Antifungal and anti-biofilm activity of patchouli essential oil (Pogostemon cablin) against oral isolates of Candida albicans

Atividade antifúngica e antibiofilme do óleo essencial de patchouli (Pogostemon cablin) frente a isolados orais de Candida albicans

Abstract

The Candida albicans species is part of the human microbiota and is associated with different types of infections. It is the most commonly isolated yeast in samples of oral cavity infections. The number of infections attributed to the Candida genus has grown significantly on a global scale and due to the rigorous and improper use of current antimicrobials, antimicrobial resistance has also increased. Pogostemon cablin Benth., popularly known as patchouli, is a medicinal herb of great relevance, with several pharmacological properties reported in the literature. The aim of this study was to evaluate the antifungal potential of P. cablin essential oil against 11 oral isolates of Candida albicans and a standard strain (ATCC90028). The chemical characterization of P.cablin was carried out using Gas Chromatography coupled with Mass Spectrophotometry (GC-MS) and 19 molecules were identified, including patchouli alcohol (15.11%), Δ-Guaiene (9.85%), α-Guaiene (8.06%) and α-Patchoulene (5.03%). The antifungal activity was analyzed using the microdilution technique to determine the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC). The mechanism of action of the possible antifungal activity of the phytocomplex in question was analyzed using the sorbitol and ergosterol assays. In addition, some virulence factors were also investigated, such as biofilm formation capacity and morphogenesis. P. cablin showed MIC and MFC values between 2 and 128 µg/mL for the strains tested. The results suggest that this activity occurs through the complexation of the oil's constituents with the ergosterol of the fungal membrane. The essential oil slightly reduced the Morphology Index (MI) of the strains tested. For the most filamentous strain, there was a significant decrease in filament length, from 285.0 µm to 220.5 µm. In the formation of biofilms, the essential oil showed a reduction of 26% to 59% at concentrations of 4 to 40 µg/mL and in the reduction of biofilms formed, the values ranged from 49% to 68%. The essential oil of P. cablin showed a complex chemical composition and significant activity on C. albicans, which was considered fungicidal for most of the strains tested. It is suggested that this antifungal action is due to the complexation of the essential oil with the ergosterol of the fungal plasma membrane. In addition, P. cablin acted on the formation and reduction of biofilm and also exerted an influence on the morphogenesis of Candida albicans.

Keywords:
antifungal agent; Candida albicans; oral candidiasis; phytochemicals; Pogostemon cablin

Resumo

A espécie Candida albicans integra a microbiota humana e é associada a diferentes tipos de infecções, sendo a levedura mais comumente isolada em amostras de infecções da cavidade oral. O número de infecções atribuídas ao gênero Candida tem apresentado um crescimento significativo em escala global e devido ao uso rigoroso e indevido dos atuais antimicrobianos a resistência antimicrobiana também cresceu. Pogostemon cablin Benth., conhecido popularmente como patchouli, é uma erva medicinal de grande relevância, com diversas propriedades farmacológicas relatadas na literatura. Diante disso, este trabalho tem como objetivo avaliar o potencial antifúngico do óleo essencial de P. cablin frente a 11 isolados orais de Candida albicans e uma cepa padrão (ATCC90028). A caracterização química do P.cablin foi realizada por meio da Cromatografia Gasosa acoplada à Espectrofotometria de Massa (CG-EM) e foram identificadas 19 moléculas, incluindo patchouli alcohol (15,11%), Δ-Guaiene (9,85%), α-Guaiene (8.06%) e α-Patchoulene (5.03%). A análise da atividade antifúngica foi realizada pela técnica de microdiluição para determinar a concentração inibitória mínima (CIM) e a concentração fungicida mínima (CFM). O mecanismo de ação da possível atividade antifúngica do fitocomplexo em questão foi analisado pelos ensaios de sorbitol e ergosterol. Ademais, alguns fatores de virulência também foram investigados como a capacidade de formação de biofilme e a morfogênese. O P. cablin apresentou valores de CIM e CFM entre 2 e 128 µg/mL para as cepas testadas. Os resultados sugerem que esta atividade ocorre pela complexação dos constituintes do óleo ao ergosterol da membrana fúngica. O óleo essencial reduziu levemente o Índice de Morfologia (IM) das estirpes testadas. Para a cepa mais filamentosa, houve uma diminuição significativa no comprimento dos filamentos, de 285,0 µm para 220,5 µm. Na formação de biofilmes, o óleo essencial apresentou redução de 26% a 59% nas concentrações de 4 a 40 µg/mL e na redução de biofilmes formados, os valores variaram de 49% a 68%. O óleo essencial de P. cablin apresentou composição química complexa e relevante atividade sobre C. albicans, sendo essa atividade considerada fungicida para a maioria das estirpes testadas. Sugere-se que essa ação antifúngica acontece pela complexação do óleo essencial ao ergosterol da membrana plasmática fúngica. Ademais, P. cablin atuou na formação e redução do biofilme e também exerceu influência sobre a morfogênese de Candida albicans.

Palavras-chave:
agente Antifúngico; Candida albicans; candidíase oral; fitoquímicos; Pogostemon cablin

1. Introduction

Oral candidiasis is an infection of the oral mucosa caused by species of the genus Candida, imbalances in the oral microbiota can favor the growth of these species and lead to the appearance of clinical symptoms (Lu, 2021; Cannon, 2022). Factors that predispose to the development of this pathology include immunosuppression, radiotherapy in the head and neck region, diabetes and prolonged administration of antibiotics and immunosuppressive drugs (Lu, 2021). Other local factors are the use of dental prostheses and decreased salivary flow, which plays an important role in mechanical cleaning, preventing C. albicans from adhering to oral epithelial cells (Ok et al., 2021).

In addition to the fragility of the individual's immune system, another relevant factor for the development of infections is the expression of virulence factors, including morphogenesis. C. albicans can change from blastoconidia to hyphae, which plays a significant role in infection and disease progression, as well as facilitating cell adhesion, penetration through the intestinal mucosal barrier and dissemination into the bloodstream in the early stages of infection (Chow et al., 2021).

Biofilm formation is also a key process in evading the immune system. This is a fundamental characteristic in the pathogenesis of Candida albicans, since most infections associated with this species occur due to the formation of biofilms on the surface of the host or on inert materials, such as implants and prostheses. The biofilm formed has a complex structure made up of various morphological forms. This resistance in the structure of the biofilm is one of the factors that contribute to its virulence and resistance to treatments, resulting in significant morbidity and mortality. (Priya and Pandian, 2020; Tsui et al., 2016).

Given this, there is an urgent need to establish new treatment options in order to intercept growing drug tolerance and control emerging microbial infections (Qadri et al., 2021; Qadri et al., 2022). Thus, a large number of antimicrobials derived from natural products have been studied as sources of new drug candidates against a variety of human infectious diseases (Ye et al., 2020; Lee et al., 2020).

Pogostemon cablin Benth., popularly known as patchouli, is a medicinal herb of great relevance, with significant potential in the fragrance industry, as well as being a key component in several traditional chinese medicines. In recent years, extensive experimental research has explored the pharmacological properties and mechanisms of action of patchouli, and several components have been reported, such as patchouli alcohol, pogostone, α-guaiene, δ-guaiene, β-caryophyllene, trans-caryophyllene, α-patchoulene, β-patchoulene, and β-elemene. (Wu et al., 2019). These studies have demonstrated a variety of beneficial effects, including antimicrobial properties, anti-inflammatory effect, antioxidant action, immune modulation, among others (Dechayont et al., 2017; Swamy and Sinniah, 2016; Junren et al., 2021).

2. Meterials and Methods

2.1. Fungal strains

For this study, we used Candida albicans oral strains, 11 clinical and 1 standard, previously identified and coded as: ATCC 90028, LABM 094, LABM 096, LABM 097, LABM 100, LABM 187, LABM 186, LABM 177, LABM 191, LABM 178, LABM 179, LABM 181. All the samples belong to the Mycology Laboratory of the Department of Pharmaceutical Sciences of the Federal University of Paraíba. All these clinical strains were previously collected (CAAE protocol: 43911715.8.0000.5188 and CAAE protocol: 23611519.6.0000.5292), stored at -2°C in Sabouraud Dextrose Broth (CDS) (KASVI®, Curitiba, Brazil) supplemented with glycerol (40% v/v), in cryotubes (2 mL). Repeats of 24-48 hours incubated at 35 ± 2 °C were used in the tests.

2.2. Substances

The test substance, Patchouli essential oil (Pogostemon cablin), was purchased commercially from Herbia ® (Joinville, Santa Catarina, Brazil).

The culture medium RPMI-1640, YPD, amphotericin B, caspofungin, fluconazole, ergosterol, tween 80, DSMO were purchased from Sigma-Aldrich® (São Paulo, Brazil) and sorbitol (D-sorbitol anhydrous) (INLAB®, São Paulo, Brazil) and Sabouraud Dextrose Agar from KASVI® (Kasv Imp and Dist e Prod) Laboratórios LTDA (Curitiba, Brazil).

2.3. Phytochemical characterization

2.3.1. Gas chromatography coupled with mass spectrometry

The characterization of the components of patchouli essential oil was carried out using a Gas Chromatograph coupled to a Mass Spectrometer (GC-MS) (Shimadzu QP2010 Ultra, Japan), using the method described by Fontes et al. (2022), with adaptations.

The analysis was carried out using helium as the carrier gas in an RTX-5MS capillary column (30 m x 0.25 mm, 0.25 µm) following the following parameters: injection temperature, 250°C; column temperature, 40°C; helium gas flow rate, 2.11 mL/min; the temperature program was started at 40°C for 2 min followed by an increase to 100°C, then the temperature was increased to 280°C for 12 min. The detected components were identified by comparing the retention time and mass spectrum data with those available in the NIST (National Institute of Standards and Technology) library.

2.4. In vitro antimicrobial tests

2.4.1. Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) tests

The minimum inhibitory concentration (MIC) of the essential oil and fluconazole was determined by the broth microdilution technique, according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2008), with some modifications. The essential oil was prepared at the time of testing, diluted in Tween 80 (2%) and sterile distilled water, obtaining an initial concentration of 2048 µg/mL. C. albicans suspensions were prepared in RPMI-1640 medium and adjusted to 10⁵ CFU/mL, according to the McFarland scale. In sterile 96-well plates, 100 µL of double-concentrated RPMI-1640 was added to each well, followed by 100 µL of the test solution in the first rows, subjected to serial dilutions in a ratio of two, obtaining concentrations of 1024 to 0.5 µg/mL for the essential oil and 64 to 1 µg/mL for fluconazole. Then, 10 µL of the fungal inoculum was added to each well.

Controls were included to verify fungal viability (RPMI-1640 and inoculum of each strain), the effect of the solvents used (DMSO, Tween), and the sterility of the medium (RPMI-1640 without the fungal suspension). The plates were incubated at 35 ± 2 °C for 24 hours, and the MIC was defined as the lowest concentration capable of visually inhibiting fungal growth compared to the control, with the tests performed in triplicate.

After reading the MIC, the Minimum Fungicidal Concentration (MFC) was determined by subculturing aliquots corresponding to the concentrations of MIC, MIC×2, and MIC×4 in Sabouraud Dextrose Agar, incubated at 35 °C for 24 hours. The MFC was defined as the lowest concentration capable of inhibiting visible colony growth, and the MFC/MIC ratio was used to classify the action as fungistatic (MFC/MIC ≥ 4) or fungicidal (MFC/MIC < 4).

Based on the methodology proposed by Alves et al. (2021), which suggests a classification to assess the antifungal potential of new compounds with pharmacological activity against Candida species, the bioactivity of the compounds was then determined using the MIC values and classified into the following categories: (a) very strong bioactivity (MIC < 3.515 µg/mL); (b) strong bioactivity (MIC between 3.515 and 25 µg/mL); (c) moderate bioactivity (MIC between 26 and 100 µg/mL); (d) weak bioactivity (MIC between 101 and 500 µg/mL); and (e) very weak bioactivity (MIC between 501 and 2000 µg/mL).

2.4.2. Action on the fungal cell wall - sorbitol test

The test was carried out using the broth microdilution technique, but the same process is carried out with RPMI in the absence of sorbitol, and with the addition of 0.8M sorbitol. Sorbitol is an osmotic protector used to stabilize fungal protoplasts. If the test product acts in any way on the fungal cell wall, it will cause cell lysis in the absence of sorbitol, but will allow fungal growth in the presence of this osmotic protector. What will be observed in practice is an increase in the MIC of the substance (Frost et al., 1995). The increase in the MIC value in the presence of sorbitol indicates that the cell wall is one of the possible targets of the test substance (Sousa et al., 2016; Freires et al., 2014).

Caspofungin is the antifungal most commonly used as a positive control for this test, at an initial concentration of 4 µg/mL (caspofungin diacetate-Sigma-Aldrich, St. Louis, MO, USA), due to its known action on the cell wall. In addition, sterility controls were carried out on RPMI 1640 with and without sorbitol, and viability of the inoculum with the addition of RPMI 1640 with and without sorbitol.

2.4.3. Action on the fungal cell membrane - ergosterol test

The test was carried out using the broth microdilution technique, but in the absence and with the addition of ergosterol 400 µg/mL to the culture medium. Some antifungal agents act on plasma membrane ergosterol, forming complexes or inhibiting membrane biosynthesis. Thus, an increase in the MIC in the presence of exogenous ergosterol will indicate an activity of the agent under study on the ergosterol present in the fungal plasma membrane (Sousa et al., 2016). For the control, the same procedure was carried out with the antifungal amphotericin B, whose mechanism of action is known and involves its binding to the ergosterol present in the fungal membrane. In addition, as previously described, sterility controls were carried out with and without ergosterol, and viability of the inoculum with and without ergosterol (Escalante et al., 2008).

2.4.4. Effect of P. cablin on biofilm adhesion

It was evaluated at the initial stage of biofilm formation, as described by Plyuta et al. (2013), with adaptations. The inoculum (10⁶ CFU/mL) of C. albicans ATCC 90028, along with the concentrations of P. cablin previously determined by the MIC values (4 µg/mL), MICx2 (8 µg/mL), MICx4 (16 µg/mL), and MICx10 (40 µg/mL), were added to 24-well plates and incubated for 48 hours at 35°C. After incubation, the planktonic cells were carefully removed from the wells by rinsing with PBS and dried for 45 minutes. The adhered cells were then stained with 0.4% crystal violet. Absorbance values were read at 595 nm using a plasma reader. The test was performed on four samples. The untreated biofilm corresponded to the growth control, while the wells with culture medium without the addition of the microorganism were the sterility controls.

2.4.5. Effects of P. cablin on biofilm reduction

Aliquots of 1000 µL of the C. albicans ATCC 90028 inoculum, containing approximately 10⁶ CFU/mL, were transferred to a 24-well microdilution plate and incubated for 48 hours at 35°C. The wells were washed with phosphate-buffered saline (PBS) to remove the loose cells, and then fresh culture medium was added. The concentrations of P. cablin EO previously determined by MIC values (4 µg/mL); MICx2 (8 µg/mL); MICx4 (16 µg/mL); MICx10 (40 µg/mL) and Fluconazole (positive control) at MIC concentrations (2 µg/mL); MICx2 (4 µg/mL); MICx4 (8 µg/mL); MICx10 (20 µg/mL) were added to the wells, followed by incubation for 48 hours at 35°C. For biofilm quantification, the wells were washed twice with PBS, air-dried for 45 minutes, and then stained with a 0.4% crystal violet solution. The absorbance was measured at 595 nm using a microplate reader. All assays were carried out in quadruplicate. The untreated biofilm served as the growth control, while the culture medium without microorganisms was used as the sterility control.

2.4.6. Morphogenesis assay

The morphogenesis test was carried out according to the technique described by Chaves et al. (2007) with some modifications. The C. albicans cells were grown overnight (18 to 24 hours) in YPD medium in the presence and absence of P.cablin. The absorbance of cellular growth was measured using spectrophotometry (600 nm), and the concentration was standardized to 1 x 10⁶ cells/mL.

To induce morphogenesis, 30 µL of the standardized growth was inoculated into YPD broth supplemented with 20% Fetal Bovine Serum (FBS) (4 mL of liquid YPD, 1 mL of FBS) in tubes and incubated at 37°C for 1 hour, followed by an additional 2 hours, totaling 3 hours of incubation.

At the end of the 1-hour period, a 500 µL aliquot of the suspension was added to 2 mL Eppendorf tubes containing 500 µL of 10% formalin for subsequent microscopic examination. The tubes containing the inocula were then incubated again to complete the 3-hour incubation period. After this period, a 500 µL aliquot was transferred to Eppendorf tubes containing 500 µL of 10% formalin and stored at 4°C for later microscopic observation.

All slides were read in triplicate. The slides from the 1-hour incubation were evaluated by counting 100 cells per slide and determining the percentage of cells that exhibited germ tube formation.

Regarding the slides from the samples subjected to 3 hours of incubation, the Morphology Index was calculated, as greater morphological variability of C. albicans cells is expected. Therefore, 100 C. albicans cells were counted on each slide, with the following morphology classifications: blastoconidia (round cells) were assigned an MI value of 1; elongated cells with a diameter twice the length were assigned MI = 2; cells resembling pseudohyphae were assigned MI = 3; and long, true hyphae with parallel sides were assigned MI = 4. The Morphology Index (MI) was determined using the following Formula 1:

$$\begin{array}{c}MorphologyIndex\left(MI\right)=\left(N°IM1x1\right)+\\ \left(N°IM2x2\right)+\left(N°IM3x3\right)+\\ \left(N°IM4x4\right)/100\end{array}$$

In this context, values close to one indicate a population of spherical yeast cells; values close to four indicate a population of true hyphae; and values between one and four suggest the presence of varied morphologies or, predominantly, pseudohyphae.

2.4.7. Measurement of Candida albicans hyphal length

The cell length of C. albicans, specifically strain 97, was measured after the induction of morphogenesis (incubation for 3 hours in YPD + 20% FBS). The NIS-Elements D software was used for this purpose. For each strain, the average length of 100 hyphal cells was determined for isolates previously cultured in the presence or absence of P. cablin.

3. Results

3.1. Gas chromatography and mass spectrometry

From the data obtained by the detector, 19 (nineteen) molecules were suggested, listed in Table 1, those with the highest similarity index with the Databases: NIST 2008, NIST 2008+Shimadzu and FFNSC 1.3.

Table 1
Chemical composition of patchouli essential oil by GC-MS.

Component	TR(min)	Area(%)
β-Patchoulene	11.997	2.88
Seychellene	12.245	0.58
Caryophyllene	12.286	2.05
α-Guaiene	12.389	8.06
Seychellene	12.511	5.50
α-Humulene	12.550	0.43
α-Patchoulene	12.608	5.03
Patchoulene	12.660	0.69
Azulene, 1,2,3,4,5,6,7,8-Octahydro-1,4-dimethyl-7-(1-methylethenyl)-,[1S-(1 α,4 α,7 α)]-	12.693	0.16
β-Selinene	12.793	0.40
Azulene, 1,2,3,4,5,6,7,8-Octahydro-1,4-dimethyl-7-(1-methylethenyl)-,[1S-(1 α,4 α,7 α)]-	12.850	2.45
Δ-Guaiene
α-Maaliene	12.905	9.85
(-)-Caryophyllene oxide	13.025	0.16
Viridiflorol	13,388	1.34
Patchouli alcohol	13.940	1.96
Terephthalic acid, di(2-ethylhecyl) ester	14.069	15.11
Terephthalic acid, di(2-ethylhecyl) ester	19.616	0.61
Terephthalic acid, di(2-ethylhecyl) ester	20.041	35.69
	20.179	7.04

The majority component indicated was patchouli alcohol 15.11%, Δ-Guaiene (9.85%), α-Guaiene (8.06%) and α-Patchoulene (5.03%) commonly found in identifications of the composition of Patchouli essential oil.

For identification, the Retention Index (RI) values of the compounds were compared with the values registered in the NIST database, as described above.

Table 2 shows that 6 (six) components had their identities confirmed through this analysis.

Table 2
Identification of the components in relation to the NIST database.

Component	TR (min)	Área (%)	IR (exp)	IR (lit)	Reference
β-Patchoulene	11.991	2.88	1404	1406	Moio et al. (2000)
α-Guaiene	12,384	8.06	1461	1456	Hudaib et al. (2001)
α-Humulene	12,545	0.43	1483	1474	Yu et al. (2004)
α-Patchoulene	12,604	5.03	1492	1486	Gudaityte and Venskutonis (2007)
Caryophyllene	12,286	2.05	1446	1440	Cardeal et al. (2006)
(-)-Caryophyllene oxide	13,384	1.34	1602	1595	Liu et al. (2006)

• TR = retention time of the component in the column; IR (exp) = experimental retention index; IR (lit) = literature retention index.

3.2. Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC)

In this study, the antifungal effect of P. cablin on 12 strains of C. albicans was observed. Table 3 shows the MIC and MFC values for P. cablin and fluconazole, used as the reference drug.

Table 3
Minimum Inhibitory Concentration (µg/mL) and Minimum Fungicidal Concentration (µg/mL) of Patchouli (P. cablin) essential oil and Fluconazole on C. albicans.

STRAINS	Pogostemon cablin				Fluconazole	Controls
	MIC	MFC	MFC	Antifungal activity	MIC	*MO	**Ste
			MIC
C. albicans ATCC 90028	4 µg/mL	>16 µg/mL	>4	Fungistatic	2 µg/mL	+	-
C. albicans LABM 094	4 µg/mL	>16 µg/mL	>4	Fungistatic	0.5 µg/mL	+	-
C. albicans LABM 096	2 µg/mL	>8 µg/mL	>4	Fungistatic	0.5 µg/mL	+	-
C. albicans LABM 097	128 µg/mL	128 µg/mL	1	Fungicidal	8 µg/mL	+	-
C. albicans LABM 100	2 µg/mL	>8 µg/mL	>4	Fungistatic	4 µg/mL	+	-
C. albicans LABM 187	4 µg/mL	4 µg/mL	1	Fungicidal	2 µg/mL	+	-
C. albicans LABM 186	2 µg/mL	>8 µg/mL	>4	Fungistatic	1 µg/mL	+	-
C. albicans LABM 177	2 µg/mL	4 µg/mL	1	Fungicidal	4 µg/mL	+	-
C. albicans LABM 191	4 µg/mL	2 µg/mL	1	Fungicidal	2 µg/mL	+	-
C. albicans LABM 178	2 µg/mL	2 µg/mL	1	Fungicidal	4 µg/mL	+	-
C. albicans LABM 179	2 µg/mL	2 µg/mL	1	Fungicidal	4 µg/mL	+	-
C. albicans LABM 181	2 µg/mL	2 µg/mL	1	Fungicidal	4 µg/mL	+	-

• MFC/MIC = ratio of MFC to MIC; *MO= viability control; **Ste = sterility control; + = microorganism growth; - = no microorganism growth. Values expressed in µg/mL. MFC/MIC> 4 indicates fungistatic activity, <4 fungicidal activity.

The essential oil inhibited the growth of 11 of the 12 C. albicans strains (approximately 91.6%) at concentrations of up to 4 µg/mL, while one strain (approximately 8.4%) was inhibited at a concentration of 128 µg/mL, these MIC values representing the lowest concentration of the essential oil capable of inhibiting the visible growth of the microorganism. For fluconazole, used as a control in the tests, the MIC values varied between 0.5 and 8 µg/mL.

In the comparative analysis between MFC and MIC expressed in Table 3, it was observed that 5 of the 12 strains tested had MFC four times higher than MIC, characterizing a fungistatic effect and the other 7 had identical MFC and MIC values, demonstrating a fungicidal effect, according to the parameters established by Siddiqui et al. (2013).

Analysis of the MFC results provides important insights into the efficacy of essential oil against C. albicans. This metric reflects the lowest concentration required to completely eliminate the microorganism, differing from the MIC, which only inhibits visible growth.

4.2. Action of P. cablin on the fungal cell wall and membrane

The tests carried out confirmed the antifungal potential of P. cablin essential oil and suggest that its effect occurs through interaction with ergosterol present in the fungal cell membrane. This is evidenced by the MIC values, which were higher in the presence of exogenous ergosterol for both strains (≥ 1024 μg/mL) compared to its absence, 4 μg/mL for the ATCC strain and 128 μg/mL for the clinical strain. There was no change in CIM values in the presence of sorbitol. (Table 4).

Table 4
MIC values (µg/mL) of Pogostemon cablin and control antifungals in the absence and presence of sorbitol and ergosterol against C. albicans ATCC 90028 and C. albicans LBM097.

Microrganisms	C. albicans	C. albicans	C. albicans	C. albicans	C. albicans	C. albicans
	ATCC 90028	LABM097	ATCC 90028	LABM097	ATCC 90028	LABM097
Substances	Control		+Sorbitol		+Ergosterol
Pogostemon cablin	4	128	4	128	≥1024	≥ 1024
Amphotericin B	0.125	0.25	-	-	4	4
Caspofungin	0.03	0.06	0.12	0.12	-	-

4.3. Morphogenesis test

The impact of P. cablin on C. albicans filamentation was analyzed using fetal bovine serum and a temperature of 37°C as inducers. After 1 hour of incubation, the ability to form germ tubes was assessed. In the absence of the phytocomplex, the average rate of germ tube formation among the strains analyzed was 94.99%. The presence of the compound did not result in a significant reduction in this capacity.

After 3 hours of incubation, the Morphology Index (MI) was calculated, since different morphological forms are more evident during this period. In the ATCC strain, a predominance of true hyphae was observed in conditions without P. cablin, with an average MI of 2.61 among the strains evaluated. However, in the presence of the compound, the predominance changed to pseudohyphae, reducing the MI to 2.51.

Strain 97 showed a high filamentous capacity, with an MI of 3.42, characterized by the formation of long, true hyphae, as well as pseudohyphae, while blastoconidia were rare. In the presence of essential oil, the Morphology Index of this strain dropped slightly to 3.38.

4.4. Measuring the hyphal length of Candida albicans

After analyzing the results obtained in the morphogenesis stage in liquid medium (3 hours), it was found that strain 97 had a higher filamentation capacity than the others. In the presence of the substance tested, a reduction in filament length was observed. Strain 97 was chosen for detailed measurements using an optical microscope and NIS-Elements D software (Eclipse Ci, Nikon, Japan).

Under conditions without the presence of P. cablin, the hyphae analyzed had an overall average length of 285.0 micrometers. However, in the presence of the EO studied, this length was significantly reduced (p<0.0001) to 220.5 micrometers.

4.5. Evaluation of the antimicrobian activity of P. Cablin essential oil on the inhibition of fungal biofilm formation

The results of the antimicrobial activity on the inhibition of fungal biofilm formation of P. cablin essential oil and fluconazole are shown in Figure 1.

Figure 1
Inhibitory effect (mean, standard deviation) of Pogostemon cablin essential oil and Fluconazole on biofilm formation of single Candida species. Results are presented as mean ± SD. (One-way ANOVA with Tukey post-test, *p<0.0001).

P. cablin reduced biofilm formation by 26% to 59% between concentrations of 4 and 40 µg/mL, respectively. Fluconazole showed a reduction of 88% to 90% between concentrations 2 and 20 µg/mL. There was a significant difference when comparing the growth control with all the groups tested (p<0.0001).

4.6. Evaluation of the antimicrobial activity of P. cablin essential oil on fungal biofilm reduction

The results of the antimicrobial activity on the reduction of the fungal biofilm of Pogostemon cablin essential oil and fluconazole are shown in Figure 2.

Figure 2
Inhibitory effect (mean, standard deviation) of Pogostemon cablin essential oil and Fluconazole on the biofilm of single Candida species. Results are presented as mean ± SD. (One-way ANOVA with Tukey post-test, *p<0.0001).

P. cablin essential oil showed a reduction of 49% to 68% between concentrations 4 and 40 µg/mL. Fluconazole showed a reduction of 74% to 87% between the respective concentrations of 2 and 20 µg/mL. There was a significant difference when comparing the growth control with all the groups tested (p<0.0001).

5. Discussion

The GC-MS of the patchouli essential oil evaluated in this study identified patchouli alcohol (15.11%), Δ-guaiene (9.85%) and α-guaiene (8.06%), compounds frequently found in P. cablin, and the same was also verified by Verma et al. (2019).

Components such as α-Guaiene and Δ-Guaiene are known for their antimicrobial properties and characteristic fragrances, which are fundamental for applications in the cosmetics and pharmaceutical industries (Jirovetz et al., 2005; Aisyah et al., 2008; Swamy and Sinniah, 2016).

Galovičová et al. (2022) identified patchouli alcohol (31.0%), α-bulnesene (21.3%), α-guaiene (14.3%) and seichelene (6.9%) as the main volatile components of P.cablin essential oil.

Kusuma et al. (2018), in their study, analyzed the essential oil of dried P.cablin leaves and identified patchouli alcohol with 53.68%, α-guaiene 11.26% and azulene 10.75% as the main components. The authors used microwave extraction without the use of a solvent, a method that can alter the composition of the essential oil compared to GC-MS, the technique used to obtain the essential oil analyzed in this study.

As proposed by Sartoratto et al. (2004), the antimicrobial activity of essential oils is classified as strong when they have an MIC of 50-500 µg/mL, moderate for MIC values between 600 and 1500 µg/mL and weak, or inactive product, for MICs above 1500 µg/mL. Webster et al. (2008) proposed a satisfactory MIC value of 1000µg/mL or less. Thus, the results of this study confirm that the essential oil of P. cablin has strong antimicrobial activity, exhibiting high inhibition against C. albicans strains.

The methodology presented by Alves et al. (2021) proposes a classification system to measure the antifungal potential of new compounds with pharmacological action against Candida species. Thus, according to the classification proposed by these authors, P. cablin EO exhibited bioactivity considered very strong and strong against almost all the clinical isolates tested, with MIC values between 2 and 4 µg/mL. Only one clinical strain showed bioactivity considered weak, with a MIC equal to 128 µg/mL.

The EO was effective against 91.6% of the C. albicans strains at concentrations of up to 4 µg/mL, which indicates its high antifungal potency. The fungistatic effect was observed in 5 of the 12 strains tested, indicating that the EO can inhibit growth without necessarily eliminating the microorganism in some strains, requiring higher concentrations to kill the fungal cells. For the other 7, it showed a direct fungicidal effect, so it not only inhibited growth, but also efficiently eradicated the microorganism.

The ability of P. cablin to act in a fungistatic or fungicidal way suggests that its efficacy may depend on factors such as the specificity of the strain, the concentration used and the exposure time, which may be advantageous, since it allows targeted applications, such as topical treatments or in combination with other antifungals to maximize efficacy, prioritizing use in situations where the fungicidal effect is desired.

The activity of this oil was also tested against other strains of the Candida genus. Cavalcante et al. (2019) obtained MIC 50 values equal to 128 µg/mL, which was the lowest concentration capable of inhibiting the growth of half of the C. tropicalis strains tested. Additionally, Alves et al. (2019) showed that P. cablin oil exerts antifungal activity against strains of C. krusei, presenting MIC values equal to 32 µg/mL.

In the assays in this study, the addition of exogenous ergosterol increased the MIC values (≥ 1024 μg/mL), suggesting that the additional ergosterol “sequesters” the active compounds of P. cablin, reducing their availability to interact with fungal membranes. This suggests that the EO acts directly on ergosterol to exert its antifungal effect.

Recently, studies have also investigated the mechanism of action of P. cablin (patchouli) essential oil as an antifungal. Zhang et al. (2024) indicated that its effects are mainly due to the presence of components such as patchouli alcohol (PA). In this study, it was shown that PA inhibited the growth of multiple strains of the Candida genus, including C. albicans, C. glabrata, C. parapslosis, C. krusei and C. tropicalis, with MICs of 64 μg/mL and CFMs of 64 to 128 μg/mL. In addition, confocal microscopy showed that the phytoconstituent inhibited biofilm formation and that the anti-Candida mechanism may be associated with damage to cell membranes and overproduction of ROS (reactive oxygen species).

On the other hand, in their study, Da Cunha et al. (2023) found that the EO of P. cablin had no change in MIC either in the presence or absence of sorbitol and ergosterol. However, in this same study, a synergistic effect was obtained when P. cablin was combined with amphotericin B, an antifungal drug whose mechanism of action has been established in the literature. This interaction is considered promising, especially for the treatment of resistant strains, as it can potentiate the therapeutic efficacy of this drug. Therefore, a more comprehensive investigation using other methodologies is necessary in order to identify the specific targets of action of this essential oil.

The study of biofilm formation is essential for evaluating the antifungal properties of natural compounds. Therefore, it was investigated whether P. cablin essential oil could influence the ability of C. albicans to form biofilms. In the present study, the essential oil tested showed a dose-dependent effect in inhibiting biofilm formation, with the reduction ranging from 26% at a concentration of 4 µg/mL, a value corresponding to the minimum inhibitory concentration (MIC), to 59% at a concentration of 40 µg/mL.

These results suggest that the EO has active compounds capable of interfering in the initial adherence or structural development of the biofilm. Although the inhibition percentages are moderate, the dose-dependent effect indicates the possibility of optimizing the concentrations. Thus, the effect observed reinforces the potential of this oil as an alternative or adjunct in the management of infections associated with Candida biofilms.

In relation to the reduction in mature biofilm, the essential oil showed a significant reduction, concentrations of 4, 8, 16 and 40 µg/mL resulted in progressive reductions in biofilm, ranging from approximately 49% to 68%. These results show the antimicrobial potential of P.cablin EO, which is possibly related to its chemical composition, rich in bioactive compounds.

Galovičová et al. (2022) indicate that P. cablin essential oil has antifungal activity against C. albicans and exhibits significant antibiofilm activity, suggesting its potential use in food preservation due to its antimicrobial properties against the various microorganisms tested and its high antioxidant power.

In addition, Zhang et al. (2024), observed that treatment with patchouli alcohol (PA) can impair the formation of C. albicans biofilms. The analysis showed that increasing the concentration of PA leads to sparser and thinner biofilms. And biofilms formed in the presence of higher concentrations of PA were associated with loss of hyphae.

The ability, to switch between yeast and filamentous hyphae forms is not only directly related to biofilm formation, but also plays a crucial role in the virulence and pathogenesis process of C. albicans (Sudbery, 2011; Noble et al., 2017). To explore the effects of P. cablin on morphogenesis, the transition from yeast to filamentous form was examined.

The results show that P. cablin essential oil has a moderate but relevant impact on the filamentation of C. albicans, especially with regard to changing the predominant morphological forms.

In the initial analysis, after 1 hour of incubation, the presence of the P. cablin compound did not generate a significant reduction in the rate of germ tube formation, indicating that the phytocomplex does not substantially interfere in the initial stages of the morphological transition induced by fetal bovine serum and the temperature of 37°C. This lack of initial effect suggests that the compound may not act directly to inhibit germ tube formation or that its action depends on cumulative factors over time.

After 3 hours, analysis based on the Morphology Index (MI) revealed changes in cell morphology. In the ATCC strain, a predominance of true hyphae was observed in the absence of the compound, with an average MI of 2.61. However, in the presence of P. cablin, the predominance changed to pseudohyphae, resulting in a reduction in MI to 2.51. This change suggests that the phytocomplex exerts a modulating action that favors a less filamentous morphology, which may be related to a reduction in the virulence of C. albicans.

Strain 97, known for its high capacity to form true hyphae (MI of 3.42), showed only a slight reduction in MI to 3.38 in the presence of the essential oil. This subtle difference suggests that, for strains with a high propensity for filamentation, the impact of P. cablin is less expressive, which may be related to genetic differences or to the strain's intrinsic robustness in the morphological transition.

In the agar-spider medium, patchouli alcohol (PA) played a significant role in inhibiting the transition from yeast to hyphae in C. albicans, the colonies showed smoother edges and fewer hyphae in the presence of PA, indicating that the compound directly affects the ability of C. albicans to form invasive structures Zhang et al. (2024).

6. Conclusion

The essential oil of P. cablin exhibited a complex chemical composition and significant activity against C. albicans, with this activity being considered fungicidal for most of the tested strains. It is suggested that this antifungal action occurs through the complexation of the essential oil with ergosterol in the fungal plasma membrane. Additionally, P. cablin acted on biofilm formation and reduction and also influenced the morphogenesis of C. albicans. These results highlight its therapeutic potential as a natural antifungal agent. However, further studies are necessary to elucidate its mechanism of action. Thus, more investigations should be conducted to evaluate its in vivo applicability, focusing on analyzing its cytotoxicity and identifying its therapeutic targets. These future studies will contribute to validating its potential use as a therapeutic alternative, aiding in the treatment of clinical manifestations of candidiasis.

Data Availability Statement

The entire data set that supports the results of this study was published in the article itself.

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Edited by

• Editor:
  Ana Paula Peron

Publication Dates

• Publication in this collection
  09 Feb 2026
• Date of issue
  2025

History

• Received
  09 Jan 2025
• Accepted
  15 Oct 2025