Antifungal effects of andrographolide and its combination with amphotericin B against selected fungal pathogens

Collection and maintenance of fungal isolates

In this study, six fungal isolates were used. Three fungal strains—Aspergillus fumigatus (ATCC 46645), Aspergillus terreus (ATCC 10690) and Candida albicans (ATCC 64124)—were obtained from American Type Culture Collection (ATCC). Additionally, three fungal isolates namely Aspergillus niger, Microsporum gypseum and Trichophyton mentagrophytes were collected from microbial stock cultures collection at the Mycology Laboratory, Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, which were further confirmed using MALDI-TOF MS. Every fungal isolate that was collected was kept at 4 °C on potato dextrose agar (PDA) medium (Mahlo, McGaw & Eloff, 2010; Mbunde et al., 2019). Prior to antifungal susceptibility testing, the pure isolates of the fungal species were subcultured on PDA medium and incubated at 30 °C for two days (C. albicans), three days (A. fumigatus and A. niger), five days (A. terreus and M. gypseum), and seven days for T. mentagrophytes.

Preparation of antifungal stock solution

Amphotericin B (MedChemExpress) and andrographolide powder (Sigma-Aldrich) were purchased from authorized distributors. Following optimization, 3.2 mg of andrographolide was dissolved in one mL dimethyl sulfoxide (DMSO) to give a stock concentration solution of 3,200 µg/mL. For amphotericin B, a stock concentration solution of 1,600 µg/mL was produced by dissolving 4.8 mg in three mL DMSO, equivalent to 1.6 mg/mL (CLSI, 2017). DMSO’s impact on test organisms was assessed preliminarily and found negligible. This was done by treating the test isolates with 2% DMSO in potato dextrose broth (PDB) medium using a 96-well microtiter plate, and incubated at 30 °C for 48 h to assess growth inhibition. Stock solutions were sealed in sterile vials and stored at −60 °C until further used (CLSI, 2017).

Preparation of fungal inoculums

The mold isolates were cultivated on PDA medium and incubated at 30 °C for 2–7 days to achieve optimal conidiation or sporulation. After being extracted, cells were washed using sterile saline and made to 0.5 McFarland equivalent yielding approximately 0.4–5.0 × 10⁶ CFU/mL (Berkow, Lockhart & Ostrosky-Zeichner, 2020; CLSI, 2017). To obtain a working inoculum suspension of approximately 0.8–1.0 × 10⁵ CFU/mL for the molds using microdilution method, this suspension was further diluted 1:50 in potato dextrose broth (PDB) medium (CLSI, 2017). For Candida albicans, colonies were obtained from a 2-day culture and suspended in sterilized saline. The turbidity was prepared to a 0.5 McFarland equivalent, yielding approximately 1–5 × 10⁶ CFU/mL (CLSI, 2022; Ohikhena, Wintola & Afolayan, 2017). Subsequently, 1:50 and 1:20 dilutions in PDB medium were made using a microdilution method to achieve a working inoculum suspension of approximately 1–5 × 10³ CFU/mL (CLSI, 2022).

Preparation of iodonitrotetrazolium chloride (INT)

Iodonitrotetrazolium chloride (INT; Sigma-Aldrich) was allowed to thaw, and 0.2 mg was dissolved using one mL sterilized distilled water to obtain 0.2 mg/mL concentration (Mawire et al., 2021). Preliminary assessment of INT’s impact on test organisms was found negative. This was done by treating the test isolates with 40 µL of 0.2 mg/mL INT in potato dextrose broth (PDB) medium using a 96-well microtiter plate, and was incubated at 37 °C for 2 h to assess colour changes due to microbial activity.

Broth microdilution antifungal assay of andrographolide

Antifungal activity was evaluated using a two-fold broth microdilution technique in 96-well sterile microtiter plates, succeeding modified protocols of Mbunde et al. (2019) and Ohikhena, Wintola & Afolayan (2017). Wells in column 1 contained 200 µL of PDB medium as sterility controls, while columns 2–11 were filled with 100 µL of the medium. Column 2 received 100 µL of andrographolide stock concentration resulting in the highest testing concentration of 1,600 µg/mL following dilution. A two-fold serial dilution (1,600 to 3.13 µg/mL) was performed across these columns by sequentially transferring 100 µL from one column to the next, discarding the final 100 µL from column 11. Column 12 contained PDB medium with DMSO, serving as growth control. Subsequently, 100 µL inoculum suspension was dispensed to wells in columns 2–12, yielding a final 200 µL volume per well. The plates were incubated for 48 h at 30 °C, followed by adding 40 µL of 0.2 mg/mL solution of INT to each well, and additional incubation for 2 h at 37 °C to observe colour changes.

Minimum inhibitory concentration determination

After incubation, 40 µL INT solution at 0.2 mg/mL concentration was introduced into the wells, and incubated for two hours at 37 °C. Presence of growth was evaluated by observing INT colour change; growth was indicated by pinkish-red formazan, whereas clear solutions indicated growth inhibition. Minimum inhibitory concentrations (MICs) were identified as lowest concentrations at which there was no colour change (Mawire et al., 2021; Mbunde et al., 2019).

Minimum fungicidal concentration determination

To ascertain the minimum fungicidal concentrations (MFCs), 20 µL of culture from colourless wells were inoculated onto fresh PDA plates, and were then incubated for 48 h at 30 °C. The lowest antifungal concentration with no or fewer than three colonies (indicates 99.0–99.5% killing activity) was considered the MFC. Agents are fungicidal if MFC/MIC ≤ 4, and fungistatic if MFC/MIC > 4 (Gamal et al., 2023; Mussin et al., 2019; Ohikhena, Wintola & Afolayan, 2017).

Antifungal effects of amphotericin B and andrographolide combination

The combination interactions between andrographolide and amphotericin B against the test isolates were evaluated using the modified microdilution checkerboard approach by Bogue et al. (2021) and Jiang et al. (2022). Briefly, two-fold serial dilutions of andrographolide (from 6.25 to 1,600 µg/mL), and amphotericin B (from 0.25 to 16.00 µg/mL) were arranged across horizontal and along vertical axes of 96-well plates, respectively. A total of 50 µL of the serially diluted andrographolide concentrations were added horizontally, and 50 µL of serially diluted amphotericin B concentrations were added in a vertical direction to the combination wells already containing 100 µL of prepared inoculum. This was incubated at 30 °C for 48 h.

Subsequently, growth inhibitions and MICs were determined using INT solution as described previously. The interaction of andrographolide with amphotericin B was referred to as fractional inhibitory concentration (FIC) index, which was evaluated using: FIC index = (MIC_ADR in combination/MIC_ADR alone) + (MIC_AMB in combination/MIC_AMB alone), where ADR and AMB represent andrographolide and amphotericin B, respectively. MIC_AMB in combination refers to the amount of amphotericin B required to inhibit growth when used alongside andrographolide, while MIC_ADR in combination represents the quantity of andrographolide needed to achieve growth inhibition under the same conditions. The fractional inhibitory concentration index (FICI) was categorized as synergy (FICI ≤ 0.5), additive (0.5 < FICI ≤ 1.0), indifference (1.0 < FICI ≤ 4.0), and antagonism (FICI > 4.0) (Alanís-Ríos et al., 2022; Ativui et al., 2022; Bidaud et al., 2022; Mussin et al., 2019; Shaban, Patel & Ahmad, 2020).

Antifungal activities of andrographolide

Andrographolide’s antifungal action was assessed by observing its minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) against various fungal isolates, including both filamentous fungi and yeasts. As illustrated in Fig. 1, the microdilution plates demonstrate the MICs for various fungal isolates using INT as an indicator. The plates show distinct MIC endpoints for the isolates tested. MIC values are marked by the white boxes, while active fungal growth, indicating no inhibition, is represented by red coloration. These findings are further supported by Table 1, which summarizes the MIC and MFC data. The ratio of MFC to MIC was determined to assess the compound’s fungistatic or fungicidal potential, with a ratio ≤ 4 indicating fungicidal activity and a ratio > 4 suggesting fungistatic action. As shown in Table 1, the MIC against Aspergillus fumigatus, Aspergillus niger, and Trichophyton mentagrophytes was 400 µg/mL, indicating that a relatively lower concentration is required to inhibit these species. In contrast, Aspergillus terreus, Microsporum gypseum, and Candida albicans required a higher MIC of 800 µg/mL, suggesting that these fungi might require higher doses for growth inhibition.

The MFC values, ranging from 800 to >1,600 µg/mL, reflect the concentration at which andrographolide exerts fungicidal effects by killing fungal cells. For most isolates, values of MFC were two to four times more than their MICs, aligning with the typical pattern for antifungal compounds. Specifically, the MFC for A. fumigatus and A. niger was 800 µg/mL, while A. terreus and T. mentagrophytes required higher concentrations of 1,600 µg/mL to achieve fungicidal action. Interestingly, the MFC values for M. gypseum and C. albicans exceeded the maximum tested concentration (>1,600 µg/mL), indicating incomplete fungicidal activity at these concentrations. This suggests a potentially fungistatic rather than fungicidal action for these isolates. There may be limited fungicidal action against these pathogens if the minimum fungicidal activity (MFC) occasionally equals or exceeds the maximum concentration tested. Nonetheless, the substance has often shown antifungal action against the pathogens at varying concentrations.

Notably, the ratio of MFC to MIC was ≤ 4 for majority of isolates, indicating fungicidal activity. Specifically, A. fumigatus, A. terreus, and A. niger exhibited an MFC/MIC ratio of 2, confirming andrographolide’s fungicidal potential. T. mentagrophytes showed a ratio of 4, still within the fungicidal range, though at a higher concentration. However, M. gypseum and C. albicans did not achieve fungicidal thresholds within the tested concentration range (MFC > 1,600 µg/mL), suggesting a possible fungistatic effect.

Antifungal activities of andrographolide and amphotericin B combination

Figure 2 illustrates the combination activity of andrographolide (ADR) and amphotericin B (AMB) with varying concentrations of AMB (annotated on the left) and ADR (annotated at the bottom), allowing visualization of the agents’ effects individually and in combination. The interactions between ADR and AMB against the fungal pathogens demonstrate notable variations in efficacy, as highlighted by their MIC values alone and in combination. The MIC_ADR alone ranged from 400 to 800 µg/mL, whereas MIC_AMB alone were between four and 16 µg/mL, reflecting AMB higher potency as a standalone agent as compared to ADR. When used in combination, the MIC values for both agents were markedly reduced; MIC_ADR decreased to a range of 50 to 200 µg/mL, and MIC_AMB was reduced to between one and eight µg/mL (Table 2). These reductions underscore the enhanced effectiveness of the combination therapy compared to the individual agents.

In addition, the combination of ADR and AMB had a synergistic effect against A. fumigatus, A. niger, T. mentagrophytes and C. albicans with the FICI values between 0.375 and 0.500 (FICI ≤ 0.5) as shown in Table 2. This implies that the combined impact of the two agents against these pathogens is more than the effects of the individual agents. To manage fungal infections caused by these fungi, the synergistic combination of andrographolide and amphotericin B shows promise as a novel therapeutic approach. This is because it may be possible to achieve therapeutic synergy at lower concentrations of both compounds, which would minimize toxicity, improve treatment outcomes, and possibly overcome resistance mechanisms. Conversely, the combination exhibited additive interaction against A. terreus and M. gypseum with FICI values of 0.75 (0.5 < FICI ≤ 1) each. This suggests that the two agents working together are just as successful as they would be alone. The outcomes of this study indicate that the combination of andrographolide and amphotericin B exhibits varying levels of efficacy depending on the fungal species. Synergistic interactions observed with A. fumigatus, A. niger, T. mentagrophytes, and C. albicans highlight the potential of this combination in treating infections caused by these pathogens, as the enhanced activity allows for lower dosages, which could reduce toxicity, especially for amphotericin B. However, the additive interactions with A. terreus and M. gypseum indicate that the combination does not offer additional benefits over individual therapies for these particular fungi.