Chemical Composition,Antifungal Activity and Molecular Docking Study of Essential oil of Rhabdocaulon lavanduloides Benth

Abstract

Introduction

The Rhabdocaulon lavanduloides species is a native plant found in the Atlantic Forest biome, which produces essential oil and belongs to the Lamiaceae family. It is popularly called Alfazema do campo.

Objective

Based on this premise the objective of this study was to elucidate the chemical composition of R. lavanduloides essential oil and evaluate its intrinsic and combined antifungal activity against Candida spp. strains.

Methodology

The essential oil components were identified by gas chromatography coupled with mass spectrometry (GC-MS). For antifungal activity, the broth microdilution method was used to obtain the cell viability curve and determine the mean inhibitory concentration (MIC). The minimum fungicidal concentration (MFC) was evaluated by subculturing in Petri dishes.

Results and Discussion

Aromatic monoterpenes were the main compounds identified as 2-methoxy-carvacrol, being the majority, followed by thymol, methyl ether, p-cymene, 2,3-dimethoxy-p-cymene, and β-caryophyllene. The antifungal results showed that the essential oil had no intrinsic action in inhibiting the strains; however, when combined with the antifungal agent, it reduced the effective concentration of fluconazole at concentrations of 8 to 32 µg/mL for the standard strain of C. albicans. A minimum fungicidal concentration of 2048 µg/mL was observed in the combination for the isolated strain of C. tropicalis. In addition, molecular docking simulations indicate that 3-isopropyl-2-methoxy-6-methylphenol exhibits a similar affinity profile to Fluconazole across all tested biological receptors, likely through competitive interaction. These findings suggest that 3-isopropyl-2-methoxy-6-methylphenol has potential therapeutic applications against these receptors.

Conclusion

The essential oil of R. lavanduloides has potential for future antimicrobial research evaluating the antifungal effect of its isolated phytoconstituents.

Keywords

Antifungal activity Lamiaceae docking

Introduction

Brazil has a mega diversity, in its different biomes, where we can highlight the Atlantic Forest, with great biodiversity and high rate of endemism.¹ The state of Paraná is home to an extensive biodiversity, including different elements and their interrelationships, which results in a vegetative mosaic in the formation of a specific ecosystem, the Atlantic Forest. As one of the first ecosystems, it is considered the largest remaining due to its latitude/altitude extension with different life forms.²

In this scenario, we find Rhabdocaulon lavanduloides (Benth.) Epling a native botanical species, also called Alfazema-do-campo, belonging to the Lamiaceae family (Figure 1). This family represents great economic importance in the various specimens with medicinal potential, aromatic, rich in essential oils, present in glandular trichomes distributed in the leaves and inflorescences with important biological activities such as antibacterial, antifungal, insecticide and antioxidant.^3–4 Besides these biological activities, the commercial cultivation of species of this botanical family, aims at the production of essential oils for the perfume industry because it contains abundant volatile aromatic oils. Examples include lavender essential oil (Lavender angustifolia), rosemary essential oil (Rosmarinus officinalis), thyme (Thymus vulgaris).⁵

Figure 1.

Rhabdocaulon lavanduloides.

The Rhabdocaulon genus is represented in Brazil by eight Brazilian species, of which three, named R. gracilis, R. erythrostachus and R. lavanduloides, occur in the Paraná state and together represent 42.83% of this diversity. The botanical species Alfazema-do-campo, presents an erect stem, branched, seridotomentosus, sessile leaf, linear-oblong, subrevolute margin, in both serid faces; racemous inflorescence, many sessile flowers, in approximate verticiles, forming a dense spike; tubular calyx, albo-viloso, fauce vilosa; little exserted corolla; 13 mm long corolla tube and 1,5-2,5 mm long calyx teeth.⁶

The Rhabdocaulon lavanduloides species produces essential oils in levels ranging from 0,46 to 2,33% and 0,98 to 3,35% in its leaves and flowers, respectively. Recent studies presented high concentration (80%) of phenolic ether 2-methoxy as the major compound. In addition, cymene (14.8%), thymol methyl ether (9.7%), the sesquiterpenes a-copaene (4.5%) and E-caryophyllene (6.5%) were also identified in considerable yields.⁷

In addition to the well-documented medicinal properties of species within the Lamiaceae family, particularly in the context of infectious diseases, it is essential to consider the limitations of conventional antifungal therapies. The widespread use of synthetic fungicides, although effective, has contributed to the emergence of resistant fungal strains, thereby compromising clinical efficacy. Moreover, growing concerns regarding the toxicity and potential carcinogenicity of certain synthetic compounds have prompted regulatory agencies and researchers to seek safer alternatives. In this regard, essential oils derived from medicinal plants have emerged as promising candidates, with numerous studies demonstrating their ability to inhibit the growth of various fungal pathogens.^8–9

Fungal infections cause approximately 6.5 million invasive cases and 3.8 million deaths globally each year, with approximately 2.5 million directly attributed to fungi. Invasive candidiasis affects approximately 1,565,000 people per year, resulting in 995,000 deaths.¹⁰ The World Health Organization¹¹ has warned of the severity of fungal infections, especially in immunocompromised individuals, highlighting the lack of attention to the issue, difficulties in diagnosis, and limitations in treatment. Among the priority pathogens are fungi of the genus Candida, classified as critical or high priority.

Candida albicans is a commensal species present in more than 80% of healthy people, but it can cause serious infections in cases of immunosuppression or microbiota imbalance. Other species, such as Candidozyma auris (Candida auris), C. tropicalis, C. parapsilosis, Nakaseomyces glabratus, and Pichia kudriavzevii, also have pathogenic potential and resistance to antifungals.¹²

Based on the fact that plants of the Lamiaceae family have been used in the treatment of infections and that they contain chemical compounds with proven antimicrobial,¹³ including antifungal,¹⁴ this study aimed to chemically characterize the essential oil from aerial parts of the species Rhabdocaulon lavanduloides (Benth.) Epling and investigate its effect on priority pathogenic fungi of the genus Candida.

Methodology

Collection and Extraction of Essential oil

The botanical material of R. lavanduloides was collected in São José dos Pinhais/PR (S 25°. 750.806 and W 49° 028.248) of native population in February 2018. Samples were collected and deposited in the Herbarium of the Integrated Spiritual Colleges and duplicates deposited in the Municipal Botanical Museum, where they were herborized under number HFIE 9.097. This research had also been registered with the Council of the National System for the Management of Genetic Heritage and Associated Traditional Knowledge—SisGen on protocol number A216E5A. After collection, the terminal branches with leaves were dehydrated in an air circulator at 40 °C for 24 h.

The essential oil from the leaves of R. lavanduloides was obtained by hydrodistillation using a Clevenger apparatus. Extraction lasted 3 h, using 35 grams of plant material per 700 mL of distilled water (1:20 ratio of plant material to water) for each hydrodistillation (3 replicates). The essential oil was separated from the aqueous phase by liquid-liquid extraction in a separatory funnel using 10 mL aliquots of double-distilled dichloromethane, with a total of 30 mL of solvent. Anhydrous magnesium sulfate (MgSO₄) was used to dry the organic phase. After simple filtration to remove the desiccant, the organic phase was transferred to a previously tared round-bottom flask (50 mL capacity). The solvent was distilled in a rotary evaporator at 45 °C to 50 °C. The oils’ masses were determined on a semi-analytical balance, and their yields were calculated relative to the mass of plant material used. They were then stored in glass vials under a nitrogen atmosphere and refrigerated until chemical characterization. The essential oil samples from the leaves were subjected to chromatographic analysis. The relative concentrations of the constituents were determined by gas chromatography with a flame ionization detector (GC-FID). The identification of the essential oil constituents was determined by gas chromatography-mass spectrometry (GC-MS). The essential oil samples were previously diluted to 1.0% (m/v) in double-distilled dichloromethane using a Shimadzu GC-FID chromatograph, model GC-2010. 1.0 μL of the diluted sample was injected using an automatic injector with a 1:20 split ratio and an Rxi 5-Sil MS capillary column (30 m × 0.25 mm × 0.25 μm). Each chromatographic analysis took 54 min, using a method adapted from Adams (2007). The injector temperature was 250 °C and the detector temperature was 280 °C. The initial oven temperature was 60 °C and the heating rate was 3 °C min-1 to 200 °C, holding for 1 min, heating at 15 °C min-1 to 250 °C and holding for 3 min. Analytical grade helium (He) was used as the mobile phase at a flow rate of 1 mL min-1. The arithmetic index (AI) of each constituent present in the EO was calculated considering the retention times of the alkanes present in a standard containing the homologous series C8-C19.¹⁵ GC-MS ANALYSIS—For this analytical technique, the essential oil and hydrocarbon standard samples were prepared similarly to that described for GC-FID. The chromatograph used was a Shimadzu brand, model GC-MS-QP2010 Plus. The method employed was the same as that described for GC-FID analyses. The capillary column used was an RTX 5 MS (30 m × 0.25 mm × 0.25 μm).

Antifungal Assays

Strains and Culture media

Fungal strains of the genus Candida (Candida albicans and Candida tropicalis) of the standard type—(CA INCQS 40006 and CT INQS 40042) were used, obtained from the Collection of Reference Microorganisms in Health Surveillance-CMRVS, FIOCRUZ-INCQS, Rio de Janeiro, and clinical isolates obtained from the URM (University Recife Mycology) Mycology Collection of the Federal University of Pernambuco. The strains were initially inoculated in Sabouraud Dextrose Agar (SDA, KASVI) and incubated for 24 h at 37 °C. In preparing the fungal suspension for testing, aliquots of yeast were transferred to test tubes containing 3 mL of sterile sodium chloride solution (0.9%) each, with the concentration standardized according to the McFarland 0.5 scale¹⁶ . Double-concentrated Sabouraud Dextrose Broth (SDB, HIMEDIA) liquid medium was used in the microdilution tests.

Drugs, Reagents, and Preparation of Solutions

The essential oil was initially diluted in dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany) at a concentration of 10% (v/v) to ensure solubility. This stock solution was then further diluted in sterile distilled water to reach the final concentrations used in the assays. All dilutions were prepared immediately prior to use to preserve the chemical integrity of the essential oil. The final concentration of DMSO in the test solutions did not exceed 1% (v/v), a level considered non-toxic for the microorganisms evaluated.¹⁷ Fluconazole (Sigma-Aldrich, St. Louis, MO, USA) was used as the reference antifungal drug. It was dissolved in sterile distilled water to prepare a stock solution, which was subsequently diluted to the desired concentrations for comparative analysis in the assays.¹⁷

Intrinsic Antifungal Effect

The broth microdilution method in 96-well plates was applied, with each well filled with 100 μL of CSD containing 10% fungal inoculum. One hundred μL of oil or fluconazole was added to the first well and serial dilution was performed, with concentrations ranging from 8192 to 8 μg/mL. The last well was used as a growth control¹⁸ (with modifications in concentrations). Controls were prepared for the product diluent (with 0.9% sodium chloride solution replacing the fungal suspension) and media sterility control. All tests were performed in triplicate. The plates were incubated at 37 °C for 24 h and then read in an ELISA spectrophotometer (Thermoplate^®) at a wavelength of 430 nm. The results obtained in the ELISA reading were used to obtain the cell viability curve and IC₅₀ of the evaluated products.¹⁹

Determination of Minimum Fungicidal Concentration (MFC)

The fungicidal or fungistatic effect of the essential oil was determined by subculturing aliquots (10 µL) from each well of the microdilution plate onto SDA and incubating for 24 h²⁰ ^{(with modifications)}. The absence of colony growth indicated a fungicidal effect, while the presence of growth was interpreted as fungistatic. In cases where fungal growth was observed at all tested concentrations, the Minimum Fungicidal Concentration (MFC) was considered to be higher than the highest concentration evaluated (8194 µg/mL).

Evaluation of the Modifying Effect of Fluconazole Action by Combination

The essential oil/drug combination was evaluated to verify any potentiating or antagonistic effects. The essential oil was used at a subinhibitory concentration (CM or CFM/16) based on the Matrix Concentration (CM) or the Minimum Fungicidal Concentration (CFM) if found (Coutinho et al 2008, with modifications).²¹ The plates were filled with 100 µL of medium + inoculum + essential oil and then microdiluted with 100 µL of fluconazole (16,384 µg/mL), with serial dilution in concentrations ranging from 8192 to 8 µg/mL. The same controls used in the intrinsic activity test were used. The plates were incubated at a temperature of 37 °C for 24 h, and the reading was performed using an ELISA spectrophotometer (430 nm) (Thermoplate^®).

Statistical Analysis

The statistical program GraphPad Prisma 6.0 was used for data analysis. A two-way ANOVA was applied to the sample. The data obtained from spectrophotometric readings were checked for normal distribution and then analyzed by ANOVA, comparing the values of each concentration of the natural product with the Bonferroni post hoc test. The IC₅₀ values were obtained by nonlinear regression for the purpose of interpolating values from standard curves.

Molecular Docking Analysis

To achieve the lowest energy state of the compounds, all ligands underwent electronic and conformational minimization²² to construct the most stable model according to the estimate in the MMFF94 (Merck Molecular Force Field) force field,²³ using the software Avogadro. The force field is a computational tool consisting of a set of metrics to determine the potential effect of structural optimization.²⁴ The molecular docking step was conducted according to the methodology established previously,²⁵ which employs specific molecular targets to enable detailed structural analysis of interactions, biological receptors for the species Candida albicans were required. Four receptors were used: Als3, Sap5, ERG11, and GlcNAc, while for Candida tropicalis, only SapT1 was used. The targets are deposited on the Protein Data Bank (PDB) website, identified by the following PDB IDs: 4LE8 (Als3),²⁶ 2QZX (Sap5),²⁷ 5FSA (ERG11),²⁸ 6AKZ (Glc-NAc),²⁹ and 1J71 (SapT1).³⁰ Fluconazole was used as a control because it is the standard azole antifungal agent. In addition to targeting ERG11, it was included for the study of other fungal virulence factors, such as SAP proteases. This broadened the analysis to a range of target proteins beyond the canonical mechanism.

Before analyzing the respective receptors, the crystal structures were run through Chimera software to remove any unrelated residues. Subsequently, all missing polar hydrogens were added to each receptor individually, followed by the application of charges to stabilize the tested targets. The Gridbox parameters are adjusted to encompass all amino acid residues present in the biological targets. Based on this premise, for the Als3 target, the size values were 125 x, 66 y, and 124 z, and axes (13.800 x, 1.570 y, and 179.101 z); the size (100 x, 99 y, and 123 z) and axes (13.380 × 24.444 y, and 25.088 z) are assigned to the Sap5 receptor; size (126 x, 118 y, and 126 z) with the axes (207.443 x, −2.012 y, and 38.565 z) are related to the ERG11 target; size (122 x, 123 y, and 121 z), with the axes (17.640 x, 17.745 y, and 55.380 z) for Glc-NAc, and finally, the Candida tropicalis target (SapT1) presented gridbox values equal to 122 x, 126 y, and 126 z (size) with the respective coordinate axes of 26.430 x, 16.430 y, and 14.580 z. Molecular docking studies consist of an execution protocol where, to determine each complex (Ligand/Protein), 50 independent simulations are performed, with 20 distinct poses arranged for each simulation. The determination of the best pose is based on the choice of greater viability, based on the best RMSD (Root Mean Square Deviation) values, with values equal to or less than 2.0 Å being essential.^31–32

Results

Yield and Chemical Composition of Rhabdocaulon lavanduloides

The essential oil obtained in 0.68% yield based on dry material, density 0,9762 ± 0,0065 (25°), was then characterized by means of GC-FID (quantification and retention index) and GC-MS (computing library search). Aromatic monoterpenes were the major compounds with 2-Methoxy-carvacrol (3-isopropyl-2-methoxy-6-methylphenol) in 78.2% yield (Table 1). The complete chemical elucidation of 2-methoxy-carvacrol derivative was confirmed by x-ray analysis in a previous paper.⁷ In addition, thymol, methyl ether (3.9%), p- cymene (3.3%) and 2,3-dimethoxy-p-cymene (2.5%) were also identified in small amounts, besides β-caryophyllene (2.1%). Previously, (6-isopropyl-2-methoxy-3-methyl-phenol) (Figure 2), a thymol derivative, was previously identified in the essential oil of Ocimum viride Willd. (Lamiaceae) in low amount (1.3%),3 being a commercial product obtained by synthesis.

Figure 2.

2-Methoxy-Carvacrol (3-Isopropyl-2-Methoxy-6-Methylphenol).

Table 1.

Chemical Composition of the Essential oil from Leaves of Rhabdocaulon lavanduloides (Benth.) Epling (Lamiaceae) Collected in São José dos Pinhais (SJP) Municipalities, Southern Brazil.

Entry	Constituents	SJP [%]	AI_exp	AI_lit	Max SD^a
1	α-thujene	0.2	927	924	0.01
2	α-pinene	0.1	935	932	0.02
3	camphene	tr	952	946	-
4	sabinene	tr	976	969	-
5	β-pinene	tr	982	974	-
6	oct-1-en-3-ol	0.2	988	974	0.01
7	β-myrcene	0.6	993	988	0.22
8	p-cymene	3.3	1028	1020	0.73
9	limonene	0.2	1032	1024	0.02
10	β-phellandrene	tr	1034	1025	-
11	γ-terpinene	-	1058	1054	0.07
12	linalool	0.2	1108	1095	0.03
13	oct-1-en-3-ylacetate	0.4	1114	1110	0.05
14	octan-3-ylacetate	0.1	1126	1120	0.01
15	thymol, methylether	3.9	1235	1232	0.31
16	carvacrol, methylether	tr	1247	1241	-
17	2,3-dimethoxy-p-cymene	2.5	1299	-	0.03
18	3-isopropyl-2-methoxy-6-methylphenol^b	78.2	1347	-	0.99
19	α-copaene	1.5	1378	1374	0.27
20	β-caryophyllene	2.1	1421	1417	0.51
21	2,5-dimethoxy-p-cymene	1.3	1425	1424	0.09
22	α-caryophyllene	0.3	1456	1452	0.08
23	γ-muurolene	0.2	1483	1478	0.10
24	δ-cadinene	0.2	1522	1522	0.06
25	β-caryophyllene epoxide	1.7	1586	1582	0.07
26	2-Mehoxy-carvacrol dimer^c	0.2	2141	-	0.09
	Total	97.5
	HM	1.1
	AHM	3.3
	OM	0.2
	OAM	86.1
	HS	4.3
	OS	1.7
	Others	0.8

Max SD (maximum standard deviation);

2-Methoxy-carvacrol (3-isopropyl-2-methoxy-6-methylphenol);

Isomer to be identified; HM: Hydrocarbon Monoterpene; AHM: Aromatic Hydrocarbon Monoterpene; OM: Oxygenated Monoterpene; OAM: oxygenated aromatic monoterpene; SH: Hydrocarbon Sesquiterpene; OS: Oxygenated Sesquiterpene. IA_lit: Kovats Index described in the literature. IA_exp: Experimental Kovats Index obtained by GC-MS;tr:traces.

The 2-methoxy-carvacrol is an isomer of thymol that is used as a preservative in halothane, anesthetics, and oral antiseptics. When used to reduce bacterial plaque and gingivitis, thymol has been found to be more effective when used in combination with chlorhexidine than when used alone. Therefore, they are compounds that contain the same molecular formula but different chemical structures, which is why they are classified as isomers. As one is an isomer of thymol, we can compare the essential oils of several plants’ rich in aromatic monoterpene thymol, as reported in the literature.^33–35 Among the various species that also presented significant percentages of thymol, in the research by Borges et al,³⁶ when determining essential oils in natura from basil (Ocimum gratissimum L.), oregano (Origanum vulgare L.), and thyme (Thymus vulgaris L.), the authors also identified thymol among the major chemical constituents.

Antifungal Assay

The intrinsic inhibitory action of the essential oil was clinically irrelevant for all strains, since a reduction in the fungal growth curve was observed (Figure 3 A, B, C and D) only at the highest concentrations evaluated (>1024 µg/mL). When combining the essential oil at a subinhibitory concentration with fluconazole, it was found that this association can cause a modifying effect on the action of the antifungal, with a potentiation of the effect against CA INCQS 40006 (Figure 3A) at concentrations ranging from 8 to 64 µg/mL and at 8 µg/mL for CA URM 4127 (Figure 3B).

Figure 3.

Inhibition of Fungal Growth by the Essential oil of Aerial Parts of Rhabdocaulon lavanduloides (EOAPRl) Alone and Combined with Fluconazole. CA: Candida albicans; CT: Candida tropicalis; INCQS: Instituto Nacional de Controle de Qualidade em Saúde; URM: University Recife Mycology; EOAPRl: Essential Oils from Aerial Parts of Rhabdocaulon lavanduloides (Benth.).

In the CT INCQS 40042 strain (Figure 1C), the effect of the combination was indifferent, with a reduction in the action of fluconazole for the CT URM 4262 strain (Figure 3D) at concentrations of 8 and 16 µg/mL, showing that the combination of oil and antifungal had no promising effect on this strain. This can also be observed in Table 2, which presents the 50% inhibitory concentration (IC₅₀) of the microorganisms for the tested products and their combination.

Table 2.

Average Inhibitory Concentration (IC₅₀—µg/mL) of the Essential oil of Aerial Parts of Rhabdocaulon lavanduloides (EOAPRl).

Treatments	CA INCQS 40006	CA URM 4127	CT INCQS 40042	CT URM 4262
EOAPRl	868.633	2464.011	5904.335	5818.034
FCZ	14.982	12.518	1.871	5.634
EOAPRl + FCZ	9.635	9.523	9.072	10.011

CA: Candida albicans; CT: Candida tropicalis; INCQS: Instituto Nacional de Controle de Qualidade em Saúde; URM: University Recife Mycology; FCZ: Fluconazole; EOAPRl: Essential oils from aerial parts of Rhabdocaulon lavanduloides (Benth.).

In this experimental procedure, the MFC was defined as the lowest concentration at which no fungal colony growth was observed following subculturing on Sabouraud Dextrose agar (Table 3). The essential oil exhibited an MFC greater than 8194 μg/mL for all Candida strains tested, indicating a fungistatic effect under the conditions of this study. For fluconazole tested alone, fungicidal activity was observed only against the Candida albicans URM 4127 strain, with an MFC of 256 μg/mL. In contrast, the remaining strains showed fungistatic responses, with MFC values equal to or exceeding 16384 μg/mL. When fluconazole was combined with the essential oil, changes in antifungal activity were noted. The C. albicans URM 4127 strain exhibited an increased MFC of 4096 μg/mL, suggesting a possible antagonistic interaction. Conversely, a fungicidal effect was observed against the Candida tropicalis URM 4262 strain, with an MFC of 2048 μg/mL, which may indicate a potential synergistic effect. For the other strains, fungal growth persisted across all tested concentrations, and the MFC remained above the evaluated range.

Table 3.

Minimum Fungicide Concentration (MFC µg/mL) of the Essential oil of Aerial Parts of Rhabdocaulon lavanduloides.

Strain	EOAPRl	FCZ	EOAPRl + FCZ
CA INCQS 40006	≥16384	≥16384	≥16384
CA URM 4127	≥16384	256	4096
CT INCQS 40042	≥16384	≥16384	≥16384
CT URM 4262	≥16384	≥16384	2048

(Benth.).

Molecular Docking Analysis

The system formed from molecular docking simulations is expressed in Figure 4(A). The complexes formed by the superposition of the poses of the ligands fluconazole (green) and 3-isopropyl-2-methoxy-6-methylphenol (lilac), through the insertion of the SAPT1 receptor, are shown. Based on the energy and RMSD metrics, it is highlighted that the reference drug (fluconazole) has an energy value of −7.3 kcal/mol, while its deviation variation was equivalent to 1.222 Å; regarding the compound 3-isopropyl-2-methoxy-6-methylphenol, it is noted that its complex exerted values equal to −5.7 kcal/mol (affinity energy) with an RMSD variation of around 1.303 Å.

Figure 4.

Molecular Docking Simulation with the SAPT1 Receptor (A) Global complex Composed of the Ligand Fluconazole (Green) and 3-Isopropyl-2-Methoxy-6-Methylphenol (Lilac) (B) Interactions Established by the Relationship Between Ligand and Protein.

When discussing the potential interaction effect resulting from complex formation, Figure 5(A) presents an overview of the drug (fluconazole)—green and the compound 3-isopropyl-2-methoxy-6-methylphenol (lilac), properly indexed in the interaction cavity of the Als3 receptor. The data from molecular docking simulations revealed that both ligands exhibited a relatively similar interaction behavior linked to a potential competitive effect. The formed complexes showed energy and RMSD values of −7.0 kcal/mol and 0.910 Å (fluconazole), respectively; while for the compound 3-isopropyl-2-methoxy-6-methylphenol, its values were equivalent to −5.6 kcal/mol (binding energy) and 1.855 Å (RMSD).

Figure 5.

Molecular Docking Study Against the Biological Target Als3 (A) Complexes Formed by Interaction with the Global Binding Cavity by the Contribution of the Ligands Fluconazole (Green) and 3-Isopropyl-2-Methoxy-6-Methylphenol (Lilac) (B) Formation of Interaction Bonds Through Amino Acid Residues.

Based on molecular docking studies, it is possible to measure in Figure 6(A) the affinity profile resulting from the relationship between the ligands fluconazole (green) and 3-isopropyl-2-methoxy-6-methylphenol (lilac) when they are directed to the SAP5 receptor. According to this study, it is noticeable that both ligands are positioned in very close regions, thus demonstrating a similarity by competitive effect. According to the analyses, the SAP5/fluconazole complex showed an interaction energy value of −6.8 kcal/mol, with an RMSD variation equal to 1.072 Å. From another perspective, the system composed of the SAP5/3-isopropyl-2-methoxy-6-methylphenol complex exerted an energy equal to −5.8 kcal/mol and a deviation of 1.018 Å equivalent to the RMSD value.

Figure 6.

Affinity Assessment by Application of the SAP5 Receptor (A) Systems Composed of the Ligands Fluconazole (Green) and 3-Isopropyl-2-Methoxy-6-Methylphenol (Lilac) (B) Current Interactions of the Respective Ligands Associated with the Amino Acid Residues.

Studies based on molecular docking analyses aim to evaluate the possible interaction profiles resulting from the relationship between ligand and protein, and in this way assess the potential effect of the ligands when associated with target enzymes. Within this premise, Figure 7(A) shows the global view of the ligands fluconazole (green) and the major compound present in the essential oil, 3-isopropyl-2-methoxy-6-methylphenol (lilac), properly indexed in the ERG11 enzyme. Through this analysis, it is pointed out that the system formed by the presence of the reference drug resulted in an energy value equal to −8.1 kcal/mol and an RMSD variation of 1.688 Å. Following this premise, it is reported that the compound 3-isopropyl-2-methoxy-6-methylphenol associated with ERG11 resulted in a complex that presented an energy value equal to −6.9 kcal/mol and 1.461 Å (RMSD). The data indicate that the proposed drug compound plays a similar role when compared to fluconazole, reinforcing the potential effect through competitive interaction.

Figure 7.

Molecular Docking Through the Implementation of the ERG11 Receptor (A) Global View of the Complexes Constructed by the Ligands Fluconazole (Green) and 3-Isopropyl-2-Methoxy-6-Methylphenol (Lilac) (B) Projections of Interactions with the Amino Acid Residues.

Analysis of molecular docking simulations, as shown in Figure 8(A), reveals the global projection of complexes formed with the ligands fluconazole (green) and 3-isopropyl-2-methoxy-6-methylphenol (lilac) in relation to the Glc-NAc receptor. Studies indicated that the Glc-NAc/Fluconazole complex exhibited an energy value equivalent to −7.2 kcal/mol and an RMSD of 1.809 Å; while the other complex, Glc-NAc/3-isopropyl-2-methoxy-6-methylphenol, presented values equal to −5.8 kcal/mol (energy) and 1.450 Å (RMSD). The global projection studies indicate which ligands exhibit similarity of interaction, and among those analyzed, it is evident that 3-isopropyl-2-methoxy-6-methylphenol demonstrated the same affinity profile when compared to the reference drug.

Figure 8.

Estimation of Interaction Formed by the Contribution of the Glc-NAc Receptor (A) General Analysis of the System with major Contributions from the Ligands Fluconazole (Green) and 3-Isopropyl-2-Methoxy-6-Methylphenol (Lilac) (B) Formation of Bonds Between the Residues of the Interaction Site Before the Amino Acid Residues.

Discussion

Natural materials obtained from plants have been an essential source for human medicine for years.³⁷ Natural products, such as essential oils, have a wide variety of structures, pharmacological and biological activities resulting from the evolution and natural selection of species.³⁸ Among this range of plant materials, the Lamiaceae family stands out for having species with various biological activities, such as antioxidant,³⁹ anti-inflammatory,⁴⁰ antifungal,⁴¹ antibacterial,⁴² and antitumor.⁴³

The genus Rhabdocaulon, belonging to this family, includes the species Rhabdocaulon denudatum (Benth.) Epling, used by communities to treat coughs and bronchitis, important symptoms in microbial infections.⁴⁴ The species R. lavanduloides, abundant in the South and Southeast regions of Brazil,⁴⁵ has little-explored potential and has shown little biological activity against species of the genus Candida, despite having compounds with proven anti-Candida activity such as: p-cymene,^46–47 thymol,⁴⁸ and β-caryophyllene.⁴⁹

Carvacrol derivatives inhibited the growth of Aspergillus niger and C. albicans in a study conducted by Lazarević et al⁵⁰ and of Alternaria solani, Botrytis cinerea, Fusarium oxysporum, Pyricularia oryzae, and Rhizoctonia solani, plant pathogens.⁵¹ Carvacrol, in turn, has been shown to be effective against planktonic cells of Candida spp with minimum inhibitory concentrations lower than 512 µg/mL and fungicidal at concentrations ranging from 128 to 512 µg/mL. In the same study, it was observed that carvacrol significantly reduced the biomass and metabolic activity of mature biofilms in various Candida species.⁵¹ These results differ from those obtained in this study, which found little or no antifungal activity despite the major component of R. lavanduloides oil being a derivative of carvacrol (2-methoxy-carvacrol).

Thus, the phytochemical composition of essential oils is responsible for their bioactivities; however, the combined action of the mixture of phytochemicals may or may not favor a particular biological activity.⁵³ In addition, this composition may change due to factors such as the plant's growing environment, the time of collection, and the method used for identification.⁵⁴

Natural products derived from plants have long served as a valuable source for therapeutic agents.³⁷ Among these, essential oils stand out due to their structural diversity and broad spectrum of pharmacological and biological activities, shaped by evolutionary pressures and natural selection.³⁸ Within this context, the Lamiaceae family is particularly notable for encompassing species with recognized antioxidant,³⁹ anti-inflammatory,⁴⁰ antifungal,⁴¹ antibacterial,⁴² and antitumor properties.⁴³

The genus Rhabdocaulon, part of this family, includes species traditionally used by local communities to treat respiratory symptoms such as cough and bronchitis, which are often associated with microbial infections.⁴⁴ R. lavanduloides, a species abundant in the southern and southeastern regions of Brazil,⁴⁵ remains underexplored despite its phytochemical profile containing compounds with documented anti-Candida activity, including p-cymene,^46–47 thymol,⁴⁸ and β-caryophyllene.⁴⁹

Previous studies have demonstrated that carvacrol derivatives can inhibit the growth of fungal pathogens such as Aspergillus niger, Candida albicans,⁵⁰ and several phytopathogenic fungi.⁵¹ Carvacrol itself has shown potent antifungal effects against planktonic cells of Candida spp., with minimum inhibitory concentrations below 512 µg/mL and fungicidal activity between 128 and 512 µg/mL. Additionally, it has been reported to significantly reduce the biomass and metabolic activity of mature Candida biofilms.⁵¹

In contrast, the present study found limited antifungal activity for the essential oil of R. lavanduloides, despite its major constituent being 2-methoxy-carvacrol, a carvacrol derivative. This discrepancy may be attributed to structural differences between carvacrol and its methoxylated form, as well as to the complex interactions among the oil's constituents. The biological activity of essential oils is not solely determined by their major compounds, but rather by the synergistic or antagonistic effects of their full phytochemical composition.⁵² Furthermore, this composition can vary significantly depending on environmental conditions, harvest timing, and extraction methods, all of which may influence the oil's bioactivity.⁵³

The essential oil tested exhibited antifungal activity against Candida spp., as evidenced by a significant reduction in cell viability in a concentration-dependent manner. The IC₅₀ values indicated a dose-dependent inhibitory effect, suggesting that the compound interferes with physiological processes essential for fungal survival. However, based on the experimental determination of the Minimum Fungicidal Concentration (MFC), which exceeded the highest concentration tested (8194 μg/mL) for all strains, the effect was classified as fungistatic. This classification is supported by the absence of colony growth in subcultures and aligns with established microbiological criteria.

Differences in susceptibility among the species evaluated may be attributed to intrinsic structural and functional variations, including cell wall architecture, membrane composition, and resistance mechanisms. These factors are particularly relevant in non-albicans strains such as Candida glabrata, C. tropicalis, and C. parapsilosis, which are known to exhibit reduced sensitivity to conventional antifungal agents and pose increasing clinical challenges.¹²

Although the essential oil alone demonstrated a fungistatic effect, its combination with fluconazole altered the antifungal profile. Notably, a fungicidal effect was observed against C. tropicalis URM 4262, and an increase in MFC was detected for C. albicans URM 4127, suggesting strain-specific interactions. These findings may indicate a potential synergistic or antagonistic modulation depending on the fungal species and highlight the relevance of combination therapies in overcoming resistance or enhancing antifungal efficacy.

Overall, the ability to suppress fungal growth, even in a fungistatic manner, may hold clinical relevance, particularly in superficial infections or in therapeutic strategies where fungal proliferation control supports host immune responses or complements the action of other antifungal agents.⁵⁴

Based on the simulation data presented in Figure 4(B), all interactions formed by the relationship between the ligand and the amino acid residues present in the interaction cavity are shown. Through this analysis, it is possible to measure that the reference drug (fluconazole) exerted two hydrophobic interactions through the contribution of the amino acid residues Val30 and Ile123. Regarding hydrogen bonds, it is possible to highlight the predominance of four bonds through the participation of the amino acid residues Asp86 (2.44 Å), Asp120 (2.70 Å), Thr222 (2.77 Å), and Thr222 (3.31 Å). Finally, the formation of a Pi-stacking interaction (Tyr84) is noticeable. These data suggest that these residues may be linked to the potential biological effect associated with the evaluated compound. Concerning the contributions formed between the amino acid residues linked to the ligand 3-isopropyl-2-methoxy-6-methylphenol, only hydrophobic interactions were observed, duly characterized by the residues Tyr84 (3.55 Å), Tyr84 (3.77 Å), Val119 (3.37 Å), and Ile123 (3.47 Å). The data highlight that the major ligand forms two similar interactions when compared to the reference drug, characterized by the residues Ile23 and Tyr84, thus indicating its potential therapeutic effect on the target in question; the remaining values referring to the interaction distances are included in Table 4.

Table 4.

Interactions Formed by the Relationship Between Ligand and Protein.

Complex	Energy (kcal/mol)	Interaction Type	Residue (Distance in Å)
SAPT1/Fluconzole	−7.3	Hydrophobic	Val30 (3.77) e Ile123 (3.58)
		H-Bond	Asp86 (2.44), Asp120 (2.70), Thr222 (2.77) e Thr222 (3.31)
		Pi-Stacking	Tyr84 (4.99)
SAPT1/3-isopropyl-2-methoxy-6-methylphenol	−5.7	Hydrophobic	Tyr84 (3.55), Tyr84 (3.77), Val119 (3.37) e Ile123 (3.47)
Als3/Fluconzole	−7.0	Hydrophobic	Val161 (3.66)
		H-Bond	Asn22 (3.08), Asn22 (2.66) e Tyr23 (3.11)
		Pi-Stacking	Tyr23 (5.18)
		Halogen Bond	Asn225 (3.32)
Als3/3-isopropyl-2-methoxy-6-methylphenol	−5.6	Hydrophobic	Val161 (3.72), Val161 (3.71), Leu167 (3.63)
		H-Bond	Tyr168 (2.35), Tyr168 (2.33) e Tyr168 (3.27)
SAP5/Fluconzole	−6.8	Hydrophobic	Ala162 (3.99) e Phe281 (3.90)
		H-Bond	Leu280 (2.66) e Gln282 (2.55)
		Halogen Bond	Arg312 (3.80)
SAP5/3-isopropyl-2-methoxy-6-methylphenol	−5.8	Hydrophobic	Ala162 (3.53), Lys257 (3.90) e Phe281 (3.63)
		H-Bond	Leu280 (2.31), Arg297 (2.49) e Arg297 (3.07)
		Pi-Cation	Arg312 (4.95)
ERG11/Fluconzole	−8.1	Hydrophobic	Thr311 (3.66) e Leu 376 (3.89)
		H-Bond	Tyr132 (2.85)
		Pi-Stacking	Tyr118 (4.66)
ERG11/3-isopropyl-2-methoxy-6-methylphenol	−6.9	Hydrophobic	Tyr118 (3.67), Tyr118 (3.74), Thr311 (3.74), Leu376 (3.79), Leu376 (3.57)
Glc-NAc/Fluconzole	−7.2	Hydrophobic	Trp127 (3.99) e Tyr371 (3.55)
		H-Bond	Asp194 (2.91), Arg254 (2.80), Gln321 (3.23) e His355 (2.88)
Glc-NAc/3-isopropyl-2-methoxy-6-methylphenol	−5.8	Hydrophobic	Trp127 (3.96), Trp127 (3.56) e Tyr371 (3.67)

Regarding the interactions formed, Figure 5(B) highlights all the established contributions. For the reference compound, only one hydrophobic interaction was observed, formed with the Val161 residue (3.66 Å). Concerning hydrogen bonds, a greater number of bonds are noticeable, related to the Asn22, Asn22, and Tyr23 residues, with the smallest distance value being 2.66 Å (Asn22), and only one Pi-stacking interaction with Tyr23 (5.18 Å). These data indicate which residues are linked to the interaction effect of the fluconazole compound, thus highlighting a possible interaction pathway to evaluate the potential competitive effect associated with the modulation profile of the target receptor. Based on the data presented in Figure W (B), the figure highlights that the compound 3-isopropyl-2-methoxy-6-methylphenol exerted three hydrophobic interactions with two distinct residues, Val161 (3.72 Å and 3.71 Å) and Leu167 (3.63 Å), and three hydrogen bonds with the same residue, Tyr168 (2.35 Å, 2.33 Å, and 3.27 Å). These data demonstrate that the compound 3-isopropyl-2-methoxy-6-methylphenol shows a potential effect associated with the observed interaction site, directly corroborating with the similar residue when compared to fluconazole (Val161). The remaining values referring to the formed distances can be observed in Table 4.

The analysis of the results from molecular docking simulations, as shown in Figure 6(B), highlights all the interactions formed through the complexes. It is emphasized that the reference drug (fluconazole) formed two hydrophobic interactions by binding to the amino acid residues Ala162 (3.99 Å) and Phe281 (3.90 Å), while for hydrogen bonds, two major residues are presented (Leu280 and Gln282), and finally, only one halogen bond with Arg312 (3.80 Å). From this perspective, it is evident that these amino acid residues may be related to the potential biological effect associated with fluconazole, given the SAP5 receptor. From the comparative analysis, it is possible to highlight that the ligand 3-isopropyl-2-methoxy-6-methylphenol formed three hydrophobic interactions characterized by the residues Ala162 (3.53 Å), Lys257 (3.90 Å), and Phe281 (3.63 Å), while for hydrogen bonds, three contributions were noticeable: Leu280 (2.31 Å), Arg297 (2.49 Å), and Arg297 (3.07 Å). These results indicate that the compound 3-isopropyl-2-methoxy-6-methylphenol showed high specificity with the interaction site when compared to the reference drug, being duly evidenced by the effective contribution of residues Phe281, Ala162, Leu280, and Arg312, demonstrating through these interactions the potential effect associated with the SAP5 receptor.

Among the interactions established between the ligands related to the amino acid residues, Figure 7(B) shows all the interactions formed by the contribution of the reference drug, which formed two hydrophobic interactions through the residues Thr311 (3.66 Å) and Leu 376 (3.89 Å), one hydrogen bond Tyr132 (2.85 Å), and one Pi-Stacking interaction (Tyr118). Regarding the interactions from the ligand 3-isopropyl-2-methoxy-6-methylphenol, five interactions with amino acid residues are noted, based on three amino acid residues: Tyr118 (3.67 Å), Tyr118 (3.74 Å), Thr311 (3.74 Å), Leu376 (3.79 Å), Leu376 (3.57 Å). The data present in the molecular docking simulations indicated that the proposed drug compound exerted relevant interactions with three similar amino acid residues when compared to fluconazole (Leu376, Tyr118, and Thr311). These results suggest that 3-isopropyl-2-methoxy-6-methylphenol demonstrates a potential biological effect associated with a competitive character when compared to the reference drug; the other distance values are presented in Table 4.

The data regarding molecular docking simulations, as shown in Figure 8(B), indicated the main interactions formed through the created complexes. Among the ligands, it is evident that fluconazole exerted two hydrophobic interactions with Trp127 (3.99 Å) and Tyr371 (3.55 Å), and four hydrogen bonds with Asp194 (2.91 Å), Arg254 (2.80 Å), Gln321 (3.23 Å), and His355 (2.88 Å). These data suggest that these amino acid residues are present in the site of relevance, serving as a crucial starting point in the ranking of potential drug candidates. From this perspective, it is noted that the compound 3-isopropyl-2-methoxy-6-methylphenol exerted only hydrophobic interactions through the implementation of Trp127 (3.96 Å), Trp127 (3.56 Å), and Tyr371 (3.67 Å). These results support the possible use of the compound 3-isopropyl-2-methoxy-6-methylphenol with the Glc-NAc receptor due to the ligand's interaction with two amino acid residues present in the reference site (Trp127 and Tyr371), thus highlighting the potential biological effect through a competition profile.

Conclusion

Although the essential oil of Rhabdocaulon lavanduloides did not exhibit intrinsic fungicidal activity, it demonstrated a fungistatic effect against all Candida strains tested. When combined with fluconazole, the oil contributed to a fungicidal response against Candida tropicalis URM 4262 and modulated the activity against Candida albicans URM 4127, indicating potential for synergistic or strain-specific interactions.

These findings support the relevance of R. lavanduloides essential oil as a candidate for further investigation, including studies with isolated phytochemicals, alternative extraction methods, and broader microbial panels. Additionally, molecular docking simulations revealed that the major compound (3-isopropyl-2-methoxy-6-methylphenol) exhibited an affinity profile comparable to fluconazole across all biological receptors, suggesting competitive binding and potential therapeutic applicability.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251406186 - Supplemental material for Chemical Composition, Antifungal Activity and Molecular Docking Study of Essential oil of Rhabdocaulon lavanduloides Benth

Supplemental material, sj-docx-1-npx-10.1177_1934578X251406186 for Chemical Composition, Antifungal Activity and Molecular Docking Study of Essential oil of Rhabdocaulon lavanduloides Benth by Wanderlei do Amaral, Ricardo Andrade Rebelo, Joara Nályda Pereira Carneiro, Taís Gusmão da Silva, Luciene Ferreira de Lima, Márcia Machado Marinho, Victor Moreira de Oliveira, Maria Flaviana Bezerra Morais Braga, Luiz Everson da Silva and Emmanuel Silva Marinho in Natural Product Communications

Footnotes

ORCID iD

Luiz Everson da Silva

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Supplemental Material

Supplemental material for this article is available online.

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