(R)-(+)-limonene: a promising antifungal agent against Candida glabrataAbstract
Fungal infections of the genus Candida are among the most common in the world and, consequently, resistance to existing antifungal agents is a very challenging reality. In this sense, medicinal plant-based substances can aid in the design of pharmacologically important products and contribute to the development of innovative therapeutic alternatives. Given this, the objective of this study was to investigate the antifungal potential of the monoterpene (R)-(+)-limonene against strains of Candida glabrata (Nakaseomyces glabrata). Antifungal activity was determined by the broth microdilution technique for the minimum inhibitory concentration (MIC) and, subsequently, the minimum fungicidal concentration (MFC) was determined. The mechanism of action of the possible antifungal activity was analyzed by sorbitol and ergosterol assays. Molecular docking was also performed to evaluate the molecular interactions of (R)-(+)-Limonene with the enzyme 1,3-β-glucanase and sterol 14α-demethylase. Based on the tests, (R)-(+)-Limonene presented a MIC and MFC of 256 μg/mL, with fungicidal characteristics, showing strong inhibitory activity for strains ATCC 9003, LM 13, and LM 16 and moderate activity for LM 46. The mechanism of action involves both the cell wall and the fungal plasma membrane. In addition, the molecular docking study pointed to an interaction between (R)-(+)-Limonene and the active sites of the proteins 1,3-β-glucanase and sterol 14α-demethylase (CYP51), the latter being more robust. As for the association test, the antifungal potential of (R)-(+)-Limonene was demonstrated when used individually, without association with fluconazole. Based on these findings, it can be concluded that (R)-(+)-Limonene is a very promising monoterpene for the development of new drugs for the treatment of candidiasis caused by Candida glabrata (Nakaseomyces glabrata).
Keywords:
candidiasis; pharmacology; monoterpene
Resumo
As infecções por fungos do gênero Candida estão entre as mais comuns no mundo e, consequentemente, a resistência aos antifúngicos existentes consiste em uma realidade bastante desafiadora. Nesse sentido, substâncias à base de plantas medicinais podem ajudar na concepção de produtos farmacologicamente importantes e contribuir para o desenvolvimento de alternativas terapêuticas inovadoras. Diante disso, o objetivo deste trabalho foi investigar o potencial antifúngico do monoterpeno (R)-(+)-Limoneno contra cepas de Candida glabrata (Nakaseomyces glabrata). A análise da atividade antifúngica foi determinada pela técnica de microdiluição em caldo para a concentração inibitória mínima (CIM) e, posteriormente, a determinação da concentração fungicida mínima (CFM). O mecanismo de ação da possível atividade antifúngica foi analisado pelos ensaios de sorbitol e ergosterol. Também foi realizado o docking molecular para avaliar as interações moleculares (R)-(+)-Limoneno com a enzima 1,3-β-glucanase e esterol 14α-demetilase. Com base na realização dos testes, o (R)-(+)-Limoneno apresentou uma CIM e CFM de 256 μg/mL, com característica fungicida, apresentando atividade inibitória forte para as cepas ATCC 9003, LM 13 e LM 16 e moderada para e LM 46. O mecanismo de ação envolve tanto a parede celular quanto a membrana plasmática fúngica. Além disso, o estudo de docking molecular apontou para uma interação entre o (R)-(+)-Limoneno e o sítios ativos das proteínas 1,3-β-glucanase e esterol 14α-demetilase (CYP51), sendo esta última de forma mais robusta. Quanto ao ensaio de associação, ficou demonstrado o potencial antifúngico do (R)-(+)-Limoneno, quando utilizado de maneira individualmente, sem associação com fluconazol. A partir desses achados, conclui-se que o (R)-(+)-Limoneno configura-se como um monoterpeno bastante promissor para o desenvolvimento de novos medicamentos para o tratamento da candidíase causada por Candida glabrata (Nakaseomyces glabrata).
Palavras-chave:
candidíase; farmacologia; monoterpeno
1. Introduction
Fungal infections are one of the main contributors to infectious disease-related deaths worldwide, with Candida infections being some of the most recurrent. In addition to the invasive form, vulvovaginal candidiasis, oral candidiasis, and breast candidiasis are also noteworthy. The most common species found are C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, and C. auris (Menezes et al., 2004; Sanches et al., 2020; Lee et al., 2021; Taylor et al., 2023).
Given this scenario, there is evidence of fungal resistance to traditionally used antifungal agents, as well as adverse effects associated with the use of these drugs. Thus, there is a need for further studies to develop technological innovations and new therapeutic alternatives. Among the existing options, the use of natural products based on medicinal plants and their derivatives is a very viable option, such as the use of terpenes (Berto et al., 2018; Rocha et al., 2021).
Among terpenes, limonene is a cyclic, unsaturated monoterpene with the molecular formula C10H16, called 4-isopropyl-1-methyl-cyclohexene, found in more than 300 plant species (Arruda et al., 2006; Zahi et al., 2015; Jongedijk et al., 2016). Preliminary studies point to limonene as a very promising antimicrobial (Millezi et al., 2014). However, there is a clear lack of research exploring the antifungal potential of limonene isomers, such as (R)-(+)-Limonene, making this substance a strong target for study. The objective of this study was to investigate the antifungal potential of the monoterpene (R)-(+)-Limonene against strains of Candida glabrata.
2. Materials and Methods
2.1. Type of research and location of laboratory tests
This is a laboratory-based experimental study with an inductive and quantitative approach. The phytoconstituent (R)-(+)-Limonene was purchased from Sigma-Aldrich Brasil Ldta. Laboratory tests to evaluate antifungal activity were conducted at the Microbiology and Biochemistry Laboratory of the Academic Unit of Biological Sciences at the Center for Health and Rural Technology of the Federal University of Campina Grande (UFCG), Paraíba, Brazil.
2.2. Preparation of material and microorganisms
The tests were performed with clinical strains of C. glabrata (LM-13, LM-16, LM-46, LM-218) and standard strains (ATCC-9003). All samples belong to the collection of the Mycology Laboratory of the Department of Pharmaceutical Sciences at the Federal University of Paraíba. The strains were kept in Sabouraud Dextrose Agar (SDA) at 4 °C. Forty-eight-hour cultures incubated at 35 ± 2 °C were used in the tests (Bona et al., 2014; Dunlap et al., 2010).
Sabouraud Dextrose Agar (SDA) was used to maintain the fungal strains, and RPMI 1640 with L-glutamine and without bicarbonate was used for antifungal activity assays. These media were commercially purchased from Difco Laboratories Ltd (USA, France) and INLAB, São Paulo, Brazil, respectively. Both media were prepared according to the manufacturers' instructions, solubilized with distilled water, and sterilized in an autoclave at 121°C for 15 minutes.
The inocula were prepared from recent cultures of each of the selected C. glabrata strains, previously grown in sterile tubes containing ASD, slanted and incubated at 35 ± 2°C for 24-48 hours to achieve satisfactory growth. In preparing the fungal strain suspensions, the recent preparation colonies were diluted in 5 mL of sterile saline solution (0.85% w/v NaCl). These suspensions were then shaken for 15 seconds using a vortex mixer (Fanem), after which the inoculum density was standardized according to the McFarland 0.5 scale to obtain 106 CFU/mL (CLSI, 2012; Hadacek and Greger, 2000).
2.3. Substances
The phytoconstituent (R)-(+)-Limonene, commercially purchased from Sigma-Aldrich® (Ponta Grossa, PR, Brazil), was used as the test product. The working solution was prepared at the time of testing by weighing the (R)-(+)-Limonene on an analytical balance (SHIMADZU) and then dissolving it in 150μL (3%) dimethyl sulfoxide (DMSO) with 50μL (1%) Tween 80 added. It was then completed with sterile distilled water (q.s.p. 5mL) to obtain an emulsion at an initial concentration of 1024μg/mL. Similarly, the drug used as a control in the execution of the methodologies was fluconazole (FLU), commercially purchased from Sigma-Aldrich® (São Paulo-SP, Brazil) and prepared according to the manufacturers' specifications and in the appropriate concentrations for each test (Bona et al., 2014).
2.4. Minimum Inhibitory Concentration (MIC) test
The antifungal activity of (R)-(+)-Limonene was determined according to the protocols of CLSI (2015) and Hadacek and Greger (2000) with minor modifications. The MIC of (R)-(+)-Limonene was determined using the broth microdilution technique in a 96-well cell culture plate (Kasvi, Kasvi Imp e Dist. Prod/Laboratório LTD, Curitiba, Brazil). One hundred μL of double-concentrated RPMI-1640-L-glutamine broth was distributed into the wells of the plate. Subsequently, 100 μL of the emulsion containing (R)-(+)-Limonene was dispensed only into the wells of the first row of the microdilution plate. Next, the substance under investigation was serially diluted at a ratio of 1:2, thus obtaining concentrations ranging from 1024 μg/mL to 4 μg/mL. Finally, 10 μL of the fungal strain suspensions (1.5 x 106 CFU/mL) were added to the wells.
Similarly, this procedure was performed for the antifungal fluconazole, evaluating the susceptibility of the strains to the standard antifungal. Microorganism growth was also controlled by placing 100μL of double-concentrated RPMI-1640, 100μL of sterile distilled water, and 10μL of the inoculum from each strain in the wells of the plate. In addition, a sterility control of the culture medium was performed, in which 200μL of RPMI-1640 was placed in a hole in the plate, in the absence of the fungal suspension.
The test was performed in triplicate. The plates were incubated at 35 ± 2 °C for 24-48 hours. After the specified time, the data were read and recorded. The MIC was considered to be the lowest concentration of the antifungal agent responsible for preventing visible growth of the microorganism, while the MIC50 was defined as the concentration responsible for inhibiting visible growth of 50% of the microorganisms, taking into account the total number of strains tested (Bona et al., 2014).
The substance was classified as having strong activity when the MIC was up to 600 μg/mL; moderate activity when the MIC ranged between 600 and 1500 μg/mL. Above 1500 μg/mL, it was classified as having weak activity or inactive (Sartoratto et al., 2004).
2.5. Minimum Fungicidal Concentration (MFC) test
After reading the MIC, aliquots of 10μL of the supernatant from the wells where complete inhibition of fungal growth occurred (MIC, MIC × 2, and MIC × 4) were subcultured in 100μL of RPMI-1640 in new 96-well plates. The system was then aseptically sealed and incubated at 35 ± 2°C for 24-48 hours. The FMC was defined as the lowest concentration at which there was no evidence of yeast growth.
The tests were performed in triplicate and the results expressed as the arithmetic mean of the MICs obtained in the three tests. Similarly, the same procedures were performed with the standard antifungal agent (Ncube et al., 2008). Antifungal activity was classified using the CFM/MIC ratio, where fungicidal activity was considered when CFM/MIC <4 and fungistatic activity when CFM/MIC ≥4 (Siddiqui et al., 2013).
2.6. Sorbitol Test - Action of (R)-(+)-Limonene on the fungal cell wall
The MIC of (R)-(+)-Limonene in the presence of sorbitol, an osmotic protector of fungal protoplasts, was determined by microdilution in 96-well plates. 100µL of RPMI-1640 supplemented with sorbitol, both doubly concentrated, was added to each well. Next, 100 µL of the (R)-(+)-Limonene emulsion was dispensed into the wells of the first row of the plate. Using serial dilution at a ratio of two, the necessary concentrations of the products were obtained in each well with a final sorbitol concentration of 0.8 mol/L.
Finally, 10 µL of inoculum from a representative strain of C. glabrata was added to the wells of the plate, with each column of the plate referring to a specific fungal strain (Frost et al., 1995; Escalante et al., 2008; Georgopapadakou, 2001).
Fungal control was performed by adding 200 µL of 0.8 mol/L sorbitol and 10 µL of the inoculum of each species to each well of the culture medium. A sterility control was also performed, where 200 µL of the culture medium was added to a well without fungal inoculum. The plates were then aseptically sealed and incubated at 35 ± 2°C for 24-48 hours. The tests were performed in triplicate, and the results were expressed as the arithmetic mean of the three experiments.
2.7. Ergosterol Assay – Action of (R)-(+)-Limonene on the fungal cell membrane
Similar to the sorbitol assay, the MIC of (R)-(+)-Limonene was determined against selected strains of C. glabrata in the presence of exogenous ergosterol by microdilution, as previously described in item 2.4 for MIC determination (Escalante et al., 2008). In this case, RPMI-1640 culture medium previously supplemented with 400 µg/mL ergosterol was used. Microorganism growth was controlled with 100 µL of culture medium and ergosterol in equal concentrations and 10 µL of each fungal inoculum. The plates were then aseptically sealed and incubated at 35 ± 2°C for 24-48 hours. The tests were performed in triplicate, and the results were expressed as the arithmetic mean of the three tests. In this way, it was possible to compare the MIC values of (R)-(+)-Limonene against C. glabrata strains in the absence and presence of exogenous ergosterol.
2.8. Association Test – Checkerboard
The association assay was performed using the checkerboard technique, with 96-well microtiter plates with conical bottoms (INLAB, São Paulo, Brazil). Initially, based on the MIC values obtained, solutions of drug A [(R)-(+)-Limonene] and drug B (FLU) were prepared in different concentrations (8XMIC, 4XMIC; 2XMIC; MIC; MIC/2; MIC/4; MIC/8). Next, 100 μL of RPMI 1640 was added to all wells of the plate. Subsequently, 50 μL of drug A was distributed horizontally in different concentrations, while 50 μL of drug B was distributed vertically in different concentrations. Thus, all different concentrations were tested and combined between the drugs. Finally, 20 μL of the inoculum was added (Huang et al., 2019).
The antimicrobial potential was measured by combining drugs and determined using the Fractional Inhibitory Concentration (FIC): MIC of the combined product ÷ MIC of the individual product. Subsequently, the Fractional Inhibitory Concentration Index was calculated using the following equation: FICI = FICA + FICB.
Thus, a synergistic association was considered when the ICIF was ≤ 0.5; an indifferent association when ICIF > 0.5 or ≤ 4.0; and an antagonistic association when ICIF > 4.0 (White et al., 1996; Tobudic et al., 2010).
2.9. In silico tests: molecular docking
Rigid molecular docking simulations were performed targeting the proteins 1,3-β-glucanase (PDB ID: 1EQC, resolution 1.85 Å) and sterol 14α-demethylase (CYP51) (PDB ID: 5TZ1, resolution 2.00 Å), whose three-dimensional structures were obtained from the Protein Data Bank (PDB) (https://www.rcsb.org/). The protein models were initially prepared in PyMOL 2.5.3, with the removal of crystallographic water molecules and co-crystallized ligands: castanospermine (CAS) and oteseconazole (VT-1161) from the above-mentioned enzymes, respectively (Schrödinger, 2025).
The ligand (R)-(+)-Limonene (LIM) was constructed in MarvinSketch 16.3.7 and subjected to sequential preparation steps: (i) geometric optimization in Avogadro 1.2.0 considering pH 7.4; (ii) energy minimization in MOPAC 2012 using the PM6 semi-empirical method; and (iii) additional optimization in Chimera 1.16 with the AM1-BCC force field, resulting in the ligand input file in .mol2 format (Pettersen et al., 2004; Hanwell et al., 2012; Stewart, 2012; Chemaxon, 2023).
Protein preparation in AutoDockTools (ADT) 1.5.4 included the addition of polar hydrogens, Kollman charge assignment, and merging of nonpolar hydrogens (Morris et al., 2009). Active sites were defined based on the position of the native ligand, establishing the center of the grid box at (32.912; 35.955; 56.222 Å), dimensions of (40 × 40 × 40) points, and spacing of 0.300 Å for 1,3-β-glucanase, and for CYP51 (70.797; 67.940; 2.545 Å), dimensions of (46 × 56 × 60) points and spacing of 0.300 Å.
Docking simulations were performed in AutoDock 4.2, using the Lamarckian genetic algorithm with 200 independent runs and standard ADT parameters. Conformations were evaluated according to Free Binding Energy (ΔG) and Inhibitory Constant (Ki), selecting those with lower ΔG as more likely.
Post-docking analysis was performed in PyMOL 2.5.3 and Discovery Studio 2021, characterizing the binding regions, interaction types, and active site residues involved. Protocol validation was performed by redocking the native ligand, considering a Root Mean Square Deviation (RMSD) value ≤ 2.0 Å acceptable (Bell and Zhang, 2019; Biovia, 2021).
3. Results
3.1. Minimum Inhibitory Concentration – MIC
Among the C. glabrata strains tested, strains ATCC 9003, LM 13, LM 16, and LM 46 were found to be sensitive to (R)-(+)-Limonene. Therefore, the concentration required to inhibit 50% (MIC50) of the microorganisms investigated was 256 μg/mL, as shown in Table 1.
Médias da CIM (µg/mL) do (R)-(+)-Limoneno e do fluconazol frente a cepas de Candida glabrata.
Thus, (R)-(+)-Limonene showed inhibitory activity classified as strong for strains ATCC 9003, LM 13, and LM 16 and moderate for LM 46 (Holetz et al., 2002; Sartoratto et al., 2004).
3.2. Minimum Fungicide Concentration – MFC
Regarding the minimum fungicidal concentration (MFC) of (R)-(+)-Limonene against the tested strains of C. glabrata, the following results were observed, as shown in Table 2.
(R)-(+)-Limonene showed fungicidal activity against the Candida glabrata strains tested, ATCC 9003, LM 13, LM 16, and LM 46.
3.3. Sorbitol and ergosterol tests
To evaluate the mechanism of action of (R)-(+)-Limonene against fungal strains, two representative strains of Candida glabrata were taken: ATCC 9003 and LM 13. For this purpose, sorbitol and ergosterol assays were performed.
Regarding the sorbitol test, which predicts interaction of the phytoconstituent through the fungal cell wall, there was inhibition with a twofold increase in MIC in the presence of this compound (Table 3). In the ergosterol test, there was an eightfold increase in MIC (Table 4).
Média da CIM (μg/mL) do (R)-(+)-Limoneno na presença e ausência de sorbitol frente a cepas de Candida glabrata.
Média da CIM (μg/mL) do (R)-(+)-Limoneno na presença e ausência de ergosterol frente a cepas de Candida glabrata.
This result reveals that the mechanism of action of (R)-(+)-Limonene against strains ATCC 9003 and LM 13 results from an interaction with both the cell membrane and the cell wall of the Candida glabrata strains tested.
3.4. Association test - checkerboard
The results obtained from the association between (R)-(+)-Limonene and fluconazole for Candida glabrata strains ATCC 9003 and LM 13 resulted in an ICF = 4.125.
Therefore, as the ICF for both strains tested was greater than 4, it can be concluded that the association presented an antagonistic interaction between (R)-(+)-Limonene and fluconazole, as shown in Table 5.
Concentração Inibitória Fracionária e o Índice da Concentração Inibitória Fracionária do (R)-(+)-Limoneno associado ao fluconazol frente a cepas de Candida glabrata.
3.5. Molecular docking: interactions of the LIM ligand with the proteins 1,3-β-glucanase and sterol 14α-demethylase
The redocking of cocrystallized ligands was performed to validate the protocol used, using castanospermine (CAS) as a control for the 1,3-β-GS enzyme and otesoconazole (VT-1161) as a control for CYP51. The RMSD values obtained were less than 2.0 Å, confirming the reliability of the methodology adopted according to Table 6.
Figures 1a and 1d show the three-dimensional overlap between the co-crystallized ligands (green) and the redocking results (blue), showing good spatial correlation and maintenance of orientation at the active site. The two-dimensional analyses (Figures 1b, 1c, 1e and 1f) indicate that CAS and VT-1161 establish critical stabilizing interactions, such as van der Waals contacts, conventional hydrogen bonds, stacked Pi-Pi, and alkyl/Pi-alkyl interactions, which reinforces the robustness of the protocol.
Redocking of castanospermine (CAS) (co-cristal da 1,3-β-GS), e oteseconazol (VT-1161) (co-cristal da CYP51).
The LIM molecule was subjected to docking against the enzymes 1,3-β-GS and CYP51 from Candida spp. The results showed binding energies of ΔG = –5.16 kcal/mol for 1,3-β-GS and ΔG = –5.85 kcal/mol for CYP51, accompanied by inhibitory constants (Ki) of 165 μM and 51.58 μM, respectively (Table 6). These findings indicate that LIM establishes relevant interactions with both active sites analyzed, but with greater inhibition potential against CYP51, as previously evidenced. Against 1,3-β-GS, LIM maintains a very favorable interaction capacity compared to the values associated with the co-crystallized ligand (Table 6).
Molecular docking analysis of LIM against 1,3-β-glucanase revealed relevant interactions with residues from the enzyme's active site. The ligand established van der Waals contacts with residues such as Glu27, Arg92, Asn146, Asn191, Glu192, His253, and Glu292, as well as alkyl and Pi-alkyl interactions with aromatic and hydrophobic residues, including Tyr29, Tyr255, Trp363, Trp373, Leu304, and Phe258 (Figure 1ab).
Surface visualization (Figure 1cd) showed that LIM fits into a cavity that collects predominantly nonpolar residues, highlighting the role of hydrophobic contacts and steric proximity in stabilizing the ligand-enzyme complex.
In relation to CYP51, LIM presented a robust interaction profile, with positioning close to the heme group, a central element for the enzyme's catalytic activity. Van der Waals contacts were observed with residues such as Thr122, Tyr132, Gly303, Gly307, Gly308, and Thr311, as well as alkyl and Pi-alkyl interactions with critical residues such as Phe126, Ile131, Leu139, Phe228, Leu300, and Ile304 (Figure 2ab). Three-dimensional representations (Figure 2cd) showed that LIM occupies the active cavity deeply, interacting along the hydrophobic channel and in direct proximity to the heme group, suggesting a strategic accommodation favored by hydrophobic complementarity between the ligand and the protein. This same phenomenon can be observed in Figure 3, in relation to the sterol 14α-demethylase protein.
Molecular docking of the LIM ligand with the sterol 14α-demethylase protein. The ligand forms multiple chemical interactions with key residues in the active site, including van der Waals bonds and alkyl/Pi-alkyl interactions. The 2D (a) and 3D (b) diagrams show the network of interactions between the LIM and sterol 14α-demethylase, while the surface models (c and d) highlight the hydrophobic complementarity and the hydrogen donor/acceptor pattern that favor the stability of the complex.
4. Discussion
The determination of the MIC and CFM of phytoconstituents is extremely important for verifying antifungal activity. In this regard, similar to the experiments conducted with (R)-(+)-Limonene, a study by Oliveira-Júnior et al. (2025) pointed to the phytoconstituent eugenol as a very promising substance when evaluating the antifungal effect against non-albicans Candida strains isolated from the oral cavity of healthy individuals. Eugenol presented MIC and MBC values of 256 μg/mL, which characterizes the strong antifungal activity of this phytoconstituent.
Araújo et al. (2018) presented a study on the antifungal activity of the phenylpropanoid o-eugenol against strains of Candida albicans, determining MIC and MBC of a variety of strains, indicating strong antifungal activity of this phytoconstituent.
The findings of the present study corroborate the hypothesis that monoterpenes have significant antifungal potential against species of the genus Candida. Similarly, Medeiros et al. (2022) demonstrated that the monoterpene linalool exhibited fungicidal activity against strains of Candida albicans. In addition, Gouveia et al. (2023) showed that the enantiomers (R)- and (S)-citronellal also exhibited fungicidal activity against strains of C. albicans.
Regarding the results found in the sorbitol and ergosterol assays, the study by Medeiros et al. (2022) also showed an increase in the minimum inhibitory concentration (MIC) of linalool in the presence of sorbitol and ergosterol, indicating that this molecule possibly affects the integrity of the cell wall and plasma membrane of C. albicans.
Gouveia et al. (2023) evaluated the molecular mechanisms that interfere with fungal cell viability by testing the (R) and (S)-citronellal enantiomers against strains of C. albicans. The study indicated that both enantiomers interacted with both the cell wall and the fungal plasma membrane.
The sorbitol and ergosterol assay demonstrated a strong interaction of (R)-(+)-Limonene with the fungal cell membrane. Fluconazole also has a similar affinity which, according to Li et al. (2025), by altering the biosynthesis of available ergosterol by inhibiting 14-α-demethylase, may justify the antagonistic effect observed in the combination of these two compounds.
However, there has been a growing increase in fungal resistance to fluconazole worldwide, especially in North America and Europe, according to studies presented by Pfaller et al. (2019) and Zarrinfar et al. (2021).
Although the combination of fluconazole and (R)-(+)-limonene had an antagonistic effect in this study, other substances may have a synergistic effect in terms of antifungal action. One such example was presented in the study by Cunha et al. (2023), which evaluated the antifungal activity of patchouli essential oil, which did not involve effects on the cell wall or plasma membrane but promoted a synergistic effect when combined with amphotericin B.
Molecular docking is a virtual computational method commonly used to predict the complex of two binding molecules, including biological macromolecules or small molecules, such as endogenous ligands and drugs. In addition, molecule docking can be used to determine ligand interactions with target proteins in structure-based drug development, as evidenced by the results of the present study using (R)-(+)-Limonene (Chun et al., 2023).
5. Conclusion
(R)-(+)-Limonene can be considered as an alternative for the development of an antifungal drug for the treatment of candidiasis caused by Candida glabrata. However, it is important that further research be conducted to elucidate mechanisms of action, toxicity, and efficacy in vivo, and it is suggested that other clinical trials be conducted using (R)-(+)-Limonene.
Acknowledgements
Professor Edeltrudes de Oliveira Lima from the Mycology Laboratory of the Department of Pharmaceutical Sciences at the Federal University of Paraíba for providing the fungal strains for the experiments.
Data Availability Statement
All data supporting the results of this study were published in the article itself.
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Edited by
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Editor:
Takako Matsumura Tundisi
