Abstract

Background and Objective: Fungal infections impose a great burden on the healthcare system. Medicinal plants are widely used in the treatment of various infectious diseases. Therefore, this study aimed to investigate the synergistic inhibitory effects of Echinophora platyloba (E. platyloba) and Mentha piperita (M. piperita) essential oils against Aspergillus flavus (A. flavus) in vitro.

Methods: The study was conducted to determine the antifungal activities of E. platyloba and M. piperita essential oils against a standard strain (Persian Type Culture Collection: 5006) and 10 clinical isolates of A. flavus. The inhibitory effect of the essential oils on the growth of A. flavus was evaluated by determining minimum inhibitory concentration (MIC) using a 96-well microplate assay according to the Clinical and Laboratory Standards Institute M38-A2 protocol.

Results: Our results showed that M. piperita essential oil exhibited the strongest antifungal activity, with MIC values mostly below 625 ppm across clinical isolates and the standard strain of A. flavus. E. platyloba essential oil demonstrated MIC values up to 1250 ppm. Notably, the combination of both essential oils produced a synergistic effect, significantly reducing the MIC values compared to each oil alone. According to the generalized estimating equations model, the combination reduced the MIC by an average of 175.52 ppm compared to E. platyloba alone (P=0.009). This indicates enhanced efficacy of the combined essential oils in inhibiting fungal growth at lower concentrations.

Conclusion: Taken together, these findings revealed that both essential oils and their combinations exhibited antifungal effects. This study suggests that M. piperita and E. platyloba essential oils may be used as a secondary treatment along with antifungal chemical drugs and may also serve as food preservatives.

Keywords: Antifungal effects, Aspergillus flavus, Echinophora platyloba, Essential oils, Mentha piperita

Introduction

Fungal infections have increased remarkably worldwide over recent decades (1). It is estimated that nearly one billion people worldwide suffer from various fungal infections, including those affecting hair, skin, and nails. Annually, approximately 1.5 million deaths are attributed to fungal diseases. The severity of these infections ranges widely—from mild superficial infections to invasive, potentially fatal systemic diseases (2). Most fungal infections are opportunistic, primarily affecting individuals with compromised health, such as those with immune deficiencies, cancer, organ failure, or those undergoing organ transplantation (2, 3).

The genus Aspergillus includes over 250 species, several of which are significant pathogens. Among them, Aspergillus flavus (A. flavus) is particularly important due to its pathogenic potential in humans and animals (4–7). A. flavus acts as a saprophytic fungus and can cause disease both directly and indirectly by contaminating agricultural products. It produces aflatoxins, carcinogenic secondary metabolites responsible for aflatoxicosis, a serious condition linked to liver cancer. Additionally, A. flavus can cause invasive aspergillosis, which leads to fatal allergic, pulmonary, and extrapulmonary complications (8). Milder forms of infections, such as keratitis, sinusitis, and cutaneous infections caused by A. flavus, have also been documented (9, 10).

Currently, synthetic antifungal agents are the primary treatment for fungal infections. Despite their effectiveness, several critical challenges limit their long-term utility. These include the increasing emergence of drug-resistant fungal strains, which reduce treatment efficacy and complicate clinical management. Additionally, many antifungal drugs exhibit toxicity and adverse side effects that restrict their prolonged or high-dose use. These limitations have heightened the urgency to explore alternative and complementary antifungal strategies, especially those derived from natural products with potentially fewer side effects and novel mechanisms of action (11).

This has increased interest in natural alternatives, particularly medicinal plants, as potential sources of antifungal compounds (12). The genus Echinophora comprises species known for diverse therapeutic properties. Echinophora platyloba (E. platyloba) is traditionally used both as a remedy and as a food seasoning and preservative (13). Previous studies have reported various pharmacological activities of E. platyloba, including antioxidant, antibacterial, antifungal, anticancer, lipid-lowering, and organ-protective effects (13–15), attributed to its phytochemical constituents (14).

Similarly, Mentha piperita (M. piperita), commonly known as peppermint, is a medicinal herb with well-documented antioxidant, antibacterial, anti-inflammatory, and antifungal properties (16, 17).

Despite extensive studies on the individual antifungal effects of these plants, limited research exists regarding their combined effects against A. flavus. Therefore, this study aimed to investigate the potential synergistic inhibitory effects of E. platyloba and M. piperita essential oils against A. flavus in vitro. This could provide a basis for developing novel, natural adjunct therapies to combat fungal infections, especially in the context of rising antifungal resistance.

Materials and Methods

The present study employed an experimental approach to investigate the in vitro antifungal activity of E. platyloba and M. piperita essential oils against A. flavus.

Chemicals

All required chemicals, including Potato Dextrose Agar, Sabouraud Dextrose Agar, Dimethyl sulfoxide (DMSO), and Roswell Park Memorial Institute, were supplied by Merck (Germany) and Sigma (USA).

Plant Material Collection

For the purpose of this study, the aerial parts of E. platyloba and M. piperita, including stems and leaves, were collected in May 2020 from mountainous areas in southern Khorramabad County, Lorestan Province, Iran. The plant samples were identified by botanists at the Lorestan Agriculture and Natural Resources Research Center, Khorramabad, Iran.

Essential Oil Extraction

After plant identification, the samples were air-dried under shaded conditions at room temperature and then ground into powder. The essential oil was extracted using hydrodistillation with a Clevenger apparatus (British Pharmacopeia) for 4 hours. To remove residual water, the essential oil was dehydrated using anhydrous sodium sulfate (Merck, Germany). Finally, the essential oil was stored in a dark vial at 4ºC.

Fungal Strains

The standard strain of A. flavus based on the Persian Type Culture Collection (PTCC: 5006) was obtained from the Medical Mycology Laboratory, Faculty of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran. Furthermore, eleven clinical isolates of A. flavus were used to evaluate the antifungal effect of E. platyloba and M. piperita essential oils. These clinical isolates were obtained from our previous study on immunocompromised patients with respiratory mycoses and were identified using molecular methods (Ethical Registration No. IR.TUMS.SPH.REC.1397.286) (Grant Registration No.97.02.38506).

Determination of Antifungal Activity

To evaluate the antifungal activity of E. platyloba and M. piperita essential oils, the broth microdilution method was used to determine minimum inhibitory concentration (MIC) and Voriconazole against A. flavus strains. The procedure was conducted according to Clinical and Laboratory Standards Institute (CLSI M38-A2) guideline. In the first step, a 96-well microplates was prepared. Then, 100 μL of RPMI 1640 medium was added to the wells. To evaluate the antifungal effects of the essential oils and their combination, two-fold serial dilutions were prepared in 1% DMSO. The selected dilutions were prepared as 5000 ppm to 19.5 ppm, respectively from the highest to the lowest concentration in the 96-well microplates. Following this procedure, two-fold serial dilutions were also prepared for Voriconazole as the antifungal agent (ranging from 100 to 0.37 μg/mL) in the 96-well microplates. After preparation of serial dilutions, the prepared concentrations of E. platyloba essential oil, M. piperita essential oil, and Voriconazole were placed in a separate row of the 96-well plate. In addition, separate rows were assigned to positive (RPMI 1640 medium + fungal suspension) and negative (RPMI 1640 medium + essential oil) control and blank (1% DMSO). Then, fungi suspension (10⁴ CFU/mL) from standard strains and clinical isolates was made in 100 μL of RPMI 1640 medium. After that, the prepared fungi suspension was added to the wells except the wells assigned to the blank and negative control. Finally, the microplates were incubated at 35ºC for 48-72 hours. All experiments were carried out in triplicate. After the incubation period, the microplates were carefully checked for turbidity due to fungi growth. The last dilution of essential oils and Voriconazole without turbidity in each well was considered as the MIC (18, 19).

Statistical Analysis

The mean MIC values were calculated. Then, the data were analyzed using SPSS (version. 21). To analyze the data, the generalized estimating equations (GEE) model with linear link function was used. P<0.05 was considered statistically significant.

Results

Antifungal Effects of E. platyloba and M. piperita Essential Oils and Their Combination Against Clinical Isolates and the Standard Strain of A. flavus

Table 1 presents the effects of the essential oils and their combination against clinical isolates and the standard strain of A. flavus. Our results showed that, except for one strain, the essential Table 2. Mean and Median Values of the Minimum Inhibitory Concentration (MIC) (ppm) of E. platyloba and M. piperita Essential Oils and Their Combination Against Clinical Isolates and Standard Strain of A. flavus
oil of M. piperita exhibited MIC values mostly below 625 ppm. Furthermore, we observed that the essential oil of E. platyloba had an MIC value

of less than 1250 ppm.

Table 1. Effects of E. platyloba and M. piperita Essential Oils and Their Combination on the Minimum Inhibitory Concentration (MIC) of Clinical Isolates and Standard Strain of A. flavus

Abbreviations: #GM, geometric mean; ## range, upper and lower range of data, MIC₅₀, minimum concentration of essential oil that can inhibit 50% of all tested fungal strains; MIC₉₀, minimum concentration of essential oil that can inhibit 90% of all tested fungal strains.

Table 2 and Figure 1 show the mean and median MIC values of E. platyloba and M. piperita essential oils and their combination against clinical isolates and the standard strain of A. flavus. The MIC value of the combination of E. platyloba and M. piperita essential oils against the standard strain was lower than their single administration.

Figure 1. Mean Values of the Minimum Inhibitory Concentration (MIC) (ppm) of E. platyloba and M. piperita Essential Oils and Their Combination Against Clinical Isolates and the Standard Strain of A. flavus

Effects of E. platyloba and M. piperita Essential Oils and Their Combination on the Minimum Inhibitory Concentration (MIC) of Clinical Isolates and the Standard Strain of A. flavus According to the Generalized Estimating Equations (GEE) Model

As shown in Table 3, the results according to the GEE model demonstrated that the effects of different essential oils were significantly (P<0.05) different from the standard medicine to treat fungal infection. According to the GEE model, assuming the strain type is constant, the MIC of M. piperita essential oil was on average 31.81 units lower than the essential oil of E. platyloba, which showed a non-significant difference (P=0.85). Our findings also revealed that the average MIC value of the combination of essential oils was 175.52 units lower than the essential oil of E. platyloba, which demonstrated a significant difference (P=0.009). These results indicated that the combination of essential oils in a lower concentration compared to the essential oil of E. platyloba alone inhibited the growth of A. flavus. Furthermore, we found that the MIC of the combination of essential oils was, on average,145.7 units lower than that of M. piperita essential oil, which indicated a non-significant differences (P=0.209).

Table 3. Effects of Essential Oils and Their Combination on the Minimum Inhibitory Concentration (MIC) of Clinical Isolates and Standard Strain of A. flavus According to the Generalized Estimating Equations (GEE) Model

Discussion

This study aimed to evaluate the antifungal effects of Echinophora platyloba and Mentha piperita essential oils, both individually and in combination, against Aspergillus flavus. We employed the broth microdilution method to determine the MICs of these essential oils against both a standard strain and clinical isolates of A. flavus. Additionally, we applied a GEE model with a linear link function to analyze the data more robustly. To the best of our knowledge, this is the first study to investigate the combined antifungal effects of E. platyloba and M. piperita essential oils against A. flavus standard and clinical isolates.

Our findings indicated that both essential oils inhibited the growth of A. flavus strains. Specifically, M. piperita essential oil exhibited antifungal activity against all clinical isolates except isolate A15 at concentrations above 156 ppm, and inhibited the standard strain at concentrations ≥19.5 ppm. Notably, M. piperita essential oil showed no antifungal effect on isolate A15 even at a concentration of 1250 ppm. The antifungal properties of medicinal plants have been extensively studied (20, 21), and the inhibitory effects of M. piperita on fungal growth have been increasingly documented (21, 22). M. piperita is known for its antioxidant, antibacterial, and antifungal effects (23) and contains key phytochemicals, such as menthone, limonene, menthol, pulegone, carvone, and 1,8-cineole (24). Several studies have confirmed the antifungal role of M. piperita essential oil (25, 26). For example, N.R. Desam et al. reported moderate antifungal activity of M. piperita essential oil against the standard A. flavus strain with an MIC of 10.0±0.06 µg/mL, attributing this effect mainly to menthol and menthone (25). Our results align with these findings, although some differences may arise due to variations in methodology, fungal strains, or the phytochemical content influenced by geographic harvesting locations.

Regarding E. platyloba essential oil, all A. flavus strains were inhibited at concentrations below 625 ppm, except isolates A15 and A24, which required concentrations below 1250 ppm. This suggests these isolates are more resistant to lower concentrations of E. platyloba essential oil. Notably, isolate A15 also demonstrated resistance to lower concentrations of M. piperita. Like M. piperita, the standard strain was most sensitive to E. platyloba, while clinical isolates exhibited greater resistance. Among clinical isolates, A9 was sensitive at 156 ppm, while others required ≥312 ppm for growth inhibition.

Several previous studies support the antifungal activity of E. platyloba. Hasanvand et al. reported an MIC of 750 ppm against the A. flavus standard strain (27), consistent with our findings, further confirming E. platyloba as a potential natural antifungal agent. Similarly, M. Hashemi et al. demonstrated antifungal activity of E. platyloba essential oil at 0.625 mg/mL against the A. flavus standard strain (28), which is consistent with our results. The antifungal effects are likely due to phytochemicals, such as p-cymene, α-pinene, limonene, β-phellandrene, and α-phellandrene, known for potent antifungal properties (28, 29).

Our study also showed that the combination of M. piperita and E. platyloba essential oils had significant antifungal effects against A. flavus. The most sensitive clinical isolate to this combination was A61, with growth inhibited at 78 ppm, similar to the standard strain. The combination's MIC value was 78 ppm, which included equal parts of M. piperita (39 ppm) and E. platyloba (39 ppm). The antifungal effect in the combination appears to be largely influenced by M. piperita, which alone has a lower MIC than E. platyloba. To date, no prior studies have investigated this specific combination against A. flavus, although inhibitory effects of other essential oil combinations have been reported (30). The reduction in MIC values in combination compared to E. platyloba alone likely results from the diverse phytochemical constituents of M. piperita enhancing the antifungal activity.

Statistical analysis revealed that MIC values for the standard strain were on average 406.3 units lower than those for clinical isolates, a statistically significant difference. This indicates that clinical isolates are more resistant, likely due to prior exposure to antifungal agents in patients and their environment.

Furthermore, the broader implications of our findings suggest that these essential oils could serve as effective adjunct treatments alongside conventional antifungal drugs. Additionally, their potential application in food preservation could provide economic and health benefits by preventing fungal contamination. Integrating these essential oils into current treatment and preservation strategies could offer more holistic approaches to managing fungal pathogens.
This study has some limitations. The main limitation is the lack of in vivo evaluation of the antifungal effects of Echinophora platyloba and Mentha piperita essential oils on laboratory animals or patients with aspergillosis. Future studies are needed to investigate the therapeutic potential and safety of these essential oils in clinical settings.

Conclusion

The evidence from this study suggests that M. piperita and E. platyloba essential oils and their combination exert antifungal effects against A. flavus in vitro. These results suggest that M. piperita and E. platyloba essential oils exhibit good anti-aspergillosis effects against A. flavus. Our results indicated that essential oils at low concentrations (< 2500 ppm) inhibited the growth of eleven clinical isolates, while lower concentrations (39-78 ppm) inhibited the growth of the standard A. flavus strain. Furthermore, we showed that the combination of essential oils in equal proportions decreased the MIC value of E. platyloba essential oil alone. This study suggests that M. piperita and E. platyloba essential oils can be used as a secondary treatment along with antifungal chemical drugs and also as food preservatives. Further studies that investigate the antifungal effects of these essential oils are required.

Conflict of Interest

The authors declared no conflicts of interest.

Author Contributions

AS conceived the research idea and MY designed the work. MA carried out the experiment and MB conducted statistical analysis. MR wrote the first draft of the manuscript. All authors reviewed and approved the final version of the manuscript.

Ethics Statement

This study was approved by the Ethics Committee of Lorestan University of Medical Sciences, Khorramabad, Iran (Ethics Approval No. IR.LUMS.REC.1400.036; approved on April 20, 2021). The study was conducted in accordance with relevant ethical guidelines. The fungal isolates used in this in vitro study were obtained from previously collected clinical samples, and no direct intervention involving human participants or animals was performed.

Data Availability Statement

The datasets generated and analyzed during the current study are available from the corresponding authors upon reasonable request. This study was conducted as part of an M.D. thesis at Lorestan University of Medical Sciences, Khorramabad, Iran.

Acknowledgment

The authors express their gratitude to the officials of Lorestan University of Medical Sciences (LUMS), Khorramabad, Iran for their support.

Consent for Publications

All authors approved the manuscript for publication.

Funding Statement

This study was funded by Lorestan University

of Medical Sciences.

Artificial Intelligence Statement

The authors used ChatGPT (OpenAI) solely to improve the English language and readability of the manuscript. All scientific content, data analysis, interpretation, and conclusions were developed and verified by the authors, who take full responsibility for the final version of the manuscript.

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