Potent and broad-spectrum anti-<i>Candida</i> activity of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one, a triterpenoid from <i>Paullinia pinnata</i>

Figures Abstract Vulvovaginal candidiasis is a common infection that affects women of reproductive age, with a high prevalence among pregnant women. When left untreated, it can lead to pregnancy complications including miscarriage. The study evaluated the activity of the ethyl acetate (EtOAc) extract of Paullinia pinnata and its isolated compound 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one against drug-resistant strains and clinical isolates of Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis and Candida tropicalis. Furthermore, the effects of the combinations of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one with voriconazole, nystatin or caspofungin on the Candida strains and isolates as well as its antibiofilm activity against the drug-resistant strains are reported in this study. The EtOAc leaves extract of P. pinnata demonstrated considerable activity against the drug-resistant Candida strains and clinical isolates with minimum inhibitory concentrations (MICs) of 31.25 to 500 µg/mL when assessed for anti-Candida activity using the microbroth dilution method. The extract was column fractionated to obtain five bulk fractions, and from the most active bulk fraction, BF4 (MIC = 3.91–31.25 µg/mL), 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one was isolated. This study is the first reported biological activity of the compound. 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one exhibited strong activity against the panel of drug-resistant Candida species with MIC ranging from 0.5 to 16 µg/mL (0.85–27.10 µM). This activity was within similar range to that of ketoconazole (MIC = 1–8 µg/mL) and amphotericin B (MIC = 0.5–2 µg/mL). When combined with voriconazole, nystatin or caspofungin using the checkerboard assay, 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one recorded synergism of 33.33% to 83.33% with the antifungals, with majority of the synergistic interactions observed against C. albicans and C. glabrata strains and isolates. 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one also demonstrated anti-biofilm activity. Its IC50 values against the formation of biofilms and preformed biofilms of the Candida species were 15.30–46.40 μg/mL (25.91–78.60 μM) and 25.40–90.84 μg/mL (43.02–153.86 μM), respectively. In conclusion, P. pinnata possesses strong anti-Candida activity and its isolated compound, 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one displayed potent anti-Candida and anti-biofilm action. These findings identify 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one as a promising anti-Candida compound warranting further investigation, including cytotoxicity in mammalian cells, before its therapeutic potential can be fully assessed. Citation: Harley BK, Ben IO, Asamoah TD, Amengor CDK, Kortei NK, Eghan P, et al. (2026) Potent and broad-spectrum anti-Candida activity of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one, a triterpenoid from Paullinia pinnata. PLoS One 21(6): e0350399. https://doi.org/10.1371/journal.pone.0350399 Editor: Vartika Srivastava, Cleveland Clinic Lerner Research Institute, UNITED STATES OF AMERICA Received: November 27, 2025; Accepted: May 11, 2026; Published: June 1, 2026 Copyright: © 2026 Harley et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting Information files. Funding: The author(s) received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. 1 Introduction Vulvovaginal candidiasis (VVC) is a common infection among women of child-bearing age caused by Candida species, particularly Candida albicans [1]. This fungal infection is depicted by vulvovaginal discharge and other inflammatory signs like itch and redness detected in the vulva and vaginal mucosa caused by an overgrowth of Candida species which usually are present as dormant vaginal commensals [2]. An estimated 70–75% of women get an episode of vulvovaginal candidiasis at least once throughout their life [3], with half of these women experiencing a second episode and 7–10% having recurrent infection [4]. Pregnant women are highly vulnerable to vulvovaginal candidiasis due to the physiological changes that occur during pregnancy such as elevated hormonal levels and increased vaginal glycogen secretion with corresponding acidification of medium that promote Candida proliferation [5]. Candida albicans remains the main aetiological specie of vulvovaginal candidiasis (85–95% of cases). However, increasing prevalence of non-albicans Candida (NAC) species including Candida glabrata, Candida krusei, Candida parapsilosis and Candida tropicalis have emerged globally [6–8]. This shift from the predominance of C. albicans in vulvovaginal candidiasis to NAC is concerning as these species often demonstrate reduce sensitivity to azole antifungal drugs [9–11]. This increase in drug resistance and limited new antifungals in the development pipeline necessitates the pressing need for the discovery of new antifungal drugs to tackle the expanding scope of Candida species. Paullinia pinnata L. (Sapindaceae) commonly referred to as ‘Toa-ntini’ in Ghana (Akan dialect) is a woody climber used widely in traditional medicine for the treatment of several diseases [12]. Across the African continent, preparations from the leaves and roots are administered for the treatment of gonorrhoea, threatened abortion, mental disorders, erectile dysfunction, skin ulcers, wounds and microbial infections [13–16]. The plant has been shown to possess diverse pharmacological activities including antibacterial [13,17,18], neuropharmacological [19,20], anthelminthic [21,22] anti-inflammatory [23,24] and wound healing [25] effects. Several bioactive triterpenoids [18,26–28] and polyphenolic [13,28–30] compounds have also been isolated from it. The present study investigated the antifungal activity of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one (Fig 1) isolated from the leaves of P. pinnata against Candida pathogens noted to cause vulvovaginal candidiasis. 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one is a lupene-type triterpenoid previously isolated from the roots of the plant but with no reported biological activity [31]. We investigated 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one for growth inhibition against drug-resistant strains and clinical isolates of C. albicans, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis and assessed its antibiofilm activity against the reference strains. Furthermore, we evaluated combinations of the compound with conventional antifungal drugs. 2 Materials and methods 2.1 Reagents and chemicals Chloramphenicol, miconazole, ketoconazole, clotrimazole, amphotericin B, voriconazole, sabouraud Dextrose Agar (SDA), XTT assay reagent, Mueller-Hinton (MH) agar and RPMI 1640 medium were obtained from Thermo Fisher (Oxoid Limited, Hampshire, UK) whereas fluconazole came from Pfizer (Pfizer Inc., New York, NY, United States) and caspofungin from Sigma Aldrich (Sigma Chemical Co., St. Louis, MO, United States). Organic solvents employed in the study were of analytical grade and were bought from BDH laboratory supplies (Merck Ltd, Lutterworth, UK). 2.2 General procedures Normal phase silica gel 60 (70–230 mesh; AppliChem, GmbH, Darmstadt, Germany) or Sephadex LH-20 (25–100 μm; Amersham Biosciences) were used as stationary phases in column chromatography (CC). Pre-coated silica gel 60 plates (0.25 mm thickness) incorporated with fluorescent indicator GF254 were employed in Thin Layer Chromatography (TLC). 1D (1H, 13C and DEPT135) and 2D (COSY, HSQC and HMBC) NMR spectra were recorded at 25 °C on a Bruker Avance-500 (500 MHz, Bruker BioSpin GmbH, Rheinstetten, Germany), the magnetic field strength (11.7 T). Sample solvent (CDCl₃). Chemical shifts (δ) were expressed in parts per million (ppm) using tetramethylsilane (TMS, δ 0.00 ppm) as internal standard and coupling constants (J) were measured in Hertz (Hz). 2.3 Plant material collection and processing The leaves of Paullinia pinnata were harvested from Sokode, Volta Region in September 2023 with permission of the landowner and identified by Mr. Alfred Agyemang, a medical herbalist at the Institute of Traditional and Alternative Medicine, University of Health and Allied Sciences (UHAS). Voucher specimen was then deposited at the herbarium facility of the of the Department of Pharmacognosy and Herbal Medicine of the same university (Voucher specimen numbers: UHAS/PCOG/2023/L005). The collected leaves were cleaned thoroughly under running water, chopped into pieces and oven dried at 45 °C for 60 hours before grinding into coarse powder. 2.4 Extraction and isolation of compound The powdered leaves of P. pinnata (950 g) were extracted by maceration with ethyl acetate (EtOAc, 2.5 L) (3 x 3 days) at room temperature with occasional agitation. Preliminary screening against the Candida reference strains was carried out with petroleum ether, ethyl acetate and methanol extracts. The results (data not shown) confirmed greater anti-Candida activity in the ethyl acetate extract compared to petroleum ether and methanol extracts The extract obtained was evaporated to dryness on a rotatory evaporator at 40 °C [32]. The weight of the extracts obtained was 14.05 g (Yield: 1.48%). A part of the EtOAc extract (12 g) was column chromatographed on silica gel eluting with dichloromethane – methanol (CH2Cl2: MeOH [v/v, from 90:1–1:1]). TLC profiling of the eluates (100 mL) enabled the bulking of five (5) fractions: BF1 (3.5 g), BF2 (0.44 g), BF3 (0.9 g), BF4 (2.08 g) and F5 (1.2 g). Bulked fraction F4 was subjected to column chromatography on silica gel with petroleum ether – ethyl acetate [Pet ether-EtOAc – 3:7–0:1] and further purification on Sephadex LH-20 with CH2Cl2: MeOH (1:1) to yield an amorphous powder (1.15 g, 0.12%) [33]. 2.5 Antifungal assay 2.5.1 Fungal strains and growth conditions. The reference Candida strains C. albicans ATCC 10231, C. glabrata ATCC 2001, C. krusei ATCC 6258, C. parapsilosis ATCC 22019 and C. tropicalis NRRL Y-12968 were purchased from American Type Culture Collection (ATCC®, Manassas, VA, USA). Forty (40) clinical isolates of Candida species were obtained from the Microbiology Laboratory unit of the Ho Teaching Hospital, Ghana. Fungal colonies of the isolates were sub-cultured separately on SDA augmented with chloramphenicol (50 μg/mL) at 37 °C for 48 h to obtain pure Candida isolates. Chloramphenicol was added to SDA to suppress bacterial contamination. The pure isolates were then cultured on HiCrome Candida Differential Agar (HiMedia Laboratories, India) for 2 days at 35 °C to produce species-specific colours. The colonies produced were identified by colour, appearance and shape (Table 1). The confirmation of the species of the Candida isolates were performed using API ID 32C strips (Biomerieux, France) according to standard microbiological methods [34]. The Candida strains and isolates were stored in glycerol stocks in −80 °C freezer. Prior to each experiment, Candida strains and isolates were retrieved from −80 °C glycerol stocks and sub-cultured on Sabouraud Dextrose Agar (SDA) at 35 °C for 48 h in a humidified chamber before use. 2.5.2 Antifungal susceptibility testing. The sensitivity of the Candida isolates to Fluconazole (25 μg), voriconazole (1 µg), miconazole (10 µg), clotrimazole (10 μg), amphotericin B (10 µg) and nystatin (100 units) was evaluated using the disc diffusion method on Mueller-Hinton (MH) agar with slight modifications [35]. Briefly, 5 mL test tubes containing 0.85% sterile saline solution were inoculated with distinct colonies of Candida isolates from the SDA plates. They were then emulsified to form a suspension of turbidity equivalent to 0.5 McFarland standard which compared well to 0.5 McF PhoenixSpec Calibrator (Becton, Dickinson and Company, USA). Thereafter, lawns of the media were seeded in three dimensions using sterile swabs dipped in the prepared inoculum. Fluconazole (25 μg), voriconazole (1 µg), miconazole (10 µg), clotrimazole (10 μg), amphotericin B (10 µg) and nystatin (100 units) loaded disks were then aseptically placed on the media lawns and incubated at 37 °C for 48 h. The zone diameters of antifungal disks were measured using a ruler. The zone diameters were interpreted according to Table 2 below [36]. 2.5.3 In vitro antifungal activity of Paullinia pinnata leaves extract, bulked fractions and compound. The antifungal activity of the ethyl acetate extract of P. pinnata leaves, the bulked fractions from column chromatography and the isolated compound were evaluated using the broth microdilution technique described in document M27 - A3 by the Clinical and Laboratory Standards Institute (CLSI) (Clinical and Laboratory Standards Institute, 2008) with slight modifications [38]. Briefly, test samples were dissolved in 10% dimethyl-sulfoxide (DMSO)/RPMI-1640 medium and a series of two-fold dilutions was carried out in 96-well microplates. Fungal inoculum (Candida reference or clinical isolates) was prepared by suspending distinct colonies from SDA plates into 0.85% sterile saline solution and adjusting the turbidity to a 0.5 McFarland standard, verified against a 0.5 McF PhoenixSpec Calibrator (Becton, Dickinson and Company, USA). The inoculum was then further diluted in RPMI-1640 medium to a final working concentration of 2 × 106 CFU/mL. 50 µL of inoculums of the Candida species standardized to 2 × 106 CFU/mL in RPMI-1640 medium were added to wells of new 96-well microplates. The wells were then treated with 50 µL of extracts/fractions (2000 µg/mL – 1.95 µg/mL), isolated compound (16 µg/mL – 0.125 µg/mL) positive controls (64 µg/mL – 0.125 µg/mL), or an equivalent volume of the DMSO vehicle (1%). The plates were then covered with a sterile plate sealer, gently shaken, and incubated at 37 °C for 72 hrs. The activity, measured in terms of minimum inhibitory concentration (MIC), was determined visually and spectrophotometrically at 490 nm using a microplate reader (Benchmark Microplate reader; Bio-Rad, CA, United States). Positive controls were voriconazole, ketoconazole and amphotericin B. The negative controls were untreated culture and DMSO vehicle. A media blank was also used to monitor for media contamination. The experiment was carried out in duplicate (two biological replicates) with three technical replicates per treatment and on different days. The ethyl acetate extract and bulked fractions were considered active when MIC was < 100 µg/mL, moderately active when MIC ranged from 100 to 500 µg/mL, and weakly active when MIC was from 501 to 1000 µg/mL. Above 1000 µg/mL it was deemed inactive [39]. The antifungal activity of the natural product was interpreted as follows: very strong bioactivity < 3.515 μg/mL; strong bioactivity 3.515–25 μg/mL; moderate bioactivity 26–100 μg/mL; weak bioactivity 101–500 μg/mL; very weak bioactivity 500–2000 μg/mL; and no activity above 2000 μg/mL [40]. To evaluate the fungicidal activity, aliquots were taken from the well that corresponded to the MIC and three consecutive wells (two higher and one lower). These were inoculated onto SDA plates and incubated at 37 °C for 24 h. The plates were then analysed for the presence or absence of growth [41]. 2.6 Checkerboard assay for antifungal combination effects The effect of the combinations of the compound with voriconazole, nystatin and caspofungin were evaluated using the checkerboard technique from EUCAST-AFST guidelines reference [42] with minor modifications. Serial dilutions (two-fold) of samples (compound and antifungal drugs) were prepared in RPMI 1640 medium in 96-well microtiter plate. 50 μL from each dilution of the extracts were transferred into new 96-well microtiter plates in the vertical direction and same quantities of antifungal drugs were added in horizontal direction to obtain various combinations of the compound with the antifungal drugs. Then, 100 μL of inoculum suspension (0.5 McFarland) of either Candida reference strain was added to each well followed by incubation at 37°C for 48 h. MIC values were determined spectrophotometrically at 490 nm using a microplate reader (Benchmark Microplate reader; Bio-Rad, CA, United States). Each experiment was carried out in triplicate (three biological replicates) with three technical replicates per treatment and on different days. The results were analyzed by determining the Fraction Inhibitory Concentration Indices (FICI) calculated as follows: FICI = FICA + FICB, where FICA = (MICCA/ MICA) and FICB = (MICCB/ MICB). MICA and MICB are the Minimum Inhibitory Concentrations (MIC) of drugs A and B alone; and MICCA and MICCB are the concentration of drugs A and B at the iso-effective combinations. The FIC Indices were interpreted as follows: Synergism: FICI ≤ 0.5, Indifference: > 0.5–4.0 and Antagonism: > 4.0 [43]. 2.7 Antibiofilm action of the isolated compound against the Candida strains and isolates The effects of the isolated compound on the biofilms of the Candida species were evaluated under two conditions using the fungal biofilm formation and susceptibility testing methods: inhibition of biofilm formation and disruption of mature biofilms. 2.7.1 Biofilm formation inhibitory assay. The inhibitory effects of the compound on the formation of biofilms by the reference Candida strains were determined using a previously described method [44]. In brief, 50 µL of RPMI 1640 was added to each well in a flat-bottom 96-well microplate together with 50 µL of the compound in column 1. Serial dilutions were carried out till 10 to obtain concentrations from 512–2 µg/mL. Then, the wells of the plates from columns 1–11 were inoculated with 50 µL of 2 × 106 cells/mL fungal suspensions. The plates were then incubated for 24 h at 37 °C. After incubation, the media in each well was carefully removed and the plates washed thrice with 100 µL PBS to remove remnant planktonic cells. Afterwards, 100 µL of XTT/menadione solution was added to each well and the plates incubated at 37oC for 2 h in the dark. 80 µL of the resulting-coloured supernatant from each well was then transferred into new microplates for reading at 490 nm using a microplate reader. 2.7.2 Disruption of mature biofilms assay. The inhibitory effect of the compound on mature biofilms of the reference Candida strains was determined as follows: 100 µL of fungal suspensions of 1 × 106 cells/mL in RPMI 1640 of each reference Candida strain and the clinical isolates were added to wells of flat-bottom 96-well plates and incubated at 37 °C for 24 h to allow for the formation of biofilms. The media from the wells were aspirated carefully so not to touch the preformed biofilms. The wells were then rinsed (2×) with 100 µL PBS to remove planktonic cells. Two-fold serial dilutions of the compound were made from 1000 to 1.95 µg/mL in another 96-well plate and transferred to the well plate containing the preformed biofilms. Afterwards, the plates were incubated for 24 h at 37 °C. The media in the wells were then removed carefully and the plate washed twice with 100 µL PBS. 100 µL of XTT/menadione solution was added to each well and the plates incubated at 37 °C for 2 h in the dark. 80 µL of the resulting supernatant from each well was then transferred into a new microplate which was read at 490 nm [45]. Percentage inhibitions in both assays were determined as follows (1): (1)The absorbances were analysed with GraphPad for Windows version 8 (GraphPad Prism Software, San Diego, USA). The experiment was replicated thrice (three biological replicates) in both assays with three technical replicates per treatment and on different days. 2.8 Statistical analysis Data from all experiments are displayed as mean ± SD. Analyses were carried out using one-way analysis of variance (ANOVA) with GraphPad Prism for Windows version 8 (GraphPad Prism Software, San Diego, USA). For the antifungal activity assays, comparisons between two groups were performed using unpaired t-tests with Welch’s correction followed by two-tailed p-value determination. For the antibiofilm assays, percentage inhibition data were plotted as sigmoidal dose-response curves using GraphPad Prism version 8, from which the half maximal inhibitory concentration (IC50) values were derived by non-linear regression analysis. For all analysis, p < 0.05 was considered statistically significant. 2.9 Ethical statement The study did not involve the use of human subjects, human tissues or organs or animals. The Candida clinical isolates used in the study were obtained from the Microbiology Laboratory unit of the Ho Teaching Hospital, Ghana. As such, there was no direct interactions with patients. Ethical approval was not required for the study under the institutional guidelines. The study used existing clinical isolates already collected during routine hospital diagnostic procedures. No identifiable patient data was used. No patient recruitment or intervention took place. No collection permits from the Ghana Forestry Commission were required for plant sample collection as Paullinia pinnata is not a protected or endangered species in Ghana. 3 Results 3.1 Identification of the isolated compound from the leaves of P. pinnata The chemical investigation of the EtOAc leaf extract of P. pinnata produced a previously isolated new lupene-type triterpenoid [31]. Its NMR spectra compared favourably with the reported literature and was identified as 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one (Fig 1). The full NMR spectra (1D and 2D NMR) of the compound is outlined in the supplementary data (S1–S6 Figs). The key 1H and 13C NMR assignment for 6α-(3′-methoxy-4′-hydroxybenzoyl)-lup-20(29)-ene-3-one are presented in Table 3 below. 3.2 Microbiological identification of fungi from pregnant women Table 4 shows that of the forty (40) Candida spp. obtained from the Ho Teaching Hospital, Ghana thirty-seven (37) were positive for five strains of Candida species, identified as C. albicans (n = 11, 29.73%), C. glabrata (n = 13, 35.14%), C. tropicalis (n = 6, 16.22%), C. parapsilosis (n = 2, 5.41%) and C. krusei (n = 5, 13.51%). 3.3 Antifungal susceptibility of Candida isolates against clinically used antifungal drugs Generally, the highest resistance rate of the Candida species was seen against fluconazole and clotrimazole (62.16%), followed by miconazole (54.05%), voriconazole (43.24%), nystatin (24.32%) and amphotericin B (5.41%). Conversely, the Candida spp. was observed to be sensitive to amphotericin B (67.57%), voriconazole (40.54%), nystatin (13.51%), fluconazole and clotrimazole (10.81%), and miconazole (8.11%) in decreasing order. Majority of the Candida spp. were also found to be susceptible dose dependent to nystatin (62.16%) (Fig 2). Apart from amphotericin B, over 50% of C. albicans isolates were resistant to all the antifungal drugs. Among the non-albicans Candida (NAC), more than 50% were resistant to fluconazole, clotrimazole and miconazole; 76.92% were susceptible dose dependent to nystatin and 69.23% were susceptible to amphotericin B. When treated with voriconazole, 46.15% of the NAC species were susceptible while 34.62% were resistant. Most of the C. glabrata and C. tropicalis were susceptible dose dependent or resistant to the antifungal drugs except amphotericin B whiles all C. krusei were resistant to fluconazole (S1 Table). 3.4 Antifungal activities of P. pinnata extract The antifungal activities of the EtOAc extract of P. pinnata against the Candida reference strains and clinical isolates are displayed in S2 Table. The extract demonstrated considerable antifungal activities against the strains and isolates with minimum inhibitory concentrations (MICs) ranging from 31.25 to 500 µg/mL and minimum fungicidal concentrations (MFCs) of 31.25–2000 µg/mL. Voriconazole, ketoconazole and amphotericin B used as positive controls also showed potent antifungal activities with MICs of 4–32 µg/mL, 1–8 µg/mL and 0.50–2 µg/mL, respectively. Although there was no significant difference in the activity of the ethyl acetate P. pinnata leaves extract against C. albicans, C. glabrata, C. tropicalis and C. krusei strains and isolates, higher concentrations of the extract were required to inhibit the C. tropicalis followed by C. albicans then C. glabrata and C. krusei strains and isolates, respectively. However, significantly (p < 0.05) higher concentrations of the extract were needed to inhibit the C. albicans, C. glabrata and C. tropicalis strains and isolates than the C. parapsilosis strains and isolates (Fig 3). Values represent the minimum inhibitory concentration (MIC) expressed in µg/mL and are presented as mean ± SD from two independent biological experiments, each performed with three technical replicates per treatment. Comparisons between two groups were carried out using unpaired t-tests with Welch’s correction followed by two-tailed p-value determination. *p < 0.05: comparison between C. albicans, C. glabrata or C. tropicalis and C. parapsilosis. 3.5 Antifungal activities of the column fractions of P. pinnata ethyl acetate extract The chromatographic separation and purification of the compound from the EtOAc extract of P. pinnata leaves was activity directed by evaluating the antifungal activities of the bulked fractions (BF1 – BF5) against the Candida reference strains. Table 5 depicts the antifungal activities of the fractions. They exhibited varying inhibitory actions against the Candida strains with bulked fraction F4 demonstrating the highest activity with MIC of 3.91–31.25 µg/mL against the panel of Candida strains. The activities of the positive controls were as earlier indicated. 3.6 Antifungal activity of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one (Fig 1) was investigated for growth inhibition using the broth microdilution technique described by the Clinical and Laboratory Standards Institute (CLSI) with slight modifications against forty-two drug-resistant strains from five (5) species of Candida of which five (5) were reference strains and thirty-seven (37) were clinical isolates. The MICs of the compound against the Candida strains investigated ranged from 0.5 to 16 µg/mL (0.85–27.10 µM). The compound’s MIC was found to range from 1 to 16 µg/mL against C. albicans, 0.5 to 4 µg/mL against C. glabrata, 1–8 µg/mL against C. tropicalis, 0.5 to 2 µg/mL against C. krusei, and 0.5 to 1 µg/mL against C. parapsilosis (S3 Table). Although the compound generally exhibited potent activities against the Candida species, significantly (p < 0.05) higher concentrations of the compound were required to inhibit the C. albicans and C. glabrata than C. parapsilosis. Again, higher concentrations (p < 0.05) were needed to inhibit the C. albicans compared to C. krusei (Fig 4). Values represent the minimum inhibitory concentration (MIC) expressed in µg/mL and are presented as mean ± SD from two independent biological experiments, each performed with three technical replicates per treatment. Comparisons between two groups were carried out using unpaired t-test with Welch’s correction followed by two-tailed p-value determination. *p < 0.05: comparison between C. albicans or C. glabrata and C. parapsilosis. #p < 0.05: comparison between C. albicans and C. krusei. 3.7 Antifungal activities of the combinations of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one with clinically used antifungal agents The effect of the combinations of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one with voriconazole, nystatin or caspofungin was investigated using the checkerboard assay. Generally, the MICs of the combinations of the compound with the antifungal drugs against the Candida species were either lower or equal to the MICs of the corresponding single agent, and most importantly, none of the combinations exhibited antagonistic interactions (S4 Table). The median fraction inhibitory concentration indices (FICIs) for combinations of the compound with the antifungal drugs were in the range of 0.39–1.30 against the Candida species with synergistic action ranging from 33.33% to 83.33%, with 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one – nystatin combination against C. albicans being the most prevalent at 83.33% (Table 6). On the other hand, the combination of the compound with nystatin against C. parapsilosis did not induce any synergistic action, with FICI range of 0.56–1.13. Around 70% of the detected synergism was against the C. albicans and C. glabrata strains and isolates. Furthermore, the three remaining Candida species demonstrated different patterns of interactions that ranged from synergism to indifference when treated with the combinations of the natural product with the antifungal drugs (Table 6). 3.8 Anti-biofilm activity of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one 3.8.1 Biofilm formation inhibition. 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one was investigated for biofilm formation inhibition against the five drug-resistant reference strains. The inhibitory activity, against all Candida species tested, measured in terms of half maximal inhibitory concentration (IC50) ranged from 15.30–46.40 μg/mL (equivalent to 25.91–78.60 μM). The compound’s IC50 for C. albicans was 46.40 ± 3.64 μg/mL (Fig 5A), for C. glabrata was 23.43 ± 1.43 μg/mL (Fig 5B), for C. tropicalis was 31.27 ± 2.50 μg/mL (Fig 5C), for C. krusei was 15.30 ± 1.39 μg/mL (Fig 5D), and for C. parapsilosis was 25.40 ± 1.89 μg/mL (Fig 5E) (Fig 5). Biofilm formation inhibition of the compound against (A) C. albicans (B) C. glabrata (C) C. tropicalis (D) C. krusei and (E) C. parapsilosis. Data are plotted as mean percentage inhibition ± SD relative to the 1% DMSO vehicle control, from three independent biological experiments. IC50 values were determined by non-linear regression analysis of sigmoidal dose-response curves using GraphPad Prism version 8. 3.8.2 Preformed biofilm inhibition. 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one was also evaluated for its tendency to disrupt preformed biofilms of the five drug-resistant reference strains. Higher concentrations of the compound were required to inhibit the preformed biofilms compared to prevention of biofilm formation with IC50 ranging from 25.40–90.84 μg/mL (equivalent to 43.02–153.86 μM). The IC50 of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one against C. albicans was 90.84 ± 8.81 μg/mL (Fig 6A), against C. glabrata was 41.99 ± 6.07 μg/mL (Fig 6B), against C. tropicalis was 54.82 ± 3.49 μg/mL (Fig 6C), against C. krusei was 25.40 ± 4.37 μg/mL (Fig 6D), and against C. parapsilosis was 33.97 ± 2.60 μg/mL (Fig 6E). Preformed biofilm inhibition of the compound against (A) C. albicans (B) C. glabrata (C) C. tropicalis (D) C. krusei and (E) C. parapsilosis. Data are plotted as mean percentage inhibition ± SD relative to the 1% DMSO vehicle control, from three independent biological experiments. IC50 values were determined by non-linear regression analysis of sigmoidal dose-response curves using GraphPad Prism version 8. 4 Discussion In our continued search for new antifungal agents, we evaluated the lupene-type triterpenoid, 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one for antifungal activity using Candida pathogens circulating in Ghanaian healthcare centres. Globally, it is estimated that about 35–40% of pregnant women may have vulvovaginal candidiasis with C. albicans responsible for more than 90% of all cases, followed by C. glabrata [46,47]. This data does no differ significantly from what is recorded in Ghana. A prevalence of 36.7% was reported in the middle belt [48] whiles 30.7% were recorded in the Ho Municipal Hospital [49] and 50.7% in the Ho Teaching Hospital in the Volta Region [5]. C. albicans and C. glabrata were the most common identified causative agents. Corroborating these statistics, the present study found that of the 40 clinical isolates obtained from the Microbiology Department of Ho Teaching Hospital, 37 were identified as Candida species with C. glabrata (35.14%) being the most abundant, followed by C. albicans (29.73%), C. tropicalis (16.22%), C. krusei (13.51%) and C. parapsilosis (5.41%). Antifungal resistance was also observed in the Candida isolates to the clinically used antifungals in Ghana in the study. The Candida species demonstrated high resistance to azole antifungals with resistance rate of 62.16% against fluconazole and clotrimazole, 54.05% against miconazole and 43.24% against voriconazole. However, they were less resistant to polyene antifungal agents with resistance rates of 5.41% and 24.32% against amphotericin B and nystatin respectively. The pattern of resistance observed in the azole antifungals, particularly fluconazole is worrying as it remains the most used first-line treatment for vulvovaginal candidiasis [50,51]. The observed resistance to azoles has been attributed to several factors including mutations in Candida ERG11, overexpression of efflux pumps and formation of biofilms [52,53]. Furthermore, the increase in resistance to other antifungal drug classes by C. albicans and non-albicans Candida species reinforces concerns about the diminishing potency of antifungal therapies and demonstrates the urgent need for the discovery of new antifungal agents capable of overcoming the resistance mechanisms in Candida pathogens. The EtOAc leaves extract of P. pinnata exhibited significant antifungal activity against the drug-resistant Candida strains and clinical isolates with minimum inhibitory concentrations (MICs) that ranged from 31.25 to 500 µg/mL and minimum fungicidal concentrations (MFCs) of 31.25–2000 µg/mL. Activity-driven chromatographic separation showed that the anti-Candida activity of the EtOAc P. pinnata extract was concentrated in bulk column fraction F4 (BF4). While the other column fractions (BF1 – BF3, BF5) exhibited moderate to no antifungal activity against the reference strains of C. albicans, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis with MIC in the range of 125–2000 µg/mL, BF4 gave MICs of 3.91 µg/mL against C. parapsilosis ATCC 22019, 7.81 µg/mL against C. glabrata ATCC 2001 and C. krusei ATCC 6258, 15.63 µg/mL against C. tropicalis NRRL Y-12968 and 31.25 µg/mL against C. albicans ATCC 10231 respectively demonstrating strong activity [40]. Further purification of BF4 led to the isolation of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one. The compound was characterized using NMR analysis and by comparing its spectra data with reported literature [31]. 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one has previously been isolated from the roots of P. pinnata. Here, we report for the first time, its isolation from the leaves of the plant. This study is also the first to investigate the antifungal activity of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one against multiple Candida species, the first reported biological activity of the compound. Our findings show that 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one exerts strong activity against C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei with MIC ranging from 0.5 to 16 µg/mL (0.85–27.10 µM). The MIC values of the compound (0.5–16 µg/mL) fell within similar range to those of ketoconazole (MIC = 1–8 µg/mL) and amphotericin B (MIC = 0.5–2 µg/mL), which were used as control. These comparisons were made under the same standardized in vitro microbiological assay conditions. In the study, the Candida isolates demonstrated resistance to the azole and polyene antifungals. The potent activity of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)- against drug-resistant Candida strains suggests it may overcome existing resistance mechanisms in the Candida species. This could reflect a mechanism of action distinct from those of conventional drugs, though this hypothesis requires experimental confirmation. The antifungal potency demonstrated by 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one in the present study compares favourably with, and in several instances surpasses, that of previously reported antifungal triterpenoids. Among the lupane-type triterpenoids, betulin and betulinic acid are the most extensively studied for antifungal activity. Betulinic acid has been reported to inhibit C. albicans with MIC values ranging from 32 to 128 µg/mL [38,54], and betulin has demonstrated MIC values of 64–256 µg/mL against Candida species [55], both of which are considerably higher than the MIC values of 1–16 µg/mL recorded for 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one against C. albicans in the present study. Among the oleanane-type triterpenoids, oleanolic acid has been reported to exhibit anti-Candida activity with MIC values typically in the range of 32–128 µg/mL against C. albicans and C. glabrata, while its 3-oxo derivative, oleanonic acid, has shown stronger activity in the range of 16–64 µg/mL [56–58]. Ursolic acid, an isomer of oleanolic acid, has similarly demonstrated MIC values of 16–64 µg/mL against Candida species [59,60]. Compared with these pentacyclic triterpenoids, the MIC values of 0.5–4 µg/mL recorded for 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one against C. glabrata and C. krusei represent a substantially higher level of potency. Within the lupane series from P. pinnata specifically, other derivatives including the 6β-epimer of the compound [26] and the 3-O-isovanilloyl lupene derivative reported by Lasisi et al. [18], have been evaluated for antibacterial activity, with no prior antifungal data reported. The present study therefore establishes 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one as one of the most potent naturally occurring lupane-type triterpenoids reported to date with respect to anti-Candida activity to the best of our knowledge. The enhanced potency relative to other triterpenoids may be attributable to the presence of the 6α-vanilloyl ester group, which introduces a polar aromatic substituent onto the lipophilic lupane scaffold. This modification may facilitate interaction with fungal membrane components by combining the membrane-partitioning properties of the triterpenoid core with the hydrogen-bonding capacity of the phenolic hydroxyl and methoxy groups of the vanilloyl moiety This structure-activity relationship hypothesis, however, requires direct experimental validation through systematic modification and testing of structural analogues. One possible mechanism through which 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one may exert its antifungal activity is through direct disruption of the fungal cell membrane, though this has not been experimentally confirmed. The triterpenoid skeleton is highly lipophilic and has been shown in multiple studies to partition readily into fungal lipid bilayers [61,62]. The rigid, planar triterpenoid scaffold is capable of intercalating between the acyl chains of membrane phospholipids, thereby altering membrane fluidity, disorganising the bilayer architecture and increasing non-specific membrane permeability [63]. This loss of membrane integrity would compromise essential membrane-dependent processes including nutrient transport, ion homeostasis and maintenance of electrochemical gradients, ultimately leading to cell death. A second possible but as yet confirmed mechanism is direct interaction with membrane ergosterol. As ergosterol is the principal sterol of the fungal cell membrane and plays a critical role in maintaining membrane fluidity, structural organisation and selective permeability, inhibition of its biosynthesis could also be another mechanism that the compound exerts its activity [64]. Some pentacyclic triterpenoids have been shown to inhibit enzymes in the ergosterol biosynthetic pathway, particularly squalene epoxidase and lanosterol synthase, both of which operate upstream of the azole target lanosterol 14α-demethylase [65,66]. Inhibition at these upstream steps would deplete ergosterol from the fungal membrane through a different enzymatic target from azoles, which would account for the potent activity of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one against azole-resistant strains in which ERG11 is mutated [11], since the compound would not depend on a functional ERG11 target to exert its effect. Furthermore, it is also hypothesized that 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one may disrupt fungal cell wall integrity through inhibition of chitin synthase or β-1,3-glucan synthase [67]. These enzymes are essential for cell wall assembly and maintenance in Candida species, and inhibition of either would weaken the cell wall, increase osmotic sensitivity and ultimately cause cell lysis. These possible mechanisms are however hypothetical and require direct experimental validation to confirm. The formation of biofilms is one of the most important virulence attributes essential for the pathogenicity of several Candida species including C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. auris [68,69]. Biofilm formation during infection has been linked to higher mortality rates when compared with infections caused by species incapable of forming biofilms [70]. The development of biofilms functions to counter host immune response by the inhibition of macrophage phagocytosis and antibody activities [68,71]. Furthermore, Candida biofilms act as a physical barrier to drug penetration and thereby contribute to their high resistance to antifungal agents [72]. 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one was evaluated for its ability to prevent the formation of biofilms and disrupt fully formed ones in the different reference Candida strains. Biofilm formation and preformed Candida biofilms were inhibited with half maximal inhibitory concentration (IC50) values ranging from 15.30–46.40 μg/mL (equivalent to 25.91–78.60 μM) and 25.40–90.84 μg/mL (equivalent to 43.02–153.86 μM), respectively. These concentrations are significantly higher than those recorded for inhibiting planktonic cells confirming the observation that when Candida species exists as biofilms, they acquire additional resistance mechanisms specific and unique to the biofilm state [4,70]. Apart from the presence of the extracellular matrix which acts as a mechanical barrier, the resistance of Candida biofilms to antifungals has also been attributed to factors such as upregulation of drug-efflux pumps which are not dependent on antifungal exposure and the presence of persister cells within the biofilm [53,73,74]. The ability of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one to inhibit both biofilm formation and preformed biofilms is of therapeutic relevance given that biofilm formation is a principal mechanism underlying treatment failure in vulvovaginal candidiasis with conventional antifungal agents demonstrating reduced efficacy against biofilm-associated Candida infections [69]. It is hypothesized that 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one may exert its antibiofilm activity by disrupting the composition of extracellular matrix through inhibition of key matrix components, retardation of efflux pump action and inhibiting persister cells. These mechanisms however are hypothetical and further studies including scanning electron microscopy should be carried out to investigate them into details as the antibiofilm activity of the compound is solely based on IC50 measurements. The use of antifungals in combination has emerged as promising therapeutic strategy to overcome resistance in fungal pathogens. Combining antifungal agents, particularly those with different mechanisms of action can increase the potency of the combined molecules resulting in synergy, and extend the spectrum of activity [75,76]. Such combinations also have the potential to reduce dosages thereby lowering toxicity, reducing mortalities and improving the overall treatment outcomes. An example is the combination of amphotericin B with 5-flucytosine used as first-line treatment for cryptococcal meningitis [77]. The effect of the combination of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one with voriconazole, nystatin or caspofungin on the Candida strains and isolates was investigated by the checkerboard assay. Voriconazole, nystatin and caspofungin were selected as representatives of the three major antifungal classes: azoles, polyenes and echinocandins [78]. Combinations of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one with the conventional antifungals showed potent synergistic (33.33% to 83.33%) activities across the Candida strains and isolates tested. This synergism was particularly evident against isolates displaying azole and polyene resistance, suggesting that such combinations may help overcome established resistance mechanisms in Candida pathogens. The high degree of synergism that 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one exhibited in combination with the antifungals also suggest that it may exert its antifungal activity through a pathway completely different from the existing antifungal drugs. Azoles act by inhibiting lanosterol 14α-demethylase, a key enzyme in the ergosterol biosynthetic pathway [79]. Polyenes exert their effect by forming pores in the cell membrane to facilitate the leakage of intracellular components through binding to ergosterol [80]. Echinocandins on the other hand interfere with fungal cell walls via inhibition of (1,3)-β-D-glucan synthase, an enzyme responsible for the biosynthesis of 1,3-β-D-glucan [81]. The difference in the mechanism of action of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one to the conventional drug classes might explain the synergistic action of the combinations of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one with voriconazole, nystatin or caspofungin. The present study found that the P. pinnata leaves possesses antifungal activity against the drug-resistant Candida species, and its isolate, 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one has demonstrated strong and broad-spectrum anti-Candida activity against standard strains and clinical isolates of Candida species implicated in yeast infections. Further studies including evaluating the efficacy and safety of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one in animal models are therefore warranted to explore this compound as a therapeutic agent for use against Candida infections especially in skin and mucosal infections. A major limitation of the study is that no cytotoxicity evaluation of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one or the EtOAc extract of P. pinnata in mammalian cells was performed. This is a significant limitation because, without cytotoxicity data, it is not possible to determine a therapeutic index and therefore the therapeutic relevance of the antifungal activity observed cannot be fully assessed. Cytotoxicity evaluation of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one against a panel of mammalian cell lines such as Vero cells and human dermal fibroblasts, given the potential application of the compound in vulvovaginal infections, as well as hepatotoxicity screening using HepG2 cells is therefore identified as the most immediate priority for follow-up investigations. A second limitation is the absence of scanning electron microscopy (SEM) data to visually confirm the morphological changes in Candida biofilms following treatment with 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one. The antibiofilm activity reported here is based solely on XTT metabolic reduction assays, which measure the metabolic activity of viable cells within biofilms as a proxy for biofilm viability. While this is a well-established and widely used method, it does not provide direct structural evidence of biofilm disruption, extracellular matrix degradation, or changes in Candida morphology such as inhibition of hyphal transition in C. albicans, which is a critical virulence attribute linked to biofilm formation. SEM studies, complemented by confocal laser scanning microscopy (CLSM) with fluorescent staining of the extracellular matrix, would provide this structural confirmation and are planned as part of subsequent investigations. Future work will therefore address these limitations. 5 Conclusion We report in this study, the anti-Candida activity of the ethyl acetate leaves extract of P. pinnata. The extract and its isolated compound, 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one have been shown to exert considerable antifungal activity against multiple drug-resistant Candida species. 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one exhibited strong antibiofilm activity by inhibiting the formation of biofilms and preformed biofilms while demonstrating strong synergistic action when combined with voriconazole, nystatin and caspofungin. The findings of the present study are based on in vitro experiments. The absence of the cytotoxicity data for 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one is a critical gap that limits the assessment of the therapeutic potential of the compound. Future studies should therefore focus on cytotoxicity evaluation in mammalian cells and subsequent in vivo efficacy studies in appropriate animal models in the continued investigation of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one as a candidate antifungal agent. Supporting information S1 Table. In vitro susceptibility patterns of the clinical strains of Candida isolates to fluconazole (25 μg), voriconazole (1 µg), miconazole (10 µg), clotrimazole (10 μg), amphotericin B (10 µg) and nystatin (100 units). SDD: Susceptible dose-dependent. https://doi.org/10.1371/journal.pone.0350399.s001 (DOCX) S2 Table. Antifungal activity of the ethyl acetate extract of the roots of P. pinnata against the Candida strains and isolates. Values are expressed in µg/mL. MIC: Minimum inhibitory concentration; MFC: Minimum fungicidal concentration; VRC: Voriconazole; KTC: Ketoconazole; AMB: Amphotericin B; EtOAc: Ethyl acetate. Experiment was carried out in duplicate with three technical replicates per treatment and on different days. https://doi.org/10.1371/journal.pone.0350399.s002 (DOCX) S3 Table. Antifungal activity of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one against the Candida strains and isolates. Values are expressed in µg/mL. MIC: Minimum inhibitory concentration; MFC: Minimum fungicidal concentration; VRC: Voriconazole; KTC: Ketoconazole; AMB: Amphotericin B. Experiment was carried out in duplicate with three technical replicates per treatment and on different days. https://doi.org/10.1371/journal.pone.0350399.s003 (DOCX) S4 Table. FIC indices for the combinations of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one with commonly used antifungal drugs against Candida strains and clinical isolates. INT: Interpretation, FICI: Fraction Inhibitory Concentration Index. S: Synergism for FICI ≤0.5, I: Indifference FICI was > 0.5 to ≤4.0, and A: Antagonism FICI >4.0. Experiment was carried out in triplicate. https://doi.org/10.1371/journal.pone.0350399.s004 (DOCX) S1 Fig. 1H NMR spectrum of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one. https://doi.org/10.1371/journal.pone.0350399.s005 (TIF) S2 Fig. 13C NMR spectrum 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one. https://doi.org/10.1371/journal.pone.0350399.s006 (TIF) S3 Fig. DEPT135 NMR spectrum of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one. https://doi.org/10.1371/journal.pone.0350399.s007 (TIF) S4 Fig. COSY spectrum of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one. https://doi.org/10.1371/journal.pone.0350399.s008 (TIF) S5 Fig. HSQC spectrum of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one. https://doi.org/10.1371/journal.pone.0350399.s009 (TIF) S6 Fig. HMBC spectrum of 6α-(3’-methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one. https://doi.org/10.1371/journal.pone.0350399.s010 (TIF) Acknowledgments We are grateful to Seeding Labs, Boston, US for the equipment used in the study and to the Microbiology laboratory of the Ho teaching Hospital, Ho for providing the Candida clinical isolates. References - 1. Satora M, Grunwald A, Zaremba B, Frankowska K, Żak K, Tarkowski R, et al. Treatment of vulvovaginal candidiasis-an overview of guidelines and the latest treatment methods. J Clin Med. 2023;12(16):5376. pmid:37629418 - 2. Willems HME, Ahmed SS, Liu J, Xu Z, Peters BM. Vulvovaginal candidiasis: a current understanding and burning questions. J Fungi (Basel). 2020;6(1):27. pmid:32106438 - 3. Rosati D, Bruno M, Jaeger M, Ten Oever J, Netea MG. Recurrent vulvovaginal candidiasis: an immunological perspective. Microorganisms. 2020;8(2):144. pmid:31972980 - 4. San Juan Galán J, Poliquin V, Gerstein AC. Insights and advances in recurrent vulvovaginal candidiasis. PLoS Pathog. 2023;19(11):e1011684. pmid:37948448 - 5. Aboagye G, Waikhom S, Asiamah EA, Tettey CO, Mbroh H, Smith C, et al. Antifungal susceptibility profiles of Candida and non-albicans species isolated from pregnant women: implications for emerging antimicrobial resistance in maternal health. Microbiol Spectr. 2025;13(7):e0078725. pmid:40454865 - 6. Nyirjesy P, Brookhart C, Lazenby G, Schwebke J, Sobel JD. Vulvovaginal candidiasis: a review of the evidence for the 2021 centers for disease control and prevention of sexually transmitted infections treatment guidelines. Clin Infect Dis. 2022;74(Suppl_2):S162–8. pmid:35416967 - 7. Sobel JD. Treatment of vaginitis caused by non-albicans Candida species. Expert Rev Anti Infect Ther. 2024;22(5):289–96. pmid:38720183 - 8. Jafarzadeh L, Ranjbar M, Nazari T, Naeimi Eshkaleti M, Aghaei Gharehbolagh S, Sobel JD, et al. Vulvovaginal candidiasis: An overview of mycological, clinical, and immunological aspects. J Obstet Gynaecol Res. 2022;48(7):1546–60. pmid:35445492 - 9. Garvey M, Rowan NJ. Pathogenic drug resistant fungi: a review of mitigation strategies. Int J Mol Sci. 2023;24(2):1584. pmid:36675092 - 10. Espinel-Ingroff A, Cantón E, Pemán J. Antifungal resistance among less prevalent candida non-albicans and other yeasts versus established and under development agents: a literature review. J Fungi (Basel). 2021;7(1):24. pmid:33406771 - 11. Whaley SG, Berkow EL, Rybak JM, Nishimoto AT, Barker KS, Rogers PD. Azole antifungal resistance in Candida albicans and emerging non-albicans Candida species. Frontiers in Microbiology. 2017;7:2173. - 12. Adeyemo-Salami OA, Ademowo OG, Farombi EO. Antioxidant and antiplasmodial activities of methanol leaf extract of Paullinia pinnata. Journal of Herbs, Spices & Medicinal Plants. 2020;26(3):315–28. - 13. Lunga PK, Tamokou J de D, Fodouop SP, Kuiate J-R, Tchoumboue J, Gatsing D. Antityphoid and radical scavenging properties of the methanol extracts and compounds from the aerial part of Paullinia pinnata. Springerplus. 2014;3:302. pmid:25279277 - 14. Nyegue MA, Afagnigni AD, Ndam YN, Djova SV, Fonkoua MC, Etoa F-X. Toxicity and activity of ethanolic leaf extract of Paullinia pinnata Linn (Sapindaceae) in Shigella flexneri-induced diarrhea in wistar rats. J Evid Based Integr Med. 2020;25:2515690X19900883. pmid:31969010 - 15. Tseuguem PP, Nguelefack TB, Piégang BN, Mbankou Ngassam S. Aqueous and methanol extracts of Paullinia pinnata (Sapindaceae) improve monosodium urate-induced gouty arthritis in rat: analgesic, anti-inflammatory, and antioxidant effects. Evid Based Complement Alternat Med. 2019;2019:5946291. pmid:31885654 - 16. Agyare C, Spiegler V, Sarkodie H, Asase A, Liebau E, Hensel A. An ethnopharmacological survey and in vitro confirmation of the ethnopharmacological use of medicinal plants as anthelmintic remedies in the Ashanti region, in the central part of Ghana. J Ethnopharmacol. 2014;158 Pt A:255–63. pmid:25446638 - 17. Ikhane D, Banwo K, Omotade O, Sanni A. Phytochemical and antimicrobial activities of methanolic extract ofpaullinia pinnataleaves on some selected bacterial pathogens. Journal of Herbs, Spices & Medicinal Plants. 2014;21(1):59–74. - 18. Lasisi AA, Ayinde BW, Adeleye AO, Onocha PA, Oladosu IA, Idowu PA. New triterpene isovanniloyl and antibacterial activity of constituents from the roots of Paullinia pinnata Linn (Sapindaceae). Journal of Saudi Chemical Society. 2015;19(2):117–22. - 19. Ajibade MA, Akhigbemen AM, Okolie NP, Ozolua RI. Methanol leaf extract of Paullinia pinnata exerts sleep-enhancing and anticonvulsant effects via a mechanism involving the GABAergic pathway. Epilepsy Res. 2022;183:106943. pmid:35636276 - 20. Ozolua R, Ajibade M, Uwaya D, Akhigbemen A, Bolanle I. A report on the antidepressant-like activity of paullinia pinnata methanol leaf extract in mice and possible involvement of monoaminergic mechanisms. Targets. 2025;3(2):22. - 21. Spiegler V, Liebau E, Peppler C, Raue K, Werne S, Strube C, et al. A hydroalcoholic extract from Paullinia pinnata L. roots exerts anthelmintic activity against free-living and parasitic nematodes. Planta Med. 2016;82(13):1173–9. pmid:27286336 - 22. Spiegler V. Anthelmintic a-type procyanidins and further characterization of the phenolic composition of a root extract from Paullinia pinnata. Molecules. 2020;25(10):2287. pmid:32414042 - 23. Tseuguem PP, Ngangoum DAM, Pouadjeu JM, Piégang BN, Sando Z, Kolber BJ, et al. Aqueous and methanol extracts of Paullinia pinnata L. (Sapindaceae) improve inflammation, pain and histological features in CFA-induced mono-arthritis: evidence from in vivo and in vitro studies. J Ethnopharmacol. 2019;236:183–95. pmid:30849505 - 24. Okwudili OS, Chimaobi NG, Ikechukwu EM, Ndukaku OY. Antidiabetic and in vitro antioxidant effects of hydromethanol extract of Paullinia pinnata root bark in alloxan-induced diabetic rat. J Complement Integr Med. 2017;15(2). pmid:29148978 - 25. Annan K, Govindarajan R, Kisseih E. Wound healing and cytoprotective actions of Paullinia pinnata L. Pharmacognosy Journal. 2010;2(10):345–50. - 26. Annan K, Houghton PJ. Two novel lupane triterpenoids from Paullinia pinnata L. with fibroblast stimulatory activity. J Pharm Pharmacol. 2010;62(5):663–8. pmid:20609071 - 27. Awouafack MD, Ito T, Tane P, Kodama T, Tanaka M, Asakawa Y, et al. A new cycloartane-type triterpene and a new eicosanoic acid ester from fruits of Paullinia pinnata L. Phytochemistry Letters. 2016;15:220–4. - 28. Miemanang RS, Krohn K, Hussain H, Dongo E. Paullinoside A and Paullinomide A: a new cerebroside and a new ceramide from leaves of Paullinia pinnata. Zeitschrift für Naturforschung B. 2006;61(9):1123–7. - 29. Abourashed E, Toyang N, Choinski J Jr, Khan I. Two new flavone glycosides from paullinia pinnata. J Nat Prod. 1999;62(8):1179–81. pmid:10479333 - 30. Lunga P-K, Qin X-J, Yang X-W, Kuiate J-R, Du Z-Z, Gatsing D. A new antimicrobial and radical-scavenging glycoside from Paullinia pinnata var. cameroonensis. Nat Prod Res. 2015;29(18):1688–94. pmid:25563339 - 31. Jackson N, Annan K, Mensah AY, Ekuadzi E, Mensah ML, Habtemariam S. A novel triterpene from the roots of paullinia pinnata: 6α-(3′-methoxy-4′-hydroxybenzoyl)-lup-20 (29)-ene-3-one. Natural Product Communications, 2015;10(4):1934578X1501000405. - 32. Harley BK, Amponsah IK, Ben IO, Mireku-Gyimah NA, Anokwah D, Neglo D, et al. Hypoglycaemic activity of Oleanonic acid, a 3-oxotriterpenoid isolated from Aidia Genipiflora (DC.) Dandy, involves inhibition of carbohydrate metabolic enzymes and promotion of glucose uptake. Biomed Pharmacother. 2022;149:112833. pmid:35316751 - 33. Harley BK, Dickson RA, Amponsah IK, Ben IO, Adongo DW, Fleischer TC, et al. Flavanols and triterpenoids from Myrianthus arboreus ameliorate hyperglycaemia in streptozotocin-induced diabetic rats possibly via glucose uptake enhancement and α-amylase inhibition. Biomed Pharmacother. 2020;132:110847. pmid:33068933 - 34. Waikhom SD, Afeke I, Kwawu GS, Mbroh HK, Osei GY, Louis B, et al. Prevalence of vulvovaginal candidiasis among pregnant women in the Ho municipality, Ghana: species identification and antifungal susceptibility of Candida isolates. BMC Pregnancy Childbirth. 2020;20(1):266. pmid:32375724 - 35. Harley BK, Quagraine AM, Neglo D, Aggrey MO, Orman E, Mireku-Gyimah NA, et al. Metabolite profiling, antifungal, biofilm formation prevention and disruption of mature biofilm activities of Erythrina senegalensis stem bark extract against Candida albicans and Candida glabrata. PLoS One. 2022;17(11):e0278096. pmid:36441750 - 36. Rati R, Patel J, Rishi S. Vulvovaginal candidiasis and its antifungal susceptibility pattern: single center experience. Int J Med Res Rev. 2015;3(1):72–8. - 37. Sarpong AK, Odoi H, Boakye YD, Boamah VE, Agyare C. Resistant C. albicans implicated in recurrent vulvovaginal candidiasis (RVVC) among women in a tertiary healthcare facility in Kumasi, Ghana. BMC Womens Health. 2024;24(1):412. pmid:39030542 - 38. Harley BK, Neglo D, Tawiah P, Pipim MA, Mireku-Gyimah NA, Tettey CO, et al. Bioactive triterpenoids from Solanum torvum fruits with antifungal, resistance modulatory and anti-biofilm formation activities against fluconazole-resistant candida albicans strains. PLoS One. 2021;16(12):e0260956. pmid:34962953 - 39. Pedroso RDS, Balbino BL, Andrade G, Dias MCPS, Alvarenga TA, Pedroso RCN, et al. In vitro and in vivo anti-candida spp. activity of plant-derived products. Plants (Basel). 2019;8(11):494. pmid:31718037 - 40. Alves DDN, Ferreira AR, Duarte ABS, Melo AKV, de Sousa DP, Castro RDd. Breakoutpoints for the classification of anti-Candida compounds in antifungal screening. BioMed Research International. 2021;2021(1):6653311. - 41. Harley BK, Neglo D, Aggrey MO, Quagraine AM, Orman E, Jato J, et al. Antifungal activities of phytochemically characterized hydroethanolic extracts of Sclerocarya birrea leaves and stem bark against fluconazole-resistant Candida albicans strains. BioMed Research International. 2022;:4261741. - 42. Bidaud A-L, Schwarz P, Herbreteau G, Dannaoui E. Techniques for the assessment of in vitro and in vivo antifungal combinations. J Fungi (Basel). 2021;7(2):113. pmid:33557026 - 43. Bidaud AL, Djenontin E, Botterel F, Chowdhary A, Dannaoui E. Colistin interacts synergistically with echinocandins against Candida auris. Int J Antimicrob Agents. 2020;55(3):105901. pmid:31954831 - 44. Pierce CG, Uppuluri P, Tristan AR, Wormley Jr FL, Mowat E, Ramage G, et al. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc. 2008;3(9):1494–500. pmid:18772877 - 45. Pierce CG, Uppuluri P, Tummala S, Lopez-Ribot JL. A 96 well microtiter plate-based method for monitoring formation and antifungal susceptibility testing of Candida albicans biofilms. J Vis Exp. 2010;(44):2287. pmid:21048668 - 46. Aguin TJ, Sobel JD. Vulvovaginal candidiasis in pregnancy. Curr Infect Dis Rep. 2015;17(6):462. pmid:25916994 - 47. Bongomin F, Gago S, Oladele RO, Denning DW. Global and multi-national prevalence of fungal diseases-estimate precision. J Fungi (Basel). 2017;3(4):57. pmid:29371573 - 48. Konadu DG, Owusu-Ofori A, Yidana Z, Boadu F, Iddrisu LF, Adu-Gyasi D, et al. Prevalence of vulvovaginal candidiasis, bacterial vaginosis and trichomoniasis in pregnant women attending antenatal clinic in the middle belt of Ghana. BMC Pregnancy Childbirth. 2019;19(1):341. pmid:31547803 - 49. Waikhom SD, Afeke I, Kwawu GS, Mbroh HK, Osei GY, Louis B, et al. Prevalence of vulvovaginal candidiasis among pregnant women in the Ho municipality, Ghana: species identification and antifungal susceptibility of Candida isolates. BMC Pregnancy Childbirth. 2020;20(1):266. pmid:32375724 - 50. Bhattacharya S, Sae-Tia S, Fries BC. Candidiasis and mechanisms of antifungal resistance. Antibiotics (Basel). 2020;9(6):312. pmid:32526921 - 51. Nishimoto AT, Sharma C, Rogers PD. Molecular and genetic basis of azole antifungal resistance in the opportunistic pathogenic fungus Candida albicans. J Antimicrob Chemother. 2020;75(2):257–70. pmid:31603213 - 52. Assress HA, Selvarajan R, Nyoni H, Mamba BB, Msagati TAM. Antifungal azoles and azole resistance in the environment: current status and future perspectives—a review. Rev Environ Sci Biotechnol. 2021;20(4):1011–41. - 53. Lee Y, Robbins N, Cowen LE. Molecular mechanisms governing antifungal drug resistance. NPJ Antimicrob Resist. 2023;1(1):5. pmid:38686214 - 54. Adongo DW, Mante PK, Kukuia KKE, Benneh CK, Biney RP, Boakye-Gyasi E, et al. Fast-onset effects of Pseudospondias microcarpa (A. Rich) Engl. (Anacardiaceae) hydroethanolic leaf extract on behavioral alterations induced by chronic mild stress in mice. PLoS One. 2023;18(2):e0278231. pmid:36730151 - 55. Makarov I, Vinogradov M, Gromovykh T, Lutsenko S, Feldman N, Shambilova G, et al. Antifungal composite fibers based on cellulose and betulin. Fibers. 2018;6(2):23. - 56. Ma F, Deng Q, Lu Y, Jia Z, Xu S, Fei Q, et al. Novel oleanolic acid derivatives containing piperazine pyrimidine moieties: design, synthesis, and antimicrobial activity. Mol Divers. 2025;10.1007/s11030-025-11398–x. pmid:41288901 - 57. Zhao H, Zhou M, Duan L, Wang W, Zhang J, Wang D, et al. Efficient synthesis and anti-fungal activity of oleanolic acid oxime esters. Molecules. 2013;18(3):3615–29. pmid:23519202 - 58. Tan J, Zhang Z, Zheng D, Mu Y, Cao B, Yang J, et al. Structure-activity relationship and biofilm formation-related gene targets of oleanolic acid-type saponins from Pulsatilla chinensis against Candida albicans. Bioorg Chem. 2024;146:107311. pmid:38547720 - 59. Jesus JA, Lago JHG, Laurenti MD, Yamamoto ES, Passero LFD. Antimicrobial activity of oleanolic and ursolic acids: an update. Evid Based Complement Alternat Med. 2015;2015:620472. pmid:25793002 - 60. Woźniak Ł, Skąpska S, Marszałek K. Ursolic acid--a pentacyclic triterpenoid with a wide spectrum of pharmacological activities. Molecules. 2015;20(11):20614–41. pmid:26610440 - 61. Gamal A, Chu S, McCormick TS, Borroto-Esoda K, Angulo D, Ghannoum MA. Ibrexafungerp, a novel oral triterpenoid antifungal in development: overview of antifungal activity against Candida glabrata. Front Cell Infect Microbiol. 2021;11:642358. pmid:33791244 - 62. Hu Q, Chen Y-Y, Jiao Q-Y, Khan A, Li F, Han D-F, et al. Triterpenoid saponins from the pulp of Sapindus mukorossi and their antifungal activities. Phytochemistry. 2018;147:1–8. pmid:29257999 - 63. Ali O, Szabó A. Review of eukaryote cellular membrane lipid composition, with special attention to the fatty acids. Int J Mol Sci. 2023;24(21):15693. pmid:37958678 - 64. Choy HL, Gaylord EA, Doering TL. Ergosterol distribution controls surface structure formation and fungal pathogenicity. mBio. 2023;14(4):e0135323. pmid:37409809 - 65. Somai BM, Belewa V, Frost C. Tulbaghia violacea (Harv) exerts its antifungal activity by reducing ergosterol production in Aspergillus flavus. Curr Microbiol. 2021;78(8):2989–97. pmid:34100987 - 66. Kamel EM, Abdelrheem DA, Salah B, Lamsabhi AM. Phytochemical inhibitors of squalene epoxidase: Integrated In silico and In vitro mechanistic insights for targeting cholesterol biosynthesis. Arch Biochem Biophys. 2025;768:110372. pmid:40054651 - 67. Wadhwa K, Kaur H, Kapoor N, Brogi S. Identification of sesamin from sesamum indicum as a potent antifungal agent using an integrated in silico and biological screening platform. Molecules. 2023;28(12):4658. pmid:37375219 - 68. Rodríguez-Cerdeira C, Martínez-Herrera E, Carnero-Gregorio M, López-Barcenas A, Fabbrocini G, Fida M, et al. Pathogenesis and clinical relevance of candida biofilms in vulvovaginal candidiasis. Front Microbiol. 2020;11:544480. pmid:33262741 - 69. Sriphannam C, Nuanmuang N, Saengsawang K, Amornthipayawong D, Kummasook A. Anti-fungal susceptibility and virulence factors of Candida spp. isolated from blood cultures. J Mycol Med. 2019;29(4):325–30. pmid:31447236 - 70. Kaur J, Nobile CJ. Antifungal drug-resistance mechanisms in Candida biofilms. Curr Opin Microbiol. 2023;71:102237. pmid:36436326 - 71. Silva NBS, Menezes RP, Gonçalves DS, Santiago MB, Conejo NC, Souza SL, et al. Exploring the antifungal, antibiofilm and antienzymatic potential of Rottlerin in an in vitro and in vivo approach. Sci Rep. 2024;14(1):11132. pmid:38750088 - 72. Nett JE, Andes DR. Contributions of the biofilm matrix to Candida pathogenesis. J Fungi (Basel). 2020;6(1):21. pmid:32028622 - 73. LaFleur MD, Kumamoto CA, Lewis K. Candida albicans biofilms produce antifungal-tolerant persister cells. Antimicrob Agents Chemother. 2006;50(11):3839–46. pmid:16923951 - 74. Lee Y, Puumala E, Robbins N, Cowen LE. Antifungal drug resistance: molecular mechanisms in Candida albicans and beyond. Chem Rev. 2021;121(6):3390–411. pmid:32441527 - 75. Khalifa HO, Majima H, Watanabe A, Kamei K. In vitro characterization of twenty-one antifungal combinations against echinocandin-resistant and -susceptible Candida glabrata. J Fungi (Basel). 2021;7(2):108. pmid:33540778 - 76. Tits J, Cammue BPA, Thevissen K. Combination therapy to treat fungal biofilm-based infections. Int J Mol Sci. 2020;21(22):8873. pmid:33238622 - 77. Fioriti S, Brescini L, Pallotta F, Canovari B, Morroni G, Barchiesi F. Antifungal combinations against Candida species: from bench to bedside. J Fungi (Basel). 2022;8(10):1077. pmid:36294642 - 78. Bouz G, Doležal M. Advances in antifungal drug development: an up-to-date mini review. Pharmaceuticals (Basel). 2021;14(12):1312. pmid:34959712 - 79. Osmaniye D, Baltacı Bozkurt N, Levent S, Benli Yardımcı G, Saglik B m N, Ozkay Y, et al. Synthesis, antifungal activities, molecular docking and molecular dynamic studies of novel quinoxaline-triazole compounds. ACS Omega. 2023. - 80. Houšť J, Spížek J, Havlíček V. Antifungal drugs. Metabolites. 2020;10(3):106. pmid:32178468 - 81. Sakagami T, Kawano T, Yamashita K, Yamada E, Fujino N, Kaeriyama M, et al. Antifungal susceptibility trend and analysis of resistance mechanism for Candida species isolated from bloodstream at a Japanese university hospital. J Infect Chemother. 2019;25(1):34–40. pmid:30401513