure of Candida albicans and the target of cell wall action in Example 3; Figure 4This is a diagram showing the in vivo pharmacodynamic effects of the wild-type C57BL / 6 mouse model of systemic Candida albicans infection in Example 4. Figure 5 This is a diagram illustrating the in vivo therapeutic effect of systemic Candida albicans infection in diabetic DB (db / db) mice in Example 5. Figure 6 This is a graph showing the blood glucose concentration in diabetic DB (db / db) mice after systemic Candida albicans infection in Example 5. exist Figures 2-6 middle P <0.05, P <0.01, P <0.001; exist Figures 1-6 In this context, 4-acetyl represents 4-acetaminobenzoic acid.

[0019] Technical terms, English abbreviations and substance definitions MEC: Minimum Effective Concentration, refers to the lowest effective concentration of a drug that completely inhibits the visible growth of fungi; IC 50 (Half-maximal inhibitory concentration) The concentration of a drug required to inhibit 50% of the growth or biological activity of microorganisms under specified experimental conditions; PDA (Potato Dextrose Agar) is a solid culture medium specifically for fungal culture and plate antibacterial experiments. Betaine, molecular formula C5H 11 NO2: Trimethylglycine, a natural quaternary ammonium alkaloid; 4-Acetamidobenzoic acid (acedoben, molecular formula C9H9NO3): also known as para-acetamidobenzoic acid, is an aromatic, low-toxicity organic acid. Candida albicans: the most common opportunistic pathogenic yeast in clinical practice, inducing mucosal and systemic invasive fungal infections; Cryptococcus neoformans: a pathogenic yeast that easily causes central nervous system infections in immunocompromised individuals; Aspergillus flavus and Aspergillus fumigatus: highly pathogenic filamentous fungi that induce invasive pulmonary aspergillosis; Sorbitol (osmotic protectant, analytical grade); YPD liquid medium (Yeast Extract Peptone Dextrose Medium): 10 g / L yeast extract powder, 20 g / L peptone, 20 g / L glucose, pH natural or adjusted to 7.0±0.2; YPD solid medium: Add 15~20 g / L of agar powder to YPD liquid medium; YPDS liquid medium: Sorbitol was added to YPD liquid medium to a final concentration of 0.8 M (approximately 145.6 g / L). YPDS solid medium: Add 15~20 g / L of agar powder to YPDS liquid medium; PBS buffer: Phosphate-buffered saline (PBS), pH 7.4, autoclaved (121°C, 20 min); DMSO cosolvent: Dimethyl sulfoxide (DMSO), analytical grade, used as a pharmaceutical cosolvent, with a final concentration not exceeding 1%; C57 mice: C57BL / 6 inbred wild-type mice, a conventional healthy animal infection model; DB mice (db / db mice): spontaneously diabetic inbred mice with leptin receptor deficiency, persistent hyperglycemia, and immunodeficiency; standard experimental animals for diabetic patients with fungal infection. SPF grade: Specific Pathogen-Free Laboratory Animals. Detailed Implementation

[0020] The present invention provides an antifungal composition comprising betaine and 4-acetaminobenzoic acid.

[0021] As one embodiment, the molar ratio of betaine to 4-acetaminobenzoic acid is (1~10):(1~10), further can be (2~8):(2~8), can also be (3~7):(3~7), and more preferably 1:1.

[0022] The present invention also provides the application of the antifungal composition described above in the preparation of antibacterial agents.

[0023] In one embodiment, the antibacterial agent of the present invention can be an antifungal agent; in another embodiment, the fungus of the present invention can be a yeast and / or a filamentous fungus; in yet another embodiment, the yeast of the present invention can be Candida albicans and / or Cryptococcus neoformans; and the filamentous fungus can be Aspergillus flavus and / or Aspergillus fumigatus.

[0024] This invention also provides the use of the antifungal composition described above in the preparation of a medicament for treating diabetes mellitus complicated with fungal infection. In one embodiment, the diabetes mellitus described in this invention can be type 2 diabetes mellitus; in another embodiment, the diabetes mellitus complicated with fungal infection described in this invention can be type 2 diabetes mellitus complicated with Candida albicans infection.

[0025] This invention also provides a medicament, which is an antibacterial drug or a drug for treating diabetic comorbidities with fungal infections, wherein the active ingredient of the medicament comprises the antifungal composition described in the above-described technical solution. As one embodiment, the medicament of this invention may also include pharmaceutically acceptable excipients; the specific type is not particularly limited and can be selected as needed.

[0026] The antibacterial composition prepared by combining betaine and 4-acetaminobenzoic acid provided by this invention has the following advantages compared with the prior art: 1. Broad-spectrum synergistic in vitro antibacterial activity, significantly superior to single-component inhibitors. This invention is the first to discover that the combined use of betaine and 4-acetaminobenzoic acid exhibits significant synergistic inhibitory effects against four clinically prevalent pathogenic fungi: Candida albicans, Cryptococcus neoformans, Aspergillus flavus, and Aspergillus fumigatus. In vitro antibacterial experiments showed that the FIC index of the combined treatment was 0.292 (far below the synergistic threshold of 0.5), and the total concentration required for the combined treatment was only 1 μg / mL, a 7-fold reduction compared to the sum of the concentrations of the two components used alone (7 μg / mL). PDA plate antibacterial experiments further confirmed that the colony diameter in the combined treatment group was significantly smaller than that in each of the single-drug groups. P <0.001). The two compounds have complementary targets, which can simultaneously interfere with fungal energy metabolism and effectively compensate for the insufficient antifungal strength of single compounds, providing a new low-toxicity compound regimen for clinical antifungal treatment.

[0027] 2. The therapeutic effect was confirmed in healthy animals, verifying its universal therapeutic value. In a systemic Candida albicans infection model in C57BL / 6 healthy mice, the combined drug administration group showed significantly better survival time and 15-day survival rate than the betaine monotherapy group, the 4-acetaminophen monotherapy group, and the model control group. P The value <0.001 indicates that the composition of the present invention also has a definite antifungal infection effect in immune-normal hosts, verifying its universal therapeutic potential as a candidate broad-spectrum antifungal drug.

[0028] 3. It has unique clinical advantages in treating fungal infections complicated with diabetes. This invention provides for the first time a combination therapy specifically targeting fungal infections in diabetic mice. Experimental results using a systemic Candida albicans infection model in db / db spontaneously diabetic mice confirm that: (a) The combined drug administration group significantly reduced the mortality rate and prolonged the survival period of diabetic mice infected with diabetes. The 15-day survival rate was close to 100%, and the survival curve was significantly better than that of the diabetic model control group, the betaine monotherapy group, the 4-acetaminobenzoic acid monotherapy group, and the first-line antifungal drug voriconazole group. P <0.001); (b) The betaine component can help regulate glucose and lipid metabolism and reduce liver and kidney damage induced by high glucose, achieving a dual therapeutic effect of "antifungal and improving basic metabolic damage in diabetes". This solves the technical problems of traditional antifungal drugs that can only inhibit fungi, cannot intervene in primary damage in diabetes, and have a high recurrence rate of infection.

[0029] These results fully demonstrate that the composition of the present invention is particularly suitable for the diabetic population, which is at the highest risk of fungal infection, and fills the long-standing technical gap in the field of fungal infection in diabetic patients.

[0030] 4. High safety profile, inexpensive and readily available raw materials, suitable for long-term administration. Betaine is a naturally occurring endogenous alkaloid in the human body with good physiological compatibility; 4-acetaminobenzoic acid has low toxicity and minimal irritation. The combined use of these two drugs can significantly reduce the effective dose of each individual drug (betaine by 8 times, and 4-acetaminobenzoic acid by 6 times), thereby reducing adverse liver and kidney reactions and meeting the needs of long-term medication for diabetic patients. Furthermore, both raw materials are mature industrial-scale products with low procurement costs, possessing promising prospects for industrial development in multiple dosage forms, including topical and oral formulations, and demonstrating significant social and economic benefits.

[0031] In summary, this invention, through the synergistic combination of betaine and 4-acetaminobenzoic acid, has for the first time constructed a combined drug system with the dual functions of "highly effective broad-spectrum antifungal activity" and "improvement of underlying diabetic lesions," achieving unexpected technical effects in both in vitro antibacterial studies and in animal models of diabetic patients with fungal infections.

[0032] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1 The MEC assays for each compound against four fungi were performed using the following steps: 1. Experimental Materials Strains: Candida albicans, Cryptococcus neoformans, Aspergillus flavus, Aspergillus fumigatus; Reagents: Betaine, 4-acetaminobenzoic acid, voriconazole, RPMI 1640 liquid medium, sterile PBS buffer, DMSO solubilizer; Consumables: 96-well cell culture plate.

[0034] 2. Experimental Methods (1) Activation of strains and preparation of bacterial culture: Four types of fungi (Candida albicans, Cryptococcus neoformans, Aspergillus flavus, and Aspergillus fumigatus) were subcultured and activated to obtain fresh mycelium / spores. The specific steps are as follows: The fungi used in the experiment were divided into two categories for activation preparation: yeasts (Candida albicans, Cryptococcus neoformans) were inoculated on solid culture medium and cultured at 37℃ for 24-48 h; filamentous fungi (Aspergillus flavus, Aspergillus fumigatus) were cultured at 35℃ for 5-7 days. After activation, yeast colonies and filamentous fungal spores were picked and dissolved in sterile physiological saline to prepare bacterial suspensions.

[0035] (2) Preparation of bacterial culture: The bacterial culture was adjusted to the experimental standard concentration using the turbidimetric method: the concentration of yeast was 5 × 10⁻⁶. 6 CFU / mL, concentration of filamentous fungi 1×10 6 CFU / mL.

[0036] (3) Gradient drug solution preparation: Betaine, 4-acetaminobenzoic acid and voriconazole were prepared into a series of dilution concentrations, and PBS and DMSO solvent control groups were set up.

[0037] (4) Micro-liquid dilution method for MEC determination: Gradient drug solution and standardized bacterial solution were added to 96-well plates, with 3 replicates per group; yeast was cultured at 37℃ for 24-48 h, and filamentous fungi were cultured at 35℃ for 48-72 h; (5) Read the results: The lowest drug concentration at which no fungal growth is visible to the naked eye is the corresponding strain MEC. Record all values.

[0038] 3. The experimental results are shown in Table 1.

[0039] Table 1. MEC of each tested compound against different strains

[0040] 4. Synergistic effect analysis of combined drug use (FIC index) FIC Calculation Method The synergistic effect of combined use of betaine and 4-acetaminobenzoic acid was evaluated using the partial inhibitory concentration index (FIC). The formula for calculating FIC is as follows: in: MEC_A, Alone: ​​The lowest effective concentration of betaine when used alone; MEC_A, combined: concentration of betaine in combined drug therapy; MEC_B, Alone: ​​The lowest effective concentration of 4-acetaminobenzoic acid when used alone; MEC_B, combined: concentration of 4-acetaminobenzoic acid in combination therapy; The judgment criteria are shown in Table 2.

[0041] Table 2 Judgment Criteria

[0042] When used in combination, betaine and 4-acetaminobenzoic acid are mixed at a concentration ratio of 1:1, and the total effective ingredient concentration of the mixture is 1 μg / mL, that is, the concentration of each component is 0.5 μg / mL.

[0043] Substituting the concentrations of each compound, both individually and in combination, into the FIC calculation formula, as follows: The results of the combined drug FIC index and synergistic effect determination are shown in Table 3.

[0044] Table 3. FIC Index and Synergistic Effect Determination of Combined Drug Use

[0045] 5. Synergy Analysis The formula for calculating the synergy factor is as follows: The results of the synergy analysis are shown in Table 4.

[0046] Table 4 Results of the Synergistic Effect Analysis

[0047] From the above results, we can conclude that: (1) When betaine and 4-acetaminobenzoic acid are used alone, they have certain antifungal activity against the four fungi, with MEC values ​​of 4 μg / mL and 3 μg / mL, respectively.

[0048] (2) When betaine and 4-acetaminobenzoic acid are used together, they show a synergistic antifungal effect. The FIC index for all tested strains is 0.292, which is less than the synergistic judgment threshold of 0.5, indicating a synergistic effect.

[0049] (3) When used in combination, the total concentration of the mixture is only 1 μg / mL, which is 7 times lower than the sum of the concentrations of the two components used alone (7 μg / mL), effectively reducing the dosage.

[0050] (4) This combination of drugs has broad-spectrum synergistic antifungal activity against Candida albicans, Cryptococcus neoformans, Aspergillus flavus and Aspergillus fumigatus, and has good prospects for clinical application.

[0051] Example 2 The antibacterial experiment on PDA plates based on MEC concentration is as follows: 1. Experimental Materials Sterile PDA solid culture medium, sterile petri dishes, betaine solution with MEC concentration measured in Example 1, 4-acetaminobenzoic acid solution, mixed drug solution, voriconazole, PBS, and DMSO.

[0052] 2. Experimental Methods (1) Prepare PDA plates containing betaine MEC, 4-acetaminobenzoic acid MEC, voriconazole MEC, betaine + 4-acetaminobenzoic acid MEC concentrations, PBS control, and solvent control, and allow them to solidify. The specific steps are as follows: Melt PDA and cool it to 45~50℃, then mix in betaine alone, 4-acetaminobenzoic acid alone, a mixture of the two, voriconazole, PBS, and DMSO. Solidify 3 parallel plates in each group.

[0053] (2) Apply Candida albicans, Cryptococcus neoformans, Aspergillus flavus and Aspergillus fumigatus solutions to the PDA plate at the corresponding concentrations and seal with sealing film.

[0054] (3) Separate culture: yeast fungi were cultured at 37°C and filamentous fungi at 35°C. The culture time was the same as in Example 1.

[0055] (4) Measure the diameter of each group of colonies, take photos and keep records, and statistically compare the antibacterial differences between the single drug and the combination drug groups.

[0056] 3. Experimental Results The colony photography results for each group are as follows: Figure 1 As shown, the colony diameter statistics are as follows: Figure 2 As shown, combined with Figures 1-2 It can be concluded that the MEC (medium-to-volume concentration) of betaine and 4-acetaminobenzoic acid used alone were 4 μg / mL and 3 μg / mL, respectively, while the MEC of the mixture used in combination was 1 μg / mL. Under these conditions, the colony diameters of the betaine single-drug group, the 4-acetaminobenzoic acid single-drug group, and the combined group were not significantly different, confirming that the two drugs have a significant synergistic antifungal effect at the MEC concentration. No inhibition zones were observed in the solvent group and the blank group, eliminating solvent interference. This synergistic effect was stably observed in four strains: Candida albicans, Cryptococcus neoformans, Aspergillus flavus, and Aspergillus fumigatus.

[0057] Example 3 Effects of 4-acetaminobenzoic acid combined with betaine on the ultrastructure of Candida albicans and verification of cell wall action targets 1. Experimental Materials Strains: Candida albicans.

[0058] Reagents: betaine, 4-acetaminobenzoic acid, sorbitol, YPD liquid medium, YPD solid medium, YPDS liquid medium, YPDS solid medium, sterile PBS buffer (pH 7.4), DMSO co-solvent.

[0059] Consumables and instruments: 96-well cell culture plates, scanning electron microscope (SEM), transmission electron microscope (TEM), microplate reader, incubator, biosafety cabinet.

[0060] 2. Experimental Methods 2.1 Strain activation and bacterial culture preparation Candida albicans was inoculated onto YPD solid medium and activated by incubation at 37°C for 24–48 h. After activation, fresh yeast colonies were picked, dissolved in sterile physiological saline, and vortexed to obtain a bacterial suspension.

[0061] 2.2 Standardization of bacterial culture The bacterial culture was adjusted to the experimental standard concentration of 5 × 10⁻⁶ using the turbidimetric method. 6 CFU / mL.

[0062] 2.3 Minimum Effective Concentration (MEC) Treatment of Candida albicans Using the MEC value from Example 1: (1) Dissolve betaine and 4-acetaminobenzoic acid in RPMI 1640 medium containing DMSO and add them to 100 μL of standardized Candida albicans culture solution (final concentration 2.5 × 10⁻⁶) according to the MEC concentration in Example 1. 6 In each group of wells containing CFU / mL, three duplicate wells are set.

[0063] (2) Place the 96-well plate in a 37℃ constant temperature incubator for 24 h.

[0064] 2.4 Scanning Electron Microscopy (SEM) Observation (1) Collect bacterial cells from each group, wash three times with sterile PBS to remove residual culture medium; (2) Fix with 2.5% glutaraldehyde fixative at 4℃ for 4 h; (3) Gradual dehydration was performed using 30%, 50%, 70%, 85%, 95%, and 100% ethanol, for 15 min each time; (4) After replacing ethanol with isoamyl acetate, critical point drying is performed; (5) The sample is attached to the scanning electron microscope stage and sputtered with gold; (6) Observe and photograph the surface morphology of the bacteria under a scanning electron microscope with an accelerating voltage of 5~15 kV.

[0065] 2.5 Observation by transmission electron microscopy (TEM) (1) Collect bacterial cells from each group and wash three times with sterile PBS; (2) Pre-fixed with 2.5% glutaraldehyde at 4℃ for 2 h, followed by post-fixation with 1% osmium tetroxide for 2 h; (3) Acetone gradient dehydration (50%, 70%, 80%, 90%, 100%). (4) Embedded in epoxy resin Epon 812, sliced ​​using an ultramicrotome (thickness approximately 70 nm). (5) Double staining with uranium acetate and lead citrate; (6) Observe and photograph the internal ultrastructure of the bacteria under a transmission electron microscope with an accelerating voltage of 80 kV.

[0066] 2.6 Sorbitol Osmotic Rescue Assay (Liquid Culture Medium Method) To verify whether 4-acetaminobenzoic acid and betaine exert their antifungal activity by acting on the cell wall of Candida albicans, an osmotic rescue experiment was conducted using a hypertonic medium containing 0.8 M sorbitol.

[0067] (1) Preparation of drug-containing culture medium: Prepare YPD liquid culture medium and YPDS liquid culture medium (containing 0.8 M sorbitol) containing a series of 2-fold diluted concentrations of 4-acetaminobenzoic acid, betaine, and a combination of the two drugs.

[0068] (2) Antimicrobial susceptibility testing: Standardized Candida albicans bacterial suspensions were inoculated into the above-mentioned drug-containing YPD and YPDS culture media, respectively, to achieve a final bacterial concentration of 2.5 × 10⁻⁶. 6 CFU / mL, incubated at 37℃ for 24 h.

[0069] (3) OD value determination: The absorbance (OD) of each well at 600 nm was measured using a microplate reader. 600 With the growth of the drug-free control group as 100%, the growth inhibition rate at each drug concentration was calculated, growth inhibition curves were plotted, and the IC50 of each group was calculated using GraphPad Prism software. 50 value.

[0070] 2.7 Sorbitol Osmotic Rescue Experiment (Solid Plate Method) To more intuitively observe the effect of a hypertonic environment on the antibacterial activity of the drug, the IC50 values ​​of each group were measured according to Example 3.3. 50 The concentration was verified using the spot inoculation method with drug-containing bacterial solution.

[0071] (1) Dilute the Candida albicans culture with sterile PBS in a serial dilution to 10⁻⁶. 6 10 5 10 4 Three concentrations of CFU / mL.

[0072] (2) Add the drug to the corresponding IC50 concentration of each bacterial culture. 50 Concentration, after mixing, to prepare a drug-containing bacterial solution: Includes IC 50 Bacterial solution containing concentrated betaine; Includes IC 50 Bacterial solution containing a combination of 4-acetaminobenzoic acid and betaine; Bacterial suspensions containing sterile PBS were used as negative controls.

[0073] (3) Take 5 μL of the above-mentioned drug-containing bacterial solution and inoculate it onto the following drug-free plates: YPD plate (normal osmotic pressure); YPDS plate (hyperosmolar environment, containing 0.8 M sorbitol).

[0074] (4) Place the plates in a 37℃ constant temperature incubator for 24 h and observe and record the growth of colonies in each group.

[0075] 3. Experimental Results 3.1 Observation results of scanning electron microscopy like Figure 3 As shown in Figure A, the surface morphology of Candida albicans showed significant differences after treatment with different drugs: PBS control group: The bacteria are typically oval or spherical, with smooth and plump surfaces, intact cell walls, and clear outlines, without obvious shrinkage or damage; 4-Acetaminobenzoic acid alone group: Some bacterial cells showed slight wrinkling on the surface, local depression of the cell wall, increased surface roughness, and a small number of cells with irregular morphology. Betaine alone group: Some bacterial cell surfaces showed wrinkles, increased intercellular adhesion, and localized cell wall damage was visible. 4-Acetaminobenzoic acid combined with betaine group: a large number of bacterial cells were severely shrunken, collapsed and deformed, the cell wall structure was obviously destroyed, the surface showed irregular wrinkles or even ruptures, and some bacterial contents were leaked out. The degree of damage was significantly more severe than that of each single drug group, and only a small number of bacterial cells with relatively intact morphology were observed.

[0076] The above results indicate that the combined application of 4-acetaminobenzoic acid and betaine can synergistically disrupt the cell wall structure of Candida albicans, resulting in severe damage to the surface morphology of the bacteria.

[0077] 3.2 Results of transmission electron microscopy observation like Figure 3 As shown in Figure B, the changes in the internal ultrastructure of Candida albicans after treatment with different drugs are as follows: PBS control group: The cell walls were of uniform thickness, the cell membranes were intact and continuous, the cytoplasm was evenly distributed, the organelles were clearly visible, and no obvious vacuoles or lysis were observed. 4-Acetaminobenzoic acid monotherapy group: Local thickening or thinning of cell wall, invagination of cell membrane, a small number of vacuolated structures in cytoplasm, and intact cell nucleus morphology; Betaine alone group: loose cell wall structure, increased gap between cell membrane and cell wall, obvious vacuoles in cytoplasm, and blurred boundaries of some organelles; In the 4-acetaminobenzoic acid combined with betaine group: the cell wall was severely damaged and broken, the cell membrane lost its continuity and multiple cracks appeared, a large amount of cytoplasm leaked out, large areas of vacuolation appeared in the cell, the cell nucleus condensed or disintegrated, and the integrity of the cell structure was severely damaged.

[0078] Transmission electron microscopy results further confirmed that the combined drug group had a significant synergistic destructive effect on the cell wall of Candida albicans, leading to leakage of cell contents and cell death.

[0079] 3.3 Results of the sorbitol osmotic rescue experiment (liquid culture medium method) like Figure 3 As shown in Table C and Table 5, the IC50 values ​​of each treatment group against Candida albicans were [not specified] in YPD normal medium and YPDS hypertonic medium (containing 0.8 M sorbitol). 50 The values ​​differ significantly: Table 5. Sorbitol Osmotic Rescue Experiment IC 50 Measurement results

[0080] As shown in Table 5: (1) In YPD normal culture medium, the IC50 of 4-acetaminobenzoic acid combined with betaine 50 The value was 1.882 μg / mL, which was significantly lower than that of the 4-acetaminobenzoic acid monotherapy group (4.607 μg / mL) and the betaine monotherapy group (4.957 μg / mL), indicating that the combination of the two drugs had significant synergistic anti-Candida albicans activity.

[0081] (2) In YPDS hypertonic medium, the IC50 values ​​of each treatment group 50 All values ​​were significantly elevated: IC50 in the 4-acetaminobenzoic acid monotherapy group. 50 The levels increased by approximately 6.73 times (from 4.607 μg / mL to 31 μg / mL) in the betaine monotherapy group, approximately 6.22 times (from 4.957 μg / mL to 30.83 μg / mL) in the betaine monotherapy group, and approximately 6.08 times (from 1.882 μg / mL to 11.44 μg / mL) in the combination therapy group.

[0082] (3) The addition of sorbitol significantly increased the IC50 of each drug group. 50 The value indicates that the hyperosmotic environment provided by 0.8 M sorbitol can reverse the antibacterial activity of the drug to a certain extent, suggesting that the antibacterial targets of 4-acetaminobenzoic acid and betaine are closely related to the cell wall integrity of Candida albicans.

[0083] 3.4 Results of the sorbitol osmotic rescue experiment (solid plate method) like Figure 3 As shown in D, the IC will be included 50 Candida albicans suspensions at various drug concentrations were inoculated onto YPD and YPDS plates, and the results were highly consistent with the liquid culture method shown in Figure C. Negative control group containing PBS: On both YPD and YPDS flat panels, 10 6 10 5 10 4 Colonies at all three CFU / mL inoculum concentrations grew vigorously, with large and dense colonies. There was no significant difference in growth between YPD and YPDS plates, indicating that 0.8 M sorbitol itself had no significant inhibitory effect on Candida albicans growth.

[0084] Includes IC 50 Bacterial suspensions containing concentrations of betaine and 4-acetaminophenyl acid: Inoculated onto YPD plates: Colony growth was significantly inhibited at all inoculation concentrations. As the inoculation concentration decreased, the number and size of colonies decreased significantly, indicating that betaine and 4-acetaminophenic acid inhibited growth at IC50 concentrations. 50 It exhibits good antibacterial effect against Candida albicans at certain concentrations; Inoculation on YPDS plates: Compared with the corresponding YPD plates, the number of colonies increased significantly and the colony volume increased significantly. Some inoculation sites even approached the growth state of the PBS control group, indicating that the hypertonic environment effectively reversed the antibacterial activity of betaine and 4-acetaminophenyl acid.

[0085] Includes IC 50 Bacterial culture group with concentration of 4-acetaminobenzoic acid + betaine combined solution: When inoculated onto YPD plates: colony growth was significantly inhibited, and the degree of inhibition was significantly stronger than that of the groups treated with betaine or 4-acetaminophen alone. At 10 5 10 4 The presence of sporadic, tiny colonies at the inoculation site at low CFU / mL concentrations indicates that the combined use of the two drugs at IC50 levels... 50 It exhibits a synergistic bactericidal effect against Candida albicans at certain concentrations; Spotting on YPDS plates: Compared with the corresponding YPD plates, the number and size of colonies were significantly increased, and the antibacterial inhibitory effect of the combination therapy group was significantly reversed. This indicates that the hypertonic environment provided by 0.8 M sorbitol can significantly protect Candida albicans, allowing it to survive at the IC50 level of the combination therapy. 50 It can still recover growth even at the highest concentration.

[0086] 4. Conclusion This embodiment confirms through scanning electron microscopy and transmission electron microscopy that 4-acetaminobenzoic acid combined with betaine can synergistically disrupt the cell wall structure of Candida albicans, leading to severe shrinkage, collapse, and cytoplasmic leakage of the bacterial surface. Sorbitol osmotic rescue experiments (liquid method and plate method) further verify that the antibacterial activity of the drug is significantly reversed under hyperosmotic conditions, IC50... 50 The value increased by about 6 times, suggesting that the combined action of 4-acetaminobenzoic acid and betaine against Candida albicans mainly targets the cell wall, and exerts a synergistic bactericidal effect by disrupting the integrity of the bacterial cell wall.

[0087] Example 4 The in vivo treatment experiment of systemic Candida albicans infection in wild-type C57BL / 6 mice was conducted as follows: 1. Experimental animals: SPF grade C57BL / 6 mice, 6 weeks old, weighing 18~22 g, acclimatized for 7 days.

[0088] 2. Modeling procedure: Activated Candida albicans was prepared into a 5×10⁻⁶ sample. 7 CFU / mL bacterial suspension, 20 μL injected into each mouse via tail vein, total inoculation volume 1×10⁻⁶. 6 CFU was used to construct a systemic fungal infection model.

[0089] 3. Group administration (10 animals per group): ① Model control group: administered physiological saline by gavage; ② Betaine monotherapy group: betaine was administered by gavage at a dose of 100 mg / kg / day; ③4-Acetaminobenzoic acid monotherapy group: 4-Acetaminobenzoic acid was administered by gavage at a dose of 125 mg / kg / day; ④ Combined administration group: Betaine was administered by gavage at a dose of 50 mg / kg / d, and 4-acetaminophen was administered by gavage at a dose of 62.5 mg / kg / d. ⑤ Voriconazole positive control group: Voriconazole was administered by gavage at a dose of 20 mg / kg / day; Each of the above groups was administered 0.2 mL by gavage once daily for 7 consecutive days. 4. Periodic observation: Mice were observed continuously for 15 days from the start of Candida albicans injection (i.e., for 7 days after the end of drug administration). Mice mortality was recorded, survival curves were plotted, and statistical analysis was performed using the Log-rank test.

[0090] 5. Experimental results are as follows Figure 4 As shown, mice in the model control group (saline) died rapidly after infection, with a very low 15-day survival rate; the two single-drug groups only slightly prolonged the survival of mice; the combined administration of betaine and 4-acetaminophen significantly improved the survival time and survival rate of mice, showing a highly significant difference compared with the single-drug groups and the model group. P The result was <0.01, demonstrating that the composition also has a synergistic therapeutic effect on fungal infections in vivo.

[0091] Example 5 The in vivo treatment experiment of systemic Candida albicans infection in diabetic DB (db / db) mice followed the steps below: 1. Screening of experimental animals: SPF-grade db / db spontaneously diabetic mice, 8 weeks old, weighing 28-35 g; after 7 days of acclimatization, fasting blood glucose was measured by blood collection from the tail tip, and mice with fasting blood glucose ≥16.7 mmol / L and typical diabetic phenotype of polydipsia and polyuria were selected for inclusion in the group.

[0092] 2. Modeling procedure: Candida albicans bacterial suspension concentration 5×10 5 CFU / mL, 20 μL injected via tail vein into each DB mouse, total inoculation dose 1×10⁻⁶. 5 CFU was used to construct a model of diabetes mellitus complicated by systemic Candida infection.

[0093] 3. Experimental groups (10 animals per group) ① Control group of diabetic infection model (administered via gavage with normal saline). ② Betaine monotherapy group: 100 mg / kg / d; ③4-Acetaminobenzoic acid monotherapy group: 125 mg / kg / d; ④ Combined administration group: betaine 50 mg / kg / d + 4-acetaminobenzoic acid 62.5 mg / kg / d; ⑤ Voriconazole positive control group: 20 mg / kg / d; Each group of mice was administered the drug once daily by gavage for 7 consecutive days; Observe the mice continuously for 15 days from the start of Candida albicans injection (i.e., observe continuously for 7 days after the end of drug administration), record the number of survivors, and plot the survival curve of DB mice; 4. Blood glucose levels were measured in each group of mice before injection of Candida albicans, 7 days after injection and after gavage, and 7 days after the end of gavage.

[0094] 5. Experimental results are as follows Figures 5-6 As shown, the survival experiment of DB diabetic mice with systemic Candida albicans infection confirmed that the use of betaine or 4-acetaminobenzoic acid alone only slightly delayed the death of model mice. Although the clinical drug voriconazole could improve the survival rate, it had hepatotoxicity and nephrotoxicity. The combination of betaine and 4-acetaminobenzoic acid could significantly prolong the survival of infected diabetic mice, with a 15-day survival rate close to 100%, which was significantly better than the model group, the single drug group, and the voriconazole control group. P <0.001); at the same time, this compound composition can mildly improve hyperglycemic metabolic disorders in mice, and has the dual effects of antifungal and regulating the basic damage of diabetes, thus possessing the unique advantage of treating diabetes complicated with systemic fungal infection.

[0095] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antifungal composition, characterized in that, It includes betaine and 4-acetaminobenzoic acid.

2. The antifungal composition according to claim 1, characterized in that, The molar ratio of betaine to 4-acetaminobenzoic acid is (1~10):(1~10).

3. The use of the antifungal composition according to claim 1 or 2 in the preparation of antimicrobial agents.

4. The application according to claim 3, characterized in that, The antibacterial properties include antifungal properties.

5. The application according to claim 4, characterized in that, The fungi include yeasts and / or filamentous fungi.

6. The application according to claim 5, characterized in that, The yeast includes Candida albicans ( Candidaalbicans ) and / or Cryptococcus neoformans ( Cryptococcusneoformans The filamentous fungi include Aspergillus flavus ( ); Aspergillusflavus ) and / or Aspergillus fumigatus ( Aspergillus fumigatus ).

7. Use of the antifungal composition of claim 1 or 2 in the preparation of a medicament for treating diabetic comorbidities with fungal infections.

8. The application according to claim 7, characterized in that, The diabetes mentioned includes type 2 diabetes.

9. The application according to claim 7 or 8, characterized in that, The aforementioned diabetic comorbid fungal infection includes type 2 diabetes mellitus complicated by Candida albicans infection.

10. A drug, said drug being an antibacterial drug or a drug for treating diabetes complicated with fungal infection, characterized in that, The active ingredient of the drug includes the antifungal composition according to claim 1 or 2.

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