In Vitro antibiofilm activity of Diplopterys pubipetala against Candida spp

Infections caused by Candida spp. Represent a significant clinical challenge, particularly due to biofilm formation associated with resistance to conventional antifungal agents. The biological diversity of the Brazilian Cerrado is a promising source of novel bioactive molecules, and the liana Diplopterys pubipetala (Malpighiaceae), native to this biome, has shown antifungal activity against planktonic cells; however, its effects on biofilms have not yet been investigated. In this study, the antibiofilm activity of the crude extract and leaf partitions of D. pubipetala was evaluated against Candida albicans, Candida tropicalis, and Candida glabrata. Biofilm minimum inhibitory concentration (MICB 50) and 50% inhibitory concentration (IC 50) were determined using biomass (crystal violet) and cell viability (MTT) assays. Amphotericin B was used as a positive control. Against C. albicans, the dichloromethane partition showed MICB 50 of 250 μg mL -1 and IC 50 of 179 μg mL -1, while the hexane partition exhibited the lowest IC 50 (69.38 μg mL -1). For C. tropicalis, the hexane and dichloromethane partitions showed MICB 50 of 500 μg mL -1 and IC 50 values of 279.2 and 337.9 μg mL -1, respectively. Overall, C. glabrata showed higher tolerance. Amphotericin B maintained high activity. Antibiofilm activity was more pronounced in the less polar partitions, particularly against C. albicans and C. tropicalis. These results suggest that less polar metabolites present in these partitions may be involved in the observed activity; however, further studies are needed for compound isolation and confirmation.

Investigations on plants from the Brazilian Cerrado have described secondary metabolites and their bioactive properties, including antioxidant, anti-inflammatory, and antimicrobial activities (Souza et al., 2012; Cruz et al., 2021; Monteiro-Alfredo et al., 2023). In this context, the liana Diplopterys pubipetala (Malpighiaceae) stands out due to its association with these activities, particularly its reported antifungal activity of the crude extract and leaf partitions against some Candida species (Sacramento et al., 2025), which is consistent with one of the major challenges faced by the scientific community, antifungal resistance.

Previous phytochemical studies conducted with D. pubipetala revealed a diverse chemical composition. UHPLC-MS/MS analyses enabled the annotation of phenolic compounds, flavonoids, and proanthocyanidins, including catechin/epicatechin, procyanidins, and flavonoid derivatives, while gas chromatography coupled to mass spectrometry analyses highlighted less polar metabolites, such as fatty acid esters, carboxylic acids, hydrocarbons, and volatile phenolic compounds (Sacramento et al., 2025). Considering that several of these chemical classes have documented antimicrobial and antifungal activity in the literature, such constituents may contribute to the biological properties previously reported for the species.

Fungi of the genus Candida have strong ability to cause disease in humans, which is intensified by their capacity to form biofilms (Assis et al., 2022; Rodrigues et al., 2017). Biofilms are characterized by a matrix containing a variety of macromolecules, including proteins, carbohydrates, lipids, and DNA (Al-Fattani and Douglas, 2006; Nett and Andes, 2020). This polymeric matrix can be as diverse as the microorganisms themselves (Mitchell et al., 2016) and acts as a protective barrier, hindering the action of antifungal agents, immune system cells, and even environmental stresses (Al-Fattani and Douglas, 2006; Lara et al., 2018).

As approximately 80% of microorganisms exist in biofilm form, it is essential to understand how they form, function, and resist antimicrobial agents. In this context, the use of natural extracts as a source for the development of new drugs represents an alternative in the control of microorganisms (Assis et al., 2022).

Although the antifungal activity of Diplopterys pubipetala against planktonic cells of Candida species has been previously reported (Sacramento et al., 2025), its effects on fungal biofilms remain unknown. This gap is particularly relevant because cells organized in biofilms exhibit distinct physiological and phenotypic characteristics compared to planktonic cells, including increased tolerance to antifungal agents and additional protective mechanisms conferred by the extracellular matrix. Thus, the evaluation of the antibiofilm activity of D. pubipetala represents a relevant approach to expand knowledge on the biological potential of this species against one of the main survival and persistence strategies of pathogenic fungi.

In this study, the susceptibility profile of Candida albicans, Candida tropicalis, and Candida glabrata was evaluated against the crude extract and leaf fractions of D. pubipetala. The hexane, dichloromethane, ethyl acetate, and methanolic fractions were obtained through fractionation based on differences in polarity, allowing the enrichment of distinct classes of secondary metabolites. In addition to determining the minimum inhibitory concentration, the ability of these samples to inhibit biofilm formation was also assessed.

Plant material and preparation

Leaves of D. pubipetala were collected in December 2021 in Nova Esperança district, Montes Claros, Minas Gerais, Brazil (16°36′07.0″S; 43°55′12.7″W). The species was botanically identified, and a voucher specimen was deposited at the Montes Claros Herbarium (No. 4033). The study was registered in the Brazilian National System for the Management of Genetic Heritage (SisGen) under number A822A14.

Leaf material was washed, dried in a forced-air oven at 35 °C–40 °C for 7 days, and ground in a Wiley mill, yielding 800 g of powder stored at 4 °C until use.

The crude extract was obtained by successive maceration using a hydroethanolic solution (70:30, v/v) at a ratio of 250 g. L -1 for 7 days at room temperature with periodic agitation. After filtration, the solvent was removed by evaporation, and three additional extraction cycles were performed, yielding a final yield of 7.75% (w/w). All extractions were performed under the same conditions to ensure methodological reproducibility.

For fractionation, 18.85 g of crude extract was resuspended in hydroethanolic solution (70:30, v/v) and subjected to sequential liquid–liquid partitioning with hexane, dichloromethane, ethyl acetate, and methanol (2 × 500 mL each). Fractions were dried at 38 °C under forced air and stored at −10 °C. The yields of the hexane, dichloromethane, ethyl acetate, and methanol fractions were 6.2%, 13.0%, 15.3%, and 12.14%, respectively. All samples were stored at −10 °C until analysis. The procedures were described by Sacramento et al. (2025).

Yeast strains used in the assay

The yeast strains used were obtained from the American Type Culture Collection (ATCC): Candida albicans ATCC 90028, C. tropicalis ATCC 13803 and C. glabrata ATCC 2001. All of them were maintained in cryotubes under cryopreservation at −20 °C in RPMI-1640 broth (Thermo Fisher Scientific, Waltham, Massachusetts, EUA) buffered with MOPS (Sigma–Aldrich, St. Louis, MO, United States), containing glycerol at 20% (v/v), as part of the culture collection of the Laboratory of Antimicrobial Testing (LEA) of the Federal University of Uberlândia (UFU).

For yeast reactivation, the thawed contents of the cryotubes were transferred partly to Sabouraud agar and partly to RPMI broth to ensure growth. From Sabouraud agar, the yeast cells were subcultured for three consecutive days prior the experiments to re-establish cell metabolism and to confirm pure colonies.

Minimum inhibitory concentration of biofilm (MICB 50) and inhibitory concentration (IC 50)

The inhibition of biofilm formation was evaluated based on the quantification of biomass and cell viability through the microdilution broth methodology recommended by the Clinical and Laboratory Standards Institute, adapted for the assay (Clinical and Laboratory Standards Institute CLSI, 2017). For this, two 96-well microplates (Nest, Wuxi, Jiangsu, China) were prepared separately in the same way (one for biomass and one for cell viability) with RPMI (Thermo Fisher Scientific) supplemented with 2% glucose. Samples were solubilized in 5% of dimethylsulfoxide—DMSO (Sigma–Aldrich) and diluted in RPMI to vary from 0.98 μg mL -1 –2000 μg mL -1 in the well and amphotericin B (Sigma–Aldrich) was used as positive control to vary from 0.0156 μg mL -1 –8 μg mL -1. The inoculum was standardized on the McFarland 0.5 scale (1 × 10 6 cells mL -1 for yeast) checked in a densitometer—Densimat (Biomérieux, Grassina, Florence, Italy), when added to the microplate, achieves the final concentration of 0.5 × 10 6 cells mL -1 in the well, without serial two-fold dilutions. Microplates were statically incubated at 37 °C for 24 h, without media replacement. After incubation, the supernatant from each well was gently aspirated and washed once with autoclaved distilled water also by pipetting and aspiration to remove cells that did not adhere to the bottom of the well.

On the plate prepared for biomass analysis, the biofilm adhered to the bottom was fixed with 200 µL of methanol (Sigma-Aldrich) for 15 min and stained with 200 µL of 1% crystal violet (Sigma-Aldrich) solution for 20 min. The microplate was washed and the adhered crystals were solubilized with 200 µL of 33% acetic acid (Sigma-Aldrich). The absorbance of the biomass was measured at a wavelength of 590 nm and the percentage of inhibition was calculated by: 1 – (A 590 of the test/A 590 of non-treated control) × 100 (Wei et al., 2006). The MICB 50 was considered the lowest concentration able to inhibit 50% or more of biofilm formation according to the calculated percentage.

The second plate prepared for cell viability analysis, 0.5 mg mL-1 of [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] tetrazolium–MTT solution (Sigma-Aldrich) was added to each well and incubated again at 37 °C for 6 h. After incubation, MTT (Sigma-Aldrich) was aspirated and the formazan product was solubilized in DMSO (Sigma–Aldrich). The absorbance resulting from cell viability was measured at a wavelength of 490 nm and the data were calculated by nonlinear regression without confidence intervals using the software GraphPad Prism version 8.0.2 to obtain the inhibitory concentration values for 50% (IC 50) (Oliveira et al., 2019). All assays were performed in triplicate.

Statistical analysis

The values obtained for biomass and cell viability were analyzed separately, but in the same way using GraphPad Prism version 8.0.2. First, they were analyzed for homogeneity by the Shapiro–Wilk test. The data did not follow a normal distribution and the mean values were statistically analyzed by Kruskal–Wallis, using P < 0.05. Subsequently, the concentrations with statistically different means were compared with the inoculum control by Dunn’s multiple comparisons test using P ≤ 0.05 identified with an asterisk () or P ≤ 0.01 identified with two asterisks (*).

The antibiofilm activity results of D. pubipetala samples and amphotericin are presented in Table 1.

In the evaluation of antibiofilm activity against C. albicans, the dichloromethane partition showed the highest efficacy, with an MICB 50 of 250 μg mL -1 and an IC 50 of 179 μg mL -1, indicating a concomitant effect on both biomass and cell viability. The hexane partition exhibited the lowest IC 50 (69.38 μg mL -1), although biomass reduction was only observed at a higher concentration (MICB 50 of 1,000 μg mL -1). Similarly, the crude extract also presented an MICB 50 of 1,000 μg mL -1, whereas the methanol and ethyl acetate partitions showed MICB 50 values of 2,000 μg mL -1, differing in their IC 50 values of 357.6, 684, and 436.7 μg mL -1, respectively (Figure 1).

The highest MICB 50 and IC 50 values among the tested antibiofilm activities were observed for C. glabrata. For the crude extract and the methanol and dichloromethane partitions, the MICB 50 values were higher than 2000 μg mL -1, while for the other two partitions they were equal to 2000 μg mL -1. The IC 50 values for the crude extract and the dichloromethane partition were also higher than 2000 μg mL -1, whereas the methanol partition showed an IC 50 of 1847 μg mL -1. The lowest IC 50 values were 1,326 and 1,026 μg mL -1 for the ethyl acetate and hexane partitions, respectively (Figure 2).

For C. tropicalis, the hexane and dichloromethane partitions were the most promising, both presenting an MICB 50 of 500 μg mL -1 and IC 50 values of 279.2 and 337.9 μg mL -1, respectively. The methanol partition also showed an MICB 50 of 500 μg mL -1, but with a higher IC 50 of 492.1 μg mL -1. The crude extract exhibited an MICB 50 of 1,000 μg mL -1 and an IC 50 of 515.2 μg mL -1. Finally, the ethyl acetate partition showed the highest MICB 50 (2,000 μg mL -1) and an IC 50 of 992.2 μg mL -1 (Figure 3).

The results obtained using amphotericin B as a positive control showed MICB 50 values ranging from 0.062 to 0.25 μg mL -1 and IC 50 values between 0.053 and 0.177 μg mL -1 for the tested Candida species.

Among the leading causes of morbidity and mortality are those associated with infections caused by Candida species, which present a wide range of clinical manifestations, varying from superficial and mucosal infections to highly disseminated systemic infections (Rodrigues et al., 2017; Sushmitha et al., 2023; Arendrup and Patterson, 2017). Over the past 3 decades, fungal biofilm formation has become an increasingly challenging issue due to its impact on treatment outcomes, which frequently fail (Andrade et al., 2020; Iñigo et al., 2012), as well as the growing incidence of nosocomial fungal infections, particularly affecting immunocompromised patients. These infections are often associated with chronic diseases and the use of medical devices, which facilitate fungal cell invasion into tissues and subsequent dissemination through the bloodstream (Serafini et al., 2021).

The results obtained in this study demonstrate relevant antibiofilm activity when compared with plant-derived agents previously reported against Candida ssp. A recent review (Dantas et al., 2025) highlights that bioactive plant compounds exhibit a wide range of antifungal and antibiofilm activities, with MICB 50 and IC 50 values frequently ranging from tens to hundreds of μg mL -1, depending on chemical composition, extract polarity, and the experimental methodology employed. In addition, other review reinforces that plant extracts rich in secondary metabolites may exhibit significant activity against Candida spp. biofilms (Adewole et al., 2026), with efficacy comparable to that observed in the most active fractions evaluated in the present study. Furthermore, evidence specific to the Malpighiaceae indicates that species within this family possess broad antimicrobial potential, including relevant antifungal and antibiofilm activities, thereby supporting their role as a promising source of bioactive compounds (Villanueva-Amador et al., 2020). From a mechanistic perspective, low-polarity compounds such as terpenes and other lipophilic metabolites are frequently associated with fungal plasma membrane disruption, increased cell permeability, and interference with the extracellular matrix of biofilms (Xu et al., 2023). These compounds may also interfere with the initial adhesion and maturation stages of biofilm development, as well as modulate quorum sensing systems and the expression of virulence factors in Candida spp. (Dantas et al., 2025; Adewole et al., 2026), providing a plausible explanation for the observed activity in the less polar fractions evaluated in this study.

The results obtained for the hexane and dichloromethane fractions obtained from D. pubipetala leaves were the most effective against C. albicans and C. tropicalis, whereas C. glabrata exhibited greater tolerance. In assays with C. albicans (hexane fraction), the low IC 50 value indicated high cytotoxic activity, although biomass reduction occurred at a higher concentration. In contrast, the proximity between MICB 50 and IC 50 values in the dichloromethane fraction suggests a simultaneous effect on both biofilm biomass and cellular viability. It is important to note that the crystal violet assay quantifies total biomass, including both living and dead cells, which may lead to an overestimation of biofilm mass. In addition, the MTT assay may underestimate cell viability, since the biofilm matrix can hinder reagent penetration and limit access to deeper cells.

Previous phytochemical studies of D. pubipetala identified low-polarity compounds in these fractions, including nonanoic acid, 2,4-di-tert-butylphenol and different fatty acid esters (Sacramento et al., 2025). These compounds have been reported in the literature as possessing antimicrobial and antifungal activities and may contribute, individually or synergistically, to the effects observed in the present study. However, the compounds responsible for the observed activity were not identified in this study, and the underlying mechanisms were not evaluated. Therefore, any associations between the metabolites present in the fractions and the observed antibiofilm effects should be considered preliminary and require confirmation through further chemical and biological studies.

Studies on the composition of the C. albicans matrix indicate the presence of lipids, including glycerolipids and sphingolipids, which exhibit both neutral and polar characteristics (Mitchell et al., 2016), supporting the possibility of enhanced antifungal effects. The diverse composition of both the biofilm matrix and the crude extract likely accounts for the observed synergistic action, as evidenced by the relatively low IC 50 and MICB 50 values among the species investigated.

C. albicans and C. glabrata are considered the main pathogenic species of the genus Candida associated with human infections (Rodrigues et al., 2017). The effects of the crude extract and fractions of D. pubipetala have previously been evaluated against planktonic cells of C. glabrata, yielding promising results (Sacramento et al., 2025). However, the comparatively high MICB 50 and IC 50 values observed here suggest a marked resistance of biofilms to the tested samples.

This behavior may be associated with heteroresistance, as C. glabrata is characterized by the presence of subpopulations within the same strain capable of growing even in the presence of antifungal agents (Berman and Krysan, 2020). This phenomenon is linked to the overexpression of efflux pumps and/or genetic mutations related to ergosterol biosynthesis, which reduce cellular permeability (Whaley and Rogers, 2016). Additionally, C. glabrata is particularly adapted for adhesion and survival without requiring pseudohyphae formation. Combined with the production of higher levels of mannose residues and adhesin-like proteins, this results in dense biofilms with complex extracellular matrices, making them less susceptible to conventional antifungal agents and natural extracts (Andrade et al., 2020).

For C. tropicalis, the hexane, dichloromethane, and methanol fractions demonstrated promising antibiofilm activity, as indicated by the proximity between MICB 50 and IC 50 values. Although these values are around 500 μg mL -1, they may suggest a positive correlation between inhibition of total biofilm growth and reduction in metabolic viability.

Given the lack of consensus in the literature regarding the classification of antibiofilm activity, the results obtained with the hexane and dichloromethane fractions against C. albicans can be considered the most promising, followed by those observed for C. tropicalis with the same fractions.

Antifungal resistance is understood as a multifactorial process involving the upregulation of efflux pumps, alterations in the cell wall, increased cell density and intercellular communication, the presence of an extracellular matrix, metabolic changes, the existence of persister cells, as well as signaling mechanisms and stress responses (Rodrigues et al., 2017). Azoles have been described as agents with low activity against Candida spp. Biofilms (Wei et al., 2006; Brilhante et al., 2015; Villanueva-Amador et al., 2020), particularly fluconazole in the treatment of infections (Arendrup and Patterson, 2017). As expected, the control substance amphotericin B used in this study maintained high activity, which can be attributed to its ability to bind to ergosterol in the fungal cell membrane and form pores, leading to leakage of intracellular components and cell death. This mechanism involves a complex biochemical pathway that may also include genetic mutations (Arendrup and Patterson, 2017; Chandra and Mukherjee, 2015).

Considering all the results obtained, it can be inferred that although Candida strains appear highly susceptible to the crude extract and fractions when tested in planktonic cells (Sacramento et al., 2025), the substantially higher MICB 50 and IC 50 values indicate significant resistance in the biofilms evaluated.

Products rich in secondary metabolites remain an important source of bioactive compounds and continue to be investigated for their potential role in addressing challenges associated with microbial resistance. Fungal biofilm formation represents a major challenge due to its impact on therapeutic outcomes and its frequent association with treatment failure, highlighting the need for new strategies for biofilm control.

In the present study, fractions from the leaves of D. pubipetala exhibited antibiofilm activity against Candida albicans and Candida tropicalis, with emphasis on the hexane and dichloromethane fractions, which showed the most pronounced effects (MICB 50 ranging from 250–500 μg mL -1 and IC 50 values as low as 69.38 μg mL -1 for C. albicans). These results suggest the presence of bioactive constituents associated with the observed activity. However, the compounds responsible were not identified, and no toxicity assessment was performed. Thus, further studies are required, including chemical characterization of the active fractions, isolation of bioactive metabolites, evaluation of toxicity profiles, as well as mechanistic investigations and assessment of potential synergistic interactions with conventional antifungal agents.