Pharmaceutical composition for inhibiting activity of fusarium solani and application thereof

Abstract

The invention discloses a pharmaceutical composition for inhibiting the activity of fusarium solani and application of the pharmaceutical composition. The active ingredients of the pharmaceutical composition are composed of plant essential oil cinnamyl aldehyde and hexokinase inhibitor lonidamine. Wherein the essential oil cinnamyl aldehyde is used as a main bacteriostatic agent, and the hexokinase inhibitor lonidamine is used as a synergistic bacteriostatic agent. Based on the characteristic that cinnamyl aldehyde exerts the bacteriostatic activity by interfering glycometabolism, the bacteriostatic activity of the natural product cinnamyl aldehyde is effectively improved by combining with hexokinase inhibitor drug combination, and potential drugs and medication strategies can be provided for treatment of fungal keratitis caused by fusarium solani.

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial drugs, and particularly relates to a pharmaceutical composition for inhibiting the activity of Fusarium solani and an application thereof. Background Art

[0002] Fungal keratitis is a common blinding eye disease. Fusarium solani is one of the main pathogens of fungal keratitis and can cause severe infectious corneal ulcers (Activation of focal adhesion kinase enhances the adhesion of Fusarium solani to human corneal epithelial cells via the tyrosine-specific protein kinase signaling pathway, 2011, Molecular Vision, 17(73):638-646). Fungal keratitis has the characteristics of slow progression and long course. Clinical treatment of fungal keratitis is mostly started with antifungal drugs, such as voriconazole and natamycin. However, there are few effective antibacterial drugs for keratitis, and long-term use will increase the drug resistance of pathogens, resulting in poor clinical efficacy and seriously affecting the patient's eye health (Analysis of pathogenic bacteria and prognosis and influencing factors of fungal keratitis, 2022, International Journal of Ophthalmology, 22(17):1892-1895). Cinnamaldehyde, a major component of cinnamon bark essential oil, a culinary spice, has been shown to have broad antibacterial activity. Furthermore, as a natural product, it is highly safe. Therefore, cinnamaldehyde has the potential to be developed as a treatment for fungal keratitis.

[0003] Hyperglycemia can induce eye diseases such as cataracts and glaucoma. Furthermore, it promotes the metabolism and growth of pathogens, enhances their virulence factors, and increases their tolerance to the host immune system. Numerous studies have shown that cancer cells undergo aerobic glycolysis through the Warburg effect, rapidly generating energy to meet their rapid proliferation needs. Lonidamine, a clinically used hexokinase inhibitor, inhibits the phosphorylation of glucose to form glucose-6-phosphate, inhibiting glycolysis and thereby blocking the glucose metabolism pathway in tumor cells, thereby enhancing the therapeutic effect of chemotherapy drugs against cancer cells. Chinese patent application CN119837864A discloses the use of lonidamine combined with apigenin to treat colorectal cancer. The pharmaceutical compositions, composed of lonidamine and apigenin in varying proportions, exhibit significantly stronger anti-tumor activity than either agent alone. Given the structural and functional unity of biological cells, providing a method for treating fungal keratitis based on the inhibition of glucose metabolism is of great research significance. Summary of the Invention

[0004] The present invention aims to provide a pharmaceutical composition for inhibiting the activity of Fusarium solani and its application, and utilizes a hexokinase inhibitor to enhance the inhibitory activity of cinnamon essential oil against Fusarium solani, thereby providing a potential drug and medication strategy for the treatment of fungal keratitis.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a pharmaceutical composition for inhibiting the activity of Fusarium solani, wherein the active ingredients of the pharmaceutical composition are composed of plant essential oil cinnamaldehyde and hexokinase inhibitor lonidamine.

[0007] Preferably, the mass ratio of cinnamaldehyde to lonidamine in the pharmaceutical composition is 0.08:0.125-0.5.

[0008] More preferably, the mass ratio of cinnamaldehyde to lonidamine in the pharmaceutical composition is 0.08:0.125.

[0009] In a second aspect, the present invention provides use of the pharmaceutical composition in inhibiting the activity of Fusarium solani.

[0010] In a third aspect, the present invention provides use of the above-mentioned pharmaceutical composition in treating fungal keratitis.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] In the pharmaceutical composition of the present invention, the essential oil cinnamaldehyde is selected as the main antibacterial agent, and the hexokinase inhibitor lonidamine is selected as the synergistic antibacterial agent. In the presence of glucose, the minimum inhibitory concentration (MIC) of cinnamaldehyde against Fusarium solani is 0.16g / L, and the MIC of lonidamine against Fusarium solani is greater than 0.5g / L. However, when cinnamaldehyde and lonidamine are used in combination, 0.08g / L cinnamaldehyde and 0.125g / L lonidamine can significantly inhibit the growth of Fusarium solani in the presence of glucose. The present invention provides a potential drug and dosage regimen for the treatment of fungal keratitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the minimum inhibitory concentration of cinnamaldehyde against Fusarium solani;

[0014] Figure 2 The effect of cinnamaldehyde on the viability of Fusarium solani spores;

[0015] Figure 3 This is a statistical graph showing the effect of cinnamaldehyde on the viability of Fusarium solani spores;

[0016] Figure 4 The effect of glucose on the inhibition of cinnamaldehyde on the viability of Fusarium solani spores;

[0017] Figure 5 This is a statistical graph showing the effect of glucose on the inhibition of cinnamaldehyde on the viability of Fusarium solani spores;

[0018] Figure 6 The effect of lonidamine on the ability of cinnamaldehyde to inhibit the viability of Fusarium solani spores;

[0019] Figure 7 This is a statistical graph showing the effect of lonidamine on the inhibition of cinnamaldehyde on the viability of Fusarium solani spores. DETAILED DESCRIPTION

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] Example 1: Minimum inhibitory concentration of cinnamaldehyde against Fusarium solani

[0022] To prepare potato culture medium: Peel 200g of potatoes, cut into small pieces, boil in boiling water for 30 minutes, and filter the potato broth. Add 20g / L of glucose and dissolve to create potato dextrose broth (PDB). Add 15g / L of agar powder to the PDB to create potato dextrose agar (PDA). Autoclave at 121°C for 20 minutes. Set aside.

[0023] Preparation of 0.1% Tween 80 as a cosolvent: add 1 mL of Tween 80 into 999 mL of reverse osmosis (RO) water, dissolve with ultrasonic for 10 minutes, and sterilize at 121° C. for 20 minutes.

[0024] Prepare 0.85% sterile saline: Add 8.5g of sodium chloride to RO water and dilute to 1L. Sterilize at 121°C for 20 minutes.

[0025] Preparation of a stock solution with a tenfold concentration of cinnamaldehyde: a certain amount of cinnamaldehyde (McLean, Cat. No. C822622) was added to a cosolvent 0.1% Tween 80 to a volume of 1 mL. The concentrations of essential oil were adjusted to 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, and 1.8 g / L, respectively. Ultrasonic solubilization was performed for 30 min.

[0026] Preparation of spore suspension: Fusarium solani X14011 (purchased from Henan Academy of Agricultural and Forestry Sciences) cultured for 7 days on PDA medium was washed with 0.85% saline, collected by centrifugation at 8000 rpm for 3 minutes, and then resuspended in saline. The spores were washed twice by centrifugation. The spores were counted using a hemocytometer to a concentration of 1 × 10 7cell / mL of spore liquid.

[0027] The MIC of cinnamaldehyde against Fusarium solani was determined using the liquid culture dilution method. 160 μL of PDB, 20 μL of spore solution, and 20 μL of cinnamaldehyde stock solution were added to a 96-well plate. A 0 g / L cinnamaldehyde group served as a positive control, and a 200 μL PDB group served as a negative control. After mixing, the 96-well plate was sealed with sealing film and incubated in a 28°C biochemical incubator for 7 days. The inhibitory effects were then observed.

[0028] According to the test results ( Figure 1 ), when the concentration of cinnamaldehyde is greater than 0.12g / L, the growth of mycelium in the pores is completely inhibited, so the minimum drug concentration of cinnamaldehyde essential oil against Fusarium solani is 0.12g / L.

[0029] Example 2: Effects of cinnamaldehyde and glucose on the antibacterial activity of Fusarium solani

[0030] To prepare a 20% sterile glucose solution, add 20g of glucose to RO water and dilute to 100mL. Sterilize the solution by filtering through a 0.22μm pore size filter. Set aside.

[0031] According to the method in Example 1, cinnamaldehyde mother solution with ten times the concentration, 1×10 7 cell / mL spore liquid, 0.85% sterile saline, and PDA solid culture medium.

[0032] Detection of the viability of Fusarium solani spores by cinnamaldehyde: 800 μL of 0.85% saline, 100 μL of spore solution and 100 μL of cinnamaldehyde stock solution were added to a 2 mL sterile centrifuge tube and mixed to adjust the cinnamaldehyde concentration to 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12 and 0.14 g / L and the spore concentration to 1 × 10 6 cell / mL.

[0033] Detection of the inhibitory effect of glucose on the spore viability of Fusarium solani by cinnamaldehyde: 700 μL of 0.85% saline, 100 μL of spore solution, 100 μL of glucose stock solution, and 100 μL of cinnamaldehyde stock solution were added to a 2 mL sterile centrifuge tube and mixed to adjust the cinnamaldehyde concentration to 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, and 0.16 g / L, and the spore concentration to 1 × 10 6 cell / mL.

[0034] Spore incubation and viability analysis: Incubate the tubes containing drug-treated spores in a shaking incubator at 180 rpm at 28°C for 24 hours. After drug treatment, centrifuge the spores at 8000 rpm for 3 minutes. Remove the supernatant, resuspend the spores in 0.85% saline, and collect the spores by centrifugation. Resuspend the spores in 1 mL of 0.85% saline, dilute the spores 500-fold, and plate 100 μL of the spores onto PDA medium. Perform three replicates for each drug treatment group. After incubation at 28°C for 3 days, determine the number of colonies.

[0035] According to the test results ( Figure 2 , Figure 3 The antibacterial activity of cinnamaldehyde against Fusarium solani was dose-dependent. No colonies grew on the PDA in the 0.12g / L cinnamaldehyde treatment group, indicating that 0.12g / L cinnamaldehyde can effectively inhibit the survival of the pathogen.

[0036] According to the test results ( Figure 4 , Figure 5 ), supplementation with glucose increased the inhibitory concentration of cinnamaldehyde against Fusarium solani from 0.12 g / L to 0.16 g / L. This experimental result shows that glucose, as a nutrient, can enhance the resistance of Fusarium solani to cinnamaldehyde.

[0037] Example 3: Effect of hexokinase inhibitors on the inhibitory activity of cinnamaldehyde against Fusarium solani

[0038] Preparation of a stock solution for hexokinase inhibition: Lonidamine (McLean, Cat. No. L849800) was selected as the hexokinase inhibitor. Dissolve lonidamine in dimethyl sulfoxide and vortex to dissolve to prepare a stock solution at a concentration of 25 g / L.

[0039] Referring to the method in Example 1, 10 times the concentration of 0.08 g / L cinnamaldehyde mother solution, 1×10 7 cell / mL spore liquid, 0.85% sterile saline, and PDA solid culture medium.

[0040] The inhibition of cinnamaldehyde on the viability of Fusarium solani spores by lonidamine was tested as follows: 700 μL of 0.85% physiological saline, 100 μL of spore solution, 100 μL of glucose stock solution, 100 μL of cinnamaldehyde stock solution and 0.5-2 μL of lonidamine stock solution were added to a 2 mL sterile centrifuge tube and mixed to adjust the concentrations of cinnamaldehyde to 0 and 0.08 g / L, glucose to 0 and 2% (m / m), lonidamine to 0, 0.125 g / L, 0.25 g / L and 0.5 g / L, and the spore concentration to 1×10 6cell / mL. Incubate at 28°C and 180 rpm for 24 hours. After drug treatment, centrifuge the spore solution at 8000 rpm for 3 minutes, wash the spores with 0.85% saline, resuspend the spores, dilute the spore solution 10-fold, and pipette 1 μL of the solution onto PDA medium. Three replicates were performed for each drug treatment group. Colony diameters were measured after incubation at 28°C for 3 days.

[0041] According to the test results ( Figure 6 , Figure 7 Compared to the blank control group, treatment with 0.125-0.5g / L lonidamine significantly reduced the colony diameter of Fusarium solani in the presence of 0.08g / L cinnamaldehyde and 0.08g / L cinnamaldehyde + 2% glucose. Glucose requires phosphorylation to glucose-6-phosphate by hexokinase for further utilization by the body. The inhibition of glucose metabolism by lonidamine potentiates the antibacterial activity of cinnamaldehyde against Fusarium solani. This technology provides a new treatment option for Fusarium solani-induced keratitis.

[0042] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for inhibiting the activity of Fusarium solani, characterized in that: The active ingredients of the pharmaceutical composition consist of plant essential oil cinnamaldehyde and hexokinase inhibitor lonidamine.

2. The pharmaceutical composition for inhibiting the activity of Fusarium solani according to claim 1, characterized in that: The mass ratio of cinnamaldehyde to lonidamine in the pharmaceutical composition is 0.08:0.125-0.

5.

3. The pharmaceutical composition for inhibiting the activity of Fusarium solani according to claim 2, characterized in that: The mass ratio of cinnamaldehyde to lonidamine in the pharmaceutical composition is 0.08:0.

125.

4. Use of the pharmaceutical composition according to any one of claims 1 to 3 in inhibiting the activity of Fusarium solani.

5. Use of the pharmaceutical composition according to any one of claims 1 to 3 in the treatment of fungal keratitis.

Images