Application of Tongkat Lactone in the Preparation of Products for Inhibiting Candida Albicans

Abstract

The present invention discloses the use of tongkat lactone in the preparation of a product for inhibiting Candida albicans, relating to the field of biomedicine. Tongkat lactone can effectively inhibit hyphae and biofilm formation of Candida albicans and reduce the adhesion of Candida albicans to cells. Low concentrations of the compound have no effect on Candida albicans growth, thereby effectively treating Candida albicans infections or diseases caused by Candida albicans infections. Tongkat lactone has great application prospects in the development of new antifungal drugs, especially drugs for treating Candida albicans infections.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of tongkat lactone in the preparation of a product for inhibiting Candida albicans. Background Art

[0002] Candida albicans is a common commensal fungus that inhabits the oropharynx, gastrointestinal tract, vagina, and skin of healthy individuals. The primary source of Candida albicans in the human body is the gastrointestinal tract, where infection occurs due to dysbiosis of the resident microbial flora, immune dysfunction, and disruption of the mucosal barrier. While Candida albicans does not typically cause disease in healthy individuals on mucosal surfaces such as the mouth and intestines, it can cause severe systemic infections in patients with compromised or suppressed immune systems (such as those undergoing chemotherapy, organ transplants, or AIDS), with a mortality rate as high as 40%.

[0003] Currently, there are limited clinical antifungal drugs available, with azoles (fluconazole) being the most widely used. Fluconazole inhibits fungal replication and exerts an antifungal effect. However, with the overuse of antibiotics, drug resistance is becoming increasingly serious. Therefore, there is a need to provide new drugs with better inhibitory effects on Candida albicans. Summary of the Invention

[0004] The present invention aims to address at least one of the technical problems existing in the prior art. To this end, the present invention provides the use of tongkat lactone or a pharmaceutically acceptable salt thereof. Tongkat lactone can effectively inhibit hyphae and biofilm formation of Candida albicans, reduce the adhesion of Candida albicans to cells and the cytotoxicity of Candida albicans, and thus effectively prevent and treat Candida albicans infection or diseases caused by Candida albicans infection.

[0005] The present invention also provides a product comprising tongkat lactone or a pharmaceutically acceptable salt thereof.

[0006] The present invention also provides a method for inhibiting the activity of Candida albicans for purposes other than disease diagnosis and treatment.

[0007] According to the first aspect of the present invention, the use of tongkat lactone or a pharmaceutically acceptable salt thereof in any one of items A1 to A4,

[0008] A1. preparing a product for inhibiting the activity of Candida albicans;

[0009] A2. Preparation of products for use against Candida albicans infection;

[0010] A3. Non-disease diagnosis and treatment destination for anti-Candida albicans infection;

[0011] A4. Preparation of products for preventing and / or treating diseases caused by Candida albicans infection.

[0012] The structural formula of tongkat lactone is shown in Formula I.

[0013]

[0014] According to some embodiments of the present invention, the use is achieved by inhibiting at least one of hyphae formation and biofilm formation of Candida albicans and reducing cell adhesion and cell toxicity of Candida albicans.

[0015] According to some embodiments of the present invention, the effective concentration of tongkat lactone or a pharmaceutically acceptable salt thereof may be 12.5 to 100 μM, for example, 12.5, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μM.

[0016] Use of tongkat lactone or a pharmaceutically acceptable salt thereof in B1 or B2 according to the second aspect of the present invention: B1, preparing a product for inhibiting the formation of Candida albicans hyphae;

[0017] B2. Non-disease diagnosis and treatment purpose: inhibiting the hyphae formation of Candida albicans.

[0018] Use of tongkat lactone or a pharmaceutically acceptable salt thereof in C1 or C2 according to the third aspect of the present invention: C1, preparing a product for reducing the adhesion of Candida albicans cells;

[0019] C2. Non-disease diagnosis and treatment destination: Reduce the adhesion of Candida albicans cells.

[0020] Use of tongkat lactone or a pharmaceutically acceptable salt thereof in D1 or D2 according to the fourth aspect of the present invention: D1, preparing a product for inhibiting Candida albicans biofilm formation;

[0021] D2. Non-disease diagnosis and treatment destination: inhibiting the formation of Candida albicans biofilm.

[0022] According to some embodiments of the present invention, the effective concentration of tongkat lactone or a pharmaceutically acceptable salt thereof is higher than 12.5 μM. Preferably, it is 12.5 to 100 μM.

[0023] According to the fifth aspect of the present invention, the use of tongkat lactone or a pharmaceutically acceptable salt thereof in E1 or E2, E1, preparing a product for reducing the toxicity of Candida albicans cells;

[0024] E2. Non-disease diagnosis and treatment destination: Reduce the toxicity of Candida albicans cells.

[0025] A product according to a sixth aspect of the present invention comprises tongkat lactone or a pharmaceutically acceptable salt thereof;

[0026] The product has at least one of the effects F1 to F7:

[0027] F1, anti-Candida albicans infection;

[0028] F2. Prevention and / or treatment of diseases caused by Candida albicans infection;

[0029] F3, inhibiting the formation of Candida albicans hyphae;

[0030] F4, reducing the adhesion of Candida albicans;

[0031] F5, inhibiting Candida albicans biofilm formation;

[0032] F6. Reduce the toxicity of Candida albicans cells.

[0033] According to some embodiments of the present invention, the product is a virulence inhibitor or a drug.

[0034] According to some embodiments of the present invention, the product further comprises a pharmaceutically acceptable excipient.

[0035] According to some embodiments of the present invention, the auxiliary material includes at least one of a carrier, a diluent, and an excipient.

[0036] According to some embodiments of the present invention, the dosage form of the product is selected from at least one of emulsion, ointment, aerosol, suspension, wettable powder, powder, granule, and aqueous solution.

[0037] According to some embodiments of the present invention, the product contains the tongkat lactone or a pharmaceutically acceptable salt thereof as an effective active ingredient.

[0038] According to some embodiments of the present invention, the effective concentration of tongkat lactone or a pharmaceutically acceptable salt thereof may be 12.5-100 μM.

[0039] The present invention has at least the following beneficial effects:

[0040] Yeast-hyphae dimorphism is a unique characteristic of Candida albicans. During infection, the free yeast state is non-pathogenic to the host, primarily functioning to adhere to receptor tissues. It then undergoes a transformation from yeast to hyphae to facilitate invasion, and then infects host tissues in the hyphae form, further exerting its virulence. The yeast-hyphae morphological transition is a crucial process for Candida albicans to exert its virulence. Tongkat lactone can effectively inhibit hyphae formation and biofilm formation, reducing cell adhesion and cytotoxicity, thereby effectively treating Candida albicans infections or diseases caused by them. Tongkat lactone is also non-toxic to human cells and is not prone to developing drug resistance. Tongkat lactone has great potential for the development of new antifungal drugs, particularly those against Candida albicans infections.

[0041] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0043] Figure 1 These are photos of Candida albicans hyphae formation in the DMSO control group (A) and the 100 μM tongkat lactone-treated group (B) of Example 1 of the present invention;

[0044] Figure 2 The effect of tongkat lactone in Example 1 of the present invention on the hyphae formation rate of Candida albicans; wherein, ** indicates a significant difference compared with the DMSO control group, p≤0.01, and *** indicates a very significant difference compared with the DMSO control group, p≤0.001;

[0045] Figure 3 The effect of tongkat lactone in Example 2 of the present invention on the adhesion of Candida albicans; wherein, * indicates a significant difference compared with the DMSO control group, p≤0.05, and *** indicates a very significant difference compared with the DMSO control group, p≤0.001;

[0046] Figure 4 This is the effect of tongkat lactone in Example 3 of the present invention on the biofilm formation of Candida albicans; *** indicates a highly significant difference compared with the DMSO control group, p≤0.001;

[0047] Figure 5 is the effect of tongkat lactone of Example 4 of the present invention on the growth of Candida albicans;

[0048] Figure 6 : The effect of tongkat lactone in Example 5 of the present invention on the toxicity of Candida albicans; wherein A is the cytotoxicity test result of tongkat lactone itself on A549 cells, and B is the test result of the effect of tongkat lactone on the toxicity of Candida albicans; wherein ** indicates a significant difference compared with the DMSO control group, p≤0.01, and *** indicates a very significant difference compared with the DMSO control group, p≤0.001;

[0049] Figure 7 5 are photos of pathological sections of the tongues of mice in each treatment group in Example 6 of the present invention. DETAILED DESCRIPTION

[0050] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0051] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0052] In the description of the present invention, the terms "comprises" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to the process, method, product or apparatus.

[0053] Eurycomalactone used in the following examples has a CAS number of 23062-24-0 and was provided by MCE.

[0054] The Candida albicans used in the following examples was the standard strain SC5314 (also known as ATCC MYA-2876). The activation step for Candida albicans was as follows: Candida albicans was inoculated onto LB solid medium (formulation: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, with double-distilled water as the solvent) for activation and incubated in a 30°C incubator overnight.

[0055] In the following examples, the method for reviving and culturing human non-small cell lung cancer cell line A549 cells is as follows: frozen-thawed A549 cells are transferred to DMEM medium (Gibco) containing 10% (v / v) FBS and cultured overnight at 37° C. and 5% CO 2 .

[0056] The formula of the GMM culture medium used in the following examples is: 6.7 g / L YNB, 0.2% glucose.

[0057] The cell maintenance medium used in the following examples was DMEM medium containing 1% (v / v) FBS.

[0058] Example 1 (Effect of Tongkat Lactone on Candida albicans Hyphae)

[0059] The test method is as follows:

[0060] The activated Candida albicans was picked and inoculated into GMM culture medium, cultured at 30℃ and 200rpm overnight, and the OD value of the culture medium was measured. 600 , dilute the bacterial solution with GMM culture medium to OD 600 =0.1. 500 μL of bacterial culture was placed in a 1.5 mL EP tube and tongkat lactone (DMSO solvent, final concentrations of 100 μM, 50 μM, 25 μM, and 12.5 μM) was added. An equal volume of DMSO without tongkat lactone was added and treated identically, serving as the DMSO control group (denoted as DMSO). Each group was repeated three times. The mixture was vortexed and incubated in a 37°C water bath for 6 h. The mixture was centrifuged at 5000 rpm for 10 min, the supernatant discarded, and the cells were resuspended in 40 μL of GMM culture medium. Mycelial formation was observed under a Zeiss Axioplan 2 microscope, and photographs were taken from different fields of view. Three photographs were taken for each sample. The number of hyphae in the photographs was counted, with the hyphae formed in the DMSO control group as 100%. The mycelial formation rate was calculated.

[0061] Photos of the effects of Tongkat Lactone on hyphae formation of Candida albicans Figure 1 The effect of tongkat lactone on the hyphae formation rate of Candida albicans is shown in Figure 2 shown.

[0062] DMSO did not affect the formation of Candida albicans hyphae. By counting hyphae, it was found that tongkat lactone had a significant inhibitory effect on Candida albicans hyphae, with a certain concentration-dependent effect. The hyphae formation rates after treatment with 50μM, 25μM, and 12.5μM tongkat lactone were 16%, 48%, and 71%, respectively. Among them, after treatment with 100μM tongkat lactone, Candida albicans basically did not form hyphae.

[0063] Example 2 (Effect of Tongkat Lactone on Adhesion of Candida albicans)

[0064] The test method is as follows:

[0065] (1) After A549 cells were revived and cultured, 0.5×10 3 Cells were seeded at a concentration of 100 cells / well in a 96-well plate and cultured overnight. When the cells covered 80% of the bottom of the 96-well plate, the culture medium was discarded and the cells were washed three times with 1× PBS.

[0066] (2) Inoculate Candida albicans in GMM culture medium, culture at 30°C, 200 rpm, and shake overnight to measure the OD value of the culture medium. 600 Then, the bacterial solution was diluted to OD 600= 0.5 to obtain a diluted bacterial solution. DMSO containing 10 mM, 5 mM, 2.5 mM, and 12.5 mM tongkat lactone, respectively, was mixed with the diluted bacterial solution at a volume ratio of 1:99 and vortexed to obtain test solution A (final tongkat lactone concentrations of 100 μM, 50 μM, 25 μM, and 12.5 μM, respectively).

[0067] (3) Add test solution A to the 96-well plate in step (1) at 100 μL / well, and set up 3 replicates for each treatment. The group not treated with tongkat lactone was used as the DMSO control group (denoted as: DMSO). The 96-well plate was placed at 37°C for incubation for 1.5 hours, and the culture medium was discarded. 100 μL of 0.5% (w / v) crystal violet solution was added to each well and allowed to act at room temperature for 45 minutes; the crystal violet solution was discarded, and the cells were washed 10 times with ice ddH2O, and 100 μL of 75% (v / v) ethanol solution was added. The cells were placed at room temperature for 30 minutes, and the OD was measured. 570 The data were processed using GraphPad Prism 6 software.

[0068] Test results such as Figure 3 shown.

[0069] Using the DMSO control group as a reference, the cell adhesion rates of Candida albicans treated with 100μM, 50μM, 25μM, and 12.5μM tongkat lactone were 40%, 68%, 77%, and 87%, respectively. Furthermore, the adhesion of Candida albicans to cells was reduced by over 50% after treatment with a final concentration of 100μM tongkat lactone. This indicates that tongkat lactone exhibits an inhibitory effect on the adhesion of Candida albicans.

[0070] Example 3 (Effect of Tongkat Lactone on Biofilm Formation of Candida albicans)

[0071] The test method is as follows:

[0072] Inoculate Candida albicans in GMM culture medium, culture at 30°C, 200 rpm, and measure the OD value of the culture medium. 600 , dilute the bacterial solution with GMM culture medium to OD 600=0.1 to obtain the diluted bacterial solution. DMSO containing 10mM, 5mM, 2.5mM, and 1.25mM tongkat lactone were mixed with the diluted bacterial solution at a volume ratio of 1:99, and the mixture was shaken to obtain test solution B (the final concentration of tongkat lactone was 100μM, 50μM, 25μM, and 12.5μM, respectively). Test solution B was added to a 96-well plate at 100μL / well, and 3 replicates were set for each treatment. The group not treated with tongkat lactone was used as the DMSO control group (denoted as: DMSO). After the 96-well plate was incubated at 37°C for 8h, the culture medium was discarded, and 100μL of 0.5% (w / v) crystal violet solution was added and allowed to act at room temperature for 45min; the crystal violet solution was discarded, and the plate was washed 10 times with pre-cooled ddH2O, and 100μL of 75% (v / v) ethanol solution was added and allowed to stand at room temperature for 30min. The OD was measured. 570 The data were processed using GraphPad Prism 6 software.

[0073] Test results such as Figure 4 shown.

[0074] Using the DMSO control as a reference, the biofilm formation rates of Candida albicans after treatment with 100μM, 50μM, 25μM, and 12.5μM tongkat lactone were 9.45%, 14.46%, 15.63%, and 21.38%, respectively. Furthermore, the biofilm formation rate of Candida albicans on polystyrene was reduced by over 90% after treatment with 100μM tongkat lactone. This indicates that tongkat lactone has a significant inhibitory effect on Candida albicans biofilm formation.

[0075] Example 4 (Effect of Tongkat Lactone on the Growth of Candida albicans)

[0076] The test method is as follows:

[0077] Inoculate Candida albicans in GMM culture medium, culture at 30°C, 200 rpm, and measure the OD value of the culture medium. 600 , dilute the bacterial solution with GMM culture medium to OD 600 =0.05 to obtain the diluted bacterial solution. DMSO containing 10mM, 5mM, 2.5mM, and 1.25mM tongkat lactone were mixed with the diluted bacterial solution at a volume ratio of 1:99, and shaken to obtain test solution C (the final concentration of tongkat lactone was 100μM, 50μM, 25μM, and 12.5μM, respectively). Test solution C was added to a 100-well plate at 300μL / well, and 3 replicates were set for each treatment. The group not treated with tongkat lactone was used as the DMSO control group (denoted as: DMSO). The 100-well plate was placed in a growth curve analyzer, and the culture conditions were 30°C and 200rpm. The OD was measured every 2h. 600The experimental results were observed after 20 h of culture, and the data were processed using GraphPad Prism 6 software.

[0078] Test results such as Figure 5 shown.

[0079] Using the DMSO control as a reference, treatment with 100μM and 50μM tongkat lactone exhibited some inhibitory effect on the growth of Candida albicans, but treatment with 12.5μM and 25μM tongkat lactone showed minimal inhibitory effect. This suggests that tongkat lactone's inhibitory effect on Candida albicans is not primarily achieved by killing fungal cells, making it less likely to induce drug resistance.

[0080] Example 5 (Effect of Tongkat Lactone on the Virulence of Candida albicans)

[0081] The test method is as follows:

[0082] (1) After A549 cells were revived and cultured, the cells were cultured at a rate of 1.5×10 4 Cells were seeded at a concentration of 100 cells / well in a 96-well plate and cultured overnight. When the cells covered 80% of the bottom of the 96-well plate, the culture medium was discarded and the cells were washed three times with 1× PBS.

[0083] (2) Inoculate Candida albicans in GMM culture medium, culture at 30°C, 200 rpm, and shake overnight to measure the OD value of the culture medium. 600 Then, the bacterial solution was diluted to OD 600 =1.0, and then diluted 10 times with cell maintenance medium to obtain the diluted bacterial solution.

[0084] (3)① Cytotoxicity assay of tongkat lactone on A549 cells:

[0085] DMSO containing 10 mM tongkat lactone was mixed with the cell maintenance medium at a volume ratio of 1:99 to obtain test solution D (final tongkat lactone concentration was 100 μM). The group not treated with tongkat lactone served as the DMSO control group (denoted as: DMSO). Test solution D was added to the 96-well plate prepared in step (1) at 100 μL / well and cultured in a cell culture incubator at 37°C, 5% CO2 for 8 h. Three replicates were performed for each treatment.

[0086] The LDH release amount of the lysate group was used as a reference (recorded as 100%) to standardize the LDH release ratio after treatment with tongkat lactone.

[0087] ②Effects of Tongkatolactone on the Virulence of Candida albicans:

[0088] DMSO containing 10 mM, 5 mM, 2.5 mM, and 1.25 mM tongkat lactone was mixed with the diluted bacterial solution at a volume ratio of 1:99 to obtain test solution E (the final concentration of tongkat lactone in test solution E was 100 μM, 50 μM, 25 μM, and 12.5 μM, respectively). 100 μL / well of test solution E was added to the 96-well plate prepared in step (1) and cultured in a cell culture incubator at 37°C, 5% CO2 for 8 h. Three replicates were used for each treatment.

[0089] (4) Cytotoxicity was determined according to the CytoTox NonRadioactive Cytotoxicity Assay protocol from Promega, and the data were subsequently processed using GraphPad Prism 6.

[0090] The LDH release amount of the DMSO control group was used as a reference (recorded as 100%) to standardize the LDH release ratio after tongkat lactone treatment.

[0091] The test results are as follows Figure 6 shown.

[0092] In the absence of Candida albicans, 100μM tongkat lactone showed no toxicity to A549 cells, indicating that tongkat lactone itself is non-cytotoxic. However, in the presence of Candida albicans, tongkat lactone exhibited a strong protective effect on the cells; cytotoxicity after treatment with 100μM, 50μM, 25μM, and 12.5μM tongkat lactone was 11%, 22%, 33%, and 46%, respectively. In particular, treatment with 100μM tongkat lactone reduced the toxicity of Candida albicans by over 80%. This indicates that tongkat lactone has a strong inhibitory effect on the cytotoxicity of Candida albicans.

[0093] Example 6 (Effect of Tongkat Lactone on Candida albicans Oral Infection in Mice)

[0094] The test method is as follows:

[0095] (1) BALB / c mice (6-8 weeks old, male, purchased from Guangdong Experimental Animal Center) were raised at the Experimental Animal Center of South China Agricultural University and randomly divided into groups of 8 mice each. The weights of the mice were recorded and each mouse was marked.

[0096] (2) Inoculate Candida albicans in GMM culture medium, shake culture at 30°C and 200 rpm overnight, centrifuge at 4°C and 3000 rpm for 5 min, discard the supernatant, wash twice with pre-cooled sterile PBS, count by hemocytometer, and prepare Candida albicans with PBS to a bacterial density of 3×10 6 CFU / mL of bacterial suspension.

[0097] (3) DMSO containing 10 mM and 5 mM tongkat lactone was mixed with PBS at a volume ratio of 1:99 to obtain test solution F (the final concentration of tongkat lactone was 100 μM and 50 μM, respectively).

[0098] (4) Mouse oral infection experiment: The mice were weighed one day before infection and injected with 0.1 mL / 10 g of hydrocortisone (22.5 mg / mL) per mouse. Before inoculation, the mice were anesthetized with 10% chloral hydrate and placed flat in a 37°C constant temperature environment. A sterile cotton ball was soaked in the bacterial suspension of step (2) for 3 minutes and then placed under the mouse tongue for 90 minutes. Mice treated with a sterile cotton ball soaked in PBS solution served as a blank control group (denoted as: PBS). On the 1st, 3rd, and 5th day, each mouse was gavaged with test solution F at 100 μL / 10 g. Mice treated with test solution F containing 0 μM tongkat lactone served as a positive control group (denoted as: +C.albicans & DMSO); the 100 μM and 50 μM tongkat lactone treatment groups were denoted as: +C.albicans & Eurycomalactone (100 μM) and +C.albicans & Eurycomalactone (50 μM), respectively. The mice were killed on the 6th day, and their tongues were dissected and further observed by pathological sections.

[0099] Test results such as Figure 7 shown.

[0100] By observing the formation of Candida albicans on the mouse tongue, it was found that a thick layer of Candida albicans hyphae formed on the tongue of the mice in the positive control group, while no hyphae were formed on the tongue of the mice in the blank control group and the tongkat lactone-treated group.

[0101] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the scope of knowledge possessed by ordinary technicians in the relevant technical field without departing from the scope of the present invention.

Claims

1. Use of tongkat lactone or a pharmaceutically acceptable salt thereof in the preparation of a product for preventing and / or treating diseases caused by Candida albicans infection, wherein the product has the functions of inhibiting Candida albicans hyphae formation, reducing Candida albicans cell adhesion, inhibiting Candida albicans biofilm formation, and reducing Candida albicans virulence.

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